脂质体瞬时转染siRNA抑制肾小管上皮细胞去乙酰化蛋白酶1的表达

中国畜牧兽医

脂质体瞬时转染siRNA抑制肾小管上皮细胞去乙酰化蛋白酶1的表达

杨洋^1,2, 吴国娟², 李培锋¹

1. 内蒙古农业大学兽医学院,内蒙古呼和浩特 010018;
2. 北京农学院动物科学技术学院,北京 102206

收稿日期:2012-02-07 出版日期:2012-08-20 发布日期:2012-08-24
通讯作者: 李培锋 E-mail:lpfneimeng@163.com; wgj9288@sina.com
作者简介:杨洋(1982－),女,内蒙古人,博士,研究方向:AAⅠ肾毒性作用受体信号通路。
基金资助:
国家自然科学基金资助项目(30972215);北京市自然科学基金资助(6092004,5102014)。

Inhibition of Liposome Transfection siRNA on HDAC1 Expression in Mouse Renal Tubular Epithelial Cells

YANG Yang^1,2, WU Guo-juan², LI Pei-feng¹

1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Animal Science and Technology College, Beijing University of Agriculture, Beijing 102206, China

Received:2012-02-07 Online:2012-08-20 Published:2012-08-24

摘要/Abstract

摘要： 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 Lipofectamine^TM 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。

关键词: HDAC1-siRNA; RTECs; FAM-siRNA; MTT

Abstract: To transfect histone deacetylases 1(HDAC1)-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)HDAC1-siRNA inhibition effect, MTT method to detect the influence of the HDAC1-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 Lipofectamine^TM2000 were optimal transfection conditions and HDAC1-374 had the most obvious interference effect. The MTT testing results of HDAC1-374 and TSA were consistent: HDAC1-siRNA could effectively restrain RTECs proliferation. The test using liposomes transiently transfected siRNA method, successfully inhibition the expression of HDAC1 in RTECs.

Key words: HDAC1-siRNA; RTECs; FAM-siRNA; MTT

中图分类号:

Q813

杨洋, 吴国娟, 李培锋. 脂质体瞬时转染siRNA抑制肾小管上皮细胞去乙酰化蛋白酶1的表达[J]. 中国畜牧兽医.

YANG Yang, WU Guo-juan, LI Pei-feng. Inhibition of Liposome Transfection siRNA on HDAC1 Expression in Mouse Renal Tubular Epithelial Cells[J]. .

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