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Animal Science Journal (2014) 85, 735–743
doi: 10.1111/asj.12206
ORIGINAL ARTICLE Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs) Huaiping SHI,* Hengbo SHI,* Jun LUO, Wei WANG, Abiel B. HAILE, Huifen XU and Jun LI College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
ABSTRACT Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.
Key words: dairy goat, human telomerase, mammary epithelial cell.
INTRODUCTION Primary mammary epithelial cells have been widely used as a model to study the function of mammary tissue. They are a typical representative of the in vivo system, maintaining organ-specific functions and signal transduction pathways. However, it is a cumbersome task to consistently obtain tissues from dairy goat mammary glands under controlled conditions (i.e., season, stage of lactation), which greatly affect the characteristics of goat mammary epithelial cells (Pantschenko et al. 2000). Recently, the established epithelial cell lines from the mammary gland have been widely accepted and used as models to study the functional regulation of lactation in cows (Kadegowda et al. 2009; Huang et al. 2013; Shao et al. 2013). Similar research on dairy goats are still required to develop stable cell lines from the goat mammary gland. In the past two decades, several bovine and goat mammary epithelial cell lines had been established by stable integration of the simian virus large T-antigen (SV40LTA) gene and other methods (Gibson et al. 1991; © 2014 Japanese Society of Animal Science
Huynh et al. 1991; Zavizion et al. 1996; German & Barash 2002; Hu et al. 2009; Anand et al. 2012). However, it was undervalued with low to no lactation specific-protein, expression levels of some cell lines, and a worst case of abnormal karyotype (Gibson et al. 1991; Huynh et al. 1991; Pantschenko et al. 2000). Studies have shown that the forced expression of exogenous human telomerase reverse transcriptase (hTERT)in normal human cells is sufficient to produce telomerase activity in the cells and prevent the erosion of telomerase and surmount the induction of both senescence and crisis. A variety of cell types can be immortalized by human telomerase. Cells immortalized with hTERT have normal cell cycles, functional
Correspondence: Jun Luo, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. (Email: [email protected]) *These authors contributed equally to this paper. Received 25 September 2013; accepted for publication 14 January 2014.
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p53 and pRB checkpoints, are contact inhibited, are anchorage dependent, require growth factors for proliferation and possess a normal karyotype (Jiang et al. 1999; Morales et al. 1999). At present, it has become popular to immortalize animal cells such as pig cells (Hong et al. 2007), and one strain of mammary epithelial cell lines from dairy goats has been established through this method (He et al. 2009). However, due to the physiological dynamics of the mammary gland in lactating dairy goats, as related to age, the results conducted on single cell lines are unreliable to represent the whole properties of mammary gland cells. This necessitates the establishment of other cell lines from a dairy goat mammary gland to compensate for its limitation. Comparative results from different goat mammary cell lines are capable of truly reflecting the goat’s lactation in vitro. In the present study, goat mammary epithelial cells (MECs) were isolated from mammary tissues of 45-day post-parturition Xinong Saanen dairy goat (2 years old) and immortalized by hTERT. Various examinations in the cells showed that the expression of exogenous hTERT efficiently extended the cell lifespan without changing the characteristic phenotypic properties of goat MECs.
dishes coated with collagen and were reversely incubated at 37°C with saturated humidity and 5% CO2. After 30 min, 2 mL culture medium was added to the culture dish. The culture medium consisted of DMEM-F12 supplemented with 10% FBS, 1 μg/mL insulin, 1 μg/mL hydrocortisone, 100 IU/mL penicillin and 100 μg/mL streptomycin. The medium was replaced with fresh medium every 48 h until the cells were migrated out of the tissue and visibly spread across the bottom of the dish. Being cultured for about five passages, MECs were obtained according to Hu et al. (2009).
Construction of MECs transfected human telomerase (hT-MECs) The pCI-neo-hTERT vector was obtained from H. Y. Wang (Northwest A&F University, Yangling, China). The vector encodes a hTERT and carries the Neo gene. When up to 80% confluence in a six-well plate was reached, MECs were transfected with pCI neo-hTERT vector by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Next day, cells were placed under selection medium (culture medium with 400 μg/mL G418 sulfate). Selecting media were changed once every 2 days. After 2 weeks, surviving colonies were isolated with cloning rings. For the routine culture, hT-MECs were subcultured to 70–80% confluence (approximately every 2–3 days) in the growth medium (culture medium with 200 μg/mL of G418) and digested by trypsase for passage.
MTT MATERIALS AND METHODS Reagents and chemicals Rabbit anti-keratin 8 was obtained from the Biosynthesis Biotechnology Company (Beijing, China). Mouse antihTERT came from Epitomics Inc. (Burlingame, CA, USA). Rabbit anti-beta-casein was purchased from Crystalline Biological Technology Co Ltd (Shanghai, China). Rabbit antiepithelial membrane antigen (EMA) and SABC-Cy3 kit were purchased from Boster Bio-engineering Limited Company (Wuhan, China). Dulvecco’s modified Eagle’s medium (DMEM)-F12, fetal bovine serum (FBS) and trypsin were purchased from Gibco (Gibco Products International Inc., Langley, OK, USA). Insulin, hydrocortisone and prolactin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Telomeric Repeat Amplification Protocol – Polymerase Chain Reaction (TRAP-PCR) kit was purchased from Keygen Biotechnology Co. Ltd. (Nanjing, China). Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Tissue collection and MECs culture MECs were obtained from mammary gland biopsy of a 2-year-old (45 days post-parturition) Xinong Saanen dairy goat. After mammary tissues were surgically removed from the dairy goat, they were placed in a sterile ice-cold D-Hank’s solution supplemented with 300 IU/mL penicillin and were immediately transported to the laboratory. The mammary tissues were trimmed of visible fat and connective tissues and washed with D-Hank’s solution several times until the solution became pellucid and devoid of milk. Tissue sections were then minced into about 1 mm3 cubes and rinsed again with D-Hank’s solution and kept up for 10 min at room temperature. The smaller pieces of tissues were put onto the cell © 2014 Japanese Society of Animal Science
MECs or hT-MECs were seeded to about 1.0 × 104 cells/well in 96-well plates in culture medium or growth medium. The number and viability of cells (MECs on passages 7, 20 and 35; hT-MECs on passages 10, 28 and 42) were determined by methylthiotetrazole (MTT) assay every day with triplicate wells for 7 days.
Karyotyping analysis Karyotype analysis was performed as follows. Briefly, hT-MECs of logarithmic phase (passage 30) were treated for 6 h with 10−5 mol/L colchicines, detached with 0.25% trypsin, washed with PBS, treated with 0.1 mol/L KCl solution for 20 min at 37°C, fixed with acetic acid and ice-cold methanol at a ratio of 1:3, dropped onto ice-cold glass slides, stained with Giemsa solution, and eventually examined under the microscope.
Immunocytochemistry hT-MECs (passage 70) grown on glass coverslips were fixed with ice-cold methanol for 30 min, rinsed three times with PBS and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Primary rabbit anti-keratin 8 and antiEMA were incubated with cells overnight at 4°C. The cover slips were washed 4 × 2 min with PBS. Secondary antibody, goat anti-rabbit IgG, was added to the coverslips and incubated for 45 min in the dark. The coverslips were washed 4 × 2 min with PBS. SABC-Cy3 was added to the coverslips and incubated for 30 min at 37°C. The coverslips were washed 4 × 5 min with PBS. Finally, the stained cells were visualized by phase-contrast microscopy.
RNA extraction, PCR and qRT-PCR hT-MECs were cultured in growth medium. When hT-MECs (passage 40) were at 90% confluence, prolactin (final Animal Science Journal (2014) 85, 735–743
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Table 1
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Primer sequence and reactive condition of RT-PCR
Gene
Primer sequence (F)
Primer sequence (R)
Reactive condition of PCR
Length (bp)
β-casein
5′-CCCAGGCACAGTCTCT AGTCT-3′ 5′-TAACAATACAGGACTCT TTCGAGGC-3′
5′-GGCTCAACTGGATAT TTAGGGA-3′ 5′-CGCTATTGGAGCTGG AATTACC-3′
95°C 4 min; 94°C 15 s, 55°C 30 s, 72°C 30 s, 45 cycles 95°C 4 min; 94°C 15 s, 55°C 30 s, 72°C 30 s, 45 cycles
196
18S rRNA
concentration 10 μg/L) was added to the growth medium. The cells were collected both at 48 h or 72 h for RNA extraction or Oil red O analysis. Total RNA were extracted with ice-cold Trizol reagent (Invitrogen, USA) and treated with deoxyribonuclease (DNase) (Tiangen, Beijing, China). Complementary DNA (cDNA) were synthesized using the PrimeScript® RT kit (Takara, Otsu, Japan). The primer sequences of beta-casein were designed based on goat mRNA homology region sequence: forward: 5′-GCAAGAGAGC AGGAAGAACTCAAT-3′; reverse: 5′-GAAAGGGACAGCAC GGACTG-3′. The PCR product of beta-casein was 500 bp. The amplified product was analyzed by electrophoresis on a 1% agarose gel. Quantitative PCR (qPCR) was performed using SYBR (SYBR® Premix Ex Taq™ II, Takara, Japan). Primers of beta-casein and 18S ribosomal RNA (rRNA) are described in Table 1. Transcript levels of beta-casein were normalized to 18S rRNA. Additionally, the primers of genes related to fatty acid metabolism, including PPARG, SREBP, FASN, SCD, ACC and GAPDH, were designed as previously described (Shi et al. 2013).
Western blot analysis hT-MECs (passage 42) were amplified and collected to detect the activity of human telomerase using TRAP-PCR kit (Keygen, Nanjing, China). At the same time, using Michigan Cancer Foundation (MCF)-7 cells as a positive control, the expression of hTERT protein was detected through Western blotting. Similarly, when hT-MECs were cultured for 72 h, the culturing supernatants were collected immediately and the expression of beta-casein protein was detected through Western blotting, using goat milk as a positive control. Then equal amounts of proteins were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels transferred onto nitrocellulose papers and cultured with antibodies against beta-casein (Crystalline Biological Technology Co. Ltd, Shanghai, China) and hTERT. Eventually primary antibodies were detected with horseradish peroxidaseconjugated secondary antibodies followed by enhanced chemiluminescence development (Pierce Chemical, Rockford, IL, USA) and light detection with Bioshine Chemi Q 2550.
Oil red O hT-MECs (passage 40) treated with prolactin were fixed for 60 min in 10% formaldehyde solution and stained with oil red O for 30 min following the protocol as described before (Koopman et al. 2001). The lipid droplets of hT-MECs were then observed under the microscope.
RESULTS MECs from dairy goat mammary tissues The isolation of primary MECs from dairy goat mammary tissues is shown in Figure 1. After the tissues were cultured for 4 to 7 days, cells were emiAnimal Science Journal (2014) 85, 735–743
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grated from mammary tissue. Fibroblast-like (Fig. 1A) and epithelial-like (Fig. 1B) cells were observed in the plate, in which most of them were mixed together. MECs were separated after about five passages and purification (Fig. 1C), and had two to four nucleoli (Fig. 1D). MECs that extended from the tissue showed a cobblestone-like shape connected tightly with a clear boundary (Fig. 1C). When low cell-density MECs were planted in the dish, they grew together and formation of a cellular island was observed (Fig. 1D).
Establishment of hT-MECs MECs were immortalized and transfected the plasmid pCI-neo-hTERT. After the transfection of pCI-neohTERT, MECs were placed under G418 (400 μg/mL) selection. Three days later, some MECs ceased to grow, and 14 days later, most of the MECs were dead and only a few of them were still alive. The remaining cells contained the pCI-neo-hTERT with G418 resistance (Fig. 2A). Then, the remaining cells were placed in the medium containing 200 μg/mL G418 and rapidly proliferated (Fig. 2B). After continual culturing to day 12, the cells showed the typical cobblestone morphology of epithelial cells containing two to four nucleoli (Fig. 2C,D).
Telomerase activity in hT-MECs The effects of human telomerase on the internal stabilities of MECs were examined through TRAP-PCR. The MCF-7 cell line was used as the positive control. After the plasmid pCI-neo-hTERT were transfected into the MECs, the hTERT protein in the hT-MECs was obviously expressed (Fig. 2E). The hT-MECs sample exhibited ladder bands, similar to the positive control, which were almost not observed in the MECs sample (Fig. 2F). This certified that the activity of telomerase was high in hT-MECs.
Proliferation assays Proliferation potential of hTERT-MECs was assessed by comparison of hT-MECs at passages 10, 28 and 42 to MECs at passages 7, 20 and 35. Briefly, all of the cell types experienced a rapid increase in cell proliferation and after the cells were planted for 48 h and reached maximal growth rate near days 5–6, they showed an ‘S’ sigmoid growth curve in the detected time (Fig. 3). Population growth rate differences among hT-MECs at passages 10, 28 and 42 was not significant. In contrast © 2014 Japanese Society of Animal Science
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Figure 1 Photomicrographs of the process of obtaining dairy goat mammary epithelial cells and morphology of epithelial cells. (A) Fibroblast cells grew from mammary tissue (×200); (B) mammary epithelial cells grew from mammary tissue (×200); (C) purified goat mammary epithelial cells (×100); (D) epithelial cells form island-like structures and the number of nucleoli range from two to four (×200). Arrows indicate nucleolus.
Figure 2 Goat mammary epithelial cells (MECs) after infection by human telomerase reverse transcriptase (hTERT). (A) Cell clone with neo resistance (×200); (B) G418 resistant colonies (×200); (C) Mammary gland cells with homogeneous morphology (×200); (D) the number of hT-MECs nucleoli range from two to four (×200); Arrows indicate nucleolus. (E) Detection of hTERT protein, lane 1 was MCF-7 cells (positive control), lane 2 was hT-MECs, lane 3 was MECs. (F) Detection of telomerase activity, lane 1 was MCF-7 cells, lane 2 was hT-MECs, lane 3 was MECs.
© 2014 Japanese Society of Animal Science
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1.2
Abscorption values
1.0 0.8 0.6 0.4 0.2 0 1
2
3
4
5
6
7 (day)
The 7th generation of normal cell The 20th generation of normal cell The 35th generation of normal cell The 10th generation of transgenic cell The 28th generation of transgenic cell The 42th generation of transgenic cell Figure 3 Growth curves of human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passages 10, 28 42 and MECs at passages 7, 20 and 35.
there was a significant difference between hT-MECs at 10, 28 and 42 passages and MECs at 7, 20 and 35 passages. In addition the growth rate of MECs at passage 35 was significantly lower than other groups. The above results indicate that the growth rate of MECs is degenerated with the increase of passage; nevertheless human telomerase is capable of extending MECs’ lifespan.
Karyotype analysis The chromosomal profile was determined employing passage 30 hT-MECs. The chromosome counts of hT-MECs were 50 to 120, and almost all of hT-MECs contained 60 chromosomes, including 29 pairs of autosomes and 1 pair of sex chromosomes (Fig. 4). Karyotype analysis of hT-MECs showed a normal modal chromosome.
Immunofluorescence Keratin 8 and EMA are two marker proteins of mammary epithelial cells. The immunofluorescence experiment was preformed to detect the existence of the two proteins in hT-MECs. The fibroblast cell from goat mammary gland was used as a negative control. As shown in Figure 5, hT-MECs had intense staining for keratin 8 and EMA (Fig. 5A,C) whereas almost no staining was detected in the fibroblasts (Fig. 5B, D). These results prove that hT-MECs still keep the characteristics of epithelial cells.
Beta-casein expression in hT-MECs Beta-casein is a marker protein representing the lactating function of MECs (Huynh et al. 1991). ExpresAnimal Science Journal (2014) 85, 735–743
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sion of beta-casein mRNA in hT-MECs (passage 40) was determined by PCR and agarose gel electrophoresis. As shown in Figure 6 an expected 500 bp band was amplified; this certified the expression of beta-casein gene in the hT-MECs through DNA sequencing. Additionally, the culturing supernatants from hT-MECs (passage 40) were employed to analyze the beta-casein protein. Figure 6B witnessed that there were betacasein proteins in the cell supernatant. The results of qRT-PCR showed that prolactin could enhance the mRNA expression levels of beta-casein in hT-MECs. The expression level of beta-casein mRNA at 72 h was higher (P < 0.05) than that of 48 h under the treatment of prolactin (Fig. 6C).
Cell lipid droplets Through staining hT-MECs (passage 40) with oil red O, we found that lipid droplets lay in the cytoplasm with different sizes. Lipid droplets in hT-MECs had no discrepancies compared with that in MECs (Fig. 7A,B). Furthermore, it was found that the mRNA level of genes (PPARG, SREBP, FASN, ACC and SCD) involved in fatty acid synthesis in hT-MECs was not different compared with MECs (Fig. 7C,D), which suggests that fatty acid synthesis in hT-MECs is unaffected by human telomerase.
DISCUSSION Lactation is an important physiological activity of dairy goats. From the beginning (onset) of lactation to peak lactation and the dry period, the lactation curve of dairy goats shows a parabolic curve (Gibson et al. 1991). In contrast to the lactation curve, the proportion of milk fat reduces with the increase of milk yield and increases with the reduction of milk yield. Goat milk is richer in medium and short chain fatty acids than cow milk, this suggests differences in mammary gland fatty acid metabolism between the two species. To understand fatty acid regulation in dairy goat mammary glands, a representative model of cells is vital. In this study, an immortal mammary epithelial cell line from a 45 days post-parturition dairy goat (2 years old) was established to meet the demand of further research in dairy goat lactation. Previous reports showed that the primary MECs spread at 6 or 25 passages (Ouyang & Qian 1999; Song et al. 2003; Li et al. 2005). However, most of the cells on 25 passages began to grow poorly with an enlarged volume; moreover, some of the cell lines were expressing none to low levels of lactation-specific proteins (Gibson et al. 1991; Huynh et al. 1991). In this communication, we found that the growth of passage-35 MECs was invariably slow, far less than that of passage 7 MECs. This is a clear indication that the cultured MECs in vitro gradually end up having degraded growth. This phenomenon is supposedly due to © 2014 Japanese Society of Animal Science
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Figure 4 Chromosome karyotype analyses. A near diploid (60, XX) number of chromosomes were detected in the majority of human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passage 30 and a typical and representative chromosome karyotype is shown.
Figure 5 Immunocytochemistry analyses. (A) Keratin 8 in human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) (×200); (B) keratin 8 in fibroblast cell (×200); (C) epithelial membrane antigen (EMA) in hT-MECs (×200); (D) EMA in fibroblast cell (×200).
damaged or induced differentiation of some cell lines in the process of culturing, which shortens the cell lifespan. Since the defective cells hardly showed the whole function of cells, it is implausible to conduct research through them. Therefore, it is very important to find ways of extending MECs lifespan and forming a stable cell line. Due to its role in extending cell lifespan, telomerase has been used to establish immortal cells (Wu et al. 2010). In this paper, we constructed stable MECs by © 2014 Japanese Society of Animal Science
employing the human telomerase (hT-MECs). The results showed that hT-MECs maintained typical phenotypic characteristics of cuboidal epithelial cells, and had a diploid karyotype with modal chromosome number 60. Furthermore, telomerase activity analysis showed that the protein expression of hT-MECs was similar to MCF-7 cells and obviously higher than that of MECs. Altogether, these data suggests that the established cell line (hT-MECs) has been protected by human telomerase. It is certified in Figure 3 that Animal Science Journal (2014) 85, 735–743
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Figure 6 Beta-casein expressions in human telomerase reverse transcriptase mammary epithelial cells (hT-MECs). (A) Beta-casein mRNA expression; (B) Beta-casein protein expression in hT-MECs (passage 40) (1 hT-MECs culture, 2 Goat milk); (C) Beta-casein mRNA expression in the presence of prolactin. The values with different letter superscripts in the histograms mean significant differences (P < 0.05) and the values with same letter superscripts mean no significant differences (P > 0.05).
hT-MECs at passage 42 still had rapid cell growth, which was similar to MECs at passage 7. The properties of hT-MECs were also investigated through molecular methods. Keratin 8 and EMA were greatly expressed in hT-MECs, which showed that hT-MECs maintained the properties of epithelial cells. hT-MECs were capable of expressing beta-casein protein and responded to prolactin. Furthermore in hT-MECs, we observed the formation of lipid droplets which resemble MECs. Lipid droplets are lipid-rich cellular organelles that regulate the storage and hydrolysis of neutral lipids. In non-adipocytes, lipid droplets are known to play a role in protection from lipotoxicity by storing free fatty acids through esterification (Ducharme & Bickel 2008; Laura et al. 2010). mRNA levels of PPARG, SREBP1, FASN, SCD and ACC related to fatty acid synthesis in hT-MECs have no Animal Science Journal (2014) 85, 735–743
significant change compared with the control. All of the results show that hT-MECs exert their physiological function normally, and can be used as a model for goat lactation research. Although some reports showed that cell senescence could not be simply overcome by ectopic expression of telomerase (Kiyono et al. 1998; Dickson et al. 2000), hT-MECs in our lab grow with normal morphology, growth characteristics, cytogenetic and secretory characteristics for 1 year (more than 100 passages). Our data agree with previous a report that a goat MECs line immortalized by hTERT had no sign of senescence surpassing 80 passages (He et al. 2009). However, testing stability of this cell line requires further experiments. In conclusion, MECs can be immortalized by hTERT. The obtained hT-MECs line had normal morphology, © 2014 Japanese Society of Animal Science
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Figure 7 Oil red staining of the mammary gland epithelial cell (200×). (A) Human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passage 40); (B) MECs at passage 7. Arrows mean lipid droplets. (C) The expressions of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) and MECs (passage 7).
growth characteristics, cytogenetic and secretory characteristics. It showed normal characteristics similar to primary goat MECs. Hence, it might represent a useful tool for studying the function of dairy goat MECs. The cell model will further help to resolve and investigate molecular mechanisms such as fatty acid and protein synthesis in dairy goat lactation.
ACKNOWLEDGMENTS This research was jointly supported by the ‘National Natural Science Foundation of China (31272409)’ and the “Special Fund for Agro-scientiic Research in the Public Interest (201103038).
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span become immortal yet retain normal growth and differentiation characteristics. Molecular and Cellular Biology 20, 1436–1447. Ducharme NA, Bickel PE. 2008. Lipid droplets in lipogenesis and lipolysis. Endocrinology 149, 942–949. German T, Barash I. 2002. Characterization of an epithelial cell line from bovine mammary gland. In Vitro Cellular & Developmental Biology – Animal 38, 282–292. Gibson CA, Vega JR, Baumrucker CR, Oakley CS, Welsch CW. 1991. Establishment and characterization of bovine mammary epithelial cell lines. In Vitro Cellular & Developmental Biology 27A, 585–594. He YL, Wu YH, He XN, Liu FJ, He XY, Zhang Y. 2009. An immortalized goat mammary epithelial cell line induced with human telomerase reverse transcriptase (hTERT) gene transfer. Theriogenology 71, 1417–1424. Hong HX, Zhang YM, Xu H, Su ZY, Sun P. 2007. Immortalization of swine umbilical vein endothelial cells with human telomerase reverse transcriptase. Molecules and Cells 24, 358–363. Hu H, Wang J, Bu D, Wei H, Zhou L, Li F, Loor JJ. 2009. In vitro culture and characterization of a mammary epithelial cell line from Chinese Holstein dairy cow. PLoS ONE 4, e7636. Huang YL, Zhao F, Luo CC, Zhang X, Si Y, Sun Z, et al. 2013. SOCS3-mediated blockade reveals Major contribution of Animal Science Journal (2014) 85, 735–743
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© 2014 Japanese Society of Animal Science
Animal Science Journal (2014) 85, 735–743
doi: 10.1111/asj.12206
ORIGINAL ARTICLE Establishment and characterization of a dairy goat mammary epithelial cell line with human telomerase (hT-MECs) Huaiping SHI,* Hengbo SHI,* Jun LUO, Wei WANG, Abiel B. HAILE, Huifen XU and Jun LI College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, China
ABSTRACT Although research on dairy goat mammary gland have referred extensively to molecular mechanisms, research on lines of dairy goat mammary epithelial cells (MECs) are still rare. This paper sought to establish an immortal MEC line by stable transfection of human telomerase. MECs from a lactating (45 days post-parturition) Xinong Saanen dairy goat were cultured purely and subsequently transfected with a plasmid carrying the sequence of human telomerase. Immortalized MECs by human telomerase (hT-MECs) exhibited a typical cobblestone morphology and activity and expression levels of telomerase resembled that of MCF-7 cells. hT-MECs on passage 42 grew vigorously and ‘S’ sigmoid curves of growth were observed. Moreover, hT-MECs maintained a normal chromosome modal number of 2n = 60, keratin 8 and epithelial membrane antigen (EMA) were evidently expressed, and beta-casein protein was synthesized and secreted. Beta-casein expression was enhanced by prolactin (P < 0.05). Lipid droplets were found in hT-MECs, and messenger RNA levels of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) were similar to MECs (passage 7). In conclusion, the obtained hT-MEC line retained a normal morphology, growth characteristics, cytogenetics and secretory characteristics as primary MECs. Hence, it can be a representative model cell line, for molecular and functional analysis, of dairy goat MECs for an extended period of time.
Key words: dairy goat, human telomerase, mammary epithelial cell.
INTRODUCTION Primary mammary epithelial cells have been widely used as a model to study the function of mammary tissue. They are a typical representative of the in vivo system, maintaining organ-specific functions and signal transduction pathways. However, it is a cumbersome task to consistently obtain tissues from dairy goat mammary glands under controlled conditions (i.e., season, stage of lactation), which greatly affect the characteristics of goat mammary epithelial cells (Pantschenko et al. 2000). Recently, the established epithelial cell lines from the mammary gland have been widely accepted and used as models to study the functional regulation of lactation in cows (Kadegowda et al. 2009; Huang et al. 2013; Shao et al. 2013). Similar research on dairy goats are still required to develop stable cell lines from the goat mammary gland. In the past two decades, several bovine and goat mammary epithelial cell lines had been established by stable integration of the simian virus large T-antigen (SV40LTA) gene and other methods (Gibson et al. 1991; © 2014 Japanese Society of Animal Science
Huynh et al. 1991; Zavizion et al. 1996; German & Barash 2002; Hu et al. 2009; Anand et al. 2012). However, it was undervalued with low to no lactation specific-protein, expression levels of some cell lines, and a worst case of abnormal karyotype (Gibson et al. 1991; Huynh et al. 1991; Pantschenko et al. 2000). Studies have shown that the forced expression of exogenous human telomerase reverse transcriptase (hTERT)in normal human cells is sufficient to produce telomerase activity in the cells and prevent the erosion of telomerase and surmount the induction of both senescence and crisis. A variety of cell types can be immortalized by human telomerase. Cells immortalized with hTERT have normal cell cycles, functional
Correspondence: Jun Luo, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. (Email: [email protected]) *These authors contributed equally to this paper. Received 25 September 2013; accepted for publication 14 January 2014.
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p53 and pRB checkpoints, are contact inhibited, are anchorage dependent, require growth factors for proliferation and possess a normal karyotype (Jiang et al. 1999; Morales et al. 1999). At present, it has become popular to immortalize animal cells such as pig cells (Hong et al. 2007), and one strain of mammary epithelial cell lines from dairy goats has been established through this method (He et al. 2009). However, due to the physiological dynamics of the mammary gland in lactating dairy goats, as related to age, the results conducted on single cell lines are unreliable to represent the whole properties of mammary gland cells. This necessitates the establishment of other cell lines from a dairy goat mammary gland to compensate for its limitation. Comparative results from different goat mammary cell lines are capable of truly reflecting the goat’s lactation in vitro. In the present study, goat mammary epithelial cells (MECs) were isolated from mammary tissues of 45-day post-parturition Xinong Saanen dairy goat (2 years old) and immortalized by hTERT. Various examinations in the cells showed that the expression of exogenous hTERT efficiently extended the cell lifespan without changing the characteristic phenotypic properties of goat MECs.
dishes coated with collagen and were reversely incubated at 37°C with saturated humidity and 5% CO2. After 30 min, 2 mL culture medium was added to the culture dish. The culture medium consisted of DMEM-F12 supplemented with 10% FBS, 1 μg/mL insulin, 1 μg/mL hydrocortisone, 100 IU/mL penicillin and 100 μg/mL streptomycin. The medium was replaced with fresh medium every 48 h until the cells were migrated out of the tissue and visibly spread across the bottom of the dish. Being cultured for about five passages, MECs were obtained according to Hu et al. (2009).
Construction of MECs transfected human telomerase (hT-MECs) The pCI-neo-hTERT vector was obtained from H. Y. Wang (Northwest A&F University, Yangling, China). The vector encodes a hTERT and carries the Neo gene. When up to 80% confluence in a six-well plate was reached, MECs were transfected with pCI neo-hTERT vector by Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Next day, cells were placed under selection medium (culture medium with 400 μg/mL G418 sulfate). Selecting media were changed once every 2 days. After 2 weeks, surviving colonies were isolated with cloning rings. For the routine culture, hT-MECs were subcultured to 70–80% confluence (approximately every 2–3 days) in the growth medium (culture medium with 200 μg/mL of G418) and digested by trypsase for passage.
MTT MATERIALS AND METHODS Reagents and chemicals Rabbit anti-keratin 8 was obtained from the Biosynthesis Biotechnology Company (Beijing, China). Mouse antihTERT came from Epitomics Inc. (Burlingame, CA, USA). Rabbit anti-beta-casein was purchased from Crystalline Biological Technology Co Ltd (Shanghai, China). Rabbit antiepithelial membrane antigen (EMA) and SABC-Cy3 kit were purchased from Boster Bio-engineering Limited Company (Wuhan, China). Dulvecco’s modified Eagle’s medium (DMEM)-F12, fetal bovine serum (FBS) and trypsin were purchased from Gibco (Gibco Products International Inc., Langley, OK, USA). Insulin, hydrocortisone and prolactin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Telomeric Repeat Amplification Protocol – Polymerase Chain Reaction (TRAP-PCR) kit was purchased from Keygen Biotechnology Co. Ltd. (Nanjing, China). Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Tissue collection and MECs culture MECs were obtained from mammary gland biopsy of a 2-year-old (45 days post-parturition) Xinong Saanen dairy goat. After mammary tissues were surgically removed from the dairy goat, they were placed in a sterile ice-cold D-Hank’s solution supplemented with 300 IU/mL penicillin and were immediately transported to the laboratory. The mammary tissues were trimmed of visible fat and connective tissues and washed with D-Hank’s solution several times until the solution became pellucid and devoid of milk. Tissue sections were then minced into about 1 mm3 cubes and rinsed again with D-Hank’s solution and kept up for 10 min at room temperature. The smaller pieces of tissues were put onto the cell © 2014 Japanese Society of Animal Science
MECs or hT-MECs were seeded to about 1.0 × 104 cells/well in 96-well plates in culture medium or growth medium. The number and viability of cells (MECs on passages 7, 20 and 35; hT-MECs on passages 10, 28 and 42) were determined by methylthiotetrazole (MTT) assay every day with triplicate wells for 7 days.
Karyotyping analysis Karyotype analysis was performed as follows. Briefly, hT-MECs of logarithmic phase (passage 30) were treated for 6 h with 10−5 mol/L colchicines, detached with 0.25% trypsin, washed with PBS, treated with 0.1 mol/L KCl solution for 20 min at 37°C, fixed with acetic acid and ice-cold methanol at a ratio of 1:3, dropped onto ice-cold glass slides, stained with Giemsa solution, and eventually examined under the microscope.
Immunocytochemistry hT-MECs (passage 70) grown on glass coverslips were fixed with ice-cold methanol for 30 min, rinsed three times with PBS and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. Primary rabbit anti-keratin 8 and antiEMA were incubated with cells overnight at 4°C. The cover slips were washed 4 × 2 min with PBS. Secondary antibody, goat anti-rabbit IgG, was added to the coverslips and incubated for 45 min in the dark. The coverslips were washed 4 × 2 min with PBS. SABC-Cy3 was added to the coverslips and incubated for 30 min at 37°C. The coverslips were washed 4 × 5 min with PBS. Finally, the stained cells were visualized by phase-contrast microscopy.
RNA extraction, PCR and qRT-PCR hT-MECs were cultured in growth medium. When hT-MECs (passage 40) were at 90% confluence, prolactin (final Animal Science Journal (2014) 85, 735–743
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Table 1
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Primer sequence and reactive condition of RT-PCR
Gene
Primer sequence (F)
Primer sequence (R)
Reactive condition of PCR
Length (bp)
β-casein
5′-CCCAGGCACAGTCTCT AGTCT-3′ 5′-TAACAATACAGGACTCT TTCGAGGC-3′
5′-GGCTCAACTGGATAT TTAGGGA-3′ 5′-CGCTATTGGAGCTGG AATTACC-3′
95°C 4 min; 94°C 15 s, 55°C 30 s, 72°C 30 s, 45 cycles 95°C 4 min; 94°C 15 s, 55°C 30 s, 72°C 30 s, 45 cycles
196
18S rRNA
concentration 10 μg/L) was added to the growth medium. The cells were collected both at 48 h or 72 h for RNA extraction or Oil red O analysis. Total RNA were extracted with ice-cold Trizol reagent (Invitrogen, USA) and treated with deoxyribonuclease (DNase) (Tiangen, Beijing, China). Complementary DNA (cDNA) were synthesized using the PrimeScript® RT kit (Takara, Otsu, Japan). The primer sequences of beta-casein were designed based on goat mRNA homology region sequence: forward: 5′-GCAAGAGAGC AGGAAGAACTCAAT-3′; reverse: 5′-GAAAGGGACAGCAC GGACTG-3′. The PCR product of beta-casein was 500 bp. The amplified product was analyzed by electrophoresis on a 1% agarose gel. Quantitative PCR (qPCR) was performed using SYBR (SYBR® Premix Ex Taq™ II, Takara, Japan). Primers of beta-casein and 18S ribosomal RNA (rRNA) are described in Table 1. Transcript levels of beta-casein were normalized to 18S rRNA. Additionally, the primers of genes related to fatty acid metabolism, including PPARG, SREBP, FASN, SCD, ACC and GAPDH, were designed as previously described (Shi et al. 2013).
Western blot analysis hT-MECs (passage 42) were amplified and collected to detect the activity of human telomerase using TRAP-PCR kit (Keygen, Nanjing, China). At the same time, using Michigan Cancer Foundation (MCF)-7 cells as a positive control, the expression of hTERT protein was detected through Western blotting. Similarly, when hT-MECs were cultured for 72 h, the culturing supernatants were collected immediately and the expression of beta-casein protein was detected through Western blotting, using goat milk as a positive control. Then equal amounts of proteins were separated by electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels transferred onto nitrocellulose papers and cultured with antibodies against beta-casein (Crystalline Biological Technology Co. Ltd, Shanghai, China) and hTERT. Eventually primary antibodies were detected with horseradish peroxidaseconjugated secondary antibodies followed by enhanced chemiluminescence development (Pierce Chemical, Rockford, IL, USA) and light detection with Bioshine Chemi Q 2550.
Oil red O hT-MECs (passage 40) treated with prolactin were fixed for 60 min in 10% formaldehyde solution and stained with oil red O for 30 min following the protocol as described before (Koopman et al. 2001). The lipid droplets of hT-MECs were then observed under the microscope.
RESULTS MECs from dairy goat mammary tissues The isolation of primary MECs from dairy goat mammary tissues is shown in Figure 1. After the tissues were cultured for 4 to 7 days, cells were emiAnimal Science Journal (2014) 85, 735–743
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grated from mammary tissue. Fibroblast-like (Fig. 1A) and epithelial-like (Fig. 1B) cells were observed in the plate, in which most of them were mixed together. MECs were separated after about five passages and purification (Fig. 1C), and had two to four nucleoli (Fig. 1D). MECs that extended from the tissue showed a cobblestone-like shape connected tightly with a clear boundary (Fig. 1C). When low cell-density MECs were planted in the dish, they grew together and formation of a cellular island was observed (Fig. 1D).
Establishment of hT-MECs MECs were immortalized and transfected the plasmid pCI-neo-hTERT. After the transfection of pCI-neohTERT, MECs were placed under G418 (400 μg/mL) selection. Three days later, some MECs ceased to grow, and 14 days later, most of the MECs were dead and only a few of them were still alive. The remaining cells contained the pCI-neo-hTERT with G418 resistance (Fig. 2A). Then, the remaining cells were placed in the medium containing 200 μg/mL G418 and rapidly proliferated (Fig. 2B). After continual culturing to day 12, the cells showed the typical cobblestone morphology of epithelial cells containing two to four nucleoli (Fig. 2C,D).
Telomerase activity in hT-MECs The effects of human telomerase on the internal stabilities of MECs were examined through TRAP-PCR. The MCF-7 cell line was used as the positive control. After the plasmid pCI-neo-hTERT were transfected into the MECs, the hTERT protein in the hT-MECs was obviously expressed (Fig. 2E). The hT-MECs sample exhibited ladder bands, similar to the positive control, which were almost not observed in the MECs sample (Fig. 2F). This certified that the activity of telomerase was high in hT-MECs.
Proliferation assays Proliferation potential of hTERT-MECs was assessed by comparison of hT-MECs at passages 10, 28 and 42 to MECs at passages 7, 20 and 35. Briefly, all of the cell types experienced a rapid increase in cell proliferation and after the cells were planted for 48 h and reached maximal growth rate near days 5–6, they showed an ‘S’ sigmoid growth curve in the detected time (Fig. 3). Population growth rate differences among hT-MECs at passages 10, 28 and 42 was not significant. In contrast © 2014 Japanese Society of Animal Science
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Figure 1 Photomicrographs of the process of obtaining dairy goat mammary epithelial cells and morphology of epithelial cells. (A) Fibroblast cells grew from mammary tissue (×200); (B) mammary epithelial cells grew from mammary tissue (×200); (C) purified goat mammary epithelial cells (×100); (D) epithelial cells form island-like structures and the number of nucleoli range from two to four (×200). Arrows indicate nucleolus.
Figure 2 Goat mammary epithelial cells (MECs) after infection by human telomerase reverse transcriptase (hTERT). (A) Cell clone with neo resistance (×200); (B) G418 resistant colonies (×200); (C) Mammary gland cells with homogeneous morphology (×200); (D) the number of hT-MECs nucleoli range from two to four (×200); Arrows indicate nucleolus. (E) Detection of hTERT protein, lane 1 was MCF-7 cells (positive control), lane 2 was hT-MECs, lane 3 was MECs. (F) Detection of telomerase activity, lane 1 was MCF-7 cells, lane 2 was hT-MECs, lane 3 was MECs.
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1.2
Abscorption values
1.0 0.8 0.6 0.4 0.2 0 1
2
3
4
5
6
7 (day)
The 7th generation of normal cell The 20th generation of normal cell The 35th generation of normal cell The 10th generation of transgenic cell The 28th generation of transgenic cell The 42th generation of transgenic cell Figure 3 Growth curves of human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passages 10, 28 42 and MECs at passages 7, 20 and 35.
there was a significant difference between hT-MECs at 10, 28 and 42 passages and MECs at 7, 20 and 35 passages. In addition the growth rate of MECs at passage 35 was significantly lower than other groups. The above results indicate that the growth rate of MECs is degenerated with the increase of passage; nevertheless human telomerase is capable of extending MECs’ lifespan.
Karyotype analysis The chromosomal profile was determined employing passage 30 hT-MECs. The chromosome counts of hT-MECs were 50 to 120, and almost all of hT-MECs contained 60 chromosomes, including 29 pairs of autosomes and 1 pair of sex chromosomes (Fig. 4). Karyotype analysis of hT-MECs showed a normal modal chromosome.
Immunofluorescence Keratin 8 and EMA are two marker proteins of mammary epithelial cells. The immunofluorescence experiment was preformed to detect the existence of the two proteins in hT-MECs. The fibroblast cell from goat mammary gland was used as a negative control. As shown in Figure 5, hT-MECs had intense staining for keratin 8 and EMA (Fig. 5A,C) whereas almost no staining was detected in the fibroblasts (Fig. 5B, D). These results prove that hT-MECs still keep the characteristics of epithelial cells.
Beta-casein expression in hT-MECs Beta-casein is a marker protein representing the lactating function of MECs (Huynh et al. 1991). ExpresAnimal Science Journal (2014) 85, 735–743
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sion of beta-casein mRNA in hT-MECs (passage 40) was determined by PCR and agarose gel electrophoresis. As shown in Figure 6 an expected 500 bp band was amplified; this certified the expression of beta-casein gene in the hT-MECs through DNA sequencing. Additionally, the culturing supernatants from hT-MECs (passage 40) were employed to analyze the beta-casein protein. Figure 6B witnessed that there were betacasein proteins in the cell supernatant. The results of qRT-PCR showed that prolactin could enhance the mRNA expression levels of beta-casein in hT-MECs. The expression level of beta-casein mRNA at 72 h was higher (P < 0.05) than that of 48 h under the treatment of prolactin (Fig. 6C).
Cell lipid droplets Through staining hT-MECs (passage 40) with oil red O, we found that lipid droplets lay in the cytoplasm with different sizes. Lipid droplets in hT-MECs had no discrepancies compared with that in MECs (Fig. 7A,B). Furthermore, it was found that the mRNA level of genes (PPARG, SREBP, FASN, ACC and SCD) involved in fatty acid synthesis in hT-MECs was not different compared with MECs (Fig. 7C,D), which suggests that fatty acid synthesis in hT-MECs is unaffected by human telomerase.
DISCUSSION Lactation is an important physiological activity of dairy goats. From the beginning (onset) of lactation to peak lactation and the dry period, the lactation curve of dairy goats shows a parabolic curve (Gibson et al. 1991). In contrast to the lactation curve, the proportion of milk fat reduces with the increase of milk yield and increases with the reduction of milk yield. Goat milk is richer in medium and short chain fatty acids than cow milk, this suggests differences in mammary gland fatty acid metabolism between the two species. To understand fatty acid regulation in dairy goat mammary glands, a representative model of cells is vital. In this study, an immortal mammary epithelial cell line from a 45 days post-parturition dairy goat (2 years old) was established to meet the demand of further research in dairy goat lactation. Previous reports showed that the primary MECs spread at 6 or 25 passages (Ouyang & Qian 1999; Song et al. 2003; Li et al. 2005). However, most of the cells on 25 passages began to grow poorly with an enlarged volume; moreover, some of the cell lines were expressing none to low levels of lactation-specific proteins (Gibson et al. 1991; Huynh et al. 1991). In this communication, we found that the growth of passage-35 MECs was invariably slow, far less than that of passage 7 MECs. This is a clear indication that the cultured MECs in vitro gradually end up having degraded growth. This phenomenon is supposedly due to © 2014 Japanese Society of Animal Science
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Figure 4 Chromosome karyotype analyses. A near diploid (60, XX) number of chromosomes were detected in the majority of human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passage 30 and a typical and representative chromosome karyotype is shown.
Figure 5 Immunocytochemistry analyses. (A) Keratin 8 in human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) (×200); (B) keratin 8 in fibroblast cell (×200); (C) epithelial membrane antigen (EMA) in hT-MECs (×200); (D) EMA in fibroblast cell (×200).
damaged or induced differentiation of some cell lines in the process of culturing, which shortens the cell lifespan. Since the defective cells hardly showed the whole function of cells, it is implausible to conduct research through them. Therefore, it is very important to find ways of extending MECs lifespan and forming a stable cell line. Due to its role in extending cell lifespan, telomerase has been used to establish immortal cells (Wu et al. 2010). In this paper, we constructed stable MECs by © 2014 Japanese Society of Animal Science
employing the human telomerase (hT-MECs). The results showed that hT-MECs maintained typical phenotypic characteristics of cuboidal epithelial cells, and had a diploid karyotype with modal chromosome number 60. Furthermore, telomerase activity analysis showed that the protein expression of hT-MECs was similar to MCF-7 cells and obviously higher than that of MECs. Altogether, these data suggests that the established cell line (hT-MECs) has been protected by human telomerase. It is certified in Figure 3 that Animal Science Journal (2014) 85, 735–743
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Figure 6 Beta-casein expressions in human telomerase reverse transcriptase mammary epithelial cells (hT-MECs). (A) Beta-casein mRNA expression; (B) Beta-casein protein expression in hT-MECs (passage 40) (1 hT-MECs culture, 2 Goat milk); (C) Beta-casein mRNA expression in the presence of prolactin. The values with different letter superscripts in the histograms mean significant differences (P < 0.05) and the values with same letter superscripts mean no significant differences (P > 0.05).
hT-MECs at passage 42 still had rapid cell growth, which was similar to MECs at passage 7. The properties of hT-MECs were also investigated through molecular methods. Keratin 8 and EMA were greatly expressed in hT-MECs, which showed that hT-MECs maintained the properties of epithelial cells. hT-MECs were capable of expressing beta-casein protein and responded to prolactin. Furthermore in hT-MECs, we observed the formation of lipid droplets which resemble MECs. Lipid droplets are lipid-rich cellular organelles that regulate the storage and hydrolysis of neutral lipids. In non-adipocytes, lipid droplets are known to play a role in protection from lipotoxicity by storing free fatty acids through esterification (Ducharme & Bickel 2008; Laura et al. 2010). mRNA levels of PPARG, SREBP1, FASN, SCD and ACC related to fatty acid synthesis in hT-MECs have no Animal Science Journal (2014) 85, 735–743
significant change compared with the control. All of the results show that hT-MECs exert their physiological function normally, and can be used as a model for goat lactation research. Although some reports showed that cell senescence could not be simply overcome by ectopic expression of telomerase (Kiyono et al. 1998; Dickson et al. 2000), hT-MECs in our lab grow with normal morphology, growth characteristics, cytogenetic and secretory characteristics for 1 year (more than 100 passages). Our data agree with previous a report that a goat MECs line immortalized by hTERT had no sign of senescence surpassing 80 passages (He et al. 2009). However, testing stability of this cell line requires further experiments. In conclusion, MECs can be immortalized by hTERT. The obtained hT-MECs line had normal morphology, © 2014 Japanese Society of Animal Science
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Figure 7 Oil red staining of the mammary gland epithelial cell (200×). (A) Human telomerase reverse transcriptase mammary epithelial cells (hT-MECs) at passage 40); (B) MECs at passage 7. Arrows mean lipid droplets. (C) The expressions of PPARG, SREBP, FASN, ACC and SCD in hT-MECs (passage 40) and MECs (passage 7).
growth characteristics, cytogenetic and secretory characteristics. It showed normal characteristics similar to primary goat MECs. Hence, it might represent a useful tool for studying the function of dairy goat MECs. The cell model will further help to resolve and investigate molecular mechanisms such as fatty acid and protein synthesis in dairy goat lactation.
ACKNOWLEDGMENTS This research was jointly supported by the ‘National Natural Science Foundation of China (31272409)’ and the “Special Fund for Agro-scientiic Research in the Public Interest (201103038).
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