长时间不同强度负压对兔BMSCs成骨分化及增殖的影响研究

Effects of long time different negative pressures on osteogenic differentiation of rabbit bone mesenchymal stem cells

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(P<0.05),低负压组与对照组比较差异无统计学意义(P>0.05);5~7d 3组间细胞数比较差异均有统计学意义(P<0.05),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(P>0.05);7d,仅高负压组与对照组比较差异有统计学意义(P<0.05);14d,3组间比较差异均有统计学意义(P<0.05),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(P<0.05)。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。

Objective To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells （BMSCs）. Methods The rabbit BMSCs were isolated and cultured by density gradient centrifugation. Flow cytometry was used to analyze expression of surface markers. The third passage cells cultured under condition of osteogenic induction and under different negative pressure of 0 mm Hg （control group）, 75 mm Hg （low negative pressure group）, and 150 mm Hg （high negative pressure group） （1 mm Hg=0.133 kPa）, and the negative pressure time was 30 min/h. Cell growth was observed under phase contrast microscopy, and the growth curve was drawn; alkaline phosphatase （ALP） activity was detected by ELISA after induced for 3, 7, and 14 days. The mRNA and protein expressions of collagen type I （COL-I） and osteocalcin （OC） in BMSCs were analyzed by real-time fluorescence quantitative PCR and Western blot. Results The cultured cells were identified as BMSCs by flow cytometry. The third passage BMSCs exhibited typical long shuttle and irregular shape. Cell proliferation was inhibited with the increase of negative pressure. After induced for 4 days, the cell number of high negative pressure group was significantly less than that in control group and low negative pressure group （P〈0.05）, but there was no significant difference between the low negative pressure group and the control group （P〉0.05）; at 5-7 days, the cell number showed significant difference between 3 groups （P〈0.05）. The greater the negative pressure was, the greater the inhibition of cell proliferation was. There was no significant difference in ALP activity between groups at 3 days after induction （P〉0.05）; the ALP activity showed significant difference （P〈0.05） between the high negative pressure group and the control group at 7 days after induction; and significant difference was found in the ALP activity between 3 groups at 14 days after induction （P〈0.05）. The greater the negative pressure was, the higher the ALP activity was. Real-time fluorescence quantitative PCR and Western blot detection showed that the mRNA and protein expressions of COL-I and OC protein were significantly higher in low negative pressure group and high negative pressure group than control group （P〈0.05）, and in the high negative pressure group than the low negative pressure group （P〈0.05）. Conclusion With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.