Recombinant Adenovirus Vector. At1rshrna The Functional Expression In C6 Cells And At1r In Shr Distribution Impact

AIM(PURPOSE): To detect the expression of AT1R gene of rat glioma cells in vitro and the distribution of AT1R protein of spontaneous hypertensive rat (SHR) in vivo through transfection with constructed Adenovirus-based shRNA expression systems which target against the rat angiotensinⅡreceptor gene , furthermore to investigate the efficacy of preformed siRNAs both in vitro and in vivo to modulate the expression of target gene in mammalian cells for antihypertensive therapy in SHR at post-transcriptional level.EXPERIMENTAL DESIGN AND METHODS: Use human embryo kidney 293 cell to culture Adenovirus-based shRNA expression systems targeting against the rat angiotensinⅡreceptor gene. The Infectivity of recombinant adenoviral vectors was determined by a TCID50 assay. And then recombinant adenoviral vectors were transfected into rat glioma cells. The cultured cells were collected at different phases. RT-PCR and western blot were performed. SHR were transfected with recombinant adenoviral vectors by caudal vein injection method. AT1R expression of major organ: include heart, liver, kidney, adrenal gland and aorta was detected by immunohistochemical method.RESULTS: 1. Adenovirus-based shRNA expression systems targeting against the rat angiotensinⅡreceptor gene were transfected into C6 successfully. C6 AT1R mRNA and protein levels behaved ultimately same. Compared to the control, C6 AT1R mRNA and protein expression could be inhibited significantly. 2. Adenovirus-based shRNA expression systems targeting against the rat angiotensinⅡreceptor gene were transfected into SHR in vivo. immunohistochemical method showed that AT1R expression of major organ decreased considerably.CONCLUSION: Adenovirus-based shRNA expression systems targeting against the rat angiotensinⅡreceptor gene were transfected successfully, the shRNAs with a 22-nt stem and a short loop were cleaved into small interfering dsRNA (siRNA) by the Dicer . Both in vitro and in vivo, the transcribed siRNA enables the effective silencing of gene expression to the target mRNA and leads to effective inhibition of translation of proteins and will lay the foundation of application of gene silencing technology to hypertensive rat for the action, curative effect and application value and be helpful to genetic therapy of the hypertension.