稳定抑制TRPM2基因表达肺微血管内皮细胞系的构建与鉴定

Construction and Verification of a Mouse Pulmonary Microvascular Endothelial Cell Lines to Inhabit TRPM2 Gene Expression with shRNA Lentivirus System

应用短发卡RNA(shRNA)慢病毒表达载体感染小鼠肺微血管内皮细胞(PMVEC),对其M2型瞬时受体电位(TRPM2)基因进行干扰,以建立稳定shRNA TRPM2 PMVEC细胞株。结果表明,正常PMVEC对胰酶的最大耐受浓度和嘌呤霉素最小致死剂量分别为0.4μg/mL和0.6μg/mL;然后加入0.6、2.0、4.0、8.0μg/mL嘌呤霉素筛选稳定抑制TRPM2表达的shRNA TRPM2 PMVEC株,结果在嘌呤霉素达到8μg/mL时该细胞株仍细胞生长良好; Semi-quantitative PCR和Western blot对获得阳性细胞株进行TRPM2基因和蛋白表达的检测显示, shRNA TRPM2阳性PMVEC的TRPM2基因和蛋白表达相对量显著低于阴性对照和正常对照组(P<0.01)。该研究通过shRNA慢病毒载体成功建立了稳定shRNA TRPM2 PMVEC细胞株,为进一步开展TRPM2在流感病毒诱导肺微血管内皮细胞损伤的作用机制奠定了基础。

To construct and verify transient receptor potential melastatin-2(TRPM2)gene silencing of mouse pulmonary microvascular endothelial cell(PMVEC),the PMVEC was infected by short sairpin RNA(shRNA)trpm2 lentivirus particles.The results showed that the maximal tolerance concentration of TPCK-trypsin to PMVEC was 0.4μg/mL and minimal lethal dose of puromycin dihydrochloride to PMVEC was 0.6μg/mL,respectively.The stable clones expressing the shRNA TRPM2 were selected by adding 0.6,2.0,4.0,8.0μg/mL puromycin dihydrochloride to kill the non-transduced PMVEC,and there was no effect on PMVEC of the stable clones expressing shRNA TRPM2 even the puromycin dihydrochloride concentration reach to maximal 8μg/mL.The result of Semi-quantitative PCR showed that the TRPM2 gene was silenced significantly in shRNA TRPM2 cell compared to shRNA control and uninfected PMVEC(P<0.01).Similarly,the protein expression of shRNA TRPM2 PMVEC was also decreased markedly compared with shRNA control and PMVEC group(P<0.01).The shRNA TRPM2 PMVEC cell line was successfully constructed by shRNA TRPM2 lentivirus particles and lie the foundation for further to investigate the mechanism of TRPM2 on PMVEC damage infected by influenza virus.