金属硫蛋白修饰人脂肪来源间充质干细胞对铅的耐受性研究

Lead tolerance of metallothionein-overexpressed human adipose-derived mesenchymal stem cells

目的构建携带金属硫蛋白(MT)基因的慢病毒载体,验证其在人脂肪来源间充质干细胞(h ADSCs)中的表达及分析其对铅中毒的作用。方法通过慢病毒载体p Lenti-CMV-h ChR 2(E123T-H134R)-EYFP系统,构建MT基因过表达慢病毒载体p Lenti-CMV-MT2A-EYFP,感染h ADSCs,构建携带MT基因的h ADSCs(MT-h ADSCs),应用免疫细胞荧光法检测金属硫蛋白的表达情况。实验分为空白对照组、空载病毒组、重组病毒感染组分析MT-h ADSCs对铅的耐受性,采用MTT法检测各组细胞的存活率。结果成功构建携带金属硫蛋白基因的慢病毒载体p Lenti-CMV-MT2A-EYFP感染h ADSCs,金属硫蛋白获得有效表达,MTT法检测结果显示重组病毒感染组细胞与空白对照组和空载病毒感染组细胞的存活率相比显著提高,具有统计学意义(P<0.05)。结论通过慢病毒载体在h ADSCs中有效表达的金属硫蛋白可提高h ADSCs对铅的耐受性,证实金属硫蛋白能降低重金属铅对细胞的毒性作用。

Objective The lentiviral vector was recombined with metallothionein（ MT） gene to identify the MT overexpression in human adipose- derived mesenchymal stem cells（ hADSCs） after transfection and then to study the lead tolerance of genetically modified hADSCs with MT（ MT- hADSCs）. Methods The recombinant plenti- CMV- MT2A-EYFP vector was constructed with pLenti- CMV- hChR 2（ E123T-H134R）- EYFP and MT2 A gene for transfecting hADSCs to obtain the MT- hADSCs. The overexpression of MT in hADSCs was identified by immunofluorescence assay. The MTT method was used to assess the cell viability of hADSCs,hADSCs transfected with empty vector,and MT-hADSCs,all of which were treated with lead acetate. Results The recombinant plenti- CMV- MT2A- EYFP was successfully constructed and transfected into hADSCs. The overexpression of MT was positively detected in the MT- hADSCs. The tolerance of MT- hADSCs to lead was significantly higher than the hADSCs and hADSCs transfected with empty vector. Conclusion MT can significantly increase the tolerance of hADSCs to lead,indicating that MT can reduce the cytotoxicity of lead. The experimental results from this study provide more evidences for further study of using MT- hADSCs to treat lead poisoning.