[Study on the specific immunity induced by dendritic cell vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell in mice].

OBJECTIVE: To explore the specific cellular and humoral immunity induced by dendritic cells (DC) vaccine loading allogenic microvascular endothelial cell bEnd.3 antigen against U14 cervical cancer cell of mice. METHODS: Mouse brain microvascular endothelial cell bEnd.3 was cultured and identified for preparation endothelial cell bEnd.3 antigen. The level of mRNA expression of vascular endothelial growth factor receptor 2 (VEGF-R2) and integrin αV was detected by reverse transcription (RT)-PCR. The BALB/c mice were immuned with DC loading bEnd.3 antigen 4 times in 4 weeks (bEnd.3-DC group), while the mice only were immuned with DC or injected with phosphate buffer saline (PBS group) as control group. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, cytotoxic T lymphocyte (CTL) response of spleen lymphocytes in vitro, the percentage of CD3+CD8+ surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and western blot. RESULTS: The expression of VEGF-R2 and integrin αV gene in bEnd.3 cells were expressed highly. After the vaccine was injected, the tumors of mice in PBS group grew faster than those in other groups, while the tumors in bEnd.3-DC group grew slowly and disappeared after 2 weeks. The volume of tumors in DC group grew slower than those in PBS group [(0.11 ± 0.13) cm³ versus (3.38 ± 0.34) cm³]. The CTL response of spleen lymphocytes in vitro showed that bEnd.3-DC cells could kill bEnd.3 cells, the special lysis rate was more than 60%. The percentage of CD3+CD8+ spleen lymphocytes in bEnd.3-DC group [(38.6 ± 0.7)%] was higher than those in other groups (P < 0.05). The titer of serum antibody of bEnd.3-DC group was 1:3200, while it was 1:800 in DC group and there were not any in PBS group. Immunocytochemistry analysis indicated there were specific antigen-antibody reaction to bEnd.3 cell in bEnd.3-DC group. Western blot analysis revealed that there were specific bands at 220,000 (VEGF-R2). CONCLUSIONS: bEnd.3-DC vaccine can inhibit the tumor growth of U14 cervical cancer cell of mice, which indicates that the special cellular and humoral immunity are induced by bEnd.3-DC antigen which maybe have some antigens in bEnd.3 cells that reacts with endothelial cell proliferation-related antigens.

[Study on the specific immunity of dendritic cell vaccine loading human umbilical vein endothelial cell antigen against U14 cervical cancer].

AIM: To explore the specific immunity of dendritic cell (DC) vaccine loading human umbilical vein endothelial cell (HUVEC) antigen.against U14 cervical cancer of mice. METHODS: Primary HUVECs were cultured, identified and made into antigen. The BALB/c mice were immunized with DCs loading HUVEC antigen 4 times a week. One week after last vaccination, U14 cervical cancer cells were injected subcutaneously into the mice. The tumor size, CTL response of spleen lymphocytes in vitro, the percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes, and the titer of serum antibody were detected. The specific immunity was examined by immunocytochemistry and Western blot. RESULTS: HUVECs with high purity were successfully cultured and by identified by immunocytochemistry and RT-PCR. After the vaccine was injected into mice, the tumors of mice in PBS group grew faster than the those in other groups. The tumors of mice in HUVEC-DC groups grew slowly and disappeared after 2 weeks and The tumors of mice in DC group disappeared after 3 weeks. The CTL response of spleen lymphocytes in vitro showed that HUVEC-DC-T cells could kill HUVEC cells. The percentage of CD3(+)CD8(+) surface markers of spleen lymphocytes in HUVEC-DC group was higher than that in other groups. The titer of serum antibody was 1:800. immunocytochemistry analysis indicated HUVEC-DC group had specific antigen-antibody reaction to HUVECs through and Western blot analysis revealed there were specific bands at 130 KDa and 220 KDa. CONCLUSION: HUVEC-DC vaccine and DC vaccine can inhibit the tumor growth of U14 cervical cancer of mice. The special cellular and humoral immunity are induced by HUVEC-DC vaccine. Furthermore, some antigens in HUVECs maybe have special immune reaction with integrin alphav and VEGF-R2.

[Effect of the microenvironment of esophageal carcinoma on dendritic cells].

AIM: To study the effect of microenvironment simulated by esophageal carcinoma homogenate supernatant on the differentiation and development of human dendritic cells (DCs) and to investigate the mechanisms of tumor immune escape for the clinical application of DC vaccines. METHODS: Fresh esophageal carcinoma and peri-cancer tissues were collected to prepare homogenate supernatant and the content of VEGF-A was detected by ELISA. The peripheral blood monouclear cells were isolated by density gradient centrifugation and cultured with RPMI1640 medium including rhGM-CSF and rhIL-4 to induce to DCs. Then the esophageal carcinoma homogenate supernatant, peri-carcinoma homogenate supernatant and VEGF-A were added on the second day and half of the medium was changed every other day. Antigen of esophageal carcinoma cell line EC9706 was added on day 4 and lipopolysaccharide (LPS) was added on day 6. DCs were collected on day 8 for further study. Checked the morphology of DCs by microscope, the immunophenotype by flow cytometry, the gene of CD1a by RT-PCR and the proliferation and killing rate of T cell by CCK-8. RESULTS: The content of VEGF-A in the homogenate supernatant of esophageal carcinoma was significantly higher than that of the peri-carcinoma (0.987+/-0.319 microg/L, 0.152+/-0.105 microg/L, P<0.05). The cell morphology in esophageal carcinoma homogenate supernatant group was inhibited. Besides, compared with normal DCs, the positive expression rate of CD86 decreased from 69+/-8 to 42+/-11, CD1a decreased from 56+/-12 to 27+/-12 and CD11c decreased from 21+/-13 to 18+/-13 (P<0.01). CD1a gene almost showed no expression. The proliferation capacity of T cells decreased from 112.53+/-7.16 to 70.18+/-3.47 (P<0.01), and their killing capacity of T cells decreased from 62.42+/-0.57 to 46.81+/-1.62 (P<0.01). However, the cells had no difference among peri-carcinoma homogenate supernatant group, VEGF-A group and normal DC group. CONCLUSION: The tumour microenvironment stimulated by the esophageal carcinoma homogenate supernatant obviously has inhibitory effect on the differentiation and function of DCs.VEGF-A may not be the key factor in the process.

[cDNA microarray-based study of gene expression profile changes in human esophageal squamous cell carcinoma].

OBJECTIVE: To investigate the differentially expressed genes between human esophageal squamous cell carcinoma (ESCC) and normal esophageal mucosa and explore an effective method with high throughput for screening the molecular markers closely correlated with the development, invasion and metastasis of ESCC. METHODS: With cDNA microarray and laser capture microdissection, T7-based amplification were used to detect the mRNA from both the primary carcinoma and the corresponding esophageal epithelium in 15 ESCC cases, and the results were analyzed by bioinformatics methods. RESULTS: Among the 886 target genes, 110 (12.42%) genes were differentially expressed commonly at least twice in all the 15 samples, including 56 (6.32%) up-regulated by at least 2 folds and 54 (6.09%) down-regulated by at least 0.5 folds. CONCLUSION: Many ESCC-associated genes were screened by the high-throughput gene chip method, and functional study of these genes may help to identify the key genes or pathways involved in the pathogenesis and development of ESCC.