hTfR报告基因慢病毒载体构建及其在神经干细胞的表达研究

Construction of human transferrin receptor gene＇s lentiviral vector and its expression in neural stem cells

目的探讨人转铁蛋白受体（hTfR）基因慢病毒载体构建方法及其在神经干细胞（NSCs）中的表达情况,为NSCs的MR分子成像提供实验基础。方法利用聚合酶链反应技术伊CRl扩增hTfR基因，并克隆到pLenti6.3载体，构建出慢病毒表达载体pLentil6．3-hTfR-IRES-EGFP。利用Lipofectin2000试剂将PLP1、PLP2、PLP-VSVG和pLenti6．3-hTfR-IRES-EGFP共转染293T细胞进行慢病毒包装，48h后收集病毒上清，体外感染NSCs。细胞流式筛选稳定表达hTfR的细胞，通过实时定量PCR和Westernboltting检测hTfR的表达，细胞免疫荧光技术对过表达的hTfR进行亚细胞的定位。结果成功构建危珊基因慢病毒表达载体，包装的慢病毒颗粒成功感染NSCs。实时定量PCR和Westernboltting鉴定出hT瓜在NSCs中过表达，hTfR基因表达相对值为2．275±0．281。细胞免疫荧光检测到过表达的hTfR主要在细胞膜上表达。NSCs分化后，hTfR在胶质细胞和神经元中稳定表达。结论本研究所用方法能成功构建hTfR慢病毒表达载体并筛选出稳定表达hTfR的NSCs系,为下一步活体内移植NSCs行MR分子成像实验研究奠定了基础。

Objective To clone the human transferrin receptor （hTfR） gene into the lentiviral expression vector plenti6.3, identify the reconstructed plasmid and detect the expression ofhTfR gene in neural stem cells （NSCs）, which will provide experimental foundation for in vivo NSCs MR molecular imaging. Methods The hTfR gene cDNA was amplified by polymerase chain reaction （PCR） and cloned into the pLenti6.3 vector to generate the lentiviral vector pLenti6.3-hTfR-IRES-EGFP. The lentiviral vector system （PLP1, PLP2, PLP-VSVG and pLentiI6.3-hTfR-IRES-EGFP） was co-transfected into 293T cells with lipofectin 2000 reagent. The packaged viruses were harvested 48 h later. NSCs were infected by lenfivirus carrying hTfR reporter gene. Transfected cells were screened by flow cytometry （FCM）. The expressions ofhTfR was confirmed by real-time quantitative PCR and Western blotting. The location ofhTfR gene was detected by cell immunofluorescence. Results Lentivirus vector containing hTfR gene was successfully constructed. NSCs could be infected by the recombinant lentivirus. The over-expressions of hTfR could be successfully detected in the infected NSCs by real-time quantitative PCR （the relative value was 2.275±0.281） and Western blotting. Cell immunofluorescence displayed that hTfR was predominantly located on cytomembrane; the expressions ofhTfR were not affected after NSCs differentiating into glial cells and neuuronal cells. Conclusion Lentiviral vector pLenti6. 3-hTfR-IRES-EGFP is successfully constructed and infected to the primary cultured NSCs, and aNSC cell line stablely expressed hTfR is successfully screened, which paves the way for further research on MR molecular imaging of NSCs transplantation in vivo.