【摘要】 【目的】为研究患者来源干细胞成骨分化的差异性,选取临床诊断为骨质疏松症患者的骨髓干细胞进行成骨分化诱导。【方法】采用密度梯度离心法,从临床6名患者(骨质疏松症3人,非骨质疏松症3人)的腰椎骨髓血液中分离获得原代骨髓间充质干细胞,并体外扩增至3-4代后进行实验。成骨诱导液的成分包括地塞米松(100 nmoL/L),二磷酸抗坏血酸(0.05 mmoL/L),β-甘油磷酸钠(10 mmoL/L)溶解于含有100 mL/L胎牛血清的低糖DMEM中。通过测定hMSC的增值与碱性磷酸酶(ALP)含量反应干细胞成骨分化活性。定量PCR检测Runx-2、ALP及成骨标志基因osteocalcin(OC)和osteonectin(ON)表达水平;通过矿化结节茜素红染色确认并定量检测钙离子浓度。【结果】比较两组干细胞增殖差异,骨质疏松组较非骨质疏松组降低,而ALP含量无显著差异。Q-PCR检测显示在第3、7天时,骨质疏松组Runx-2、ALP的mRNA表达水平均显著低于非骨质疏松组。骨质疏松组的成骨相关基因OC在3、7、14 d时mRNA表达水平均显著低于非骨质疏松组,而ON仅在第7天表达具有显著差异;矿化结节染色及钙离子定量检测显示,较之非骨质疏松组,骨质疏松组矿化结节染色浅,钙定量降低。【结论】使用成骨诱导干细胞分化方案(地塞米松、二磷酸抗坏血酸、β-甘油磷酸钠)结果提示骨质疏松症患者的干细胞成骨分化能力较非骨质疏松患者降低。因此,选取有骨质疏松症患者自身的骨髓间充质干细胞作为治疗的种子细胞可能并不适用于临床。更多还原

【Abstract】 【Objective】 To demonstrate differences in osteogenic differentiation, we test the capacity of hMSC between the osteoporosis and the non-osteoporosis patients. 【Method】 Six cases of bone marrow derived from patients with lumbar operation, in which three patients were diagnosed with osteoporosis and three with non-osteoporosis. For each patient, hMSC were divided into control group and osteogenic induction group. hMSC were separated from fresh bone marrow by density centrifugation and expanded.Osteogenic differentiation of hMSC was induced by treatment with dexamethasone(100 nmoL / L), ascorbic acid(0.05 mmoL / L), β-glycerol phosphate(10 mmoL / L) and low glucose DMEM medium with 0.1 volume fraction fetal bovine serum. The hMSC proliferation was evaluated after the induction by simultaneous monitoring of CCK-8 value and alkaline phosphatase activity(ALP). RT-qPCR assay was used to identify the differentially expressed osteogenic gene Runx-2, ALP, osteocalcin(OC) and osteonectin(ON). The formation of mineralized nodule was verified by staining with Alizarin red S as well as calculated by calcium quantitative assay.【Result】 The values of proliferation were in creased signicantly in non-osteoporosis group,while the ALP indices showed to significant differences between the groups. RT-qPCR assay indicated that, compared to osteoporosis group, Runx-2 and ALP were up-regulated on day 3 and 7, OC was up-regulated on day 3, 7 and 14, ON was up-regulated on day 7. The calcium nodules staining and quantitative assay showed that the capacity of hMSC in osteoporosis group was suppressed during the process of osteogenic induction,in comparison to the non-osteoporosis group. 【Conclusion】 Our data suggest multipotency of hMSC were suppressed by osteoporosis in osteogenic differentiation, which is induced by dexamethasone, ascorbic acid, and β-glycerol phosphate. Hence, it may not suitable for applying hMSC from osteoporosis patient in treatiment of their own disease.更多还原