| Purpose1998, Fire and his colleagues found a phenomenon called RNA interference (RNAi). This phenomenon refers to the gene targeting specificity post - tran-scriptional gene silencing ( PTGS) induced by the double - stranded RNA ( dsRNA) which is injected into the Caenorhabditis elegans. During the past few years, substantial improvements about RNAi were gained worldwide. RNAi became a new gene inhibiting technique, and can successfully interfere the express of the specific genes using RNAi in vitro - cultured mammalian cells such as from human or rat. This provides a new possibility for human gene therapy.Angiotensin II type 1 receptor ( AT1R) is the specific receptor of angiotensin II which is the key factor of renin ?angiotensin system ( RAS) , and plays critical role on the effect of Angll. Now it was found that ATI R can express in vascular smooth muscle cells ( VSMCs) , myocardial and fibroblast ceils, and be very important in the reconstruction of heart ?vessels system. In the present study we constructed plasmids with rat AT1R short hairpin RNA (shRNA) and transfect the plasmids into rat VSMCs to investigate the changes of the expression of AT1R mRNA and protein respectively, and the effect on hyperplasia of VSMCs in rat.MethodsExperiment I . Plasmid construction containing the rat short hairpin RNA of angiotensin II type 1 receptorFinding the sequence of rat angiotensin fl type 1 receptor mRNA in the GeneBank, putting into the corresponding software and designing the primerrank. The fragments amplificated by PCR were inserted to the pGEM?-T vector, and were then selected and identified.Experiment 31. Inhibition of angiotensin fl type 1 receptor expression by short hairpin RNA in mammalian cellsThe plasmids containing the shRNA of AT1R were constructed, and the change of mRNA and protein were detected by RT - PCR and Western blot after transfecting vascular smooth musle cells.Experiment ffi. Effect of the plasmid containing the short hairpin RNA of AT1R on hyperplasia of VSMCs in ratThe plasmids containing the shRNA of AT1R were constructed, and trans-fected vascular smooth musle cell to detect the effect on the ATI R expression by RT - PCR and Western blot, observe the shape of VSMCs by the inverted phase contrast microscope, and detect the hyperplasia of VSMCs by trypan blues tai-ning and MTT.ResultsExperiment I . Plasmid construction containing the rat short hairpin RNA of angiotensin II type 1 receptorThe result of confirmation test is consistent with the prediction, and the plasmid was certified to be in the right rank.Experiment H . Inhibition of angiotensin fl type 1 receptor expression by short hairpin RNA in mammalian cellsAfter transfecting cells, there was significant difference (P<0.01) in the expression of AT1R mRNA between the gene transfected group (pATIR - shRNA! 1. 371 ?. 148; pATIR - shRNA2l. 453 ?. 123) and the control group (2.088 ?.258) ; and there was significant difference (P <0. 01) in the expression of AT1R protein between the gene transfected group ( pATIR - shRNA, 1.121 ?. 037; pATIR - shRNA2l. 202 ?. 068) and the control group (3. 168 ?.21). It is shown that pATIR - shRNA can decrease the expression of AT1R mRNA and protein.Experiment HI. Effect of the plasmid containing the short hairpin RNA ofAT1R on hyperplasia of VSMCs in ratAfter transfecting cells, there was significant difference (P <0.01) in the expression of AT1R mRNA between the plasmid transfected group (pATIR -shRNA, 1. 371 ?. 148; pATIR - shRNA21.453 ?. 123) and the control group(2.088 ?.258) , and there was significant difference (P <0.01) in the expression of AT1R protein between the gene transfected group (pATIR -shR-NA, 1.121 ?. 037; pATIR - shRNA2l. 202 ?.068) and the control group (3.168 ?.21). It is shown that pATIR - shRNA can decrease the expression of AT1R mRNA and protein. There was significant difference ( P <0.01) in the Cell number between the plasmid transfected adding Ang II group ( pATl R -shRNA, 5.483 ?.436; pATIR -shRNA2 5.55 ?0.446) and the Angflcon-trol gro...
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