冲击波诱导人骨髓基质细胞成骨分化及机制的研究

Extracorporeal shock wave promotes differentiation of human bone marrow stromal cells into osteoprogenitors

目的 观察出生后人骨髓基质细胞 (hMSCs)在体外培养条件下增殖与分化的特点 ;研究适宜能量冲击波对出生后hMSCs成骨分化的作用及机制。方法 抽取健康自愿者髂骨骨髓 ,采用密度梯度离心法进行hMSCs体外培养。设冲击波组 (SW组 )与对照组 ,应用不同能量级冲击波对SW组原代细胞进行处理 ,根据细胞活力测定与集落形成数量确定适宜的冲击波能量值。应用适宜的冲击波能量处理hMSCs原代细胞并传代培养 ,采用倒置显微镜观察、细胞增殖活力测定、ELISA法检测细胞分泌TGF β1、茜素红染色、钙钴法染色、四环素荧光标记、细胞分泌碱性磷酸酶测定和逆转录 聚合酶链反应 (RT PCR)检测骨钙素mRNA表达等方法 ,对SW组和对照组的各代细胞形态、增殖与分化及其机制进行探讨。结果 冲击波处理体外原代培养hMSCs的适宜能量为10kV(5 0 0 ) ,SW组细胞在冲击波处理后早期分泌TGF β1显著高于对照组(P <0 .0 0 1)。SW组与对照组细胞在形态学方面第 3代前无明显差别 ;SW组各代细胞分泌碱性磷酸酶显著高于对照组(P <0 .0 1) ;茜素红染色、钙钴法染色、四环素荧光标记等显示SW组细胞的成骨作用明显优于对照组 ;SW组细胞经冲击波处理后第 10天应用RT PCR方法可以检测到骨钙素mRNA的表达 ,与对照组比较差异有统计学意义 (P <0 .

Objective To study the characteristics in differentiation and proliferation of postnatal human bone marrow stromal cells (hMSCs) and ESW to promote hMSCs to differentiate into osteoprogenitor cells.Methods hMSCs were source of the bone marrow collected from healthy human donors and cultured according to Majumdar's method in vitro.An optimal dose of SW was selected to treat primary MSCs on the authority of hMSCsproliferation and colony-forming unit (CFU-F).hMSCs differentiated into osteoprogenitor cells were evaluated by morphology,TGF-β1 and Alkaline phosphatase in the culture supernatants,calcium node formation,alkaline phosphatase Ca-Co staining,osteocalcin mRNA expression.Results The optimal energy of ESW treating MSCs was 10 kV (500),which had the best promoting effect on the CFU-F and cell proliferation.There was no difference in cell morphology between SW group and control group before 3th subculture,but the cells in SW group tended to osteoblast in morphology after 4th subculture.It was found that alkaline phosphatase activity in SW group was increaseed as compared with that in control group in every subculture (P<0.01).It was showed that osteogenesis was clear in SW group by histochemistry and tetracycline fluorescence staining.The expression of osteocalcin mRNA was detectable in 10 days and TGF-β1 was induced after SW treatment.Conclusion ESW could promote postnatal hMSCs differentiation into osteogenic lineage associated with induction of TGF-β1.The optimal dose of ESW is 10 kV (500).