Chin J Integr Med

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ORIGINAL ARTICLE Effects of Ginsenoside Rg-1 on the Proliferation and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells YIN Li-hua (殷丽华), CHENG Wen-xiao (程文晓), QIN Zi-shun (秦子顺), SUN Ke-mo (孙可墨), ZHONG Mei (钟 梅), WANG Jia-kui (王家奎), GAO Wei-yue (高维岳), and YU Zhan-hai (余占海) Objective:: To evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic ABSTRACT Objective differentiation of human periodontal ligament stem cells (hPDLSCs) and to explore the possible application on the Methods:: To determine the optimum concentration, the effects of ginsenoside Rg-1 alveolar bone regeneration. Methods ranging from 10 to 100 μmol/L were evaluated by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide, alkaline phosphatase activity and calcium deposition. Expressions of runt-related transcription factor 2, collagen alpha-2(I) chain, osteopontin, osteocalcin protein were examined using real-time polymerase chain reaction. Results:: Compared with the control group, a certain concentration (10 μmol/L) of the Rg-1 solution Results significantly enhanced the proliferation and osteogenic differentiation of hPDLSCs (P 1×10 6 cells) were washed and resuspended in phosphate buffered saline (PBS). For indirect immunostaining, cells were incubated on ice for 20 min, with 2.5 μg of STRO-1 antibody (R&D Systems, Minneapolis, MN, USA). After washing, samples were incubated with phycoerythrin (PE)conjugated secondary antibody (R&D Systems) on ice for 20 min, and then washed with PBS once again. After that, cells were analyzed using a flow cytometer (Becton Dickinson, Mountain View, CA, USA). CD146, CD34 and CD45 were detected as described above for STRO-1 with corresponding primary antibodies.

Alizarin Red S Staining-Based Calcium Deposition Analysis At the 21st day after culturing, the medium was removed and the cells were rinsed twice with PBS. Calcium deposition area of each group was examined using an Alizarin Red S Staining (Genmed Scientifics Inc., USA).

Preparation of Rg-1-Containing Culture Rg-1 (Bio-function, Beijing, China) was

dissolved in α-MEM containing 10% FBS and 0.05‰ dimethyl sulfoxide (DMSO, Sigma, USA) to obtain different concentrations (10 nmol/L, 100 nmol/L, 1 μmol/L, 10 μmol/L and 100 μmol/L).(13)

In vitro Experiments hPDLSCs were divided into 5 induced groups and 1 blank control. The induced groups were treated with specific concentrations (10, 100 nmol/L, 1, 10 and 100 μmol/L) of Rg-1 dissolved in α-MEM mineralized solution (90% α-MEM, 10% FBS, 10 nmol/L dexamethasone, 0.05 mmol/L ascorbic acid, 10 mmol/L β sodium glycerol phosphate). The blank control was treated by the mineralized solution only.

Proliferation Assays 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-diphenytetrazoliumromide (MTT) assay (Sigma, USA) was used to examine the proliferation of induced hPDLSCs. hPDLSCs were seeded in 96-well plates at a density of 2×103 cells/well and cultured at 37℃ with 5% CO2. At 24 h post seeding, the medium was replaced with the Rg-1 containing medium. Then cell culturing was terminated by adding 20 μL MTT to each well. After 4 h, 150 μL DMSO were added to assay cell proliferation through day 1 to day 5. The absorbance was determined at 490 nm using an enzyme linked immunosorbent assay reader (Elx 800, Bio-Tek, Winooski, VT, USA).

Measurement of Alkaline Phosphatase Activity The hPDLSCs (passage three) were seeded in 96well plates at a density of 2×103 cells/well and cultured for 24 h (37 ℃, 5% CO2) in α-MEM supplemented with 10% FBS. Then, the medium was removed and the cells were kept in Rg-1 containing medium for 3, 5 and 7 days, respectively. Cells were lysed by 0.1% Triton X-100 overnight at 4 ℃. The alkaline phosphatase (ALP) activity was measured by p-nitrophenyl phosphate (pNPP) using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The absorbance was determined at 520 nm using an enzyme linked immunosorbent assay reader.

Expression of Osteogenic Relative Genes The hPDLSCs at passage three were seeded in 6-well plates at a density of 2 ×104 cells/well and cultured for 24 h at 37 ℃ in 5% CO2. The medium was then removed and the cells were kept in Rg-1 containing medium for 7 days. The total RNA was extracted and cDNA was synthesized using real-time polymerase

Chin J Integr Med

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chain reaction (RT-PCR) machine (Real Time PCR System, ABI 7300, Applied Biosystems, USA) with Trizol, PrimeScript RT Master Mix and Premix Ex TagTM (all by Takara Bio Inc., Japan). The expression of runt-related transcription factor 2 (RUNX2), collagen alpha-2(Ⅰ) chain (COL1A2), osteopontin (OPN) and osteocalcin (OCN) were examined using the antisense primers listed in Table 1 (synthesized by Takara). Table 1.

RUNX2

As shown in Figure 2, the isolated cells were strongly positive for STRO-1 and CD146 (90.9% and 91.8%, respectively), which are the surface markers for PDLSCs.(5) They were unlikely the hematopoietic blood cells because of the lack of CD34 and CD45 markers (2.2% and 1.4%, respectively). Taken together, the data suggested that these cells isolated from PDL tissues were hPDLSCs.

RT-PCR Primers (Human)

GenBank accession

Gene

became adherent at 5–7 days and single cell colonies were observed 14 days after single-cell suspensions were cultured. The adherent single cells first showed a fusiform shape (Figure 1A) and then displayed directivity (Figure 1B) and eventually propagated into whirlpool-like confluence (Figure 1C).

Primer

Hs00231692

F5'-CACTGGCGCTGCAACAAGA-3' R5'-CATTCCGGAGCTCAGCAGAATAA-3'

COL1A2 Hs00164004

F5'-GAGGGCAACAGCAGGTTCACTTA-3' R5'-TGGGCCAATGTCCACAAAGA-3'

NM001040060 F5'-GATGAATCTGATGAACTGGTCACT-3'

Clones of cells presented cartilage matrix as examined by toluidine blue staining (Figure 3A). At 21 days after alizarin red staining, the wells were fully covered with mineralized deposits, suggesting successful osteogenic differentiation (Figure 3B). With regard to adipogenic differentiation, cells showed a larger number of clusters of lipid droplets after 21-day adipogenic stimulation (Figure 3C).

R5'-GGTGATGTCCTCGTCTGTAGCA-3' NM199173

F5'-GACGAGTTGGCTGACCACA-3' R5'-CAAGGGGAAGAGGAAAGAAGG-3'

Statistical Analysis SPSS 18.0 was used to analyse data. All results were expressed as mean ± standard deviation and statistical significance was determined by one-way analysis of variance (ANOVA). P -values less than 0.05 were considered to be significant.

Effect of Rg-1 on the Proliferation of hPDLSCs Using MTT assay, the effects of Rg-1 (10 nmol/L to 100 μmol/L) were examined on the proliferation of hPDLSCs. Along with the increase of concentration, the OD values increased substantially (Figure 4, P

Effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells.

To evaluate the effects of ginsenoside Rg-1 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and...
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