| Objective:Colorectal cancer is one of the most common malignant gastrointestinal tumors in the world.The current main treatments are surgery,radiotherapy and chemotherapy with better therapeutic effect for early colorectal cancer.However,the recurrence and mortality of colorectal cancer are still high,effective and specific treatment technology is needed urgently.In recent years,targeted therapy is not only an rapid development of tumor therapy technology,but also will be an important development direction in the future.In the early stage of our laboratory,anti-p21Ras intracellular antibody was constructed with the support of the National Natural Science Foundation of China and the Yunnan Provincial Science and Technology Project.The antibody was aim to block the ras signal transduction pathway and target the high expressed tumor ras gene.Early studies have shown that the antibody has significant inhibitory effect on the growth of high p21Ras expressed tumor cell lines and transplanted tumor of nude mice.However,the antibody has no self-proliferative capacity and tumor targeting.Therefore,the wild-type adenovirus vector was altered by genetically engineering.The essential region for replication of adenovirus E1 A and E1B promoters were replaced by tumor specific promoter hTERT and HRE respectively;Fiber of type 5 adenovirus was replaced by type 35 adenovirus fibrer,as could contribute more efficiently on infecting CIK cells.The obtained targeted tumor-specific proliferating adenovirus is called KGHV400.Next,insert p21 Ras single-chain antibody into KGHV400,obtained tumor-specific proliferative adenovirus KGHV500,which is also both targeted and carried the anti-p21Ras single chain antibody therapeutic gene.Early experimental studies have shown that the virus has good effect on the treatment of breast cancer.The main purpose of this study:1,Construct a tumor-specific proliferative adenovirus KGHV500 which containing anti-p21ras single chain antibody gene.2,Study the effect of KGHV500 on human colorectal cancer SW480 cell through vitro experiments.3.Study the effect and safety of KGHV500 combined with CIK cells in the treatment of colorectal cancer through vivo experiments.And ultimately,achieve p21ras single chain antibody express sustainably and safely in colorectal cancer cells,provide a new method for colorectal cancer targeted therapy.Methods:Firstly,the package,amplification and purification of recombinant adenovirus KGHV500:The adenovirus shuttle plasmid pXC1-hTERT-ScFv-HRE recombinate with the cytoplasmic plasmid pBHGE3-F5/35 in HEK293 cells,packaging into KGHV500 which carrying the anti-p21Ras single chain antibody gene.The amplification of the recombinant adenovirus was performed in vitro.Double cesium chloride density gradient centrifugation was performed to purifie the adenovirus.TCID50 method was used to determine the virus‘titer.Secondly,vitro experiments:1,SW480 cells were infected by KGHV500:To ensure KGHV500 can infect SW480 cells,detect the expression of CD46 in’ SW480 cells at first.Transmission electron microscopy was used to determine whether KGHV500 can infected with SW480 cells successfully.The optimal MOI value of KGHV500 infected SW480 was determined by different MOI values infection.The changes of tumor cell killing,invasion,migration and apoptosis were detected by MTT assay,Transwell invasion chamber test,cell scratch test and TUNEL apoptosis method respectively.3,CIK cells loaded KGHV500:Separate human peripheral blood mononuclear cells in vitro,and induced by cytokines.Co-culture with recombinant adenovirus KGHV500,and detect the infection efficiency.Thirdly,vivo experiment:The colorectal cancer transplanted tumor model was established by using human colorectal cancer cell line SW480,and were divided into 5 groups randomly:tail vein injection with CIK cells carrying KGHV500 group,CIK cells carrying KGHV400(no anti-p21ras single chain antibody gene)group,CIK cells group,KGHV500 group and PBS group.The vivo experiments were divided into two aspects:1,Measure the size of tumor size every 3 days,and evaluate the effect of KGHV500 combined with CIK cells in vivo by drawing the tumor growth curve.2,Sacrificed the nude mice after treatment of 1d,2d,3d,5d and 7d,then detecte the safety of KGHV500 combined with CIK cells in vivo treatment by immunohistochemical method.In addition,the efficacy of KGHV500 in combination with CIK cells in vivo was also investigated by Western blotting.Result:The recombinant adenovirus KGHV500 was amplificatd in vitro and purified by double cesium chloride density gradient.The final titer was 5.0 × 109 pfu/ml.Vitro experiments showed that there is a large number of CD46 protein expression(KGHV500 receptor)on the surface of SW480 cells.Transmission electron microscopy(TEM)showed that KGHV500 could infect SW480 cells successfully,and the optimal MOI value of KGHV500 infected with SW480 cells was 100.The cell scratches showed that the percentage of cell scratches were(1.89 ± 3.41)%at 24h and 8.81 ± 4.04%at 48h after SW480 cells were infected by KGHV500.The percentage of control group was(10.52 ± 1.40)%at 24h and(22.58 ± 4.21)%at 48h,The difference between experimental group and control group was statistically significant(P<0.05),indicating that KGHV500 could inhibit the migration ability of SW480 cells effectively.Transwell invasion chamber showed that the number of cells transferred into the lower layer was much lower than the control group.The number of cells was 27 ± 8.73 for the experimental group.while the control group was 151.57± 29.90,Compared with the groups,statistical significance was significant(P<0.01),indicating that recombinant adenovirus KGHV500 can inhibit the invasive of colorectal cancer cells effectively.MTT experiments showed that when colorectal cancer cells were infected with KGHV500,the absorbance were 1.64 ± 0.13,1.45 ±0.15,1.11 ± 0.23,0.90 ± 0.12 and 0.49 ± 0.12 at 1d,2d,3d,4d,5d respectivly.While the control group was 1.68 ± 0.13,1.80 ± 0.08,1.71 ± 0.14,1.95 ± 0.13 and 1.91 ±0.18,The absorbance of the experimental group was increasing with the passage of time,while the absorbance of the tumor cells in the control group began to increase at first,and became stable gradually.The results showed that KGHV500 can kill SW480 cells effectively.TUNEL apoptosis test showed that the number of apoptotic cells was significantly increased after infecting with SW480 cells(76.34 ± 10.05%).The control group,which was not infected with the virus,almost has no apoptotic cells,the percentage of apoptosis was(96.68±.25)%.The difference between the experimental group and control group was statistically significant(P<0.01),which proved that the VI virus could promote the apoptosis of colorectal cancer cells significantly.In addition,human peripheral blood mononuclear cells were inducted into CIK cells by cytokine in vitro.The expression of CD46 was abundant on the surface of CIK cells,and the positive rate was almost 100%.Finally,CIK cells were successfully loaded with recombinant adenovirus KGHV500 and the efficiency of CIK cells infected with KGHV500was 63.15%.Vivo experiments showed that the tumor tissue size of tail vein injection with KGHV500+CIK cells group was stable relatively,the changes of tumor size was not obvious,while the tumor grew faster for the control group.The tumor size of group was the fastest,followed by the injection of KGHV500 group,CIK cells group and KGHV400 + CIK cells group.Compared with the control group,the tumor size of tail vein injection with CIK cells which loaded with KGHV500 was the smallest,indicating that the recombinant adenovirus KGHV500 and CIK cells had a significant inhibitory effect on tumor growth.TUNEL results of tumor tissues showed that the percentage of apoptotic cells was significantly higher than the other groups(75 ±16.68)%.The percentage of apoptotic cells in the CIK cells group and the KGHV500 group was significantly lower than that in the control group(P<0.05)(56 ± 10.52)%,(28 ± 7.65)%and(23 ± 8.76)%,respectively.There were almost no apoptotic cells in tumor tissues of nude mice with tail vein injection of PBS,and the percentage of apoptotic cells was(5±3.36)%.Those data demonstrated that recombinant adenovirus KGHV500 combined with CIK cells could promote the apoptosis of SW480 cells significantly.The results of safety test showed that,tail vein injection with CIK cells which carrying KGHV500 group was detected a large number of adenoviruses expression in the tumor site.The Hscore of adenoviruses expression was 62.72 ±6.78,130.25±22.58,179.53±42.58,200.07±48.63,250.67±52.68 and the percentage of positive cells was(15.52±6.59)%,(32.56 ±12.19)%,(42.53±18.63)%,(60.07± 12.54)%,(78.67±18.53)%,respectively。With the increase of treatment time,the expression of adenovirus is getting higher and higher.Othewise,small amounts of adenovirus was found in the spleen,and no adenovirus expression was detected in other organs.The adenovirus was detected in tumor of nude mice injected with KGHV500 group.The Hscore was 18.2±8.59,31.62±15.86,65.73±28.59,100.52±32.86,180.65±23.58 and the percentage of positive cells was(5.78±7.52)%,(12.57±8.46)%,(25.73±13.59)%,(39.52±14.56)%,(45.65±17.82)%,respectively.With the increase of treatment time,the expression of adenovirus is getting higher and higher.The expression of adenovirus also showed a gradual increase trend,but weaker relatively.Heart,liver,spleen and lung tissues of nude mice injected with KGHV500 group also were detected the occurance of adenovirus,which indicated that CIK cells carrying KGHV500 has a better security.In addition,The Hscore of ScFv was 1.47±8.78,100.84±12.60,130.47±42.54,177.47±35.48,220.59±46.28 and the percentage of positive cells was(16.48±11.59)%,(42.54±18.24)%,(45.67±8.15)%,(60.65±12.12)%,(78.05±20.54)%,respectively。With the increase of treatment time,the expression of ScFv is getting higher and higher.The ScFv was detected in tumor of nude mice injected with KGHV500 group.The Hscore was 12.24 ±2.59,28.44 ±14.60,50.95 ±11.92,83.25±32.20,130.64±40.23 and the percentage of positive cells was(4.28±3.12)%,(12.54±8.50)%,(18.83±13.25)%,23.64±15.21)%,(40.65±19.20)%,respectively.With the increase of treatment time,the expression of ScFv is getting higher and higher.The expression of adenovirus also showed a gradual increase trend,but weaker relatively.Western blot results also show that the experimental group only tumor and spleen were found the presence of ScFv,the rest of the tissue were all negative results.In the control group,nude mice of KGHV500 group were injected intravenously into the tail vein,and the expression of ScFv was detected except the brain tissue.Conclusion:1,Achieved the CIK cells carrying tumor-specific proliferative adenovirus KGHV500 which contain the anti-p21ras single chain antibody gene through the construction of recombinant adenovirus KGHV500 and the successful infection with CIK cells;2,Confirmed that the recombinant adenovirus KGHV500 can inhibit tumor cell SW480 growth,invasion and migration,and promote tumor cell apoptosis effectively through cell scratches,Transwell invasion,MTT and TUNEL apoptosis and other vitro experiments;3,Confirmed that recombinant adenovirus KGHV500 combined with CIK cells can inhibit the growth of tumor tissue and promote tumor cell apoptosis effectively.The treatment also has a better security.This study combined with targeted therapy,immunotherapy and gene therapy,provide a new method for the treatment of colorectal cancer. |
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