| [Objective]Malignant tumor is one of the diseases with the highest mortality and serious harm to human health.Gastric cancer is one of the most common malignant gastrointestinal tumors.Surgery,chemotherapy and radiotherapy is a common method for the treatment of malignant tumors,but surgical treatment has limitations,the effect of surgical treatment for advanced,metastatic or recurrent tumor is not good,and the effect of radiotherapy and chemotherapy also have great side effect on the human body.Targeted therapy is a new direction for the treatment of malignant tumors.The targeted therapy is to study the therapeutic antibody and combined with antigen to achieve antitumor effect.Our laboratory constructed anti p21Ras scFv gene,the antibody can pass through the cell membrane into the cell,blocking the Ras signaling pathway,targeting the tumors with high expression of p21Ras protein,and the antibody was confirmed that has obvious antitumor effect of high expression of p21Ras in malignant tumor.However,the antibody has no self-proliferative capacity and tumor targeting.Therefore,the transformation of the wild type Ad5 adenovirus vector was constructed to recombinant adenovirus KGHV400 containing human telomerase reverse transcriptase gene promoter(hTERT)?hypoxia response element(HRE)and F3 5 pili gene,and then inserted into the anti-p21 Ras scFv gene was previously constructed oncolytic adenovirus KGHV500.KGHV500 can specifically replicate in tumor cells,and can efficiently infect CIK cells with tumor targeting;the main purpose of this paper is:1.In vitro experiments,the effects of KGHV500 on human gastric adenocarcinoma cell SGC-7901;2.In vivo experiments,to investigate the effect and safety of CIK cells combined with KGHV500 mediated by anti p21Ras antibody treatment of gastric carcinoma implanted in nude mice.Finally,the anti p21ras single chain antibody in tumor cells can be expressed sustained and safely,so as to provide a new method for tumor targeted therapy.[Methods]1.The amplification,concentration and purification of recombinant adenovirus KGHV400 and KGHV500:The virus was amplified in vitro and the recombinant adenovirus was purified by double cesium chloride density gradient centrifugation.The titer of the virus was measured by TCID50 method.2.Envision immunohistochemical method to detect the expression of the KGHV500 receptor CD46 protein that in the surface of gastric cancer cell.KGHV500 infected SGC-7901 gastric cancer cells with different MOI values to determine the optimal MOI value.The infection of KGHV500 to SGC-7901 cells was observed by electron microscopy.3.The killing ability,invasiveness,migration and cell apoptosis of tumor cells were detected by MTT colorimetric assay,Transwell invasion chamber test,Cell scratch test and TUNEL apoptosis detection.4.Isolation and identification of CIK cells:human peripheral blood mononuclear cells were isolated and induced to be CIK cells in vitro by cytokines.The KGHV500 receptor CD46 protein on the surface of CIK cells was identified by immunohistochemical Envision method.5.The establishment of human gastric cancer cell line SGC-7901 in nude mice model and treatment group:subcutaneous injection of SGC-7901 gastric cancer cells in right subaxillary of nude mice.The nude mice were randomly divided into five groups for treatment,experimental group intravenous injection with KGHV500 CIK cells,the four control groups were intravenously injected with KGHV400 CIK cells,CIK cells,KGHV500,PBS treatment.6.In vivo experiment:continuous dynamic monitoring of the nude mice transplantation tumor growth and size,and draw the tumor growth curve;apoptosis of tumor tissue were detected TUNEL apoptosis after treatment.The experimental group received intravenous injection of CIK+KGHV500 and control group were injected with KGHV500 nude mice were compared in treatment after 1d,2d,3d,5d and 7d respectively,The tumor and the heart,liver and spleen,lung,kidney,stomach,pancreas,small intestine,large intestine,brain and other organs made of paraffin section,and the immunohistochemical method was used to observe the distribution of CIK cells carrying the KGHV500 distribution in vivo,to study the safety of CIK cells combined with KGHV500 in the treatment of tumor.[Results]1.The recombinant adenovirus was amplified by double cesium chloride density gradient,and the viral titers of recombinant adenovirus KGHV400 and KGHV500 were 5.0 ×109 pfu/ml and 2.0 × 109pfu/ml.2.The results of immunohistochemistry showed that the expression of KGHV500 receptor CD46 protein on the surface of SGC-7901 gastric cancer cells was mainly located on the cell membrane,the positive rate was about 100%.The optimal MOI value of adenovirus infected SGC-7901 gastric cancer cells was 100.It was observed that KGHV500 infected SGC-7901 cells could enter the tumor cell membrane and nucleus by electron microscope.3.In vitro experiments,cell scratch experiments was showed that KGHV500 infection of SGC-7901 cellsi in the experimental group after 48 hours,the cell migration percentage was(8.25+5.20)%,while the control group of uninfected SGC-7901 cells in 48h,the cell migration percentage was(22.06+6.15)%,showed that the migration ability of SGC-7901 cells was inhibited by KGHV500.Transwell invasion assay showed that the experimental group KGHV500 infected SGC-7901 cells compared to the control group,the number of cells transferred to lower microporous membrane decreased,and the invasion number of experimental group and the control group in the 24h cells were 15.0+6.73 and 108.57+25.90,statistical analysis of difference between the two is obvious,with statistical significance(P<0.01),show that KGHV500 can effectively inhibit the invasion ability of SGC-7901 cells.MTT assay showed that the absorbance of experimental group were significantly reduced with time,while absorbance of SGC-7901 cells uninfected KGHV500 decreased slowly,the absorbance were measured in 1d,2d,3d,4d,5d of the experimental group were 1.37 + 0.32,0.36 + 0.15,0.14 + 0.06,0.08 +0.03 and 0.03 + 0.01,the control group were 1.39 + 0.43,1.32 + 0.37,1.19 + 0.29,1.05 + 0.21 and 0.82 + 0.15,the results showed that KGHV500 had a strong effect on SGC-7901 gastric cancer cells.TUNEL apoptosis assay showed that the number of apoptotic cells in experimental group was significantly higher than that in control group,and the apoptosis rate was(68.36+5.15)%and(12.62±3.41)%,these results indicated that KGHV500 could effectively promote the apoptosis of SGC-7901 cells.4.Human peripheral blood mononuclear cells were isolated and cultured by a variety of cytokines into CIK cells,CD46 protein was identified positive expression on the CIK cells,and the expression rate was higher,mainly in the cell membrane.5.The model of human gastric cancer in nude mice was established.The experimental group and four control groups were divided and were injected with CIK+KGHV500,CIK+KGHV500,CIK cells,KGHV500 and PBS into the tail vein.6.In vivo,continuous dynamic monitoring of growth state and the size of the transplanted tumors in nude mice,the tumor growth curve showed that the experimental group and the control group were injected with CIK+KGHV500,CIK+KGHV400,CIK,KGHV500,PBS cells,in thirty-fourth days after treatment,the tumor size reached 124.82 + 50.23,255.87 + 49.42,915.23 + 75.68,1112.08 + 150.45,1389.85 + 205.81,the experimental group of tumor with slow growth,and have no significant changes,while the control group tumor grew faster,and prone to decay.TUNEL apoptosis detection have found that the apoptosis rate of tumor tissues in experimental group were injected with CIK+KGHV500 was(82.68+11.15)%,the number of apoptotic cells was significantly higher than the control group.The results showed that the CIK cells combined with KGHV500 has obvious effects in promoting apoptosis of transplanted tumor in nude mice.Immunohistochemical staining results showed that in addition to a small amount of adenovirus expression in nude mice was found in spleen,while other organs were not detected positive expression of adenovirus in the experimental group,but the heart and liver,spleen,lung,kidney and stomach tissues were detected in adenovirus expression with injection of KGHV500.Western-blotting results showed that in addition to a small amount of expression of anti-p21Ras scFv protein in nude mice were found in spleen,while other organs were not detected positive expression of protein in the experimental group,but the heart and liver,spleen,lung,kidney and stomach tissues were detected in protein expression with injection of KGHV500.That good safety of CIK cells with recombinant adenovirus carrying KGHV500 transplantation for the treatment of gastric cancer in nude mice.[Conclusion]In summary,in vitro experiments,we demonstrated that KGHV500 can inhibit the migration,invasion and cell viability of gastric cancer cells SGC-7901,and promote apoptosis of SGC-7901.In vivo experiments,CIK carrying KGHV500 has obvious antitumor effect on tumor growth,CIK can be used as a carrier of KGHV500,and targeting of tumor safety and effectively.The results showed that the treatment of CIK cells and KGHV500 in gastric cancer xenografts were effective and safe,and provided a new method for gene targeted therapy. |
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