| Ovarian cancer is one of the common malignant tumors which seriously threaten to women’s health. In China, the incidence of ovarian cancer takes the third place among the gynecological tumors, and its mortality comes first. The important cause responsible for failure in treatment of ovarian cancer is the primary or secondary multidrug resistance of cancer cells.More and more research found that tumor microenvironment plays an important role in the formation and development of tumor and its resistance to drugs. Hypoxia, one of the basic characteristics of the microenvironment, exists widely in all kinds of solid tumors. It has been proved in existing research that hypoxia can induce tumor to resist to drugs through various channels. As a cytopathic hypoxia sensor, the hypoxia-inducible factor takes center place in hypoxia regulation for its mediation role in most tumor hypoxia stress response and becomes the therapeutic targets for reversal of tumor resistance.In the Hypoxia-inducible family, HIF-1α and HIF-2α are the main members to control hypoxia stress. They are similar in components and construction, and control many same downstream genes. However, many studies show that both of them have differences in the distribution and function. Now, the comparative studies of both have been carried out in some aspect in different tumors, like structure, distribution and function. But in ovarian cancer, there is still the lack of a large sample of data. It is not clear whether there are differences about both of HIF-1and HIF-2distribution in ovarian cancer, what is the cause and impact of resistant ovarian cancer cells formation,Based on the peculiar microenvironment of tumor hypoxia, our study was designed to preliminarily demonstrate whether HIF-1α and HIF-2α are related with expression of P-gp by detecting the expression of HIF-1α, HIF-2α and P-gp in epithelial ovarian tumors. Meanwhile, we constructed the hypoxia model of ovarian cancer SKOV-3cell in vitro with the CoCl2simulation and detected the inhibiting effects of cisplatin on SKOV-3cell. By detecting the effect of different concentration of CoCl2and different hypoxia time on the expression of HIF-la and HIF-2a, we explored effection of HIF-la and HIF-2a on resistance drug of ovarian cancer under chronic hypoxia microenvironment.Chapter One Expression of HIF-la, HIF-2a and P-gp in epithelial ovarian tumorsObjective:to detect the expression of HIF-la, HIF-2a and P-gp in epithelial ovarian tumors, to explore the relationship of HIF-1α, HIF-2a and P-gp with the clinical pathologic characteristic of epithelial ovarian tumors and the correlation between each other.Methods:Strictly in accordance with the access standard, we collected specimen of total175cases diagnosed as primary epithelial ovarian tumors after operation at Second Xiangya Hopsital from Jan.2005to Dec.2008. Through immunohistochemist-ry methods, we detected the expressions of HIF-la, HIF-2a and P-gp in the epithelial ovarian tumors.Results:①In benign, borderline and malignant epithelial ovarian tumor, the positive rate of HIF-la was46.15%,61.90%and78.72%respectively, with statistically significant difference (P<0.05), but its expressing strength showed no obvious statistical difference (P>0.05). The positive rate of HIF-2a was respectively7.69%,47.62%,58.16%in benign, borderline and malignant cells, also with statistically significant difference (P<0.05), and its expressing strength also showed statistical difference(P<0.05). The positive rate of P-gp was respectively30.77%,38.10%and60.28%, displaying statistical differences, but their differences in expressing strength had no statistically significance (P>0.05)②In serum, Mucinous and endometrioid malignant epithelial ovarian cancer, there were no statistical differences between the positive rate of HIF-1α, HIF-2a and P-gp (P>0.05), so were in high, medium and low differentiated malignant epithelial ovarian cancer (P>0.05). In the Ⅰ-Ⅱ stage and Ⅲ-Ⅳ stage of malignant epithelial ovarian cancer, positive rate of HIF-la and P-gp had no statistical difference(P>0.05), but positive rate of HIF-2a was41.79%in I-II stage and73.24%in Ⅲ-Ⅳ stage of malignant epithelial ovarian cancer, with statistical differences (P<0.05).③In malignant epithelial ovarian cancer, positive rate of HIF-la and HIF-2a were78.72%and58.16%, respectively, which had statistical differences (P＜0.01). In the tissues surrounding the necrotic area, the nucleus positive rate of HIF-la was31.31%, while that of HIF-2a was43.48%; differences were statistically significant (P＜0.05).④In the malignant epithelial ovarian cancer, expression of HIF-la and HIF-2a both were positively correlated with expression of P-gp(P<0.01), the correlation coefficients are0.604and0.575respectively.Conclusion:①Expression of HIF-la and HIF-2a were related with the malignant degree of epithelial ovarian cancer; HIF-2a expression showed a more close relation with it.②HIF-2a was related with the clinical stages of malignant epithelial ovarian cancer.③HIF-la and HIF-2a were differently expressed in tissues of malignant epithelial ovarian cancer.④Expression of HIF-la and HIF-2a both were positively correlated with expression of P-gp in malignant epithelial ovarian cancer. Chapter two Construction of the hypoxia model of epithelial ovarian cancer SKOV-3cell in vitro and the effect of cisplatin to SKOV-3in hypoxiaObjective:to construct the hypoxia model of ovarian cancer SKOV-3cell in vitro with the CoCl2simulation, and detect its inhibiting rate effected by cisplatin under hypoxia condition.Methods:The SKOV-3cell lines of human ovarian cancer were cultivated by using the DMED medium with0,25,50,100,150,200and250μmol/L CoCl2. MTT method was used to detect the inhibiting effects of different concentrations of CoCl2on SKOV-3cell in ovarian cancer; Blood gas analyzer were used to monitor the changes of PO2, PCO2and pH in the hypoxia model before and after improved; and Western blot method was used here to detect separately the expression of HIF-1α and HIF-2a under hypoxia and normal condition. MTT method was employed to detect the inhibiting rate of SKOV-3cell by cisplatin separately under hypoxia and normal condition.Results:①As the concentration of CoCl2increased, the inhibiting rate of SKOV-3cells raised under hypoxia condition.②Compared with normal group, expression of HIF-la and HIF-2a increased significantly after hypoxia treatment, with statistically significant differences (P＜0.05).③Compared with normal group, the inhibiting rate of SKOV-3cells was reduced from83.7%to53.5%after cultivated with cisplatin under hypoxia condition (P＜0.05)Conclusion:①The hypoxia model of ovarian cancer SKOV-3cell in vitro was successfully constructed with the CoCl2.②Hypoxia induced ovarian cancer SKOV-3cell to be resistant to cisplatin. Chapter Three Effects of Hypoxia on the expression of HIF-la and HIF-2a and formation of ovarian cancer’s drug resistanceObjective:To detect the expression of HIF-la and HIF-2a in ovarian cancer cell SKOV-3after treated with different concentrations of CoCl2and the expression of HIF-1α, HIF-2a and P-gp under different hypoxia time. To explore the effect of chronic hypoxia on the expression of HIF-la and HIF-2a and the role of HIF-la, HIF-2a and P-gp in the formation process of drug resistance of ovarian cancer.Methods:DMED medium with0,25,50,100,150,200and250μmol/L CoCl2were used to cultivate the SKOV-3cell lines of human ovarian cancer. mRNA and total protein were extracted after cultivated for8h. In hypoxia model group, mRNA and total protein were extracted separately when SKOV-3cell lines cultivated for0,2,4,8,12,24and48h. Through the western blot and RT-PCR detection, both protein and mRNA expressions of HIF-la and HIF-2a and P-gp in the SKOV-3cell were detected.Results:①The protein and mRNA expressions of HIF-la and HIF-2a raised along with the increase of CoCl2concentrations;②In early stage of chronic hypoxia (<8h), protein and mRNA expressions of HIF-1α, HIF-2a and P-gp were up-regulated. In the late stage of chronic hypoxia (>8h), HIF-2a and P-gp were stably expressed in both protein and mRNA level, but the protein and mRNA expression of HIF-la decreased.Conclusion:①Hypoxia induced by different concentration of CoCl2made impact on expression of HIF-1α and HIF-2a;②Under continuous hypoxia condition, expression of HIF-la and HIF-2a showed differences in time phase; expression of HIF-2a presented a better stability than the expression of HIF-1α;③In the early stage of chronic hypoxia (<8h), the expression of P-gp were up-regulated was related with expression of HIF-la and HIF-2a; In the late stage of chronic hypoxia (>8h), the high expression of P-gp was related with expression of HIF-2a. |
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