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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 channel1,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24 h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24 h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-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 use a vector-mediated RNAi technique to target the expression of voltage-dependent anion channel 1 (VDAC1) in immortalized rat choroid plexus Z310 cells and construct a low expression of VDAC1 gene Z310 cell line , And detected the effect of lead acetate on its proliferation, which provided the experimental basis for further study on the function and role of VDAC1 protein in Z310 cytotoxicity induced by lead acetate. Methods Four specific VDAC1-miRNA (GFP) interfering vectors were constructed by molecular cloning technology, screened, sequenced and amplified. The recombinant plasmids were transfected into Z310 cells by Lipofectamine LTX transfection kit. After transfection for 24 h, the transfected cells were observed by fluorescence microscopy. After Blasticidin screening, the expression of target gene and target protein in transfected cells was detected by real-time fluorescence quantitative PCR and Western blotting. The effects of lead acetate (1, 5, 10, 20, 50, 100, 200 and 400 μmol / L) for 24 h on the proliferation of VDAC1 low expression Z310 cells were detected by CCK- Results The most effective interference vector 13MR0047-3-1 was screened out in this study. Compared with the empty vector group, the silencing efficiency of the target gene was 52.62%. Compared with the untreated Z310 group, the silencing efficiency of the knockdown gene was 81.28% VDAC1 expression. The results of CCK-8 assay showed that lead acetate at a dose of 20μmol / L could inhibit the proliferation of Z310 cells with low VDAC1 expression, and there was a dose-effect relationship. Compared with Z310 cells in the same exposure dose, (200 ~ 400μmol / L) was more toxic to VDAC1 low expressing Z310 cells, the difference was statistically significant (P <0.05). Conclusion The targeted suppression of voltage-dependent anion channel 1 in immortalized Z310 cells in rat cerebral choroid plexus was successful. The low expression of VDAC1 gene Z310 cell line was successfully constructed. The low expression of VDAC1 makes Z310 cells more sensitive to the toxic effects of lead acetate, and its mechanism deserves further study.