Experimental Study Of Fcy::Fur/5-Flurocytosine Suicide Gene System On Cervical Cancer

cervical cancer is one of the commonest malignant tumor in female reproductive system. Now epidemiological investigation shows that the patients are becoming more youthful along with the increase of human papillomavirus(HPV) infection and the change of social life. Although the prognosis of cervical cancer had been obviously improved by combined therapy including operation, radiotherapy and chemotherapy, the treatment of advanced stage and recurrence cervical cancer are not satisfied. Lymph node metastasis is not only the key route of distant metastasis, but also a vital prognostic factor in cervical cancer. In recent years, lymph node metastasis is being focused on in malignant tumor research because of the development of specific marker of endothelial of lymphatic vessel. But the mechanism research and treatment targeting to cervical cancer lymphatic metastasis were hard to breakthrough due to the lack of corresponding lymphatic metastasis model.As a method of malignant tumor treatment, gene therapy was paid close attention to in recent years, and suicide gene therapy became one of the most promising strategy in the areas of tumor gene therapy research due to its bystander effect. Cytosine deaminase/5-fluorocytosine(CD/5-FC) system is one of the popular suicide gene system at present. Fcy::Fur gene was fused by two genes, Fcy and Fur. Gene Fcy encodes yeast cytosine deaminase and gene Fur encodes uracil phosphoribosyltransferase. Fusion gene Fcy::Fur had stronger activity that transformed 5-FC into 5-fluorouracil (5-FU) than CD gene. This project successfully established the lymphatic metastasis model of cervical cancer in Kunming(Km) mice, and investigated the effect of Fcy::Fur/5-FC suicide gene system in cervical cancer in vitro and in vivo. we hope this can provide an ideal model for the research of cervical carcer metastasis mechanism and antitumor lymphatic transfer therapy and a theoretical and experimental basement for the clinical application of Fcy::Fur/5-FC suicide gene therapy on cervical cancer.CHARPTER ONE:THE INHIBITION EFFECT OF FCY::FUR /5-FC SUICIDE GENE SYSTEM ON CERVICAL CANCER CELLS PROLIFERATIONOBJECTIVE: To investigate the cells proliferation effect of Fcy::Fur/5-FC suicide gene system .METHODS: Human cervical cancer HeLa cells were transfected by Fcy::Fur gene and then treated with 5-Flurocytosine, the influence on proliferation and apoptosis were investigated by MTT assay and AO/EB staining respectively.RESULT: After transfected with Fcy::Fur gene, human cervical cancer HeLa cells were treated with 5-FC.The inhibition rate of cell proliferation were 71.23%, 48.48%, 22.99% and 2.94% respectively at 24h in 10mM, 1mM, 100uM and 10uM group by MTT assay, 88.39%, 64.48%, 53.66% and 19.97% at 72h, 98.74%, 74.13%,70.46% and 22.24% at 120h. Treated with 1mM 5-FC, apoptosis rate of HeLa cell was 15.33%, 40% and 78.67% at 24h, 48h, 72h respectively in AO/EB staining experiment.CONCLUSIONS: These results demonstrated that the Fcy::Fur/5-FC suicide gene system can efficiently suppress cells proliferation and enhance the apoptosis in human cervical cancer HeLa cellsCHARPTER TWO: ESTABLISHMENT OF CERVICAL CANCER LYMPHATIC METASTASIS MODEL AND INITIALLY EXPLORATION OF CLINICAL SIGNIFICANCE OF PROX-1OBJECTIVE: To establish the cervical cancer model of lymphatic metastasis using Km mice , and initially explore the clinical significance of Prox-1.METHODS: The lymphatic metastasis model were established by injecting cervical cancer cells into left nail pads of female Km mice, detected the tumor formation rate , tumor growth and lymph node metastasis status. After 5,10,15,20 days of inoculation, one group mice were killed to get the specimen including lymph nodes and tumors. Then HE dyeing and immunohistochemistry stain of cytokeratin were used to detect weather lymphatic metastasis occurred , fluorescent quantitive polymerase chain reaction (FQ-PCR) and Western-blot assay were used to detect the expression of VEGFR-3 and Prox-1.RESULTS: The tumor formation rate was 100%; the lymph node metastasis were correlated with inoculation time; FQ-PCR and Western blot demonstrated that Prox-1 mRNA and protein in distant lymphatic metastasis group were more than that in sentinel node metastasis group and non-lymphatic metastasis group. The differences among these three groups were statistically significant(P<0.05);VEGFR-3 mRNA and protein in sentinel node metastasis group was no significantly difference compared with distant lymphatic metastasis group and non-lymphatic metastasis group respectively(P>0.05).CONCLUSION: The cervical cancer lymphatic metastasis model can be successfully established through the nail pad hypodermic injection in Km mice. It is an ideal model for the study of cervical cancer metastasis mechanism and antitumor lymphatic transfer therapy; Prox-1 may be a sensitive index for predicting tumor lymph node metastasis and antitumor lymphatic transfer. CHARPTER THREE : THE EFFECTS OF FCY::FUR/5-FLUOROCYTOSINE SUICIDE GENE SYSTEM ON CERVICAL CANCER IN VIVOPART I:EXPERIMENTAL STUDY OF FCY::FUR /5- FC SUICIDE GENE SYSTEM ON CERVICAL CANCEROBJECTIVE: To investigate the antitumor efficacy of Fcy::Fur / 5-FC suicide gene system on cervical cancer in Km mice which has normal immunity and its side effects .METHODS: The cervical cancer NO.27 (U27) cells were injected into exograft cervix in left foot pads in female Km mice. Tumor model were achieved and divided into three groups: control group ,gene therapy group and 5-FU therapy group. The volumes, weights, doubling times and inhibiting rates of the tumor, lymph node metastasis rate, lymphatic microvessel density(LMVD), mice suvival period and the side effects of gastrointestinal tract were calculated.RESULTS:(1)At the end of treatment , the tumor volumes and weights in gene therapy group and 5-FU group were less than those in control group respectively(P<0.05);doubling times of tumor in control group were 44.33±2.33h, prolonged to 50.04±2.71h and 49.52±3.21h in gene therapy group and 5-FU group respectively(P < 0.05); the tumor-inhibiting rate was 49.83% in gene therapy group and 47.51% in 5-FU group .But there was no significantly different between gene therapy group and 5-FU therapy group in volumes, weights ,doubling times and inhibiting rates of tumor(P>0.05) ; (2) The lymph node metastasis rate was 100%,20% and 50% in control group,gene therapy group and 5-FU therapy group respectively; the LMVD in gen therapy group was obviously lower than that in control group. (3) The mice suvival periods in gene therapy group and 5-FU therapy group were obviously prolonged compared with those in control group respectively. (4)5-FU group had obvious side effects in gastrointestinal tract ,while the gene therapy group was the same as control group. The difference of mice weight increase between gene therapy group and control group was not significant(P>0.05), but which in 5-FU group was significantly lower than these two groups (P<0.05).CONCLUSION: The Fcy::Fur/5-FC suicide gene therapy system can inhibit cervical cancer growth, reduce lymphatic metastasis of tumor , prolong mice survival period and has little gastrointestinal tract side effects, it may be a good method for advanced cervical cancer. PART II:THE EXPERIMENTAL STUDY OF FCY::FUR/5-FC SUICIDE GENE SYSTEM ON THE IMMUNE FUNCTION IN CERVICAL CANCER MICE WITH LYMPHATIC METASTASISOBJECTIVE: To investigate the change of Th1/Th2 cytokine of Fcy::Fur/5-FC gene therapy in the tumor-burden mice. To ascertain the effects of Th1/Th2 cell by Fcy::Fur/5-FC suicide gene system.METHODS: To establish the tumor models with U27and the normal control group without implantation. After 12days treatment, ELISA methods were used to observe the cytokine expression level including IFN-γand IL-4.The expression of IFN-γmRNA and IL-4mRNA were measured by FQ-PCR.RESULTS:(1) It was founded that Fcy::Fur/5-FC gene therapy can reverse the Th1/Th2 and enhance the immune function of anti-tumor by enhancing the secretion of IFN-γ, decreasing the secretion of IL-4;(2) The result of FQ-PCR showed that the expression of IFN-γmRNA in gene therapy group was markedly higher than that in control group, however, expression of IL-4mRNA decreased obviously compared with that in control group.CONCLUSION: It showed that Fcy::Fur/5-FC gene therapy can improve the immune function of mice through inducing the host anti-tumor immune activity and reverse the Th1/Th2 in mice with tumor.