miRNA-192慢病毒表达载体构建及其抗高糖刺激氧化损伤作用

Construction of Lentivirus Expression Vector of MiRNA-192 and Its Action Against Oxidative Damage Stimulated by High Glucose

[目的]构建miRNA-192慢病毒过表达载体,并初步评价其在糖尿病肾病中的潜在作用.[方法]检测人肾系膜细胞（HMC）中miRNA-192的含量,以判断载体过表达miRNA-192的程度.采用不同浓度高糖刺激过表达miRNA-192的HMC,检测细胞中谷胱甘肽过氧化物酶（GSH-PX）和超氧化物歧化酶（SOD）活性,评价其过表达拮抗高糖刺激的损伤作用.[结果]酶切和DNA测序证实miRNA-192基因序列被正确克隆入pTLOX载体并能有效的包装成病毒颗粒;病毒滴度达4×107 TU/mL,实时定量RT-PCR检测显示其过表达水平相对对照载体组达376倍,证明目的基因已在转染细胞中成功表达.不同浓度高糖处理组中GSH-PX和SOD活性均比正常对照组下降（P〈0.01）.[结论]成功构建了miRNA-192病毒过表达载体和高滴度病毒颗粒,并能有效稳定感染HMC;过表达miRNA-192能改善高糖对GSH-PX和SOD活性的抑制作用,拮抗高糖对HMC的氧化应激损伤.

[Objective]To construct the lentivirus expression vector of miRNA-192 and evaluate its potential role in diabetic nephropathy. [Methods]The content of miRNA-192 in human mesangial cell（HMC） was detected for as-sessing the degree of over-expression vector of miRNA-192. Different concentration of high glucose stimulated the HMC with overexpression of miRNA-192. Glutathione peroxidase（GSH-PX） and superoxide dismutase（SOD） activi ties in cells were detected. The effect of the overexpression of miRNA-192 on the damage stimulated by high glucose was evaluated. [Results] Restriction enzyme analysis and DNA sequencing confirmed that the gene sequence of miR NA-192 was cloned into pTLOX vector correctly and enveloped to be virus particles. The virus titer was up to 4 × 107TU/ml. Real-time quantitive RT-PCR showed that the overexpression of miRNA 192 of virus vector was 376 times of that of control vector group, which indicated that goal gene was expressed in transfected cells successfully. Compared with normal group, the activities of GSH-PX and SOD in the high-glucose treatment group decreased（ P〈 0. 01）. [Conclusion]The overexpression virus vector and high titer virus particle of miRNA-192 have been successful-ly constructed. Overexpression of miRNA can improve the inhibition of GSH-PX and SOD by high glucose and pro-tect the HMC against high-glucose induced oxidative damage.