| Purpose:Lung cancer is one of the highest incidence and mortality of malignant tumors in world,and the treatment methods include surgery,radiotherapy,chemotherapy,molecular targeted therapy,biological therapy and traditional Chinese medicine treatment.In recent years.Targeted therapy plays a significant role in lung cancer treatments,and has become an important method.Ras/Raf/MEK/MAPK signaling pathway plays an important role in the differentiation,proliferation and death of lung cancer cells.The ras gene is the key of the Ras/Raf/MEK/MAPK signaling pathway and regulate the upstream switch of the signaling pathway.Therefore,targeting ras gene has become a hot spot in targeted therapy of lung cancer.We have constructed KGHV500,a tumor-specific proliferative adenovirus,with following characteristics:? equipped with anti-ras single chain antibody gene for blocking the ras signaling pathway and inhibiting ras gene expression;?carrying tumor-specific promoters hTERT and HRE,can be replicated in tumor cells but not in the normal cells;?carrying green fluorescent protein gene,easy to observe the virus infection and its proliferation in tumor cells;?transformed the fibrin gene,so that it can efficiently infect CIK cells.Use the CIK cells as the body carrier to target tumor tissue.Previous experiments demonstrated that KGHV500 could target tumor tissue by using CIK cells as a vivo vector,effectively inhibiting the growth of breast cancer xenografts in nude mice(unpublished data).In order to further confirm the targeting and therapeutic effect of KGHV500 on other tumors,we used human lung adenocarcinoma cell line A549 to study the effect of KGHV500 on the migration,proliferation and apoptosis in vitro,and research the targeting and therapeutic effect of KGHV500 on lung adenocarcinoma in vivo,providing a new method for the targeted therapy of lung cancer.Methods:Firstly,Detection of ras gene and protein expression in A549 cells:1.DNA of A549 cells was extracted and 12 exons of ras gene were amplified by PCR.Sequencing was performed to detect ras gene status in A549 cells.2.The expression of ras protein in A549 cells was detected by immunofluorescence.3.A549 cells were made to detect the expression of A549 cells on the surface of A549 cells to determine whether KGHV500 could infect A549 cells.Secondly,vitro experiments:1.Amplification and purification of recombinant adenovirus KGHV500,determine the titer of KGHV500 by TCID50 method.Electron microscopy to observe whether KGHV500 can infect A549.KGHV500 infects A549 cells with different MOI values to determine the optimal MOI value.2.The effects of KGHV500 on the migration,proliferation and apoptosis of A549 cells were detected by scratches.MTT and TUNEL.3.Isolated mononuclear cells from peripheral blood,induced into CIK cells and identified by immunohistochemistry phenotype,determined the efficiency of KGHV500 infection in CIK cells.Thirdly,vivo experiment:1.Nude mice transplanted tumor model was established by human lung adenocarcinoma cell line A549.The rats were divided into 5 groups.The experimental group was injected with CIK cells of KGHV500 by tail vein injection,there were 4 groups in the control group,divided into tail vein injection KGHV500 group,tail vein injection CIK cell group,tail vein injection load KGHV400 CIK cell group and the same amount of PBS group.2.The vernier caliper measured the changes of tumor volume every 3 days,and observed the therapeutic effect of KGHV500 on lung adenocarcinoma in vivo.The expression of adenovirus was detected by immunohistochemistry and WB on the 1st day,the 2nd day,the 3rd day,the 5th day and the 7th day after the tail vein injection to research the targeting and safety of KGHV500 in nude mice.The tumor tissues of the mice were examined on the 1st day,the 2nd day,the 3rd day,the 5th day and the 7th day after the tail vein injection.Immunohistochemistry was used to detect the expression of single chain antibody in tumor tissue to study the expression of single chain antibody gene in lung adenocarcinoma xenografts.The tumor tissues of the mice were selected and the apoptosis of the tumor tissues was detected by Tunel test.Results:A549 cells were found to be k-ras mutants by sequence alignment and high expression of ras protein.The expression rate of CD46 on A549 cells was close to 100%,which indicated that KGHV500 could infect A549 cells efficiently.In vitro experiments,KGHV500 purified titer of 5.0×109pfu/ml.In the electron microscope,we can observe a large number of virus particles in the nucleus of A549,indicating that KGHV500 can infect A549 cells.The optimal MOI value of KGHV500 to A549 cells was 100.In the scoring experiment,the total mobility of 48 hours in the experimental group was 22.07%± 1.26,while the control group was 44.50%±0.71 and the P value was<0.01,which indicated that KGHV500 could significantly inhibit the migration of A549 cell line.In the MTT experiment,the absorbance of A549 cells increased slightly from the first day to the second day after infection with KGHV500 in the experimental group A549 cells,and then decreased to 0.08±0.01 on the fifth day.The absorbance of A549 cells in the control group without KGHV500 increased from the first day to 1.05±0.07 on the fifth day,and there was significant difference(p<0.01)in the control group compared with the experimental group,Indicating that KGHV500 is lethal to A549 cells and can significantly inhibit the proliferation of A549 cells.In the Tunel experiment,the apoptotic rate was 81.6 ± 7.2 in the experimental group and 7.4±1.2(P<0.01)in the control group,indicating that KGHV500 could significantly promote the apoptosis of A549 cells.In vivo experiments:lung adenocarcinoma in nude mice transplanted tumor formation rate of 74%.The results showed that the tumor growth rate of KGHV500 + CIK group,KGHV400 + CIK group,CIK group and KGHV500 group increased gradually,and the tumor tissue size of KGHV500 + CIK group did not change significantly,While the tumor volume of the PBS group increased by more than 10 times.The results showed that KGHV500 + CIK could significantly inhibit the growth of lung adenocarcinoma in nude mice.Immunohistochemically detection of KGHV500 + CIK targeting lung adenocarcinoma in nude mice showed that KGHV500 reached the tumor tissue on the first day of KGHV500 + CIK group,and the expression of KGHV500 increased with the passage of time.The expression of adenovirus was also detected in the tumor tissue of only KGHV500 group,but the amount was small and irregular.In the other organs(heart,liver,lung,kidney,stomach,pancreas,large intestine,small intestine),KGHV500 + CIK group 1-7 days were not found KGHV500,the presence of KGHV500 alone injection of heart,liver,Lung,kidney,stomach,pancreas,large intestine,small intestine 1-7 days can be detected in the presence of adenovirus.It is suggested that KGHV500 can target tumor tissue with CIK cells as an in vivo carrier.Immunohistochemically detection of single-chain antibody in nude mice in the expression of tumor tissue showed that KGHV500 + CIK group 1-7 days were single-chain antibody expression,and with the passage of time-increased expression,the single chain antibody gene is expressed as replication of the adenovirus.The expression of single chain antibody was also detected in KGHV 500 group,but the expression of single chain antibody could be expressed with the replication of adenovirus.The expression of single chain antibody in nude mice was detected by WB,showed that KGHV500 + CIK group only in the tumor and spleen detected in the expression of single-chain antibody,and in KGHV500 group all tissues expressed single antibody except brain.The expression of single chain antibody in tumor tissue in KGHV500 + CIK group was significantly higher than that in KGHV500 group.5.TUNEL experiments showed that,the apoptotic rates of KGHV500 + CIK group,KGHV400 + CIK group,CIK group,KGHV500 group and PBS group were 96.9±23.4,80.3±16.48,37.3 ± 12.3,11.5 ± 1.2 and 1.3 ± 0.3 respectively,and the apoptosis of KGHV500 +CIK group(P<0.01),indicating that KGHV500 + CIK can promote the apoptosis of lung adenocarcinoma.Conclusion:In summary,in vitro experiments,we demonstrated that KGHV500 could inhibit proliferation,migration,and promote apoptosis of A549.Experiments in vivo showed that the tumor-specific proliferative adenovirus KGHV500 could target the tumor tissue through CIK cells,and inhibit the growth of the tumor with safety and therapeutic effect.In addition,the antitumor activity of tumor-specific proliferative adenovirus,CIK Cells and p21Ras single chain antibody was combined together and improved the suppressing the effect to lung adenocarcinoma transplanted tumor of nude mice.Finally,this study laid experimental foundation for the targeted therapy to the ras gene-driven tumor. |
PDF Full Text Request