| Lung cancer has become one of the most serious cancers that threaten human life and its annual death toll is above 1 million.According to the International Bureau of Hegemony(IACR),it is estimated that the number of lung cancer deaths due to lung cancer will increase by 10 million by 2030,whose deaths account for 19% of the global cancer deaths annually.So lung cancer is often called "king of cancer." Cancer is one of the health issues of most concern in the world,but it faces many challenges in the treatment of cancer.With the in-depth study of cancer,some new treatments have been found.Among them,gene therapy and cell therapy have become effective new therapies.As an oncolytic virus,adenovirus has a certain ability of inhibiting tumor growth,and it can act as a viral vector to carry other anti-tumor effects by generating foreign genes.Methods to engineer adenoviral vectors currently include gene deletion mutations,modification of viral structural proteins,and insertion of specific promoters and specific genes.Currently,many cancer researchers are working on oncolytic therapies for the treatment of cancer,but there are still some problems with the therapeutic effect.HN protein is one of the six proteins encoded by Newcastle disease virus(NDV),which synergizes with F protein in NDV to facilitate the cytopathic effect of NDV virus.It has been reported that NDV virus can selectively replicate in tumor cells and promote the death of cancer cells in the absence of normal cells.The hTERTp promoter is a tumor-specific promoter that initiates the replication or expression of certain genes such as the E1 A gene and HN only in tumor cells,giving them the ability to specifically replicate and kill.The existence of these two proteins can effectively enhance the tumor-targeting effect of oncolytic adenovirus,making the treatment effect of the tumor obviously improved.In this study,the four recombinant adenovirus(Ad-HN-hTERTp-E1 a,Ad-h TERTp--E1 a,Ad-HN and Ad-mock)were constructed by us lab.Ad-HN-hTERTp-E1 a contains a tumor-specific promoter(hTERTp,human telomerase reverse transcriptase)to initiate the E1 A gene(essential for viral replication)and a cytomegalovirus(CMV)promoter to initiate HN gene.Therefore,Ad-HN-hTERTp-E1 a is a dual-specific oncolytic adenovirus that has both tumor-specific and tumor-specific replication capabilities;the remaining three are control viruses.Objective:To investigate the inhibitory effect of Ad-HN-hTERTp-E1 a on A549 cells in vitro and in vivo.Contents:1.Inhibitory effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells.2.Inhibitorypathway of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells.3.Inhibitory effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells in vivo.Methods:1.Inhibitory effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells.Study the cytotoxicity effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells through MTS staining and crystal violet staining experiments.2.Inhibitory mode of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells.Verify the inhibition pathway of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells according to the morphological change of mitochondrial membrane potential(JC-1 staining),cell nuclear(DAPI staining)and cell apoptosis(AnnexinV-FITC/PI staining).3.Inhibitory effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells in vivo.Study the Inhibitory effect of recombinant adenovirus Ad-HN-hTERTp-E1 a on A549 cells in vivo through establishment of nude mice model of tumor,combined with recording the volume of the tumor and the survival rate after intratumoral injection of recombinant adenovirus.Results:1.The results of crystal violet staining and MTS detection showed that Ad-HN-hTERTp-E1 a,Ad-hTERTp-E1 a and Ad-HN have inhibitory effect on A549 cells,while the inhibitory effect of Ad-mock on A549 was not significantly different from that of the control group(P>0.05);The three virus had significant dose-effect and time-effect.The inhibitory rates of recombinant adenovirus on A549 cells was: Ad-HN-hTERTp-E1a> Ad-hTERTp-E1a> NH> Ad-Mock.2.The results of JC-1 staining to detect mitochondrial membrane potential,DAPI staining to detect nuclear fragmentation and Annexin V-FITC / PI staining to detect apoptosis,we found that the recombinant adenovirus Ad-HN-hTERTp-E1 a,Ad-hTERTp-E1 a and Ad-HN all play an inhibitory effect on A549 cells by inducing apoptosis.The ability of recombinant adenovirus to induce apoptosis of A549 cells was:Ad-HN-hTERTp-E1 a > Ad-hTERTp-E1a>Ad-HN> Ad-Mock.3.The results of establishment of nude mice model of tumor,ombined with recording the volume of the tumor and the survival rate after intratumoral injection of recombinant adenovirus showed that the recombinant adenovirus Ad-HN-hTERTp-E1 a,Ad-hTERTp-E1 a and Ad-HN could inhibit tumor growth and prolong the survival of nude mice.The in vivo inhibition ability of recombinant adenovirus on A549 cellswas: Ad-HN-hTERTp-E1a> Ad-hTERTp-E1a>Ad-HN.Conclusion:1.Recombinant adenovirus Ad-HN-hTERTp-E1 a has a significant inhibitory effect on A549 cells.2.Recombinant adenovirus Ad-HN-hTERTp-E1 a mainly through the induction of apoptosis,to achieve inhibition of A549 cells.3.Successfully established a nude mouse tumor model,and found that the recombinant adenovirus Ad-HN-hTERTp-E1 a has a significant inhibitory effect in vivo. |
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