ECSOD体外转染恒河猴骨髓间充质干细胞的实验研究

Transfection of ECSOD to Rhesus Bone Marrow Mesenchymal Stem Cells In Vitro

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(P<0.01),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(P>0.05)。结论:AD-ECSOD能够高效转染R-BMMSC,对其生物学特性无明显影响。

This study was aimed to investigate the biological effects of Rhesus bone marrow mesenchymal stem cells （R-BMMSC） transfected by adenovirus beating extracellular superoxide dismutase gene （AD-ECSOD）. Using density gradient centrifugation and adherent culture way, the R-BMMSC transfected by AD-ECSOD and reporter gene EGFP were isolated, cultured and purified; the transfection efficiency was detected by flourescence microscopy and flow cytomatry; the ECSOD protein expression in cell culture supematant were detected by ELISA; the surface antigens on R-BMMSC （ CD34, CD29, CIM5, CD90, HLA-DR） were detected by flow cytometry; and differentiation capability of transfected R-BMMSC were detected by oil red O and alizarin staining; the proliferation capability of R-BMMSC was assay by MTT method. The results showed that the transfection efficiency of AD-ECSOD （MOI 500, 1 000, 1 500 and 2 000） for R-BMMSC was 〉95%. At 24 h after transfection, the ECSOD protein could be detected in cell culture supernatant, and its level was significantly higher than that of control group （P 〈0.01 ）. At 48 h after transfection, the expression level of ECSOD protein on MOI 1 500 and 2 000 was the highest. The proliferative capability, surface antigen expression and mulfidirective differentiation ability of transfected R-BMMSC were similar to non-transfected R-BMMSC. It is concluded that the AD-ECSOD can effectively transfect the R-BMMSC without influences on its biological features.