摘要
目的研究质粒表达型短发夹RNA(smallhairpinRNA,shRNA)对CHL-3A4转基因细胞的细胞色素P4503A4(cytochromeP4503A4,CYP3A4)基因表达的影响。方法设计并构建3条shRNA真核表达载体(CYP3A4Ⅰ、CYP3A4Ⅱ、CYP3A4Ⅲ),脂质体转染CHL-3A4转基因细胞。转染48h后,TR-PCR和WesternBlotting分别观察3条shRNA对CYP3A4mRNA和蛋白表达的影响;噻唑蓝(MTT)观察shRNA抑制环磷酰胺对CHL-3A4转基因细胞毒性作用。结果CYP3A4Ⅲ表达载体的shRNA分别能抑制CHL-3A4细胞的CYP3A4mRNA表达(70%)及蛋白表达(75%),抑制环磷酰胺对CHL-3A4细胞(75%)细胞毒作用。结论RNA干扰能显著抑制CYP3A4基因的表达,RNA干扰技术为肝细胞色素P450的研究提供了新的方法。
Objective To investigate the inhibitory effect of the eytoehrome P450 3A4（CYP3A4） gene expression and function in CHL-3A4 cells lines by vector-eepressed small hairpin interfering RNA（shRNA）. Methods The shRNA expression vectors targeting CYP 3A4 gene（ CYP3A4 Ⅰ , CYP3A4 Ⅱ , CYP3A4 Ⅲ ） were designed and constructed. The inhibitory effect of shRNA was detected by Western blot and RT-PCR analysis. The inhibitory effect of cytotoxie of cyclophosphamide using shRNA was measured by MTT assay. Results CYP3A4 Ⅲ shRNA expressing vector significantly reduced the CYP3A4 mRNA （70%） and protein expression levels （75 % ） of the CYP3A4 gene in CHL3A4 cells suppression of CYP3A4 gene expression by CYP3A4 Ⅲ largely reversed cyclophosphamide cytotoxieity（75 % ） in CHL-3A4 cells. Conclusion Vector-based RNAi could suppress CYP3A4 gene expression and function,and the use of RNAi to inhibit CYP3A4 gene expression in mammalian cells was a promising new tool for the study of cytoehrome P450 gene function.
出处
《中国基层医药》
CAS
2006年第4期543-545,共3页
Chinese Journal of Primary Medicine and Pharmacy
