Netrin-1慢病毒表达载体的构建及延缓TGF—β诱导HK-2细胞上皮-间充质转化的初步研究

目的：构建netrin-1基因的慢病毒表达载体，初步研究netrin-1在肾小管纤维化的作用。方法：将扩增的netrin-1表达片段克隆入FUW慢病毒表达载体，鉴定重组质粒正确性。利用HEK-293T细胞包装慢病毒，病毒感染人肾小管上皮HK-2细胞，Westernblot检测重组病毒netrin-1在真核细胞内表达表达；TGF-β刺激过表达netrin-1的HK-2细胞，利用Westemblot检测其纤维化指标的变化。结果：FUW—netrin-1慢病毒表达载体测序正确，并在感染病毒的细胞中检测出特异性条带，TGF-β刺激感染netrin-1慢病毒HK-2细胞的E-cadherin的下降水平比未感染病毒组低。结论：成功构建了netrin-1的慢病毒表达载体，发现netrin-1可能延缓肾小管内皮细胞发生纤维化。

Construction of Lentiviral Vector with Netrin-1 Gene and Attenuates EMT of HK-2 Cells that is Induced by TGF-β

Objective： To construct a lentiviral vector expressing Netrin-1 gene, and to investigate the effect of netrin-1 on the process of renal tubule fibrosis. Methods： The gene fragment of netrin-1 was cloned into lentiviral vector FUW. Then the recombinant plasmid was confirmed by sequencing. The recombinant lentivirus was packaged in HEK-293T cells, and the expression of netrin- 1 in HK-2 cells was determined by Western blotting. The expression of fibrosis markers in HK-2 cells treated with TGF-β was investigated. Results： The recombinant plasmid FUW-netrin-1 was constructed and identified. The expression ofnetrin-1 in HK-2 cells was detected by Western blowtting. The loss of E-cadherin expression decreased significantly in the HK-2 cells infected by netrin-1 virus particle after TGF-β treatment compared with contronl group uninfected with netrin-1 virus. Conclusion： The lentivirus vector FUW-netrin-1 has been constructed successful, and netrin-1 may attenuate the progression of renal tubule fibrosis.