ECSOD体外转染恒河猴骨髓间充质干细胞的实验研究

发布时间：2018-08-19 12:37

【摘要】：本研究旨在探讨载带超氧化物歧化酶基因的腺病毒(AD-ECSOD)感染恒河猴骨髓间充质干细胞(R-BMMSC)生物学特性的影响。用含有ECSOD和报告基因EGFP的腺病毒感染经梯度密度离心和贴壁培养法分离、培养并纯化的R-BMMSC,用倒置荧光显微镜及流式细胞仪观察检测感染效率,ELISA法检测细胞培养上清中的ECSOD蛋白表达水平;流式细胞仪检测感染后R-BMMSC的表面抗原(CD34,CD29,CD45,CD90,HLA-DR);油红O和茜素红染色观察R-BMMSC的成骨及成脂诱导分化能力;MTT法检测AD-ECSOD转染对R-BMMSC增殖能力的影响。结果表明,不同滴度AD-ECSOD(感染滴度为每个细胞500,1 000,1 500和2 000 pfu)对R-BMMSC的感染效率均95%。转染后24 h即可在细胞上清液中检测到ECSOD蛋白表达,且明显高于对照组(P0.01),转染后48 h蛋白表达量最高。AD-ECSOD转染后R-BMMSC的增殖能力及其表面抗原表达和多向分化能力与未转染组比较无统计学差异(P0.05)。结论:AD-ECSOD能够高效转染R-BMMSC,对其生物学特性无明显影响。
[Abstract]:The aim of this study was to investigate the biological characteristics of rhesus monkey bone marrow mesenchymal stem cells (R-BMMSC) infected by adenovirus carrying superoxide dismutase gene (AD-ECSOD). The adenovirus containing ECSOD and reporter gene EGFP was isolated by gradient density centrifugation and adherent culture. The purified R-BMMSC was cultured and purified by inverted fluorescence microscopy. The infection efficiency was observed by flow cytometry, and the expression of ECSOD protein in cell culture supernatant was detected by ELISA; the surface antigens of R-BMMSC (CD34, CD29, CD45, CD90, HLA-DR) were detected by flow cytometry; the osteogenesis and adipogenic differentiation of R-BMMSC were observed by oil red O and alizarin red staining; and the proliferation of R-BMMSC by AD-ECSOD transfection was detected by MTT. The results showed that the infection efficiency of R-BMMSC was 95% with different titers of AD-ECSOD (500,1,1,500 and 2,000 PFU per cell). The expression of ECSOD protein was detected in the supernatant 24 hours after transfection, which was significantly higher than that in the control group (P 0.01). The expression of ECSOD protein was the highest at 48 hours after transfection. Conclusion: AD-ECSOD can transfect R-BMMSC efficiently and has no significant effect on its biological characteristics.
【作者单位】：军事医学科学院放射与辐射医学研究所;南昌市疾病预防控制中心;
【基金】：国家自然科学基金面上项目(编号81172594)
【分类号】：R329.28

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