| Objective: 1.To establish stable NOD/SCID murine model of human hepatocellular carcinoma and reconstruction of human immune system (HCC-hu-PBL-NOD/SCID); 2.To investigate the therapeutic effect and mechanism of human interleukin 12 gene on the treatment of hepatocellular carcinoma in the murine models.Method: 1.To establish the HCC-hu-PBL-NOD/SCID model, NOD/SCID mice were injected with HepG2 cells subcutaneously, and then with human PBL; 2.The mice were randomized into 3 groups after tumorigenesis including treatment group, empty vactor group and control group.On day 1,4,7 the mice in the first group were injected intratumorally with irradiated lethally 9597/IL-12 cells, the mice in the secend group with irradiated lethally 9597/plasmid cells, and the third group with PBS; 3.Observe the changes in tumor volume of mice, as well as the body weight and general conditions. Furthermore the murine peripheral blood was collected on day 7, 14, 21, 28. On day 28 the mice were sacrificed and the tumors were taken. Count the amount of peripheral white blood cells, and detect the level of ALT/AST with ELISA; 4.Detect the level of Th1/Th2/Th17 cytokine and IL-12 in murine peripheral blood and tumors with CBA and ELISA; 5.Detect cell cycles and apoptosis of the tumors locally with Flow cytometry; 6.Detect immune cells and microvessel densities in the tumors with immunohistochemistry.Results: 1.Tumors developed in all the NOD/SCID mice after injected with HepG2 cells subcutaneously and then with human PBL intraperitoneal. Histologic examination with HE staining showed the histologic and cytologic features in the murine tumors similar to the features in human HCC. The presence of human lymphocytes (CD3+ T cells: 5.63±3.48 cells/HP,CD4+ T cells: 3.21±2.83 cells/HP,CD8+ T cells: 1.18±2.20 cells/HP) and cytokine (IL-2: 4.11±0.80pg/ml,IL-4: 4.24±0.98pg/ml , IL-6: 3.12±0.43pg/ml , IL-10: 2.86±0.54pg/ml , TNF: 5.32±0.63pg/ml , IFN-γ: 11.19±2.36pg/ml , IL-17A: 85.37±12.21pg/ml) was revealed; 2. The tumor growth was inhibited after injection intratumorally in the treatment group whose volumes of tumor were 956.51±257.27mm~3 on day 28 which was less than the other two groups. 3.There was no difference in the body weight between the treatment group and the other groups during the experimental period; 4.The amount of peripheral WBC in the treatment group was 9.32±1.93×10~9cells/L which was more than the amount in the other two groups on day 7 after injection intratumorally, and the treatment group(8.04±1.46×10~9cells/L) and the empty vector group(7.97±1.20×10~9cells/L) more than the control group(6.42±1.68×10~9cells/L) on day 21. Finally, there was no difference on day 28(P>0.05); 5. Diffenrence did not lie on the level of AST among the three groups(P>0.05). But the level of ALT in the treatment group rose on day 14 which was 78.76±20.33IU/L, and recoveried on day 28(35.56±16.20IU/L); 6. The level of peripheral IL-12 in the treatment group was 13.82±9.15pg/ml on day 7, 9.08±3.41pg/ml on day 14, 3.13±2.36pg/ml on day 21,and 2.24±1.44pg/ml on day 28 which were all higher than the other two groups.The levels of IL-2 and IFN-γin the treatment group were 4.94±2.66 pg/ml and 5.63±1.97 pg/ml which were higher than the other two groups on day 21; 7.In the tumors microenvironment there were more IL-2(5.71±1.94pg/ml), IFN-γ(12.61±4.29pg/ml) and IL-12(3.06±1.05pg/ml) in the treatment group than those in the other two groups. However it was on the contrary on the level of IL-17A (67.40±16.51pg/ml). Futhermore more CD3+ T cells (9.18±3.72 cells/HP), CD4+ Th cells(5.62±2.15 cells/HP), IFN-γ+ Th1 cells(4.22±1.59 cells/HP), S-100+ DCs(10.24±3.39 cells/HP) infiltrated in the microenvironment of tumors in the treatment group in which MVD was lower at 4.32±2.04 vessels/HP; 8. The cells in the tumors in the period of G0/G1 increased and in the period of S decreased which were different with the control group significantly (P<0.05). More apoptosis occured in the neoplasms in the treatment group(25.54±7.13%) than those in the other two groups.Conclusions: 1. HCC-hu-PBL-NOD/SCID model can be established with Injecting with HepG2 cells subcutaneously, and then with human PBL intraperitoneal to simulate the microenvironment of human hepatocellular carcinoma; 2.Injecting intratumorally with human IL-12 gene contributes to inhibite the growth of tumors in the murine models; 3.The side effects associated with IL-12 gene therapy are light including transient increase of ALT and mild diarrhea merely.
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