Study On The Molecular Mechanisms Of The Change Of Natural Killer Cell Immune Activity In Patients With Ovarian Cancer

Ovarian cancer is a kind of serious gynecological malignancy, which can severely threaten women's health. During the past more than ten years, the incidence of ovarian cancer shows a tendency of ascending year after year. Because of the obscure early symptoms and the absence of effective measures of universal inspection and early diagnosis of ovarian cancer, more than 70% of the ovarian cancers have reached an advances stage when they were diagnosed, with little hope to get good curative effect and pregnosis. Currently, ovarian cancer is the first leading cause of gynecological cancer-related deaths in China. The synthesized therapy mainly including operation and chemo-therapy is still a basic curative method.Immune functions and the occurrence and expanding of cancer have many affinities. Natural killer (NK) cells represent a cell subset belonging to the innate immune system and have the ability both to lyse target cells and to provide an early arouse of immuno-regulatory cytokines. Unlike cytolytic T lymphocytes (CTLs), NK cells exert their cytolytic function swiftly at very early stage, without the need for previous in vitro or in vivo activation and are independent of a MHC restricted manner. They are thought to represent an important defense mechanism against various intracellular pathogens, such as viruses and tumors. NK cells are now a hotspot in immunological researches and are considered as promising reagents in adoptive transferring treatment of many cancers. On the other hand, to investigate the molecular immune-escape mechanisms of cancer must be of great importance in our research on looking for the ways to overcomethe immune-escape phenomenon.At present, some research work have been done on NK cells and gynecological malignancy immunity, considering that the immune function is lower than normal. But most of those investigations did not make deeper study on the mechanisms but only stick to the change of the amount and cytotoxity of NK and the cytokins. To date, we can find few reports on the study of natural killer cell receptor and their ligands in ovarian tumors, let along the regulating mechanism.In this experiment, we planned to combine the research work on clinical samples and on cell lines of ovarian cancer, for the purpose of going deep into the molecular mechanisms of the change in NK cell activity and the regulating factors, providing new clues and theoretic proofs for improving adoptive immunotherapy of cancer in ovarian cancer patients.PART ⅠSTUDY OF NATURAL KILLER CELL RECEPTORS NKG2A, NKG2D IN PATIENTS WITH OVARIAN CANCER AND RELATED LIGANDS ONOVARIAN CANCER TISSUESObjective: To explore the status of natural killer cells in patients with ovarian cancer, including NK cell cytotoxity and the receptor expression, and the expression of their correspondent ligands on tumor tissues, and to explore the functions of those in immune surveillance and the immune escaping of ovarian cancer.Methods: MTT assay was used to evaluate the cytotoxity of NK cells and flow cytometry analysis is used to detect the expression of NKG2D and NKG2A in the peripheral blood of ovarian tumor patients, the expressions of HLA-E and MICA correspondent tissues were examined by means of half-quantified reverse transcription-polymerase chain reaction (RT-PCR). Make an analysis on these results with clinical factors of ovarian cancer. Results:1. In ovarian cancer, the cytotoxity of NK and the expression of NKG2D decline obviously, whereas the rate of NKG2A expression increased significantly. This tendency gets more evident with the stages of ovarian cancer goes up. The expressions of NKG2A rise evidently in low differentiation tissues.2. The rate of MICA mRNA expression in ovarian carcinomas was significantly higher. Although there are some trends, no evident relation was found between the expression of MICA mRNA and clinical factors.3. There are no evident differences in the expression of HLA-E mRNA in ovarian carcinomas and benign tumors. And no evident connection was found between the expression of HLA-E mRNA and clinical factors.4, IL-15 has the function of promoting NKcells in inhibiting the growth of cancer cells.Conclusion:1. The activity of NK cells and the anti-cancer cellular immunity level reduce in patients with ovarian cancer, the balance between the two receptors NKG2D and NKG2A may play an important role in regulating NK cell activity. The drop of NKG2D and the rise of NKG2A may be an crucial reason for the descend of the activity of NK cells and the decline of immune surveillance.2. The examination of the activity and the receptor of natural killer cells may be of some clinical value in understanding the status of immunity of patients and may be useful in the observation and prognosis estimating on ovarian cancer.3. MICA mRNA expression is relative to malignant transformation, one of the approach ovarian cancer escape the immune-survey probably through the release of soluble MICA.PART ⅡTHE EFFECTS OF MICA EXPRESSION IN OVARIAN CANCER CELLS ON THE SENSITIVITY TO NK CYTOTOXITY AND THE REGULATING ROLE OF IL-15Objective: To make further research on the relation between MHC expression and NK cell activity. Through observing the effects made by IL-15 to both NK92 cell and ovarian cancer cell lines, try to clarify the molecule mechanism how IL-15 regulate NK cells.Methods: MTT assay and flow cytometry analysis was used to evaluate the cytotoxity and receptors of NK92 cells, half-quantified RT-PCR to detect the expressions of HLA-E and MICA in 4 kinds of ovarian cancer cell lines, the MICA molecule was detected by means of flow cytometry analysis and immunofluorescence, Measure above indexes after using anti-MICA to block MICA, and after NK92 was stimulated by IL-15. The IFN-γ concentration was checked by way of ELISA.Results:1. The MICA and MICA molecules expressed on ovarian cancer cell lines is not parallel with each other, The HLA-E mRNA was found only on 0VCAR3.2. The sensitivity of ovarian cancer cell lines decline evidently after MICA molecule was blocked, in the cell line with HLA-E+, this change even more evident.3. After stimulated by IL-15, NK92 excrete more IFNγ, and shows higher cycotoxity.4. After treated by IL-15, the expression of MICA on ovarian cancer cells improve with different degree, except that of 3AO. All the ovarian cancer cell lines shows heighten sensitivity to NK cells, but the extents are not the same.Conclusion:1. The expression of MICA mRNA is not parallel with MICA molecule on ovarian cancer cell lines. The expression of MICA molecule holds a crucial effect in the sensitivity.2. NK92 excrete more IFNγ, and shows higher cycotoxity after stimulated by IL-15, this provide the utility of NK92 in adoptive immunotherapy in ovarian cancer with theoretic basis and new direction.3. The use of IL-15 may be of great value in improving curative effect, inthe field of immune therapy, the hope for IL-15 to be a new way is promising.