PEI-Et输送特异性shRNA对大鼠肝细胞AGT基因表达的影响

目的:研究交联小分子量聚乙烯亚胺衍生物PEI-Et对大鼠肝细胞(BRL-3A)的细胞毒性、转染效率和携带高血压相关基因血管紧张素原(AGT)短发卡RNA(shRNA)沉默AGT表达的能力。方法:MTT法检测PEI-Et/shRNA复合物对BRL-3A细胞的毒性,流式细胞术检测PEI-Et/shRNA复合物对BRL-3A细胞的转染效率,RT-PCR和Western blot检测PEI-Et/shRNA对AGT的基因沉默效果。结果:在相同质量比(w/w)时PEI-Et/shRNA的细胞毒性小于PEI 25kDa/shRNA(P0.01),PEI-Et/shRNA在w/w为30时达到最高转染效率,高于PEI 25 kDa(P0.01),PEI-Et/shRNA能高效沉默BRL-3A细胞中AGT基因的表达。结论:PEI-Et在BRL-3A细胞中是一种低细胞毒性、高转染效率的非病毒基因载体(与商业化的PEI 25kDa比较),能携带AGT shRNA高效沉默BRL-3A细胞中AGT基因的表达,通过用PEI-Et/AGT shRNA来抑制AGT的表达将为高血压的基因治疗提供一种新的思路。

Effects of Delivery of Specific shRNA using PEI-Et on AGT Gene Expression in Rat Liver Cells

Objective： To synthesize cross-linked small-molecular-weight polyethylenimine derivative PEI-Et and investigate its cytotoxicity, transfection efficiency and ability to delivery hypertension related gene angiotensinogen （AGT） short hairpin RNA （shRNA） to silence AGT expression. Methods： MTT assay was used to measure the cytotoxicity of PEI-Et/shRNA complexes. Flow cytometry was performed to investigate transfection efficiency of PEI-Et/shRNA in BRL-3A cells. RT-PCR and Western blot were used to detect the AGT gene silencing effect of PEI-Et/shRNA. Results： PEI-Et/shRNA showed lower cytotoxicity than PEI 25kDa/shRNA at the same weight ratio （w/w）. Transfection results indicated that PEI-Et/shRNA displayed the highest transfection efficiency at w/w 30, which was higher than PEI 25kDa/shRNA （P〈0.01）. PEI-Et/shRNA could efficiently inhibit the expression of AGT in BRL-3A cells. Conclusion： PEI-Et was a non-viral vector with much lower cytotoxicity and enhanced transfection efficiency than PEI 25kDa in BRL-3A cells, and it could delivery AGT shRNA to efficiently silence AGT expression in BRL-3A cells. Therefore, PEI-Et/AGTshRNA would be a promising tool for delivering AGT shRNA to BRL-3A cells for hypertension therapy.