周期性压力培养对兔骨髓间充质干细胞增殖及基质金属蛋白酶-2的影响

发布时间：2018-11-26 21:24

【摘要】： 目的研究周期性压力培养对兔骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)增殖活性及基质金属蛋白酶-2(matrix metalloproteinase 2, MMP-2)的影响,探索影响BMSCs增殖的最佳力学参数,达到大量扩增的目的。方法密度梯度离心法分离培养新西兰大白兔BMSCs,取生长良好的第3代细胞,按4×104个/孔接种于6孔板,设立5组:5.32 kPa, 10.64 kPa, 21.28 kPa, 29.26 kPa (1 kPa=7.5 mmHg)压力组、对照组(正常大气压培养)。分别给予对应的压力培养,每日加压6 h×3 d;正常培养组细胞只接受正常大气压刺激,不予额外加压。通过四甲基偶氮唑(MTT)比色试验,流式细胞仪(flow cytometer, FCM)鉴定细胞表面抗原阳性表达及检测细胞增殖周期,同时收集培养液作MMP-2质量浓度检测。观察不同周期性压力对BMSCs增殖及MMP-2的影响。结果6 h/d压力干预BMSCs,3 d后细胞形态无异常变化,呈集落样生长。随着压力的增加,细胞吸光度值逐渐升高,以21.28 kPa压力组最高,但升高至29.26 kPa压力时吸光度值明显降低(F=3.731, P=0.008)。与正常培养组比较,5.32, 10.64, 21.28 kPa压力组细胞周期发生改变,增殖指数(proliferation index, PI)均明显升高(P 0.05或0.01),但29.26 kPa压力组PI则明显下降。细胞培养上清液MMP-2质量浓度以21.28 kPa压力组最低,29.26 kPa压力组最高,组间比较差异有显著性意义(t=213.214,P 0.001)。结论5.32～21.28 kPa的周期性压力可以提高BMSCs的增殖能力,特别是21.28 kPa压力刺激促增殖作用尤为明显,并降低培养液中的MMP-2的浓度。但压力过高则抑制细胞增殖,培养液中的MMP-2的浓度升高,导致细胞死亡增多。
[Abstract]:Objective to study the effect of cyclic pressure culture on (bone marrow mesenchymal stem cells, BMSCs) proliferation activity and matrix metalloproteinase-2 (matrix metalloproteinase 2 (MMP-2) of rabbit bone marrow mesenchymal stem cells (BMSCs), and to explore the best mechanical parameters affecting the proliferation of BMSCs. To achieve the purpose of a large number of amplification. Methods New Zealand white rabbit BMSCs, cells were isolated and cultured by density gradient centrifugation. The third passage cells were inoculated into 6 well plates according to 4 脳 104 cells per well. The cells were divided into 5 groups: 5.32 kPa, 10.64 kPa, 21.28 kPa,. 29.26 kPa (1 kPa=7.5 mmHg) pressure group and control group (normal atmospheric pressure culture). The cells in the normal culture group received only normal atmospheric pressure stimulation, but no additional pressure. The positive expression of cell surface antigen and cell proliferation cycle were identified by flow cytometry (flow cytometer, FCM) and tetramethyl azo (MTT) colorimetric assay. Meanwhile, the culture medium was collected to detect the mass concentration of MMP-2. The effects of different periodic pressures on BMSCs proliferation and MMP-2 were observed. Results there was no abnormal change in cell morphology after 6 h / d pressure intervention on BMSCs,3 d, and the cells grew like colony. With the increase of the pressure, the absorbance value of the cells increased gradually, especially in the 21.28 kPa pressure group, but the absorbance value decreased significantly when the pressure reached 29.26 kPa (FN 3.731, P0. 008). Compared with the normal culture group, the cell cycle of 5.32, 10.64 and 21.28 kPa stress groups changed, and the proliferation index (proliferation index, PI) increased significantly (P 0.05 or 0.01), but the PI decreased significantly in 29.26 kPa pressure group. The concentration of MMP-2 in the supernatant of cell culture was the lowest in 21.28 kPa pressure group and the highest in 29.26 kPa pressure group. There was a significant difference between the two groups (t _ (213.214) P _ (0.001). Conclusion the periodic pressure of 5.32 ~ 21.28 kPa can increase the proliferative ability of BMSCs, especially when stimulated by 21.28 kPa, and decrease the concentration of MMP-2 in the culture medium. However, high pressure inhibited cell proliferation and increased MMP-2 concentration in the culture medium, resulting in the increase of cell death.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.28

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