Eukaryon Expression Of Gene A200711 And Effect On H7402 Cell Apoptosis

The data show that more than 45%of morbility and 42%of mortality of hepatoma cases in the world occured in China.As a common cancer,hepatic cellular cancer has ranked the sixth in global cancer cases and becomes a major hazard to public health.The signal regulation mechanism of tumor cell apoptosis is similar to the Trichinella spiralis encystment mechanism.Recent studies show that the process of encystment of Trichinella spiralis mimic host muscle cell repairing process regulated by apoptosis.During the early stage of infection,Trichinella spiralis produce a sort of protein named "parakines",which can regulate IGF,TGF-β,death receptor and mitochondrion signal conducting pathways.It also convert basophilic cytoplasm to acidity.The change of cytoplasmic provides a advantageous environment to sustain the balance of apoptosis and anti-apoptosis regulation protein in infected host muscle cell and satellite cell,to initiate the formation of nurse cell.Evolution analysis of Trichinella spiralis shows that it is a species with conservative gene sequence and low evolution rate.As a result of that,we may suspect that there are some apoptosis regulative protein exsiting in Trichinella spiralis,which may contribute enormously to the biotherapy of tumor.Human hepatoma carcinoma cell H7402 and human leukemic cell K562 was used to screen target sequence in Trichinella spiralis AD and Trichinella spiralis NBL T7 phage display libs.Target sequence was amplificated by PCR and obtained a DNA fragment of 457 bp.A eukaryotic expression plasmid of A200711 gene was successfully constructed.The recombinant of pEGFP-N1-A200711 were transfected into H7402 cell.The proteins of 16.74kD were detected by SDS-PAGE,showing that the target protein was successfully expressed.Cell proliferation and apoptosis variations were detected after the recombinant of pEGFP-N1-A200711 were transfected into H7402 cell,with H7702 cell as control group.Chromatin pycnosis,nucleus gather and apoptosis characteristics were observed in pEGFP-N1 group by Hochest-PI dipl-staining.MTT and flow cytometry detection show that expression of target gene successfully induced H7402 cell apoptosis,the disparation is significal in statistic(p<0.05).It had not induce H7702 apoptosis in contrast.The transcraption and expression of Caspase 3 and Caspase 9 in H7402 cell were analysed by半定量RT-PCR,Western-blot and immunofluorescence.The results evidence that the expression of target gene can upregulate the expression of Caspase3 and Caspase9,which may contribute to H7402 cell apoptosis.In this study,eukaryotic expression plasmid of A200711 gene was successfully constructed,and the target gene was successfully expressed in H7402 cell.The recombinant of pEGFP-N1-A200711 can effectively inhibit the proliferation of H7402 cell,and can upregulate the transcraption and expression of Caspase 3 and Caspase 9 to induce H7402 cell apoptosis.The study supports the hypothesis that target gene could activate the mitochondrion apoptosis pathway of H7402 cell.The study will be a scientific referential to the investigation of anti-tumor biotherapy.