Study On Induction Of Apoptosis Of Human Cervical Carcinoma Cells By Recombinant Oncolytic Adenovirus Via Mitochondrial Pathway

Cervical cancer is the second most common malignant tumor in the world after breast cancer.The high incidence age of cervical cancer is generally 30 to 55 years old.In recent years,the incidence of cervical cancer has become younger.Oncolytic adenoviruses are very promising cancer gene therapy drugs because they have the characteristic of selective replication in malignant tumor cells,which can lead to the lysis and inflammation of tumor cells.The recombinant oncolytic adenovirus Ad-VT used in this study was constructed by our own laboratory,which was based on the apoptotic gene Apoptin,the tumor-specific promoter hTERTp,and the viral replication essential gene E1 A.The oncolytic adenovirus with tumor-specific replication and tumor-specific killing ability is constructed by the RAPAd.I adenoviral vector system.A large number of studies have shown that Ad-VT has significant inhibitory effects on various tumors such as gastric cancer,liver cancer,lung cancer,melanoma,colorectal cancer and prostate cancer,but has no effect on normal human cells.Objective To investigate the inhibitory effect of recombinant oncolytic adenovirus on human cervical cancer Hela cells and the effect of recombinant oncolytic adenovirus on Hela cell apoptosis.Further explore the ways in which recombinant oncolytic adenovirus can induce Hela cell apoptosis,the mitochondrial apoptosis pathway of Hela cells.Through this study,a preliminary understanding of the pathway of tumor cell apoptosis induced by recombinant oncolytic adenovirus was conducted.Method 1 Hela cells’ mitochondria extraction,function verification and activity verification The mitochondria of Hela cells were extracted by Minute mitochondrial separation kit.The COX IV protein levels in mitochondria were detected by Western Blot to verify the purity of mitochondria.The function and activity of mitochondria was verified by detecting the ATP levels and reactive oxygen species levels of mitochondria.2 Recombinant oncolytic adenovirus inhibits Hela cells The inhibitory effect of recombinant oncolytic adenovirus on Hela cells was confirmed by CCK-8 assay and lactate dehydrogenase release assay.The apoptosis of Hela cells induced by recombinant oncolytic adenovirus was investigated by Hoechst staining and Annexin V-FITC/PI flow assay.The effects of recombinant oncolytic adenovirus on migration and invasion of Hela cells were investigated by Transwell migration assay and Biocoat invasion assay.Apoptosis-associated proteins were detected by Western Blot assay.That was a preliminary study on the pathway of recombinant oncolytic adenovirus-induced apoptosis in Hela cells.3 Recombinant oncolytic adenovirus induces apoptosis of Hela cells via mitochondrial pathway The effects of recombinant oncolytic adenovirus on mitochondrial membrane potential of Hela cells were investigated by JC-1 staining and JC-1 flow cytometry.The mitochondria of Hela cells infected with recombinant oncolytic adenovirus were extracted by Minute mitochondrial separation kit;The mitochondria were tested for ATP level and reactive oxygen species level to investigate the effect of recombinant oncolytic adenovirus on mitochondrial function of Hela cells.The expression of mitochondrial apoptosis pathway-associated protein was detected by Western Blot,and the Mitochondrial apoptosis pathway of Hela cells induced by recombinant oncolytic adenovirus was studied.Result 1 The mitochondria with high purity,good function and good activity were successfully extracted by Minute mitochondrial separation kit.2 Through CCK-8 assay and lactate dehydrogenase release test,it was confirmed that recombinant oncolytic adenovirus can inhibit Hela cells and the inhibition has significant time effect and dose effect.The inhibition rate of recombinant adenovirus to Hela cells is: Ad-VT>Ad-T>Ad-Vp3>Ad-Mock.Through hoechst staining and Annexin V-FITC/PI flow assay,it was confirmed that recombinant oncolytic adenovirus inhibits Hela cells by inducing apoptosis,and the ability of 4 recombination oncolytic adenovirus to induce apoptosis of Hela cells was: Ad-VT>Ad-T>Ad-Vp3>Ad-Mock;Through Transwell migration assay and Biocoat invasion assay,it was confirmed that recombinant adenovirus could influence Hela cell migration and invasive ability,and the degree of influence is: Ad-VT>Ad-T>Ad-Vp3>Ad-Mock;Through the detection of apoptosis-related proteins by Western Blot,it was confirmed that recombinant oncolytic adenovirus can regulate the apoptosis of Hela cells by affecting a variety of Hela cell’s apoptosis pathway proteins.3 Clearing recombinant oncolytic adenovirus can reduce the mitochondrial membrane potential and induce early apoptosis of Hela cells by JC-1 staining,and through JC-1 flow clarification,it express a significant time effect for early apoptosis of Hela cells.The effect of the four recombinant oncolytic adenoviruses on the early apoptosis of Hela cells was: Ad-VT>Ad-T>Ad-Vp3>Ad-Mock;it was confirmed that recombinant oncolytic adenovirus has a significant effect on the function and activity of mitochondria in Hela cells by ATP and ROS assays;Western Blot was used to preliminarily confirm that recombinant oncolytic adenovirus induces Hela cells apoptosis by regulating mitochondrial apoptosis pathway related proteins such as AIF and Arts Conclusion Recombinant oncolytic adenovirus can inhibit the Hela cells by inducing apoptosis;and it can induce apoptosis of Hela cells through various pathways;the most important pathway is mitochondrial pathway,Recombinant oncolytic adenovirus regulates apoptosis of Hela cells by affecting the expression of mitochondrial apoptosis pathway-associated proteins.