Construction Of Adenoviral Expression Vector Carrying FLIP Gene And Its Anti-apoptosis In AE2 Cell And PVEC

Objective: ALI/ARDS is one of the common diseases. In the past 10-15 years, although the mortality is decreased, it is almost 40%. At present, treatment of ARDS is mainly supportive because the pathogenesis of ARDS is not understood clearly. So, clinical specialists are focused on new ways to cure ALI/ARDS. FLIP is an inhibitor of apoptosis. It is identical in length with caspase-8 and also contains N-terminal tandem DEDs, but its caspase domain ,which critical amino acid residues are substituted by a tyrosine residue, render it with effect of inactivating enzyme. FLIP could compete with caspase-8 in combining with FADD; inhibit conformation of DISC and cell apoptosis. In recent years, researchers have made contributions on the pathomechanism and clinical treatment strategy of ALI/ARDS. It is generally accepted that ALI/ARDS is a severe inflammation which caused by severe trauma or sepsis. There are a lot of cells (PNM, AEC, and PVEC) and cytokines involved in the pathomechanism. Respiratory membrane consisted of PVEC and AEC mainly, was damaged and its permeability increased, which plays a very important role in the pathogenesis of ALI/ARDS. The research pays attention to the vital role of the apoptosis of PVEC and AEC during ALI/ARDS. We constructed an adenovirus vector carrying FLIP gene by Ad-Max system, and then examined the hypothesis that FLIP, a competitive inhibitor of caspase-8, can inhibit AE2 cell and PVEC apoptosis to block the pathological process of ALI/ARDS.Method1. Constructed an adenovirus vector carrying Rat FLIP gene by primitive FLIP T-vector and AdMax system. Identifed Ad-FLIP with EcoRI+NheI restriction enzyme digesting and PCR method, then augmented the virus and measured the MOI;2. Cultivated the rat AE2 cells (purchased from ATCC); infected the cell with Ad-FLIP according to the MOI. After 24 hours, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to investigate the expression of mRNA and protein of FLIP; induced the cell apoptosis by cytotoxic Fas antibody. Flow cytometry was adopted to analyze apoptosis; cell viabilities were measured by MTT; assay of caspase-3 activity in two groups of cells after CH-11 treatment was performed with Caspase-3 activity kit; the expression of Bax were investigated by Western blot .3. Primary cultivated the rat PVEC with a classic primary culture strategy. Indentified the cell by immunofluorescence staining withⅧfactor monoclonal antibody. Cells was infected with Ad-FLIP according to the MOI. After 24 hours, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were used to investigate the expression of mRNA and protein of FLIP; Flow cytometry was adopted to analyze apoptosis after CH-11 treatment.Result 1. Constructed an adenovirus vector carrying Rat FLIP gene with AdMax system, the EcoI+NheI restriction enzyme digesting and PCR results showed that the sequence is correct, and the V.P. index is 2.3×1011,TCID50 is7.1×109;2. After transfection of Ad-FLIP into AE2 cell, the mRNA and protein level of FLIP-L elevated markedly. Ad-FLIP infected AE2 cell showed more resistance to Fas antibody inducing apoptosis than control cells. Cell survival rates examined by MTT were increased by Ad-FLIP treatment(p<0.05). FLIP overexpressing AE2 cell are more resistant to CH-11 inducing apoptosi(sp<0.05). Caspase-3 activation was completely inhibited in FLIP overexpressing AE2 cells compared with control cells.Further analysis showed that FLIP significantly down-regulated the expressions of Bax in AE2 cells.3. Primary cultivation of Rat PVEC was using a classic primary culture strategy. The cells had a characteristic morphology consistent with an endothelial origin and was positive to immunofluorescence staining withⅧfactor monoclonal antibody. Semiquantitative RT-PCR analysis and western-blot were utilized to observe the different expression of FLIP gene in Ad-FLIP infected cells and control cells. The results indicated that the expression of FLIP was elevated markedly by Ad-FLIP infection in Rat PVEC. For further investigation on the anti-apoptosis functions of FLIP, we used flow cytometry to investigate the characteristic of the transfected cells with Ad-FLIP. Compared with control group, the apoptosis rate of transfected cells decreased obviously ( p<0.05).Conclution1. Transfection with Ad-FLIP could elevate the mRNA and protein level of FLIP in Rat AE2 cell and PVEC markedly.2. Our study indicated that FLIP exerted inhibitory effects on apoptotic pathways in rat AE2 cell and PVEC, which significantly protected AE2 cell from the lethal effect of death receptor Fas by suppressing caspase-3 activity and downregulating the expression of Bax.