Zhao Bowen, Zhang Hongwei, Xu Qiang, Ge Quanhu, Li Bolong, Peng Xinyu, Wu Xiangwei
No.1 Department of General Surgery, The First Affiliated Hospital, College of Medicine, Shihezi University, Shihezi Xinjiang, 832008, P.R.China.
No.1 Department of General Surgery, The First Affiliated Hospital, College of Medicine, Shihezi University, Shihezi Xinjiang, 832008,
Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2017 May 15;31(5):594-599. doi: 10.7507/1002-1892.201701095.
To investigate the effects of long time different negative pressures on osteogenic diffe-rentiation of rabbit bone mesenchymal stem cells (BMSCs).
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.
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 ( <0.05), but there was no significant difference between the low negative pressure group and the control group ( >0.05); at 5-7 days, the cell number showed significant difference between 3 groups ( <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 ( >0.05); the ALP activity showed significant difference ( <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 ( <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 ( <0.05), and in the high negative pressure group than the low negative pressure group ( <0.05).
With the increase of the negative pressure, the osteogenic differentiation ability of BMSCs increases gradually, but the cell proliferation is inhibited.
探讨长时间不同负压对兔骨髓间充质干细胞（BMSCs）成骨分化的影响。
采用密度梯度离心法分离培养兔BMSCs。运用流式细胞术分析表面标志物表达。将第三代细胞在成骨诱导条件下，分别置于0 mmHg（对照组）、75 mmHg（低负压组）和150 mmHg（高负压组）（1 mmHg = 0.133 kPa）不同负压环境中培养，负压时间为30 min/h。在相差显微镜下观察细胞生长情况并绘制生长曲线；诱导3、7和14 d后，采用酶联免疫吸附测定法（ELISA）检测碱性磷酸酶（ALP）活性。通过实时荧光定量PCR和蛋白质印迹法分析BMSCs中Ⅰ型胶原（COL-I）和骨钙素（OC）的mRNA及蛋白表达。
流式细胞术鉴定培养细胞为BMSCs。第三代BMSCs呈典型的长梭形且形态不规则。细胞增殖随负压增加而受到抑制。诱导4 d后，高负压组细胞数量显著少于对照组和低负压组（P<0.05），但低负压组与对照组之间差异无统计学意义（P>0.05）；5~7 d时，3组细胞数量差异有统计学意义（P<0.05）。负压越大，对细胞增殖的抑制作用越强。诱导3 d时，各组间ALP活性差异无统计学意义（P>0.05）；诱导7 d时，高负压组与对照组ALP活性差异有统计学意义（P<0.05）；诱导14 d时，3组间ALP活性差异有统计学意义（P<0.05）。负压越大，ALP活性越高。实时荧光定量PCR和蛋白质印迹检测显示，低负压组和高负压组COL-I和OC蛋白的mRNA及蛋白表达均显著高于对照组（P<0.05），且高负压组高于低负压组（P<0.05）。
随着负压的增加，BMSCs的成骨分化能力逐渐增强，但细胞增殖受到抑制。
