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.
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 kPa）（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、14 d 采用 ELISA 法检测细胞 ALP 活性，14 d 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 4 d，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）；5～7 d 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 3 d 各组间比较差异无统计学意义（ P>0.05）；7 d，仅高负压组与对照组比较差异有统计学意义（ P<0.05）；14 d，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 mRNA 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。.
Keywords: Bone marrow mesenchymal stem cells; cell proliferation; negative pressure; osteogenic differentiation; rabbit.