| Uterine cervical carcinoma is one of the most common gynecologic malignant tumors in the world, and its occurrence ratio has taken the second place next to breast cancer. It increases by 493,000 cases every year in the world, death about 230,000, and continues rising year by year. The uterine cervical carcinoma, which is becoming a severe threat to our health and life, has a low rate in cervical cancer emergence, but due to the large population of the country, there are about 140,000 new cases annually. The traditional therapies of uterine cervical carcinoma are surgery, chemotherapy and radiotherapy etc. which are with poor prognosis. With the development of molecular biology and intensive study of the immunology, pathogeny and molecular mechanism of tumor, the gene therapy of tumor achieved fantastic development and provide a new way to the therapy of malignant tumor. The fact that adenovirus infects most cell types with no requirement for cell division, combined with the high titers and high efficiency of gene transfer obtainable with recombinant adenovirus, make it one of the most widely used vector in gene therapy.Lactoferrin (LTF) is a multifunctional glycoprotein with antitumor effects and can inhibit the occurrence of tumours, the development and transfer including esophageal carcinoma, oral cancer, lung cancer, liver cancer, colon carcinoma and blander cancer. But molecular mechanism of human lactoferrin induced tumor growth inhibition is incompletely understood. Moreover, the adenovirus mediated human lactoferrin gene therapy on cervical cancer has not yet been characterized. Therefore, the effects of Ad-hLF mediated by replicationdeficient recombinant adenovirus and its mechanisms on uterine cervical carcinoma had been systemly researched in this study by using MTT method, immunohistochemistry SP method, flow cytometry (FCM), Western blot, Enzyme Linked Immunosorbent (ELISA). We obtained the following results by studying the effects of hLF on body’s immune system, cell cycle and apoptosis of the tumor tissue cells. The results showed as follows:(1) High titers of recombinant adenoviral particles after propagation of Ad-hLF/Ad-GFP in HEK293 cells were obtained, 2.1×10~9pfu/mL and 3.4×10~9pfu/mL respectively. This is a prerequisite to the next step experiment.(2) The impact of Ad-hLF on the reproduction and mechanisms of HeLa cells. In the study, we infected the HeLa with Ad-hLF and determined the impact of Ad-hLF on the HeLa cells by using MTT method, flow cytometry (FCM), Cells scratch test,analysis of cell cycle and RT-PCR. We discovered that Ad-hLF had a significant inhibition of reproduction and transfer of HeLa cells (P<0.05) ; Ad-hLF hindered the cell cycle of HeLa cells and arrested HeLa cells in G0/G1 phage, induced cell apoptosis and down regulated the expression of Bcl-2 mRNA in HeLa cells but the expression of Bcl-2 mRNA up regulated and lowed the ratio of Bcl-2 mRNA to promote the apoptosis.(3) The study of growth and mechanism of tumor bearing mice treated with Ad-hLF. The growth of U14 solid tumor was significantly inhibited (P<0.05). The weight inhibition rate was 53.51% and the life span was efficiently increased. The analysis of flow cytometry (FCM) turned out that concentration of free Ca2+ was significantly increased in tumor tissue cells of tumor-bearing mice treated with Ad-hLF(P<0.05); the expression of CyclinE down regulated; P21, P27 up regulated and tumor tissue cells were arrested in G0/G1 phage. Besides, the results of Western blot showed that the expression of Fas in tumor tissue cells of tumor-bearing mice treated with Ad-hLF was up regulated; the expression of anti-apoptosis protein Bcl-2 down regulated; pro-apoptosis protein Bax up regulated; Caspase-3 was actived and the tumor tissues were induced to apoptosis through death receptor pathway and mitochondrial pathway.(4) Through the evaluation of immune function of the tumor bearing mice, we found that Ad-hLF induced the anti-tumor immunological effect of the body of the tumor-bearing mice and raised the activity of NK cells. Ad-hLF counld increase the proportion of peripheral blood CD4+ and CD8+ T lymphocyte subpopulation significantly(P<0.05),It could also up-regulate the levels of serum IFN-γand IL-2(P<0.05), but down-regulate the level of serum IL-4 (P<0.05), then reverse the shift of Th1/Th2 toward Th2 partially. Meanwhile, Ad-hLF treatment could also decrease the level of serum TNF-αsignificantly, which deprived the necessary conditions of the growth and metastasis of tumor cells. Ad-hLF treatment could also suppress the expression of the VEGF of tumor tissues. |
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