Construction And Identification Of Transfection Efficiency In Volvo Of Rap1 ShRNA Expression Vector And Its Interference Role To Liver Regeneration In Rat

Objective:1.To contruct three kinds of Rap1 shRNA expression vector with RNA interference technique.2.To study the interference role of efficient Rap1 shRNA to liver cells in vivo and choose the mostly efficient siRNA.3.To Study the protection effect role of Rap-1 shRNA to liver regeneration.Methods:1.Beginning with the AUG start codon,scan the length of Rap1 mRNA for AA sequences Record the AA and downstream 19 nucleotides and compare the sequence to the appropriate genome database to eliminate any sequences with significant homology to other genes.In this study,we selected three target sequences for testing and one corresponding negative control vector(HK),and controlled the GC content between 30%～60%.Rap1 siRNA and HK was synthesized in vitro and cloned into the EGFP reporter plasmid pGenesil-3. Sequence analysis were identified after double endonuclease digestion.2.Forty KunMing mouses weighing 18～22g were randomly divided into 4 groups:groupⅠ,HK was transfected(n=10);groupⅡ, Rap1 shRNA 1 was transfected(n=10);groupⅢ,Rap1 shRNA2 was transfected(n=10);groupⅣ,Rap1 shRNA3 was transfected(n=10); Synthesized shRNA was injected into mouses' intraperitoneal at 0h,16h,24h.The animals were killed at 48h after transfection,we collected cells,We used RT-PCR and immunofluorescence to determine the expression level of Rap-1a mRNA and protein.We selected the mostly efficient shRNA to do subsequently experiment according to RT-PCR and S-P result.3.Thwity KunMing mouses weighing 18～22g were randomly divided into 3 groups:groupⅠ,partial hepatectomy(PH)(n=10)and groupⅡ,Rap1 shRNA + PH(n=10).In groupⅠandⅡ,the animals were anesthetized with intraperitoneal 10%chloral hydrate 350(mg/kg)and confirmed with middle lobectomy and left lobectomy.In groupⅡRap1 shRNA solution(2.0～2.5 mg/kg)was given inject intraperitoneal at 0h,16 h and 24h after PH,while in groupⅠPBS solution was given instead of Rap1 shRNA solution.In groupⅠ,Ⅱ,the animals were killed at 48 h after PH.Livers were immediately removed and sliced.We used RT-PCR and immunofluorescence to determine the expression level of Rap1 mRNA and protein,ERK protein,cycline D1 proteine,Ki67 protein.Results:1.Rap1 siRNA expression vector was successfully constructed and identified by double endonuclease digestion.Sequence analysis of the inserted fragment revealed the salne sequence as that of the synthesized siRNA oligonucleotides.2.Three siRNA expressing recombinants and the corresponding negative control vector were constructed and transfected into liver cell successfully.The transfection efficiency of groupⅠ,Ⅱ,Ⅲ,Ⅳis more than 50%.Rapl mRNA and protein expression of groupⅠis obvious higther than groupⅡ,Ⅲ,Ⅳ(p＜0.05).Rap1 mRNA and protein expression of groupⅡis significantly degrader than groupⅢ,groupⅣ(p＜0.05).3.expression level of Ki67,ERK,cyclineD1,Rap1 protein in groupⅡis higher than in groupⅠ,expression level of Rap1 mRNA in groupⅠis higher than in groupⅡ.Conclusions:1.Target siRNA were successfully designed and synthesized by adopting bioinformatic techique.2.Rap1 shRNA transfect into liver cell successfully.Three Rap1 shRNA can degrade the expression level of Rap1 mRNA and protein, Rap1 shRNA1 is the mostly effective inference role,established a favourable foundation for further experiment of liver regeneration.3.Rap1 shRNA can inhabit expression of Rap1 mRNA,and enhance liver regeneration.