CAJ | 학술논문

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（ hADSCs）中的表达及分析其对铅中毒的作用。方法通过慢病毒载体pLenti－CMV－hChR 2（E123T－H134R）－EYFP系统，构建MT基因过表达慢病毒载体pLenti－CMV－MT2A－EYFP，感染hADSCs，构建携带MT基因的hADSCs （ MT－hADSCs），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－hADSCs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pLenti－CMV－MT2A－EYFP感染hADSCs，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hADSCs中有效表达的金属硫蛋白可提高hADSCs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。
목적：구건휴대금속류단백（ MT）기인적만병독재체，험증기재인지방래원간충질간세포（ hADSCs）중적표체급분석기대연중독적작용。방법통과만병독재체pLenti－CMV－hChR 2（E123T－H134R）－EYFP계통，구건MT기인과표체만병독재체pLenti－CMV－MT2A－EYFP，감염hADSCs，구건휴대MT기인적hADSCs （ MT－hADSCs），응용면역세포형광법검측금속류단백적표체정황。실험분위공백대조조、공재병독조、중조병독감염조분석MT－hADSCs대연적내수성，채용MTT법검측각조세포적존활솔。결과성공구건휴대금속류단백기인적만병독재체pLenti－CMV－MT2A－EYFP감염hADSCs，금속류단백획득유효표체，MTT법검측결과현시중조병독감염조세포여공백대조조화공재병독감염조세포적존활솔상비현저제고，구유통계학의의（ P＜0．05）。결론통과만병독재체재hADSCs중유효표체적금속류단백가제고hADSCs대연적내수성，증실금속류단백능강저중금속연대세포적독성작용。
Objective The lentiviral vector was recombined with metal-lothionein ( MT) gene to identify the MT overexpression in human adi-pose-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 ( E123 T -H134R)-EYFP and MT2A 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 re-combinant 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.