Construction Of RNA Interference Expression Plasmid Of Smad3 And Primary Study On Its Relation With Glycosyltransferase Family PpGalNAc-Ts

Object:Smad3 is one of the most important proteins in the TGF-β/Smads pathway. It performs a significant role during the whole process of hepatic fibrosis through introducing the signal of TGF-βand activiating hepatic star cells . This thesis aims to a construct and screen a optimal RNAi-vector of Smad3 and study the relationship btween Smad3 and beta-3 glycosyltransferase family ppGalNAc-Ts.Methods:To follow the principle of selecting RNA interference target sequence, to utilize the web resource,design three potential small interference RNA (siRNA) of Smad3 mRNA. Subcloned this siRNA to the RNAi eukaryotic expression plasmid pRNAT-U6.1/Neo and obtained three RNAi-vectors of Smad3.Then selected a optimal RNAi-vector among the three vectors through RT-PCR and Western-Blot .Using the optimal RNAi-vector of Smad3 to knockdown Smad3 ,detected the mRNA of ppGalNAc-Ts .At the same time ,using the two RNAi-vector of ppGalNAc-T2 which had been constructed by our lab to knocdown ppGalNAc-T2 ,detected the mRNA and protein of Smad3. Studied the relationship between Smad3 and glycosyltransferase family ppGalNAc-Ts.Results: Constructed and Selected a optimal RNAi-vector which could effectively inhibited the mRNA and protein expression of Smad3.As the knockdown of Smad3, the mRNA expression of ppGalNAc-Ts were lightly influenced .Besides, as the knockdown of ppGalNAc-T2 ,the mRNA and protein expression were neraly not influenced .Conclusion:Constructed and Screened a optimal RNAi-vector,The knockdown model of Smad3 gene was successfully consructed and provide a surport of the RNAi method to the hepatic fibrosis curing.As the the knockdown of Smad3, the mRNA expression of ppGalNAc-Ts were lightly influenced .Besides, as the knockdown of ppGalNAc-T2 ,the mRNA and protein expression were neraly not influenced .The rusults indicated that there is nearly no relation between glycosyltransferase family ppGalNAc-Ts and Smad3. The ralationship between Smad3 and other glycosyltransferase need further research.