| Apoptosis is a ubiquitously biological phenomenon in the body.It also plays a very important role in the evolution of organisms,the maintenance of homeostasis of internal environment and the development of systems.Recent studies have shown that Caspase family plays an important role in the regulation and execution of apoptosis.Therefore,establishing a rapid method to detect Caspase protease's activity is conducive to early judgment of apoptotic status of cells,and can provide a convenient and fast way for screening drugs that can treat diseases.Tumor is one of the main causes of human death.In recent years,the discovery of oncolytic virus has provided a new way for the treatment of cancer.By combining radiotherapy and chemotherapy,the abilityof selective replication and infection of the virus can be enhanced,which can induce the necrosis or apoptosis of cancer cells.Newcastle disease virus(NDV)is favored by virologists because of its oncolytic ability.At present,a large number of studies have shown that viral infection can induce oxidative stress in cancer cells and then promote cell apoptosis,but whether Newcastle disease virus can induce oxidative stress to promote cell apoptosis has not been studied.In this paper,four amino acids,Asp-Glu-Val-Asp(DEVD),the recognize motif of Caspase-3,were introduced into the middle of the Fluc-C and N fragment.Subsequently,the recombinant gene were cloned into the N and C terminal end of the split intein,respectively,and named as pFluc-DEVD.Then plasmids were transfected into cells and renilla luciferase was co-transfected in each sample as an internal control for transfection efficiency.Then the apoptosis level was detected by the double luciferase reporter gene and the Western-blot analysis.Subsequently,apoptosis detection cell lines were constructed by using lentivirus packaging method.The results appear as shown below:(1)Western-blot detection indicated that the Fluc level was significantly increased in pFluc-DEVD transfected group when pre-treated by apoptosis stimulants.Dual luciferase assay showed that the content of firefly luciferase expressed in the pFluc-DEVD plasmid transfected group was significant upregulation.(2)The STS which can induce apoptosis and Newcastle disease virus SH15 were used to stimulate HeLa cells.The results showed that the expression of luciferase was significantly up-regulated.In this study,SH15 virus was used to infect HeLa and LoVo cells.Then samples were collected at different time.Flow cytometry(FCM)and indirect immunofluorescence assay(IFA)were used to detect ROS expression in cells and mitochondria.Catalase,superoxide dismutase,glutathione peroxidase,hydrogen peroxide,nitric oxide and MDA detection kits were used to detect the level of various intracellular enzymes.Quantificational real time PCR and indirect immunofluorescence assay were used to explore the pathway of producing the ROS.Finally,Western-blot was used to detect the expression of Caspase-3,PARP and NP protein to explore the changes of apoptotic level of cells when oxidative stress was inhibited.The results were shown as follows:(1)Newcastle disease virus SH15 strain can induce oxidative stress in tumor cells in a time-dependent and dose-dependent manner.The expression of NO,H2O2 and MDA was uprelated,while the expression of antioxidant enzymes such as CAT,SOD and GSH-Px decreased in the later stage of infection.(2)Newcastle disease virus SH15 strain can induce tumor cells to produce mROS through mitochondrial pathway and NADPH oxidase pathway.(3)By inhibiting the production of ROS,the apoptotic level was downregulated and the replication of viruses was decreased.In conclusion,we successfully constructed a cell line stably expressing Fluc-DEVD gene,which facilitated the subsequent detection of apoptosis.It was also confirmed that NDV infection could induce oxidative stress in tumor cells.Furthermore,when the production of ROS was inhibited,the replication of NDV and the number of apoptotic cells were decreased. |
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