体外负压培养对骨髓间充质干细胞成骨活性的影响

摘要

Objective:To investigate effects of intermittent negative pressure on osteogenesis in human bone marrow-derived stroma cells (BMSCs) cultured in vitro. Methods:The third passage cells were divided into negative pressure treatment group and control group. The cells in the treatment group were induced by negative pressure intermittently (pressure: 17 kPa, 30 min per time, and four times of each day). The cells in the control group were cultured in conventional condition. The osteogenesis of BMSCs was examined by phase-contrast microscopy. The alkaline phosphatase (ALP) activities were determined. The expression of collagen type I was detected by immunohistochemistry method. The Mrna expressions of osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL) in BMSCs were analyzed by real-time polymerase chain reaction (PCR). Results: BMSCs showed a typical appearance of osteoblast after 2 weeks of induction by intermittent negative pressure. The activity of ALP increased significantly, and the expression of collagen type I was positive. In the treatment group, the Mrna expression of OPG increased significantly (P<0.05) and the Mrna expression of OPGL decreased significantly (P<0.05) after 2 weeks, compared with the control. However, 3 days after the exposure to 2-week negative pressure, these were no significantly different from that of the control group(P>0.05). Conclusion: Intermittent negative pressure could promote osteogenesis in BMSCs in vitro.%目的:探讨体外负压培养对骨髓间充质干细胞(bone marrow-derived stroma cells,BMSCs)成骨活性的影响.方法:取第3代BMSCs分为实验组和对照组,实验组进行间歇性负压培养,设置压力为17 kPa,每次30 min,每日4次,干预2周；对照组于普通CO2培养箱中常规培养.倒置显微镜下观察细胞形态,检测ALP活性,免疫组织化学检测Ⅰ型胶原的表达,RT-PCR检测2周后以及终止负压后l、2、3d骨保护素(osteoprotegerin,OPG) mRNA和骨保护素配体(osteoprotegerin ligand,OPGL) mRNA表达水平.结果:诱导2周后,BMSCs呈现出显著的成骨细胞特性,与对照组比较,ALP活性显著增加,Ⅰ型胶原表达阳性,实验组细胞OPG mRNA表达水平显著提高,OPGL mRNA表达水平显著降低,且差异均有统计学意义(P＜0.05).负压终止后3d,两组细胞OPG mRNA和OPGLmRNA表达水平差异无统计学意义(P＞0.05).结论:体外负压培养可以提高BMSCs成骨活性.