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目的:观察重组逆转录病毒人血管内皮生长因子165(retrovirus pLXSN/hVEGF165)基因转染对人骨髓基质细胞(hM-SCs)成骨能力的影响。方法:从健康志愿者全骨髓中分离培养人hMSCs,体外扩增纯化后随机分为4组:①retrovirus pLXSN/hVEGF165组:培养液中加入人血管内皮生长因子基因重组逆转录病毒1×1010OPU/ml,孵育24h后换普通培养液继续培养;②retrovirus pLXSN组:培养液中加入逆转录病毒空载体,其余处理与retrovirus pLXSN/hVEGF165组相同;③阳性对照组:培养液中添加地塞米松、抗坏血酸和β-甘油磷酸钠;④空白对照组:不给予特殊处理。各组细胞处理后2周,免疫组化观察VEGF表达,Von Kossa染色检测人hMSCs中骨胶原结节形成,全自动生化分析仪检测培养上清碱性磷酸酶活性。结果:经多次换液传代,人hMSCs呈均一梭形形态。处理后retrovirus pLXSN/hVEGF165组与阳性对照组细胞形态逐渐趋于扁平,突起减少。retroviruspLXSN组和空白对照组形态无明显变化。免疫组化染色见VEGF主要在胞浆内表达,retrovirus pLXSN/hVEGF165组VEGF表达明显强于其他3组。Von Kossa染色retrovirus pLXSN/hVEGF165组与阳性对照组可见大量红色骨胶原结节形成,显著多于retrovirus pLXSN组和空白对照组。retrovirus pLXSN/hVEGF165组与阳性对照组培养上清液碱性磷酸酶活性显著高于retroviruspLXSN组和空白对照组。结论:基因重组逆转录病毒人血管内皮生长因子转染对人hMSCs成骨能力具有促进作用。
OBJECTIVE: To observe the effect of recombinant retrovirus pLXSN / hVEGF165 gene transfection on the osteogenic potential of human bone marrow stromal cells (hM-SCs). Methods: Human hMSCs were isolated and cultured from whole bone marrow of healthy volunteers, expanded and purified in vitro and randomly divided into 4 groups: ① retrovirus pLXSN / hVEGF165 group: Human vascular endothelial growth factor gene recombinant retrovirus 1 × 1010 OPU / ml , And incubated for 24 hours, then cultured for normal culture; ② retrovirus pLXSN group: retroviral empty vector was added to the culture medium, and the rest treated with retrovirus pLXSN / hVEGF165 group; ③ positive control group: dexamethasone, ascorbic acid and β-glycerophosphate; ④ blank control group: do not give special treatment. The expression of VEGF was detected by immunohistochemistry and the formation of collagen in human hMSCs by Von Kossa staining. The activity of alkaline phosphatase in culture supernatant was detected by automatic biochemical analyzer. Results: The human hMSCs showed a uniform spindle shape after multiple fluid changes. After treatment, the cell morphology of retrovirus pLXSN / hVEGF165 group and the positive control group gradually became flat and the number of protrusions decreased. The morphology of retroviruspLXSN group and blank control group showed no significant change. Immunohistochemical staining showed that VEGF was mainly expressed in the cytoplasm. VEGF expression in retrovirus pLXSN / hVEGF165 group was significantly stronger than the other three groups. Von Kossa stain retrovirus pLXSN / hVEGF165 group and the positive control group showed a large number of red collagen nodules were significantly more than the retrovirus pLXSN group and the blank control group. The activity of alkaline phosphatase in the retrovirus pLXSN / hVEGF165 group and the positive control group was significantly higher than those in the retroviruspLXSN group and the blank control group. Conclusion: Recombinant retroviral human VEGF gene transfection can promote the osteogenic potential of human hMSCs.