In Vitro Study On The Immune Effect Of Human Skin Fibroblasts After Transfected By Recombinant Adenovirus Vector Mediated CTLA4Ig And CD40Ig

It has been found that the immune rejection of heterogenic skin transplantation is acute cellular rejection, and activated T cells play a vital role in this progress.Moreover,the costimulation signals were essential for T cell full-activation.Blocking the costimulation signals will induce immune tolerance and prolong grafts survival.It was reported that blocking the costimulation signals using antibody could induce immune tolerance,but it is faced with problems such as high cost which is a obstacle for its extensive use.As we all know that Adenoviral vectors(Ad)have been widely applied in gene therapy and gene functional study, owing to their feature of safety and easily to culture.Using adv we can import exogenous gene into target cell,and it can not only express the treated protein but also block the costimulation signals,hope to reduce cost.Fibroblasts are easily to get and culture,so they and are usually used as the target cell of gene therapy.In this paper we transfect fibroblasts using Adv-CTLA4Ig and Adv-CD40Ig,then we check transfection efficiency,the cytotoxicity of Adv,also we will study study the immune effect of human skin fibroblasts after transfected by recombinant adenovirus vector mediated CTLA4Ig and CD40Ig.The results of this study are as follows:1.The transfection efficiency is tested using Flow cytometry after human skin fibroblasts transfected by Adv.The result shows that the transfection efficiency of Adv-EGFP at MOI 50 and 100 were 51.7±7.24%,69.99±3.26%.We also find that the shape and vigor have no change with the nomal.So we use 100 as the MOI.2.The cytotoxicity of Adv is tested by shape observation and MTT test.The result shows that Adv-CTLA4Ig,Adv-CD40Ig,Adv-CTLA4Ig+Adv-CD40Ig at MOI 100 is no significant cytotoxicity and the cell shape is similar between different groups. 3.The expression of CTLA4Ig and CD40Ig is tested by ELISA.The result shows that when Ad transfect human skin fibroblasts at MOI 100,there were CTLA4Ig and CD40Ig expressed respective in the culture medium,and the quantity is CTLA4Ig 2.79±0.08ug/ml,CD40Ig 1.25±0.11ug/ml,CTLA4Ig 2.14±0.21ug/ml and CD40Ig 1.00±0.04ug/ml。4.The immune effect of human skin fibroblasts after transfected by recombinant adenovirus vector mediated CTLA4Ig and CD40Ig were checked by co-cultured with PBMs.The result shows after the Adv-CTLA4Ig,Adv-CD40Ig,Adv-CTLA4Ig+Adv-CD40Ig transfect human skin fibroblasts at MOI 100,the proliferation depression of PBMs is significant lower than the contrast group(single Adv P＜0.05,mixed Adv P＜0.01).Adv-CTLA4Ig+Adv-CD40Ig group(31.67±2.25%)is higher than Adv-CTLA4Ig group,Adv-CD40Ig group(P＜0.05). AdvCTLA4Ig group(23.41±1.23%)is higher than AdvCD40Ig(19.83±4.71%)(P＜0.05).Conclusion:Adv can infect human skin fibroblasts efficiently and the infected cells could express aim protein efficiently.Adv is no significant cytotoxicity and the cell shape is no significant different contrast to normal.Adv-CTLA4Ig,Adv-CD40Ig,Adv-CTLA4Ig+Adv-CD40Ig can block the BT/CD28,CD40/CD40L co-stimulation signals efficiently and depresse the proliferation of PBMs.The immunosuppression effect is Adv-CTLA4Ig+Adv-CD40Ig(31.67±2.25%)＞Adv-CTLA4Ig(23.41±1.23%)＞Adv-CD40Ig (19.83±4.71%).So the recombinant adenovirus vector mediated CTLA4Ig and CD40Ig can induce immune tolerance and be used in HFB gene therapy.