Study On The Effect Of Defensin 2 Cooperating With Vesicular Stomatitis Virus Matrix Proteins On Colon Cancer

OBJECTIVE: ?-Defensin 2(MBD2)is an important member of the defensin family and is an important molecule for natural immunity.It has a bridge between acquired immunity and natural immunity.Induction of dendritic cell(DC)maturation by immunochemotaxis enhances the body's anti-tumor immunity.Vesicular stomatitis virus' s matrix protein(VSVM)is one of the constituent proteins of vesicular stomatitis virus and is a key molecule for VSV virus-induced tumor cell lysis.VSVM can induce tumor cell apoptosis while inhibiting the growth of tumor blood vessels and activating cellular immune responses.Based on this,we speculate that when MBD2 is co-expressed with VSVM,MBD2 can induce dendritic cell maturation by immune chemotaxis and enhance the immune and anti-tumor effect induced by VSVM.For this purpose,he eukaryotic expression vectors p VSVM,p MBD2 and the co-expression vector p VITRO2-VSVM-MBD2 were constructed.the role of MBD2 combined with VSVM in the treatment of colon cancer was explored through the effects of each of the above expression vectors on the growth of mouse colon cancer cell CT26 and mouse colon cancer xenograft tumors.Methods: The MBD2 and VSVM genes were cloned into the p VITRO2 vector to obtain three eukaryotic expression plasmids of p VSVM,p MBD2 and p VSVM-MBD2.In vitro cell growth assay,using Lip2000 transfected colon cancer cell CT26 cells,cell proliferation inhibition rate was observed by MTT assay,PI staining and Annexin V-PE/7-AAD double staining were used to detect the apoptosis rate.The chemotaxisof DC cells was used to observe the activity of MBD2 in the supernatant of CT26 cells transfected with each plasmid.In vivo experiments,BALB/c mouse colon cancer(CT26)tumor models were established.Tumor-bearing mice were randomly divided into 6 groups: p VSVM group,p MBD2 group,p VSVM+p MBD2 double plasmid group,and p VSVM-MBD2 group,Lip2000 group,PBS group.The p VSVM,p MBD2,p VSVM+p MBD2,p VSVM-MBD2,and p VITRO2 were respectively configured as a working solution with Lip 2000 and injected intratumorally into each mouse transplanted tumor.The PBS group was injected with only PBS solution.According to the above method,injection was performed every 3 days and continuous injection was performed 6 times;after treatment,the volume of tumor nodules in each group of mice was compared to observe the survival time and toxicity of the mice.Result: After transfecting CT26 cells with the recombinant plasmids p VSVM,p VSVM-MBD2,p VSVM,and p MBD2 double plasmids,they all inhibited the growth of CT26 cells and promoted apoptosis,which was time-dependent;Compared with the control group,there was no significant change in the proliferation and apoptosis of p MBD2 transfected cells;The chemotaxis of dendritic cells induced by p VSVM-MBD2 recombinant plasmid transfected with CT26 cells was significantly higher than that of p VSVM and liposome transfection group,but there was no significant change with p MBD2 transfection group.In the tumor-bearing mice model,the tumor growth of p VSVM group,p MBD2 group,p VSVM+p MBD2 double plasmid group,and p VSVM-MBD2 group was significantly slower than that of PBS group and Lip2000 group,and the tumor growth rate was the slowest in the p VSVM-MBD2 treatment group.The survival rate of tumor-bearing mice was as high as 70% and survival time was longer than that of other groups.Conclusion: 1.the study confirmed by in vitro cell experiments,p VSVM,p VSVM-MBD2plasmid can induce apoptosis of tumor cells.2.The establishment of a tumor xenograft model confirmed that complex tumors formed by p MBD2,p VSVM,p VSVM-MBD2 and liposomes could inhibit the growth of tumor-bearing mice.3.MBD2 can induce dendritic cell maturation by immune chemotaxis and enhance the VSVM-induced immune anti-tumor effect.