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目的:比较不同种类特定序列寡核苷酸(Oligonucleotide,ODN)对人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hBMSCs)增殖能力的影响。方法:密度梯度离心法分离培养hBMSCs,传至第3代进行成骨、成脂及成软骨诱导,鉴定其多向分化能力;将hBMSCs以不同密度接种(3.0×103/cm2、6.0×103/cm2、1.2×104/cm2、2.4×104/cm2),采用CCK-8连续检测7d,确立最佳铺板密度;依据ODN种类(MT01、FC003、Sa05f)和终浓度(0.5mg/L、1.0mg/L、2.0mg/L、4.0mg/L)分为12个实验组和1个对照组(加等量PBS),采用CCK-8连续检测3d,明确不同种类及浓度的ODN对hBMSCs增殖能力的影响。结果:分离培养的hBMSCs具有成骨、成脂和成软骨的多向分化能力;以6.0×103/cm2的接种密度铺板可获得良好的细胞生长曲线;hBMSCs在加入每种不同浓度的ODN后,与对照组相比,终浓度为0.5mg/L的ODN FC003在第1天检测到光密度值显著升高(P<0.01);终浓度为1.0mg/L和4.0mg/L的ODN Sa05f在第1天检测到光密度值均显著降低(分别为P<0.01,P<0.05);终浓度为2.0mg/L的ODN MT01在连续3d内的光密度值均明显升高,差异具有统计学意义(第1天P<0.01,第2,3天P<0.05)。结论:终浓度为2.0mg/L的ODN MT01可显著促进hBMSCs的增殖。
Objective: To compare the effects of different types of oligonucleotide (ODN) on the proliferation of human bone marrow mesenchymal stem cells (hBMSCs). Methods: The hBMSCs were isolated and cultured by density gradient centrifugation. The hBMSCs were passaged into the third passage to induce osteoblastic, adipogenic and chondrogenic differentiation. The hBMSCs were seeded at different densities (3.0 × 103 / cm2 and 6.0 × 103 / cm2, 1.2 × 10 4 / cm 2 and 2.4 × 10 4 / cm 2). The optimal plating density was determined by CCK-8 for 7 days. The optimum plating density was determined according to the ODN types (MT01, FC003 and Sa05f) /L,2.0mg/L,4.0mg/L) were divided into 12 experimental groups and 1 control group (plus equal amount of PBS), CCK-8 was used to detect the cells for 3 days, and the proliferative ability of hBMSCs with different kinds and concentrations of ODN Impact. Results: The hBMSCs cultured in vitro showed the ability of osteogenic, adipogenic and cartilage differentiation. Good cell growth curve was obtained when the hBMSCs were plated at the density of 6.0 × 103 / cm2. After adding hBMSCs with different concentration of ODN, Compared with the control group, ODN FC003 at the final concentration of 0.5mg / L showed a significant increase in optical density (P <0.01) on day 1; ODN Sa05f at the final concentrations of 1.0mg / L and 4.0mg / On the first day, the optical density values were significantly decreased (P <0.01, P <0.05, respectively); ODN MT01 at a final concentration of 2.0 mg / L increased significantly in three consecutive days with statistically significant differences Significance (P <0.01 on day 1, P <0.05 on days 2 and 3). CONCLUSION: ODN MT01 at a final concentration of 2.0 mg / L can significantly promote the proliferation of hBMSCs.