| [Objective]The effect of recombinant adenovirus KGHV500 on glioma U251 cells was studied in vitro and in vivo experiments were performed to investigate the efficacy and targeting of anti-p21Ras scFv against nude mice glioma xenografts mediated by CIK-carrying recombinant adenovirus KGHV500.To achieve sustained and long-term expression of anti-p21Ras single-chain antibody in glioma,provide experimental basis and new methods for the treatment of glioma.[Methods]1.Amplification,identification,concentration,and purification of recombinant adenoviruses KGHV500 and KGHV400:The recombinant adenovirus KGHV500 was extensively amplified in HEK293 cells in vitro.Detection of F35 cilia fragments and single-chain antibody fragments by PCR to identify whether it is KGHV500;The recombinant adenoviruses were purified by double-barium chloride density gradient centrifugation and assayed by TCID50 method.KGHV500 infected human glioma U251 cells with different MOI values to identifiy KGHV500 infection of human glioma U251 cells multiplicity of infection;2.In vitro:IHC results showed that the positive expression of CD46 protein on human glioma U251 cell membrane;Transmission electron microscopy was used to observe the infection of human adenoviruses KGHV500 and KGHV400 against human glioma U251 cells;Cell scratch test to detect the effect of KGHV500 on human glioma U251 cell migration;The effect of KGHV500 on the viability of human glioma U251 cells was detected by MTT assay;Transwell invasion chamber assay was used to detect the effect of KGHV500 on the invasive ability of human glioma U251 cells.TUNEL apoptosis assay was used to detect the pro-apoptotic effect of KGHV500 on human glioma U251 cells.3.Isolation and identification of CIK cells:Human peripheral blood mononuclear cells were isolated and induced into CIK cells by co-culture with various cytokines;The CD3 and CD56 proteins of the CIK cell surface were detected by immunohistochemical Envision method to identify CIK cells.The immunohistochemical Envision method was used to identify the KGHV500 receptor-CD46 protein on the surface of CIK cells.Detection of KGHV500's infection efficiency on CIK cells by immunohistochemistry ENVIISION assay;Electron microscope was used to observe the status of the CIK cells carrying the recombinant adenoviruses KGHV500 and KGHV400.4.The establishment of nude mice xenograft tumor model of human glioma U251 cells and group the tail vein for injection therapy and in vivo experiments;A nude mouse model of glioma xenograft was established by subcutaneous injection of human glioma U251 cells into the right axilla subcutaneous of nude mice,Tumor-bearing nude mice were randomly divided into 5 groups for treatment.The experimental group received CIK cells carrying KGHV500 via the tail vein and the four control groups were intravenously injected with CIK cells carrying KGHV400,KGHV500,CIK cells,and PBS treatment.Dynamically monitor the growth status of each group of nude mice and transplant tumor size,draw the tumor growth curve;The experimental group received intravenous injection of KGHV500+CIK and control group were injected with KGHV500 were compared in treatment after 1st,3rd,5th,and 7th days respectively.Paraffin sections were prepared from liver,spleen,lung,kidney,stomach,pancreas,large intestine,small intestine,and brain tissues and sectioned for HE staining.Envision immunohistochemistry was used to observe the expression of recombinant adenovirus and its expressed single-chain antibody in nude mice.Intratumor expression,Envision immunohistochemistry to observe the expression of recombinant adenovirus hexon in each organ of nude mice;Western Blot was used to detect the expression of scFv in nude mice implanted with KGHV500+CIK in tail vein and KGHV500 in tail vein of control group at the late stage of treatment.Apoptosis in the transplanted tumor tissues at the late stage of treatment was detected in each group by TUNEL reagent.The expression of apoptotic genes in the transplanted tumor tissues at the late stage of treatment was detected by RT-qPCR.[Results]1.The recombinant adenovirus KGHV500 was electrophoresed to prove that the recombinant adenovirus contains ScFv fragments,F5/35 fragments,identified as the recombinant adenovirus KGHV500,after amplification and double cesium chloride density gradient centrifugation,dialysis purification,the final reorganization The titers of adenoviruses KGHV500 and KGHV400 were 1 ×109 pfu/ml and 1.5×1010 pfu/ml,respectively.KGHV500 infected human glioma U251 cells had the multiplicity of infection of 100.2.In vitro:(1)The immunohistochemical Envision assay was used to detect the positive expression of KGHV500 receptor CD46 on the surface of human glioma U251 cells.The positive expression of CD46 protein was located at the cell membrane;(2)Electron microscopy showed that the recombinant adenoviruses KGHV500 and KGHV400 infected glioma U251 cells and entered the cells.The virus particles were found in U251 cells cytoplasm.(3)The cell scratch test showed that KGHV500 can inhibit the migration of human glioma U251 cells.The cell migration rate of KGHV500-infected U251 cells at 24h and 36h was(12.28±2.51)%,(26.25(±2.48)%;The cell migration rates of KGHV400-infected U251 cells in the control group at 24 h and 36 h were(27.27±3.24)%,(43.32± 1.7)%;The cell migration rates of the U251 cells in the control group that were not infected with the recombinant adenovirus at 24h and 36h were(39.54± 1.38)%,(89.48±0.24)%.(4)Transwell invasion chamber experiments showed that KGHV500 can significantly inhibit the invasive ability of human glioma U251 cells.After 20 hours of infection,the number of cells transferred to lower microporous membrane of U251 cells infected with experimental group was 1.75± 1.03;the number transferred to lower microporous membrane of U251 cells infected with KGHV400 group was 16.83±2.54.The number of the U251 cells in the control group that had not been infected with the recombinant adenovirus transferred to lower microporous membrane after 20 hours was 144.50±29.12.(5)The results of MTT colorimetric assay showed that the absorbance of U251 cells infected with KGHV500 at the 1st,2nd,3rd,4th,and 5th days was 0.56±0.026,0.24±0.021,0.16±0.029,0.11 ±0.032,0.06±0.033,respectively.Absorbance of control U251 cells infected with KGHV400 at days 1,2,3,4,and 5 was 0.58±0.033,0.57±0.05,0.60±0.078,0.84±0.023,and 0.99±0.057,respectively.In another control group,the absorbance of U251 cells not infected with the recombinant adenovirus at days 1,2,3,4,and 5 were 0.59±0.025,0.67±0.038,0.81 ±0.06,1.15±0.083,and 1.37±0.18,respectively.KGHV500 was shown to significantly inhibit the viability of human glioma U251 cells.(6)Apoptotic TUNEL assay showed that the apoptosis rate was(73.10±3.14)%in glioma U251 cells infected with KGHV500 in the experimental group;the apoptosis rate was(18.46±2.32)%in the control group after KGHV400 infection in glioma U251 cells.The glioma U251 cell apoptosis rate in the control group without recombinant adenovirus was(1.44±0.03)%.The results showed that KGHV500 has obvious pro-apoptotic effects on human glioma U251 cells.3.Human peripheral blood mononuclear cells were isolated and induced into CIK cells by co-culture of various cytokines.The positive rate of CD3 and CD56 protein on the cell membrane of CIK cells was approximately 30%after identification by immunohistochemistry,and it was identified as CIK cells.The positive rate of CD46 protein in the cell membrane was about 100%,and it was positively located in the CIK cell membrane;Immunohistochemical Envision assay detects the infection rate of KGHV500 against CIK cells is approximately 90%;The CIK cells infected with the recombinant adenoviruses KGHV500 and KGHV400 were observed by transmission electron microscopy.It was seen that the CIK cell membrane adhered to adenovirus particles.4.In vivo experiment:(1)A xenograft model of human glioma U251 cell in nude mice was established.(2)In the experimental group,KGHV500+CIK was injected into the tail vein of the nude mice.The control group was injected with KGHV400+CIK cells,KGHV500,CIK cells,and PBS buffer.(3)Dynamically monitor the growth status of each group of nude mice and transplant tumor size,tumor growth curve showed on the 34th day after the tail vein injection treatment,the volume of tumor(mm3)of the above five groups reached 350.30±93.30,1111.80±145.23,1650.00±241.91,2325.30±332.04 and 3222.60±407.2 respectively.The growth rate of transplanted tumor tissue in the experimental group was slow,while that of the control group was faster.(4)HE slices showed no obvious morphological differences between the experimental group and the control group..(5)The experimental group received intravenous injection of KGHV500+CIK and control group injected with KGHV500 were compared in treatment afrter 1st,3rd,5th,and 7th days by examined of the hexon protein of adenovirus and single-chain antibody expression in nude mouse transplantation tumor,the expression gradually increased with time,the positive in the experimental group was stronger than that in the control group;(6)The adenovirus hexon in the heart,liver,spleen,lung,kidney,stomach,pancreas,large intestine,and small intestine of the nude mice in the control group were all positive.In the experimental group,nude mice in the experimental group had positive expression of adenoviruses and single-chain antibodies in the spleen,kidney,and liver,and no adenovirus positive expression was found in heart,lung,stomach,pancreas,large intestine,small intestine,and brain tissues.(7)The results of Western Blot showed that:strong anti-p21Ras single-chain antibody expression,spleen,liver and kidney also have a certain amount of single-chain antibody expression,heart,lung,stomach,pancreas,brain,large intestine,small intestine,no single-chain antibody expression.In the control group,single-chain antibody expression was observed except for brain tissue.(8)TUNEL apoptosis experiments showed that the experimental group was injected with KGHV500+CIK and the control group injected with KGHV400+CIK,KGHV500,CIK cells,and PBS respectively.The apoptosis rate of each group was(78.20±3.52)%,(27.23±3.21)%,20.47± 1.04)%,(8.83±0.78)%,(2.100±0.99)%,the apoptosis rate of experimental group was significant increased.(9)The expression of apoptotic gene was detected by RT-qPCR in all groups after transplantation.The expression levels of pro-apoptotic genes Caspase-3,Caspase-7 and p53 were higher in the experimental group,and the expression of anti-apoptotic genes Bcl-2 and Survivin was lower,indicating that the KGHV500 can significantly promote the apoptosis of xenograft tumor in nude mice.[Conclusion]The in vitro results showed that the tumor-specific adenovirus KGHV500 can significantly inhibit the migration and invasion of glioma U251 cells,inhibit the viability also,and promote the apoptosis of U251 cells.In vivo experiments,the tumor growth curve showed that the CIK-carrying KGHV500 has a significant growth inhibitory effect on glioma xenografts;and CIK cells carrying KGHV500 can effectively target the transplanted tumor tissue.In vivo and in vitro experiments demonstrated that CIK cells and KGHV500 co-mediated the efficacy and better targeting of anti-p21ras single-chain antibody in treating glioma xenografts. |
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