慢病毒介导I型胶原短发夹RNA转染对大鼠系膜细胞生长行为的影响  

Effect of lentivirus -mediated shRNA targeting collagen type I gene on biological behaviors of rat mesangial cells

作　　者：单毅[1] 周春华[2] 赵海丹[2] 

机构地区：[1]海军总医院重症医学科,北京100048 [2]海军总医院肾内科,北京100048(

出　　处：《中国医药》2010年第8期723-725,共3页China Medicine

基　　金：基金项目：北京市自然科学基金资助项目（7063094）

摘　　要：目的通过观察慢病毒介导的I型胶原（COLI）短发夹（sh）RNA感染大鼠系膜细胞后其增殖、凋亡及细胞周期的变化，探索未来应用于动物实验，甚至临床时RNA干涉（RNAi）药物对靶细胞生长行为的影响。方法利用感染增强剂（对照组）、空慢病毒载体及感染增强剂（pSC-GFP组）、COLIshRNA慢病毒表达载体及感染增强剂（pSC—GFP／COLI组）分别感染大鼠系膜细胞，用噻唑蓝法检测细胞增殖，利用AnnexinV／PE法检测细胞凋亡，利用流式细胞仪检测细胞周期，进而观察慢病毒表达载体对细胞上述能力的影响。结果pSC—GFP／COLI组和pSC—GFP组均能显著抑制细胞增殖，且慢病毒表达载体抑制能力又显著高于空病毒，分别于感染后24h、48h和72h计算抑制效率为22．52％、28．57、33．33％。凋亡实验结果表明，慢病毒感染细胞后一定程度诱导了细胞凋亡，但作为载体携带的COLIshRNA未引起进一步的细胞凋亡加剧。细胞周期实验发现慢病毒引起细胞周期阻滞于G2／M期。而在72hCOLIshRNA对s期的阻滞逐渐突出。结论慢病毒介导I型胶原短发夹RNA转染大鼠系膜细胞后，在一定程度上影响了细胞生长行为，但总体来说仍是安全的，为进一步动物实验及药物研制奠定基础。Objective By observing the change of proliferation, apoptosis and cell cycle about RMC infected by lentivirus-mediated COL I shRNA, we explore the physiological impact on the target cells by RNAi medicine. Methods We observed the infected RMC by Infection enhancer（ control group） , lentivirus vector（ pSC-GFP group） and lentivirus-mediated COL I shRNA（ pSC-GFP/COL I group）, cell proliferation was measured with MTT method, apoptosis was measured with Annexin V/PE method, cell cycle was measured with flow cytometry and the impact on the above-mentioned ability of cells infected by lentiviral expression vector was observed. Results Both pSC-GFP/ COL I group and pSC-GFP group can significant inhibit cell proliferation. Suppression ability of pSC-GFP group was significantly higher than pSC-GFP group. 24 hours,48 hours and 72 hours after infection showed inhibition efficiency of 22.52%, 28.57%, 33.33%, respectively. The result of apoptosis show that apoptosis was induced to a certain degree after lentivirus-mediated shRNA infected RMC, but RNAi didnt induce further aggravation. The result of cell cycle experiment showed that lentivirus induces G2/M cell cycle arres. To 72 houres after RMC infected by lentiviral expression vector, increasingly prominent showed that COL I shRNA induced S cell cycle arrest. Conclusions M- ter transfection of rat mesangial cells, COL I shRNA affects cell proliferation at a certain degree. But on the whole, it＇s a safe way to lay the foundation for further animal experiment and drug development.

关 键 词：细胞周期 慢病毒属 细胞增殖 细胞凋亡 胶原I型 

分 类 号：R349.53[医药卫生—基础医学]