Enhanced Antitumor Effects Of TK-MCP-1 Fusion Gene Against Intraperitoneally Transplanted Ovarian Carcinoma Models With Immune Reconstitution In SCID Mice

Part 1Development and Evaluation of Intraperitoneally Transplanted Human Ovarian Carcinoma Model with Immune Reconstruction in Severe Combined Immunodeficient MiceObjective: To establish an intraperitonealy transplanted human ovarian carcinoma model in humanized SCID mice providing a better understanding of ovarian carcinoma.Methods: SKOV3 cells were Cultivated routinely, infected with the retroviral particles expressing vectors of PLXSN, pLXSN/TK, pLXSN/MCP-1 or pLXSN/TK-MCP-1 respectively, and selected by G418. Reverse transcription-polymerase chain reaction(RT-PCR) was used to confirm the express of HS V-TK and MCP-1. Enzyme-linked immunosorbent assay(ELISA) for the IgG of C.B17/SCIDS in serum was performed to eliminate immune leakage. Human Peripheral Blood Mononuclear cells(PBMC) for immunologic reconstitutionwere obtained by Ficoll. 24 hour later, SKOV3/neo, SKOV3/HSV-TK, SKOV3/MCP-1 or SKOV3/TK-MCP-1 cell lines were inoculated by intraperitoneal injection. Enzyme-linked immunosorbent assay(ELISA) for human immunoglobulins G(IgG) in C.B17/SCIDs' serum was performed to confirm immune reconstruction. The biological behavior of the intraperitoneally transplantated human ovarian carcinoma model with reconstruction in SCID mouse, tumor growth , morphology and pathology were detected.Results: According to the results, cell lines which could highly express either or both of these genes were named SKOV3/HSV-TK, SKOV3/MCP-1 or SKOV3/ TK-MCP-1. The cell line SKOV3/neo which was transfected by vector pLXSN was taken as a control. 3 weeks after intraperitoneally transplantation of PBMC, immune reconstruction are successfully established in SCID mouse . The ratio of successful tumor transplantation was 100%, the tumor was widespread in peritoneal cavity, mainly in diaphragm, liver and mesentery. Pathological detection indicates the tumor had the characteristics of serous and papillary adenocarcinoma of human ovarian carcinoma.Conclusion: An intraperitonealy transplanted human ovarian carcinoma model has been successfully established in humanized SCID mice that simulates the biological behavior of human ovarian carcinoma disseminating intraperitoneally in patients with immune function and may function as an ideal animal model for preclinical research of ovarian carcinoma treatment.Part 2Enhanced Antitumor Effects of TK-MCP-1 Fusion Gene against HumanOvarian Carcinoma in vivoObjective: To investigate the effect of TK-MCP-1 fusion gene against ovarian cancer in humanized SCID mice intraperitonealy transplanted human ovarian carcinoma.Methods: We injected GCV 75mg/kg.d intraperitoneally for 5 days after tumor transplantation, then, observed the biologic characteristics of SCID , such as spirit, appetite and abdominal bulge. The survival periods of 4 SCID mouse selected randomly from each groups were recorded from successfully transplanted human ovarian carcinoma cells to death natural. The rest 6 SCID mouse of each groups were sacrificed as soon as the appearance of death in the control group, and detected the dissemination of tumor. Flowcytometry examinated the number of macrophages infiltrating the tumor sites. Reverse transcription-polymerase chain reaction(RT-PCR) was used to confirm the express of TNF-α, indicating the effects of macrophages against tumor.Results: The reduction of abdominal bulge and the improvement of spirit and appetite of TK-MCP-1 group were greater than TK or MCP-1, the condition of control group had no amelioration. The survival period of TK-MCP-1 group was significantly longer than TK or MCP-1 group, followed by the Control group(P<0.05). There was no significant difference between TK and MCP-1 groups(P>0.05). TK-MCP-1 group shrinked ovarian tumors significantly, followed by TK or MCP-1 group, otherwise , Control group was still widespread in peritoneal cavity and cavitas pelvis(P<0.05). There was no significant difference between TK and MCP-1 groups(P>0.05). Flow cytometry examination revealed that the number of macrophages infiltrated the tumor tissues in the control group, TK or MCP-1 group and TK-MCP-1 group increased in order(P<0.05). There was no significant difference between TK and MCP-1 groups(P>0.05). The result of Reverse transcription-polymerase chain reaction(RT-PCR) demonstrated that the express of TNF-α of the control group, TK or MCP-1 group and TK-MCP-1 group increased in order(P<0.05). There was no significant difference between TK and MCP-1 groups(P>0.05).Conclusion: TK-MCP-1 fusion gene inducing nonspecific and specific antitumor immunity lead to a significantly enhanced efficacy of suicide gene therapy in vivo. Furthermore, this new effective approach may open novel therapeutic strategy for the treatment of ovarian cancer.Research Significance: Gene therapy is currently being investigated as a novel treatment for numerous malignancies including ovarian cancer. We studied the efficacy of TK-MCP-1 fusion gene against ovarian cancer in humanized SCID mice intraperitonealy transplanted human ovarian carcinoma. This research may provide novel therapeutic strategy for ovarian carcinoma and powerful evidence for preclinical research of ovarian carcinoma treatment.