The Study Of Inhibiting Renal Fibrosis By Transgene Of Antisense Vascular Smooth Muscle Alpha Actin

Objective(1)To construct adenovirus vector of antisense vascular smooth muscle alpha actin (pAdanti- a -SMA).(2)To observe whether the tubular epithelial cell(TEC) transfected by pAdanti- a -SMA can tolerate the tubular epithelial-myofibroblast Transdifferentiation (TEMT) induced by transforming growth factor betal(TGF- P l).(3)To study the renal fibrosis(RFB) progression of 5/6 nephrectomized rats transfected by pAdanti- a -SMA. In all, to verify whether the pAdanti- a -SMA can inhibit RFB and to discuss the mechanism of the inhibition.MethodsPart I Adenovirus vector construction and identification. The anti- a -SMA gene was extracted by primer designed by primer premir 5.0. The pGEM-T-Easy-anti- a -SMA plasmid, pShuttle-CMV-anti- a -SMA plasmid and the pAdanti- a -SMA were constructed in sequence and identified. Multiplicity of infection(MOI) of pAdanti- a -SMA was measured.Part II Experiment in vitro. The TEC lines was cultured with high concentration TGF- ?1 and pAdanti- a -SMA of different MOI. The growth and the phenotypic change of the cells were observed. The a -SMA immunofluorescence positive cells were counted and the a -SMA protein was measured by western bolt.Part III Experiment in vivo. To transfect 5/6 nephrectomized rats by the pAdanti- a -SMA. Rats survival rate, weight, renal function and renal tissues pathology were assessed. Expression of a -SMA, TGF- ?1 and PDGF-BB in kidney were detected by immunohitochemistry and analyzed by Tiger 920.ResultsPAdanti- a -SMA of different MOI was successfully constructed. PAdanti- a -SMA was identified by sequencing, enzyme digestion, RT-PCR and passed the safety detection. TEC was successfully transfected by pAdanti- a -SMA. High concentration of TGF- ? induced most of TEC transdiffere to a -SMA+ cells, a -SMA+ cells of TEC transfected by pAdanti-a-SMA( MOI=100) were sharply decreased and kept on decreasing in TEC transfected bypAdanti- a -SMA (MOI=400). Western bolt detection found the similar results. 5/6 nephrectomized rats was successfully transfected by pAdanti- a -SMA. The survival rate and rising weight of 5/6 nephrectomized rats transfected by pAdanti- a -SMA is higher than that of rats which were not transfected. The renal function and renal tissues pathology of the transfected rats was better than that of rats which were not transfected. Quantity of a -SMA+ cells in rats transfected is higher than that in rats which were not transfected , while the quantity of the TGF-P T and PDGF-BB+ cells both show no difference in the same experiment.Conclusion1. The pAdanti- a -SMA is a safe adenovirus vector with high transfection efficiency.2. TGF- P 1 can induce the tubular epithelial-myofibroblast transdifferentiation. TEC tranfected by pAdanti- a -SMA can tolerate the transdifferentiation in a dose-dependent manner.3. The RFB progression of 5/6 nephrectomized rats transfected by pAdanti- a -SMA is inhibited. The inhibition is free of TGF- ?1 and PDGF.4. To inhibit the a -SMA remodeling is a possible way to therapy RFB.