The Experiment Of The Protective Effect Of Zinc Finger Protein A20 On Endothelial Cell In Rat Model Of Acute Liver Allograft Rejection

Objective ① To generate a recombinant adenoviral vector encoding zinc figer protein A20 (rAdEasy-A20); ② To study the mechanisms involved in the suppressed rat liver allograft rejection by zinc finger protein A20 with rAdEasy-A20 or an empty Ad vector (rAdEasy) intravenous injection into the penile vein; This study was conducted to elucidate the molecular mechanisms involved in the protective effect of zinc finger protein A20 on liver allograft. Methods ① The Nco I →Sal I fragment (2332 bp) of A20 gene, gotten from the plasmid pCAGGS-FLAGmA20 which carrying the whole mouse A20 cDNA, was cloned into the shuttle plasmid pAdTrack-CMV. With the resultant plasmid and the backbone plasmid pAdEasy-1, the homologous recombination took place in the Escherichia coli BJ5183and the recombinant adenoviral plasmid was generated. The adenovirus was packaged in the 293 cells and the recombinant adenovirus rAdEasy-A20 was generated. The empty Ad vector (rAdEasy) was generated as the same principle. ② Allogeneic liver transplantation was performed using a combination of Wistar rat with SD rat with the two-cuff method. Rats were randomly divided into three groups: liver transplantation group, rAdEasy-A20 treatment group (rAdEasy-A20 group) in which rAdEasy-A20 (5 × 10~9 pfu rAdEasy-A20/rat) was injected intravenously through the penile vein into donor Wistar rats 24h before liver transplantation, and rAdEasy treatment group (rAdEasy group) in which rAdEasy ( 5 × 10~9 pfu rAdEasy/rat) was injected intravenously through the penile vein into donor Wistar rats 24h before liver transplantation. Liver graft and blood samples were harvested at 7 days after liver transplantation or on the brink of death. Recipient survival time was recorded. Histopathological characteristics of liver allograft were detected with HE staining. Plasm levels of alanine aminotransferase (ALT), total bilirubin (TBIL), and albumin (Alb) were measured with an automatic biochemical analyser. Expression of VCAM-1 in liver allograft was measured with immunohistochemical staining. Apoptosis of hepatocytes was detected with TUNEL staining.Results ①The Nco I →Sal I fragment (2332 bp) of mouse A20 gene was cloned into the shuttle plasmid pAdTrack-CMV. The recombinant adenoviral plasmid was generated. The adenovirus was packaged inthe 293 cells and the recombinant adenovirus rAd Easy-A20 was generated. High level expression of A20 mRNA and A20 protein in 293 cells transfected with rAdEasy-A20 was detected by immunohistochemical staining, respectively. Whereas A20 mRNA and A20 protein were not detected in normal 293 cells and 293 cells transfected with rAdEasy. ?Allogeneic (Wistar rat-*SD rat) liver grafts demonstrated moderate to severe pathology-proven acute rejection 7 days after transplantation or on the brink of death. There was a significant increase of ALT and TBIL level and marked decrease of Alb level, the liver function was markedly impaired. The recipient rats without any immunosuppressive therapy died from allograft rejection 5 ~ 20 days after liver transplantation. rAdEasy-A20 significantly suppressed liver allgraft rejection and prolonged liver allograft survival, only 1/5 cases of liver allograft transfected with rAdEasy-A20 showed pathological features of I stage rejection, and thus rAdEasy-A20 prolonged recipient survival to 11~33 days. Immunohistochemical staining showed markedly suppressed expression of VCAM-1 in liver allograft treatment with rAdEasy-A20 compared to liver allograft without any therapy or treatment with rAdEasy. TUNEL staining appeared significantly decreased apoptosis index of hepatocyte in liver allograft treatment with rAdEasy-A20 (4.34 + 3.41%) compared to liver allograft without any therapy (10.25+ 5.14%) or treatment with rAdEasy (15. 36 + 5. 51%). Conclusions ?Zinc finger protin A20 can protect the endothelialcell, and it suppress lymphocyte activation by inhibition of NF- k B activation. (2) rAdEasy-A20 treatment can suppress liver allograft rejection and prolong recipient survival time by inhibiting expression ofVCAM-1 in liver allograft.