Preliminary Study Of Orf Virus Inhibiting The Growth Of Gastric Cancer Cells And Its Mechanism

Orf virus(ORFV),also known as sheep aphthous virus,can cause infectious impetigo in sheep(infectious pustular dermatitis of sheep).ORFV is a potential oncolytic virus.It has strong immunoregulatory activity,mild symptoms after infection,fast recovery,and high safety.At present,some scholars have begun to conduct preliminary oncolytic effects of ORFV the study.In recent years,more viruses have been used as oncolytic viruses for the treatment of tumors,and oncolytic virus therapy has achieved good results,which has brought new hope to the majority of cancer patients,and gastric cancer Gastrointestinal malignancies lack the ideal treatment.The purpose of this project is to explore the inhibitory effect of ORFV on the growth of gastric cancer cells and to explore its oncolytic mechanism.Firstly,to investigate whether ORFV has oncolytic effect on gastric cancer cells,we infected AGS gastric cancer cells with ORFV and observed whether the virus could infect gastric cancer cells under electron microscope.The results showed that after ORFV infection,ORFV virus particles and virus particles could be observed in AGS cells The number is much higher than the complex number of viral infections.This result proves that ORFV can infect gastric cancer cells and can replicate in gastric cancer cells,which satisfies the important premise of becoming an oncolytic virus.To explore the effect of ORFV on gastric cancer cells,we infected different gastric cancer cell lines with ORFV,and observed the effect of ORFV on the growth of gastric cancer cells through the CCK-8 experiment.The results of CCK-8 showed that ORFV can inhibit the growth of each gastric cancer cell line,and It has different inhibitory effects on the growth of gastric cancer cells of different strains,and it has a significant inhibitory effect on the growth of AGS,MFC,BGC823 cells,and it inhibits the growth of SGC7901 when ORFV MOI=5 after 72h infection,but there is no obvious inhibitory effect on the growth of HGC27 and MKN45 cells.Secondly,in order to investigate whether ORFV has oncolytic effect on tumor cells,we first infected different tumor cells with ORFV,and observed the effect of ORFV on different tumor cells through CCK-8 experiment.The results of CCK-8 showed that ORFV could inhibit tumor cells growth,and the effect of inhibiting the growth of different tumor cells is different,which is very sensitive to AGS(gastric cancer cells),Hela(cervical cancer cells)and other cells,and very insensitive to 5637(bladder cancer cells).This result suggests that ORFV also has an oncolytic effect on other tumor cells.In the next experiment,we mainly explore the effect of ORFV on gastric cancer cells.After ORFV infection,we observed that the cell proliferation rate was significantly slowed down under the microscope.We suspect that ORFV may inhibit cell growth by inhibiting the cell cycle process.Thirdly,to investigate whether the cell cycle changes after ORFV infection,flow cytometry was used to detect the change of cell cycle after ORFV infection of gastric cancer cells with different infection multiplicity.In the control group,the proportion of G2/M phase also increased with the increase of the infection number;Western blot detection of cell cycle-related proteins,the expression of G2/M phase-related proteins all showed a downward trend.This result suggests that ORFV inhibits the growth of cells by blocking the gastric cancer cell cycle in the G2/M phase,thereby making it more conducive to virus replication.Fourthly,research on the killing mechanism of ORFV,because after ORFV infection,we also observed under the microscope that the cells became larger and rounder,similar to apoptosis.We suspected that apoptosis may occur after ORFV infection.1.To investigate the occurrence of apoptosis,flow cytometry was used to detect the apoptosis rate,and it was found that when MOI=5 and MOI=10 infected gastric cancer cells,the apoptosis rate was lower at 24h.After prolonged infection time,the apoptosis rate increased significantly,The rate of apoptosis is time-dose dependent;Western blot detection of the expression of caspase-3 and PARP spliceosome,after prolonging the time of ORFV infection and increasing the number of infections,spirosomes can be detected,that is,ORFV infected cells have apoptosis at a later stage.2.In order to further study the mechanism of apoptosis,we used Western blot to detect the expression of related proteins in the caspase activation pathway after ORFV infection.The results showed that the cleavage of caspase8 and caspase9 increased significantly after ORFV infection,suggesting that ORFV may be through caspase Pathways induce apoptosis.Therefore,we used the caspase inhibitor Z-VAD-FMK to inhibit the activation of the caspase pathway,and at the same time used flow cytometry to detect the incidence of apoptosis,and Western blot to detect the expression of apoptosis-related proteins.The results showed that the caspase activation pathway was inhibited,the rate of apoptosis was significantly reduced.This result proves that ORFV induces apoptosis through the caspase activation pathway.Since apoptosis and autophagy are often activated together,we are curious about whether autophagy also occurs in cells.Fifthly,to investigate the occurrence of cell autophagy,uing real-time quantitative fluorescence detection of mRNA levels of autophagy-related genes after ORFV infection,real-time quantitative fluorescence experiment detected that the expression level of Beclinl has increased and the expression level of BCL-2 has decreased.The trend suggests that the free Beclinl in cells may increase after ORFV infection,which will induce the occurrence of intracellular autophagy;Western blot detected the expression of autophagy-related proteins at different time points after ORFV infection.The results showed that LC3Ⅱ increased significantly at the early stage of ORFV infection,and P62 also showed a decreasing trend when the infection time was extended to 36h,suggesting that autophagy occurred in cells.After the cells were transfected with mcherry-EGFP-LC3 plasmid,the distribution of LC3 protein in the cells was observed under a confocal microscope.In AGS cells infected with ORFV,LC3 was distributed in autophagolysosomes,indicating that the intracellular autophagy flow was activated.Electron microscopy observed that some cells infected with ORFV had nuclear fragmentation,mitochondrial shrinkage,mitochondrial swelling and other apoptosis phenomena,and most cells had autophagosomes.The results of electron microscopy were further It is proved that ORFV can induce apoptosis and the occurrence of autophagy in gastric cancer cells.This study demonstrated in vitro that ORFV can inhibit the growth of tumor cells.When gastric cancer cells were selected for further research,the results showed that ORFV can infect gastric cancer cells and replicate in gastric cancer cells.At the same time,it can inhibit the growth of cells by blocking the cycle of gastric cancer cells in G2/M phase.And by inducing cell apoptosis to kill gastric cancer cells,in addition to the above results,we also detected the occurrence of autophagy in the cell.This laid the foundation for exploring the oncolytic effect of ORFV on gastric cancer and ultimately for the treatment of gastric cancer.