构建高效抑制细胞色素P450 2E1基因转录位点的siRNA表达载体  

Construction of siRNA expressive vector to the optimal point depressing transcription for cytochrome P450 2E1 gene

作　　者：徐芸[1] 万志红[2] 李继昌[1] 

机构地区：[1]郑州大学第一附属医院消化科,450052 [2]解放军第三军医大学西南医院感染科,400038

出　　处：《肝脏》2007年第4期271-274,共4页Chinese Hepatology

摘　　要：目的寻找高效抑制细胞色素P450 3E1(CYP2E1)基因转录的位点,构建该位点的siRNA表达载体。方法用PCR的扩增物作为表达框架,直接转染E47细胞,产生siRNA抑制CYP 2E1基因转录,筛选高效抑制CYP 2E1基因表达的位点,并构建BS/U6/CYP2E1的表达质粒,转染E47细胞,观察该质粒在E47细胞中发生RNAi效果。结果4个备选位点的抑制效率最高,用该位点构建BS/U6/CY2E1的表达质粒可有效地、特异地抑制CYP2E1基因的表达。结论用PCR产物作为表达框架,在体内产生双链的siRNA作为筛选高效的RNA干扰位点的方法,既快速又简便。用该位点的构建BS/U6/CYP2E1的表达质粒可用于CYP2E1基因的功能研究、酒精性脂肪肝病和非酒精性脂肪性肝病的发病机制及肿瘤发生的研究等领域中。Objective To screen out the optimal point depressing transcription for P450 2E1 （CYP 2E1 ） gene, and construct siRNA expressive vector to the point . Methods The siRNA expresion cassettes was constructed by PCR, and the PCR products was directly transfected into E47 cells resulting in functional expression of siRNA to depress expression for CYP2E1 gene, to screen out the optimal point to depress transcription for CYP 2E1 gene,and constructed a BS/U6/CYP2E1 which was a DNA vector-based approach to achieve RNAi in E47 cells. Results The point 1233 - 1251 was the potimal ones in the four preliminary points, and BS/U6/CYP2E1 could show gene-specific suppression of CYP 2E1. Conclusion a facile PCR based strategy for rapid synthesis of siRNA expression units were tested. The siRNA expression unit are constructed by PCR, and the PCR products are directly transfected into mammalian cells resulting in funtional expression of siRNAs. This approach may prove useful for identification of optimal siRNA-target combinations. Constructed BS/U6/CYP2E1 could be used in studying the function of the CYP2EI,the pathogenesis of alcoholic and nonalcoholic fatty liver disease and carcinogenesis.

关 键 词：细胞色素P450 2E1 小干扰RNA RNA干扰 

分 类 号：R346[医药卫生—基础医学]