VDAC1基因低表达型Z310细胞系的建立与乙酸铅对其增殖的影响  

Establishment of VDAC1-deficient Z310 cell line and the effect of lead acetate on its proliferation

作　　者：刘羽[1,2] 董一文[2] 胡红[1,2] 敬海明[2] 李煜[2] 谭壮生[2] 王红梅[2] 尤育洲[2] 李明[2] 焦智浩[2] 赵超英[2] 马玲[1,2] 宁钧宇[1,2] 李国君[1,2] 

机构地区：[1]首都医科大学公共卫生学院,北京100069 [2]北京市疾病预防控制中心/北京市预防医学研究中心卫生毒理所,北京市食物中毒诊断溯源技术重点实验室

出　　处：《毒理学杂志》2014年第1期19-25,共7页Journal of Toxicology

基　　金：北京市卫生系统高层次卫生技术人才培养项目(2011)

摘　　要：目的运用载体介导的RNA干扰技术靶向抑制电压依赖性阴离子通道1（voltage—dependent anion channell，VDAC1）在永生化大鼠脑脉络丛Z310细胞中的表达，构建VDAC1基因低表达型Z310细胞系，并检测乙酸铅对其增殖的影响，为进一步研究VDAC1蛋白在乙酸铅对z310细胞毒性机制中的功能和作用提供实验基础。方法利用分子克隆技术构建4种特异性VDAC1-miRNA（GFP）干扰载体，筛选、测序验证并扩增。通过Lipofectamine LTX转染试剂盒将重组质粒转染Z310细胞，转染24h后，以荧光显微成像技术观察细胞的转染效果。经Blasticidin筛选后，通过实时荧光定量PCR法和蛋白印迹法（Western blotting）检测转染后细胞中靶基因和靶蛋白的表达情况。并采用CCK-8法检测乙酸铅（1、5、10、20、50、100、200和400μmol／L）染毒24h对VDAC1低表达Z310细胞增殖的影响。结果本实验筛选出最有效的干扰载体13MR0047—3—1，与空载体组比，其对靶基因的沉默效率为52．62％；与未处理的Z310细胞组比，其下调了81．28％VDAC1蛋白的表达。CCK-8法结果显示，20μmoL／L以上剂量的乙酸铅可抑制VDAC1低表达Z310细胞的增殖，存在剂量一效应关系；且与相同染毒剂量组的空载体对照Z310细胞比，高剂量乙酸铅（200～400μmol／L）对VDAC1低表达Z310细胞的毒性更大，差异具有统计学意义（P〈0．05）。结论大鼠脑脉络丛永生化Z310细胞电压依赖性阴离子通道1的靶向抑制成功，VDAC1基因低表达型Z310细胞系构建成功。VDAC1的低表达促使Z310细胞对乙酸铅的毒性作用更为敏感，其机制值得进一步研究。Objective To establish VDACl-deficient Z310 cell line through RNA interference, investigate the influence of lead acetate on its proliferation, and thus lay the basis for the further study on the function of VDAC1 in the neurotoxicity of lead. Methods Molecular cloning technology was used to construct four specific VDACI-miRNA （GFP）interference vectors. The resultant correct plasmids were transfected into the Z310 cells by Lipofectamine LTX, after 24 h, the transfection was identified by fluorescence microscope. After Blasticidin resistant selection, the RNA and protein expression levels of VDAC1 gene in Z310 cells were detected by real-time RT-PCR and western blotting so as to verify the effect of interference. After VDACl-deficient Z310 cells were treated with lead acetate （0, 1, 5, 10, 20, 50, 100, 200 and 400 μmol/L） for 24 h, cell survival rate were examined by CCK-8 assay. Results 13MR0047-3-1 was selected as the most effective interference vector, and its target gene silencing efficiency was 52.62% ,and western blotting showed that the protein expression level of VDAC1 was knocked down 81.28%. CCK-8 assay showed that the proliferation of VDACl-deficient Z310 cells was significantly inhibited by lead acetate with a dose-effect relationship, the survival rate decreased as dose increase, and there were statistically significant difference from 20 μmol/L to 400 μmol/L as compared to the control （ P 〈 0.05 ）. The toxicity of lead acetate （200 and 400 μmol/L） to VDACl-deficient Z310 cells was greater than the cells transfected with empty vector （P〈0.05）. Conclusion The RNA interference of VDAC1 gene in Z310 cells was high effective and VDACl-deficient Z310 cell line was successfully established. The decreased expression of VDAC1 induces higher toxic effect of treatment with lead acetate in Z310 cells and the mechanism is worth of further studying.

关 键 词：永生化Z310细胞 电压依赖性阴离子通道1 乙酸铅 增殖 

分 类 号：R114[医药卫生—卫生毒理学]