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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 .0
Objective To observe the characteristics of postnatal human bone marrow stromal cells (hMSCs) proliferation and differentiation under in vitro culture conditions and to investigate the effect and mechanism of appropriate energy shock wave on osteogenic differentiation of hMSCs after birth. Methods The iliac bone marrow of healthy volunteers was collected and the hMSCs were cultured in vitro by density gradient centrifugation. The shock wave group (SW group) and the control group were treated with shock wave of different energy levels to treat the primary cells of SW group, and the suitable shock wave energy value was determined according to the cell viability and colony formation. Primary cells of hMSCs were treated with appropriate shock wave energy and subcultured. The cells were stained with inverted microscope and measured by cell proliferation assay. The expression of TGFβ1, alizarin red, calcium and cobalt staining, Phosphatase and reverse transcriptase polymerase chain reaction (RT PCR) were used to detect the expression of osteocalcin mRNA. The morphological, proliferation and differentiation of SW and control groups were also explored. Results The appropriate energy of shock wave treatment of primary cultured hMSCs in vitro was 10 kV (500). The levels of TGF β1 secreted by SW cells in the early stage after shock wave treatment were significantly higher than those in the control group (P <0.01). There was no significant difference in morphology between the SW group and the control group before passage 3; the alkaline phosphatase (AKP) secreted by all the SW groups was significantly higher than that of the control group (P <0.01); alizarin red staining, Staining and tetracycline fluorescent labeling showed that the osteoblastic effect of SW group was better than that of control group. The expression of osteocalcin mRNA was detected by RT-PCR method in SW group on the 10th day after shock wave treatment, compared with the control group Statistical significance (P <0 .0