建立有效的脂质体瞬时转染法,将小干扰RNA(siRNA)转染至小鼠肾小管上皮细胞(renal tubular epithelial cells,RTECs),从而抑制其乙酰化组蛋白酶1(histone deacetylases 1,HDAC1)的表达.使用阳离子脂质体法瞬时转染小鼠肾小管上皮细胞,用荧光标记siRNA(FAM-siRNA)筛选转染比例,实时荧光定量PCR(Real-time PCR)确定最优条件并检测不同位点(374,525和822)HDAC1-siRNA的抑制效果,噻唑蓝(MTT)法检测不同时间点HDAC1-siRNA对RTECs增殖的影响,并设立曲古霉素处置组作为试验对照.荧光表达和Real-time PCR结果显示30 pmol siRNA∶1.5 μL LipofectamineTM 2000为最佳转染条件,HDAC1-374的干扰效果最明显.MTT检测结果显示,HDAC1-374与TSA一致,证实HDAC1-siRNA能有效抑制RTECs的增殖.本试验利用脂质体瞬时转染siRNA法成功抑制了RTECs中HDAC1的表达.%Abstract: To transfect histone deacetylases KHDAC1)-small interferencing RNA (siRNA) into mouse renal tubular epithelial cells (RTECs) by liposome transfection and to investigate its inhibitory effect on HDAC1. It was transient into RTECs by liposome transfection. Fluorescently labeled siRNA (the FAM-siRNA) to filter the proportion of transfected. Real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) to determine the optimal conditions and testing the various sites(374, 525 and 822)HDACl-siRNA inhibition effect. MTT method to detect the influence of the HDACl-siRNA on suppression RTECs proliferation in different time points, and trichostatin A(TSA) disposal group as a test control. Expression of fluorescence and Real-time PCR results showed that 30 pmol siRNA :1. 5 mL LipofectamineTM2000 were optimal transfection conditions and HDAC1-374 had the most obvious interference effect. The MTT testing results of HDAC1-374 and TSA were consistent: HDACl-siRNA could effectively restrain RTECs proliferation. The test using liposomes transiently transfected siRNA method, successfully inhibition the expression of HDAC1 in RTECs.
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