| I. The expression of B7-H3in hepatocellular carcinomaObjective:To detect the expression of B7-H3in serum and tissues of patients with hepatocellular carcinoma (PHC) and to investigate its role in the occurrence and progress of PHC.Methods:63samples of serum and liver tissue were collected from PHC patients,5adjacent normal tissues of hepatic hemangioma were collected as control, and the other50healthy people serum samples were also collected in the same time. Immunohistochemistry was performed to analyze the expression of B7-H3on normal liver and PHC tissues. The expression of soluble B7-H3(sB7-H3) in serum from PHC patients and healthy people was detected by ELISA. Details are as follows:(1) Immunohistochemistry was performed to analyze the expression of B7-H3on63PHC tissues. And according to the stained area and for the intensity of staining, the result was divided into positive and negative.(2) Collect the clinical pathological parameters, such as the patients’gender, age, histological type, lymphatic metastasis and distant metastasis, and then analyze the relationship between the expression of B7-H3and those clinical pathological factors mentioned above.(3)ELISA was performed to analyze the expression of sB7-H3in collected serum and relationship between the level of sB7-H3from PHC patients and clinical pathological parameters was analyzed, such as the histological type, clinical stage, size of tumor lymphatic metastasis and distant metastasis. Meanwhile, the correlation analyze was made between the levels of sB7-H3and AFP, CA19-9and CEA in PHC patients(P<0.05),Results: (1) B7-H3was widely expressed on PHC tissues, of all the cancer tissues,57samples was positively expressed B7-H3. The positive rate of B7-H3expression on PHC samples was90.5%;(2) B7-H3expression on PHC samples was not related with the patients’ gender, age, histological type, clinical stage, size of tumor lymphatic metastasis and distant metastasis.(3) The level of sB7-H3from PHC patients was (4143.47±976.27) pg/ml, which was significantly higher than that from healthy people(2076.18±605.42) pg/ml; Otherwise, the level of sB7-H3was related to clinical stage, distant metastasis and the positive expression of B7-H3in PHC tissues (P<0.05), while there was no significant difference between its expression and the other clinical pathological parameters, such as the patients’age, gender, histological type, lymphatic metastasis and size of tumor. Furthermore, a positive correlation was found between the levels of sB7-H3and CA19-9in PHC patients(P<0.05), but it was not associated with AFP and CEA.Conclusion:B7-H3was highly expressed on PHC tissues, while the benign hepatic hemangioma tissues demonstrated negative or very weak background immunostaining; The level of sB7-H3in PHC patients was significantly higher than that in healthy people, and B7-H3expression was correlated with clinical pathological index.Our study above shows that B7-H3may participate in tumor progression in a novel way, and it may serve as a potential molecule target for tumor therapy.II. The expression and signification of co-stimulatory molecule B7-H3on HepG2cellsObjective:To detect the effects of B7-H3, which was expressed on HepG2cells, an PHC cell line, on cells’ proliferation, adhesion, migration, invasion capacity and the regulation of HepG2-mediated on CD8+T cells, via silencing the expression of B7-H3with plasmid.Methods: (1) The expression of B7-H3was detected by RT-PCR, FCM and Laser scanning confocal microscopy (LSCM).(2) Specific shRNA of B7-H3was transfected into HepG2cells via liposome method. Then the expression level of mRNA and protein of B7-H3was detected by RT-PCR and FCM respectively, after being transfected48h.(3)The proliferation and adhesion capacity of HepG2cells was detected by CCK-8assay; the migration and invasion capacity of HepG2cells under the culture conditions of DMEM-HG complete medium, were evaluated by wound-healing assay and a transwell cell culture system.(4)T cells were isolated from healthy human peripheral blood by gradient centrifugation, the CD8T cells were sorted with immunomagetic beads and cultured with HepG2cells, then stimulated with PHA or PMA.(5)The effect of B7-H3on human primary hepatocellular carcinoma cell line HepG2mediating regulation on activation, cell cycle and IL-17secretion of human peripheral blood CD8+T cells.(6) FCM was used to analyze the expression of the early activation phenotype CD69on CD8+T cells and the cell cycle of CD8+T cells; IL-17secretion of T cells was assayed by intracellular staining.Results:(1) B7-H3was found to be highly expressed in HepG2cells both on the mRNA and protein level.(2) The PGPU6/GFP/neo-B7-H3shRNA could be effectively transfected into HepG2cells and efficiently suppress the expression of B7-H3on HepG2cells.(3) After silencing the expression of B7-H3, the proliferation of HepG2cells was reduced, especially at48h and72h after transfection. The difference of HepG2cells adhesion capacity between B7-H3silenced groups and control group was no statistical significant after0.5h and1h inoculation, but after3h inoculation, the adhesion capacity of HepG2cells was significantly higher, in the B7-H3silenced group, than that in the control group (p<0.05); After silencing the expression of B7-H3, the number of migration and invasion of HepG2cells were reduced. (4) Blockade B7-H3on HepG2cells could significantly attenuate the inhibitory effects of HepG2cells on the expression of CD69, the early activation phenotype of T cells. Likewise, blockade B7-H3on HepG2cells apparently reversed the inhibitory effects of HepG2cells on CD8+T cell cycle through down-regulating the cell number in G0/G1phase and up-regulating the cell number in S phase; Moreover, HepG2cells caused a sharp increase of IL-17which was secreted by CD8+T cell and the level of IL-17was further up-regulated after blocking down B7-H3.Conclusion:B7-H3takes part in the proliferation, adhesion, invasion and migration ability of HepG2cells, as well as the immunoregulation HepG2cells mediated on T cells activation, cell cycle and IL-17secretion. |
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