重组腺病毒Ad-人基质金属蛋白酶1体外转染大鼠BMSCs研究

RECOMBINANT ADENOVIRUS Ad-HUMAN MATRIX METALLOPROTEINASE 1 TRANSFECTING BONE MARROW MESENCHYMAL STEM CELLS OF RATS IN VITRO

目的利用重组腺病毒Ad-人基质金属蛋白酶1(human matrix metalloproteinase 1,hMMP-1)体外转染大鼠BMSCs,为BMSCs联合hMMP-1基因移植治疗肝纤维化奠定实验基础。方法取2～3周龄SD大鼠骨髓采用全骨髓贴壁法分离培养BMSCs,传至第3代并进行鉴定;用携带增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)标记的重组腺病毒Ad-hMMP-1-EGFP体外转染第3代BMSCs,荧光显微镜下观察转染情况,流式细胞仪检测转染效率并确定最佳感染复数(multiplicity of infection,MOI)。实验分为未转染BMSCs组(A组)、Ad-EGFP转染BMSCs组(B组)及Ad-hMMP-1-EGFP转染BMSCs组(C组),采用MTT法检测转染后细胞增殖情况,RT-PCR及Western blot分别检测转染后各组细胞内hMMP-1基因和蛋白表达情况,ELISA法检测上清中蛋白hMMP-1分泌水平,hMMP-1酶活性荧光定量试剂盒测定其活性。结果重组腺病毒转染BMSCs后24 h可见绿色荧光表达,72 h时荧光强度最高,最佳MOI为200。MTT检测示3组细胞均于培养3 d进入对数生长期,6 d后进入平台期;各时间点3组间细胞吸光度(A)值比较,差异均无统计学意义(P>0.05)。RT-PCR、Western blot及ELISA检测示,A、B组均无hMMP-1基因和蛋白表达,C组hMMP-1基因和蛋白均高效表达。荧光定量试剂盒测定A、B组均未检测到hMMP-1酶活性,C组hMMP-1酶活性为1.24 nmol/(mg.min)。结论利用重组腺病毒Ad-hMMP-1成功将外源基因导入大鼠BMSCs并高效表达,为BMSCs联合hMMP-1基因移植治疗肝纤维化奠定了实验基础。

Objective To transfect bone marrow mesenchymal stem cells （BMSCs） of rats by recombinant adenovirus Ad-human matrix metalloproteinase 1 （hMMP-1） in vitro so as to lay the experimental foundation for the treatment of 1 iver fibrosis with a combination of BMSCs and hMMP- 1 gene transplantation. Methods BMSCs were isolated from bone marrow of 2-3 weeks old Sprague Dawley rats by whole bone marrow adherence method and identified, then transfected by recombinant adenovirus Ad-hMMP-1 carrying enhanced green fluorescent protein （EGFP） marker in vitro. The green fluorescent expression was observed by fluorescence microscope and the transfection efficiency was detected by flow cytometry to determine the optimum multiplicity of infection （MOI）. BMSCs at passage 3 were divided into 3 groups： untransfected BMSCs group （group A）, Ad-EGFP transfected BMSCs group （group B）, and Ad-hMMP-1-EGFP transfected BMSCs group （group C）; the gene and intracellular protein of hMMP-1 were detected by RT-PCR and Western blot; the ELISA assay was used to detect the supernatant protein expression, and the hMMP-1 activity was measured by fluorescent quantification kit. Results The green fluorescent was observed in BMSCs transfected by recombinant adenovirus at 24 hours after transfection; the fluorescence intensity was highest at 72 hours; and the optimum MOI was 200. The cells of 3 groups entered the logarithmic growth phase on the 3rd day and reached plateau phase on the 6th day by MTT assay; no significant difference was found in the cell proliferation rate among 3 groups （P 〉 0.05）. RT-PCR, Western blot, and ELISA assay showed high expressions of the hMMP-1 gene and protein in group C, but no expression in groups A and B. The hMMP-1 activity was 1.24 nmol/（mg.min） in group C, but hMMP-1 activity was not detectable in groups A and B. Conclusion The exogenous hMMP-1 gene is successfully transfected into BMSCs of rats via recombinant adenovirus and can highly express, which lays the experimental foundation for the treatment of liver fibrosis with a combination of BMSCs and hMMP- 1 gene transplantation.