Experimental Study On The Treatment Of Breast Cancer By Anti-p21Ras Single-chain Antibody Mediated By CIK Cells And Tumor-specific Adenovirus KGHV50

An important direction for cancer therapy is gene therapy,immunotherapy and targeted therapy.A number of features unique to adenoviruses contribute to their utility as gene-transfer vectors,including tranducing both dividing and non-dividing tumor cells,not integrate into the cell genome,no insert mutagenicity,high safety,high efficiency,low toxicity,high-capacity,high specificity,and low degradation in cells.Cytokine-induced killer cells(CIK)by mononuclear cells in vitro co-stimulation by a variety of cytokines,induction culture to produce a non-immunogenic and having tumor targeting of cells,tumor adoptive immunotherapy the common effector cells.We previous successfully constructed carrying anti p21Ras single-chain antibody tumor-specific replicating adenovirus Ad-2P/scFv,by intratumoral injection in vivo experiments described Ad-2P/scFv Ras protein can inhibit the expression of tumor growth;but the body is unable by deep tumors intratumoral injection of treatment.Next,the laboratory successfully constructed a recombinant adenovirus efficiently infected CIK cells Ad-2P/2F,in vivo experiments show that CIK cells can be safely and accurately carry the virus to the tumor tissue site.In this experiment,adenovirus as a vector,CIK cells for the two carriers jointly mediated anti p21Ras single chain antibody therapy in nude mice.The laboratory will be constructed successfully carrying anti-p21Ras single-chain variable antibody(scFv)gene and two tumor-specific promoter of adenovirus shuttle plasmid pXC2P-scFv and chimeric backbone plasmid pBHGE3-2F cells were co-transfected HEK293 homologous recombination of carrying anti p21Ras single chain antibody genes and tumor-specific promoter,and adenovirus can infect CIK cells KGHV500.After successful viral packaging by PCR,a large number of amplifying the recombinant adenovirus,the virus was collected and concentrated,purified by dialysis,the TCID50 assay KGHV500 dialyzed recombinant adenovirus had a titer of 2.0×1011pfu/ml.The recombinant adenovirus KGHV500 multiplicity of infection of 10,100,300 and 500 multiplicity of infection(MOI)infected CIK cells,to determine the optimal MOI for infection efficiency of CIK cells is 100.Each nude mice injected subcutaneously 1×107 high-protein expression p21Ras of MDA-MB-231 human breast cancer cells,breast cancer in nude mice xenograft model,select a nude mice were randomly divided into two groups,one for the tail vein KGHV500 infected injection of CIK cells in the experimental group,another group of intravenous injection of the same amount of PBS control group.Vernier caliper monitoring tumor size,tumor growth curve,the experimental group showed a significant inhibitory effect(P<0.05)than the control group.Tail vein injection mice were sacrificed on day 25,whichever is the brain,heart,liver,spleen,lung,kidney,stomach,large intestine,small intestine,pancreas and tumor tissue.HE staining changes of the cells:tumor tissue was found in the experimental group than in the control group,fewer mitotic figures,slow proliferation,apoptosis,less necrotic cells,no significant difference compared to other tissues in the experimental group and the control group;Immunohistochemistry and Western Blot CIK cells were observed carrying adenovirus KGHV500 expression in nude mice organs of replication and scFv,showed KGHV500 arrive safely at the tumor site and replicate in tumor cells accompanied by the expression of scFv,lymphatic spleen there are few positive cells also expressed presumably with CIK cells in peripheral lymphoid organs homing sex-related,but not found in other organs KGHV500;real-time PCR detection of tumor tissue and organs in the virus replication-essential gene E1A,the expression of E1B and single-chain antibody genes,the results show:only the experimental group tumor tissue expression of a gene of interest,indicating KGHV500 viral replication proliferation only in the tumor tissue of the experimental group and expressed single chain antibodies,and other normal tissues viruses not copied while not expressing a single chain antibody.TUNEL apoptosis detection results show that the experimental group in the tumor tissue of tumor apoptotic cells was significantly more than the control group,the experimental group,the apoptosis rate was 58.3 ± 6.2,control group apoptosis rate was 18.6± 3.6(P<0.05).Real-time PCR detection of tumor tissue Caspase-3,Bcl-2 and other apoptosis-related gene expression,the results show:the experimental group tumor apoptosis execution factor Caspase-3,Caspase-7 and the pro-apoptotic factor p53 far higher than the expression level(P<0.05),whereas apoptosis gene Bcl-2,no significant difference(P>0.05),indicating a single chain antibody induces apoptosis of tumor cells.In summary,this experiment successfully constructed p21Ras carrying anti-tumor-specific single chain antibodies replicating adenovirus KGHV500,and KGHV500 viruses use CIK cells successfully reached the targeted tumor tissue by expression of anti p21Ras single chain antibody inhibited xenograft tumor growth.In this study,tumor-specific replicating adenovirus replication in tumor cell proliferation and to achieve a long-term expression p21Ras single chain antibody using CIK cell proliferation and tumor-specific targeting of the adenovirus to achieve in vivo safety of the treatment,the use of CIK cells,tumor-specific replicating adenovirus and p21Ras each single chain antibody synergistically increased anti-tumor activity against breast cancer xenograft tumor suppression treatment for ras genes that drive tumor targeted therapy experimental basis.