化学合成小干扰RNA体外转染大鼠支持细胞的实验研究

目的:观察化学合成siRNA阻断大鼠支持细胞内源性基因表达的可行性,并优化转染条件。方法:体外取SD大鼠睾丸,分离、培养支持细胞,应用化学合成的不同浓度siRNA在阳离子脂质体Lipofectamice TM 2000介导下转染原代支持细胞。RT—PCR检测大鼠支持细胞中GAPDH mRNA水平的变化,MTT法检测支持细胞的活性。结果:转染终浓度为150nmol/L的siRNA能特异性有效的阻断GAPDH基因的表达,细胞毒性随浓度提高而增强,终浓度为200nmol/L的siRNA对细胞有明显毒性。结论:siRNA能在大鼠支持细胞内启动RNA干扰,150nmol/L的siRNA能特异有效地阻断内源基因表达并保持较高的细胞活性。

The Empirical Study of in Vitro Transfection by Chemical Synthesized siRNA in Rat Sertoli Cells

Objective:To observe the feasibility of chemical synthesized siRNA in blocking the endogenous gene expression in rat sertoli cells,and optimize transfection conditions.Methods:The testes were removed from SD rats,and the sertoli cells were separated and cultured in vitro.Chemical synthesized siRNA of different concentrations were transferred into primary ser- toli cells using cationic liposome Lipofectamine TM 2000.The changes of GAPDH mRNA level in rat sertoli cells were detec- ted by RT-PCR.And the cytoactive of sertoli cells was examined using MTT.Results:siRNA of transfection concentration at 150umol/L could block the GAPDH gene expression specifically and effectively,and the cytotoxicity strengthened with the in- crease of the concentration,siRNA of final concentration at 200nmol/L showed overt toxicity to cells.Conclusion:siRNA can switch on RNA interference in rat sertoli cells,which of 150nmol/L can specifically and effectively block the endogenous gene expression without sacrificing cytoactive.