The Research Of HUVEC Vaccine Anginst Angiogenesis In Human Squamous Cell Carcinoma

Due to eating habits,living environment and genetic factors,China has the highest incidence and mortality rate of ESCC in the world.Although surgery,radiotherapy and chemotherapy are widely used in the treatment of esophageal cancer,the five-year survival rate of esophageal cancer treatment is still very low.Therefore,we need to investigate new strategies to treat esophageal cancer.The study found that the growth of the tumor must be suatained by the formation of new blood vessels to meet the needs of oxygen and nutrients,when the tumor grows to a certain size.At present,there are more than 30 factors related to tumor angiogenesis,which can be used to block tumor angiogenesis by inhibiting the related pathways and factors.Bevacizumab is the first inhibitor of tumor angiogenesis approved by FDA in 2003,and then a variety of anti-angiogenic monoclonal antibodies are gradually applied to clinical treatment.Because of the variety of tumor angiogenesis and the complex signaling pathway,the effect of drug therapy for single target is very poor.Tumor immunotherapy is a new treatment method,which can control and kill tumor cells by stimulating and enhancing the immune function of the host and thenatural defense mechanism of the host.It is safe,effective,low side effects,and gradually become an important means of tumor treatment after surgery,radiotherapy and chemotherapy.The endothelial cells of tumor tissue are in a highly active proliferative state,while the endothelial cells of normal blood vessels are generally at rest.The method of active immunotherapy with tumor vascular endothelial cells as the target aims to produce a comprehensive inhibition of tumor angiogenesis by targeting multiple antigens related to proliferation.Human umbilical vein endothelial cells(HUVEC)cultured in vitro are proliferating endothelial cells,which can express high proliferation related antigen,These antigens are the same as those expressed in the proliferating tumor endothelial cells in vivo.In this study,we constructed a human NOD/SCID mouse model of esophageal cancer xenograft,and explored the effect of HUVEC vaccine on the inhibition of human esophageal cancer and anti-tumor angiogenesis through the human immune system,which provide the basis for anti angiogenesis in tumor immunotherapy.Methods1.Blood samples were obtained from 24 cases of NOD/SCID mice.To detect whether the NOD/SCID mice had the phenomenon of "immune leakage",ELISA assay was used to detect the content of IgG in the serum of NOD/SCID mice.The IgG content in serum of mice was calculated by the standard curve.2.PBMC was isolated from human peripheral blood by density gradient centrifugation,and then NOD/SCID mice were injected with 4 × 106 PBMC intraperitoneally.After 4 weeks,the blood samples were obtained from posterior orbital vein of the mice,and the expression of CD45+ T lymphocytes in the peripheral blood of the mice was detected by flow cytometry.3.The mice were randomly divided into two groups which were intraperitoneally injected and successful immune reconstitution.One group mice(n=8)were immunized with 5×106HUVEC vaccine on the left armpit weekly for five consecutive weeks to stimulate the immune system,and the other group(n=8)wereimmunized with PBS,mice without immune reconstitution(n=8)were injected with PBS as blank control group.4.The mice were injected with 6×106 esophageal cancer cells 9706 in PBS group,PBMC group and PBMC+HUVEC group subcutaneously.Tumor growth was measured every other day after subcutaneous tumors became palpable,and the mice were sacrificed 28 days later,tumor specimens were harvested for imaging and weighting.The serum of three groups of mice were detected by ELISA anti-HUVEC antibody titer assay,and the expression of anti-tumor endothelial cell antibody in serum of mice was detected by measuring the value of 450 nm optical density.Subsequently,the tumor tissue was grinded for hemoglobin assay,and the concentration of hemoglobin in the supernatant of tumor tissue was detected by Drabkin’s reagent Kit at 540 nm.To estimate the microvascular density(MVD)within the tumors,the tumor specimens were fixed in 10 % formalin immediately after resection,embedded in paraffin and cut into 4 μm sections.MVD was investigated by the vascular endothelial antibody against CD31 through immunohistochemical technique.5.The spleens tissue of mice were stripped and spleen specimens were harvested for imaging and weighting.The spleen tissue of mice was grinded,filtered and lysed with red blood cell lysate,the expression of CD45+T lymphocytes in the spleen and peripheral blood of three groups of mice were detected by flow cytometry.The RNA of tumor tissue was extracted and the expression of CD144 and VEGFR2 gene was detected by Q-PCR method.In order to explore the reason of inhibiting tumor growth,the protein of tumor tissue was extracted and the expression of CD144 and VEGFR2 antigen in tumor tissue were detected by Western blot.The expression of CD144 and VEGFR2 antibodies in serum of 3 groups of mice were detected by Western blot,and the resistance of anti-HUVEC antibody in serum of 3 groups of mice was detected by ELISA,these method were used to explore the reason of HUVEC vaccine anginst tumor angiogenesis.Results1.The content of IgG in peripheral blood of mice were less than 2 ng/ml,far less than the standand 1μg/ml.The result concluded that there was no "immune leakage" in all of the mice in the postnatal environment.2.The expression of CD45+T lymphocytes in peripheral blood of mice was analyzed by flow cytometry in fourth week.The result showed that the content of CD45+ T lymphocytes was more than 0.1%,which proved that the immune reconstitution was successful.3.After removing the tumor,the weight of tumor tissue of the three groups were PBS group>PBMC group >PBMC + HUVEC group(p<0.01),serum anti-HUVEC antibody titer in three groups of mice:PBS group<PBMC group<PBMC + HUVEC group(p<0.05),hemoglobin content in tumor tissue:PBS group>PBMC group >PBMC + HUVEC group(p < 0.05),the microvessel density in tumor tissue sections:PBS group>PBMC group >PBMC + HUVEC group(p<0.05).4.After removing the spleens,the weight of spleens tissue of the three groups were PBS group<PBMC group<PBMC + HUVEC group(p<0.05),The result of CD45+T lymphocytes expression in spleens and peripheral blood of mice that was detected by flow cytometry: PBS group<PBMC group<PBMC + HUVEC group(p<0.001).5.The results of detection of CD144 and VEGFR2 genes in tumor tissues : PBS group>PBMC group>PBMC + HUVEC group(p<0.05);the expression of CD144 and VEGFR2 antigen in tumor tissue:PBS group>PBMC group>PBMC + HUVEC group;the expression levels of CD144 and VEGFR2 antibodies in serum:PBS group<PBMC group<PBMC + HUVECgroup;The ability of anti-HUVEC antibody in serum of three groups of mice was measured by ELISA: PBS group<PBMC group<PBMC + HUVECgroup(p<0.001).Conclusions1.The NOD/SCID mice were successfully reconstructed as the human immune system;2.HUVEC vaccine can inhibit the growth of human esophageal cancer xenografts by reducing the angiogenesis.