长时间不同强度负压对兔BMSCs成骨分化及增殖的影响研究

发布时间：2019-01-22 12:22

【摘要】：目的探讨长时间不同强度负压对兔BMSCs成骨分化及增殖的影响。方法取4~6月龄新西兰大白兔股骨骨髓,采用密度梯度离心法分离培养BMSCs,并行流式细胞术及成骨诱导鉴定。取第3代细胞采用成骨诱导培养基诱导培养,并加载不同强度负压,分别为0、75、150 mm Hg(1 mm Hg=0.133 k Pa)(对照组、低负压组、高负压组);负压时间为30 min/h。倒置显微镜下观察细胞生长状态,绘制细胞生长曲线;诱导3、7、14d采用ELISA法检测细胞ALP活性,14d采用实时荧光定量PCR及Western blot检测各组Ⅰ型胶原、骨钙素(osteocalcin,OC)基因及蛋白表达。结果经流式细胞仪及成骨诱导鉴定所培养细胞为BMSCs;第3代BMSCs为典型的长梭形和不规则形。诱导4d,高负压组细胞显著少于对照组及低负压组(P0.05),低负压组与对照组比较差异无统计学意义(P0.05);5~7d 3组间细胞数比较差异均有统计学意义(P0.05),负压越大细胞增殖受抑制越明显。ALP活性检测示,诱导3d各组间比较差异无统计学意义(P0.05);7d,仅高负压组与对照组比较差异有统计学意义(P0.05);14d,3组间比较差异均有统计学意义(P0.05),负压越大ALP活性越高。实时荧光定量PCR及Western blot检测示,低负压组及高负压组Ⅰ型胶原和OC m RNA及蛋白表达量均显著高于对照组,高负压组高于低负压组,比较差异均有统计学意义(P0.05)。结论随负压增高,兔BMSCs成骨能力逐渐增强,而细胞增殖能力逐渐受抑制。
[Abstract]:Objective to investigate the effect of different intensity negative pressure on osteogenic differentiation and proliferation of rabbit BMSCs for a long time. Methods the bone marrow of 4 and 6 months old New Zealand white rabbits was isolated and cultured by density gradient centrifugation. Flow cytometry and osteogenesis induction were used to identify the bone marrow. The cells of the third passage were induced by osteoblast induction medium and loaded with negative pressure of different intensities, which were 0 ~ 75150 mm Hg (_ 1 mm Hg=0.133 k Pa) (control group, low negative pressure group and high negative pressure group, respectively, and the negative pressure time was 30 min/h.. The state of cell growth was observed under inverted microscope and the cell growth curve was drawn. The activity of ALP was detected by ELISA assay and the expression of type I collagen, osteocalcin (osteocalcin,OC) gene and protein were detected by real-time fluorescence quantitative PCR and Western blot on the 14th day after induction. Results the cultured cells were identified by flow cytometry and osteoblast induction to be typical fusiform and irregular shape of BMSCs in the third passage of BMSCs;. After induction for 4 days, the number of cells in the high negative pressure group was significantly lower than that in the control group and the low negative pressure group (P0.05), but there was no significant difference between the low negative pressure group and the control group (P0.05). There were significant differences in the number of cells between the three groups on the 7th day after induction (P0.05), and the more negative pressure the cells were inhibited, the more obvious the cell proliferation was. The ALP activity test showed that there was no significant difference between the three groups on the 3rd day of induction (P0.05). At 7 days, there was significant difference between the high negative pressure group and the control group (P0.05), and the difference between the 14 day group and the control group was statistically significant (P0.05). The higher the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot analysis showed that the expression of type I collagen, OC m RNA and protein in low negative pressure group and high negative pressure group were significantly higher than that in control group, and that in high negative pressure group was higher than that in low negative pressure group, the difference was statistically significant (P0.05). Conclusion with the increase of negative pressure, the osteogenic ability of rabbit BMSCs is gradually enhanced, and the proliferation of rabbit cells is inhibited gradually.
【作者单位】：石河子大学医学院第一附属医院普外一科;
【基金】：国家自然科学基金资助项目(31271458) 兵团应用基础研究计划资助项目(2016AG019)~~
【分类号】：R687

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