摘要
目的:观察针对TGF-βRⅡ的干扰RNA对TGF-βRⅡ表达的抑制作用,探讨RNA干扰TGF-βRII的方法对肾间质纤维化的抑制作用。方法:PCR方法扩增TGF-βRⅡ的cDNA序列,将全长的TGF-βRⅡcDNA插入pcDNA3中,构建TGF-βRⅡ的真核表达载体,酶切、测序及间接免疫荧光鉴定;根据TGF-βRⅡ的设计针对TGF-βRⅡ的干扰RNA,构建针对TGF-βRⅡ的RNA干扰质粒pSUPER-TGF-βRⅡ并进行酶切及测序鉴定;脂质体介导针对TGF-βRⅡ的RNA干扰质粒和TGF-βRII的真核表达载体共转染293T细胞,western-blot检测293细胞内TGF-βRⅡ的表达。结果:pcDNA3-TGF-βRⅡ测序结果显示TGF-βRⅡ基因未发生突变。间接免疫荧光检测TGF-βRⅡ在293T细胞内的表达;测序显示RNA干扰质粒pSUPER-TGF-βRⅡ序列正确,RNA干扰质粒明显抑制293T细胞内TGF-βRⅡ的表达。结论:成功构建了TGF-βRⅡ的真核表达载体及针对TGF-βRⅡ的RNA干扰质粒,RNA干扰质粒能够抑制TGF-βRⅡ的表达,为进一步研究针对TGF-βRⅡ的RNA干扰对肾纤维化的预防及治疗作用提供有用工具。
Objective： To observe the inhibition of TGF-βRⅡ gene expression by RNA Interference and evaluate the inhibitory effect of RNA interference method on renal fibrosis. Methods： The gene sequence encoding TGF-βRⅡ was amplified by polymerase chain reaction （PCR） method and cloned into the vector pcDNA3 to construct a eukaryotic vector expressing the TGF-βRⅡ. According to the gene sequence of the TGF-βRⅡ, the interfering RNA for TGF-βRⅡ were designed and cloned into the vector of pSUPER to construct the plasmid for interfering the expression ofTGF-βRⅡ . Both of the vectors were analyzed by enzyme digestion and sequence analysis. Results： The sequence analysis of the pcDNA3-TGF-βRⅡ and pSUPER-TGF-βRⅡ demonstrated that none mutation hap- pened in both the sequence ofTGF-βRⅡ and the interfering RNA for TGF-βRⅡ . With the indirect immunofluorescence method, the expression of TGF-βRⅡ in 293Tcells was detected. Results showed that the expression of TGF-βRⅡ can be obviously inhibited by TGF-βRⅡ interfering RNA. Conclusion： A eukaryotic vector expressing the TGF-βRⅡ and a plasmid interfering the expression of TGF-βRⅡ were constructed successfully which laid a foundation for investigating the treatment and prevention on renal fibrosis with the RNA interfering technique.
出处
《现代生物医学进展》
CAS
2009年第1期43-45,83,共4页
Progress in Modern Biomedicine
关键词
TGF-βⅡ型受体
真核表达
RNA干扰
Type Ⅱ Receptor Of TGF-β（TGF-βRⅡ ）
Eukaryotic Expressing
RNA Interference
