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[目的]探讨体外负压培养对hBMSCs成骨活性的影响。[方法]取第3代hBMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17 kPa,30 m in/次,4次/d,干预2周;对照组于普通CO2培养箱中常规培养。倒置显微镜下观察细胞形态,MTT法和流式细胞仪检测细胞增殖和凋亡,检测ALP活性,茜素红染色观察钙节结形成,免疫组织化学检测Ⅰ型胶原和VEGF的表达。[结果]诱导2周,实验组细胞增殖能力下降,S期细胞百分比为(5.14±1.56)%,较对照组(13.45±3.51)%约下降62.4%,细胞凋亡增加。实验组ALP活性为(15.68±1.97)mU/mg,对照组为(6.34±1.21)mU/mg,两组比较差异有统计学意义(P<0.05);与对照组比较,实验组钙结节形成增多,VEGF表达较对照组显著提高;实验组Ⅰ型胶原表达呈阳性,对照组呈阴性反应。[结论]负压能抑制hBMSCs增殖,促进细胞凋亡,但可以提高细胞成骨活性。
[Objective] To investigate the effect of negative pressure culture in vitro on osteogenic activity of hBMSCs. [Method] The third generation hBMSCs were divided into experimental group and control group. The experimental group was subjected to intermittent negative pressure culture, and the pressure was 17 kPa, 30 mins / time, 4 times / d for 2 weeks. CO2 incubator routine culture. Cell morphology was observed under inverted microscope. Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. ALP activity was detected by ALT assay. Calcium nodal formation was observed by alizarin red staining. Expression of collagen type Ⅰ and VEGF was detected by immunohistochemistry. [Results] After 2 weeks of induction, the cell proliferation in the experimental group was decreased, the percentage of cells in S phase was (5.14 ± 1.56)%, which was about 62.4% lower than that in control group (13.45 ± 3.51)%, and the cell apoptosis increased. The ALP activity was (15.68 ± 1.97) mU / mg in the experimental group and (6.34 ± 1.21) mU / mg in the control group, with significant difference between the two groups (P <0.05). Compared with the control group, The formation of increased, VEGF expression was significantly increased compared with the control group; type Ⅰ collagen positive expression in the experimental group, the control group was negative. [Conclusion] Negative pressure can inhibit the proliferation of hBMSCs and promote cell apoptosis, but can increase the osteogenic activity of hBMSCs.