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Influence of Gonadotropin-releasing Hormone Agonist on the Effects of Chemotherapy Upon Ovarian Cancer and the Prevention of Chemotherapy-induced Ovarian Damage:an Experimental Study with Nu/nu Athymic Mice

Author: LinQiongYan
Tutor: WangYiFeng
School: Guangzhou Medical College
Course: Obstetrics and Gynaecology
Keywords: Gonadotropin-releasing hormone agonist Cisplatin Ovarian function Ovarian tumors Nude mice Tumor models Apoptosis Immunohistochemistry Transmission electron microscopy
CLC: R737.31
Type: Master's thesis
Year: 2010
Downloads: 53
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Abstract

Background ovarian cancer is women's leading cause of death in gynecological malignancies. Ovarian cancer is increasingly younger, and most when diagnosed at a late stage. Traditional surgery, radiotherapy and chemotherapy and biological therapy treatments have limited efficacy, adverse reactions, and the 5-year survival rate is hovering at about 30%. Cisplatin drugs for the treatment of first-line chemotherapy of ovarian tumors. Cisplatin is not only killing effect on tumor cells, also normal ovarian tissue, especially oocyte damage, leading to the decline of ovarian function and even premature aging (premature ovarian failure). Reported 80% of ovarian cancer cells expressing GnRH receptors in vitro experiments confirmed that of GnRH analogues, in combination with GnRH receptors play a direct inhibition of tumor growth and anti-proliferation, induction of apoptosis. Experimental Study of gonadotropin-releasing hormone agonist (gonadotropin-releasing hormone agonist GnRHa) may inhibit the hypothalamic - pituitary - gonadal axis, to prevent the recruitment of primordial follicles and further maturation, protection of ovarian function in cancer chemotherapy process. There is little research about ovarian cancer chemotherapy process applications GnRHa effect of chemotherapy. So through animal trials research cisplatin chemotherapy process application of GnRHa on tumor cell proliferation and apoptosis and whether the protective effect of ovarian function, seeking both to improve the quality of life in patients with ovarian cancer without affecting tumor effect of chemotherapy treatment, have important clinical significance. Objective To establish nude mice with epithelial ovarian cancer xenograft tumor animal models, and applications cisplatin chemotherapy combined GnRHa (triptorelin), to explore the impact of GnRHa effect of cisplatin chemotherapy and ovarian function protection. (A) build animal models and explore GnRHa ovarian function protective effect 1. Ovarian cancer cell line OVCAR-3 high-sugar type DMEM medium cultured to logarithmic phase, 0.25% trypsin digestion 3min, the reaction was terminated prepared as a single cell suspension, cell counts, and adjusting the cell density of 2 × 107/ml. 2.4 nude mice tumor pre-experiment. In the the SPF environment clean bench, with 75% alcohol disinfection abdominal skin, were inoculated intraperitoneally with 0.2ml of cell suspension were observed animal tumorigenicity. 3. Observed in January after the tumor tissue the HE staining pathological examination and abdominal tumors in primary culture, pathological findings as poorly differentiated ovarian serous adenocarcinoma and primary cell cultures OVCAR-3 line. And then take 24 mice were randomly divided into four groups according to the pre-experimental methods animals inoculated 24 mice were randomly divided into four groups: the control group [intraperitoneal injection of normal saline (NS), tumor defendant 0.2ml / w × × 4w), the combination therapy group (GnRHa0.3mg subcutaneous injection, 2 Zhou Houshun platinum intraperitoneal injection 5mg / w × 4w); each group of six. Week said the weight of nude mice. 4 ovarian tissue morphology observation and counting with the normal structure of primordial follicles and growth of the number of follicles. Serum anti-Mullerian hormone (AMH) ELISA detection. Results of 10 days after the inoculation of nude mice can be observed slightly bulging abdomen, reduced activity, intraperitoneal needle seen at the grain - like the size of nodules, see photo body weight growth curve; open the abdominal cavity, visible tumor nodules located in the bowel peritoneal surface, mesenteric mainly a diameter of about 3-15mm (see photo). Just a control group in a pale yellow ascites of approximately 0.4 ml of nude mice autopsy found. Assembly tumor was 96%. Abdominal tumor primary culture cell morphology and OVCAR-3 cell morphology. Pathological examination results: poorly differentiated ovarian serous adenocarcinoma. Tumor tissue lumps violations of adipose tissue and striated muscle tissue; nucleus nuclear cytoplasm ratio increased cytoplasm transparent vacuoles, some of the red dye; nuclei were oval or irregular in shape, see nucleolus and scattered withered apoptotic cells (Figures 2, 3). Ovarian tissue after treatment in nude mice in the control group of normal ovarian morphology after intraperitoneal injection of cisplatin naked eye view of ovarian tissue shrinkage, uneven surface. The weight of both ovaries the original ovarian follicles and growing follicles in Table 1. Significantly reduce ovarian weight of the cisplatin group, a statistically significant difference with the other three groups (P lt; 0.05); pairwise comparison of the combined treatment group and the control group, GnRHa group, the difference was not statistically significance (P gt; 0.05). 4 nude mice ovarian primordial follicles, the growth of the number of follicles difference was statistically significant (P lt; 0.05). Cisplatin group, the number of primordial follicles, the growth of the number of follicles was significantly less than the other three groups, a statistically significant difference (P lt; 0.05). But the combined treatment group with the control group, respectively, compared with twenty-two of GnRHa group, the difference was not statistically significance (P gt; 0.05). After treatment, four groups of nude mice serum AMH levels are shown in Table 1, in each group, the difference being statistically significant (P lt; 0.05). Cisplatin group was significantly lower than the other three groups, a statistically significant difference (P lt; 0.05); However, the combined treatment group with the control group, respectively, of GnRHa group pairwise comparisons, the difference was not statistically significance (P gt; 0.05). Conclusion human ovarian cancer cell lines OVCAR-3 build intraperitoneal xenografts of ovarian cancer in nude mice simple 4 × 106/0.2ml that it can become a tumor, can be more complete to maintain the characteristics of human ovarian serous cystadenocarcinoma; Shun platinum nude mouse ovarian function has the damaging effects of ovarian function damage caused by cisplatin in nude mice; GnRHa not only has a protective effect early in chemotherapy can significantly reduce chemotherapy-induced side effects of weight loss. (B) to explore the method of GnRHa cisplatin chemotherapy effect a nude mouse tumor weight of the treated group and the proliferation antigen ki67 immunohistochemistry rating. Transmission electron microscopy specimen preparation and observation of tumor cell apoptosis in tumor half cysteine ??protease protein a -3 (Caspase-3) and 1.4 NFkb test results in nude mice tumor weight and proliferation antigen ki67 immunohistochemistry score are shown in Table 2,4 nude mice tumor weight difference was statistically significant ( P lt; 0.05), the cisplatin group with tumor weight of the combined treatment group was significantly lower than the control group and GnRHa group, a statistically significant difference (P lt; 0.05); cisplatin group and the combined treatment group difference was not statistically significant ( P gt; 0.05); the control group GnRHa group, the difference was not statistically significance (P gt; 0.05). The control group did not undergo apoptosis of ovarian cancer cell membrane, mitochondria, nucleus and other organelles normal; three groups via cisplatin, GnRHa, GnRHa cisplatin treatment, medication group showed morphological changes of apoptosis, cytoplasmic concentrated , deeply stained nuclei, chromatin condensation into blocks and edge set, which for the most characteristic changes of the early, middle and late apoptotic. Early apoptosis: the nucleus chromosomes occurs set of edges, irregular nuclear shape, nuclear membrane surface irregularities; apoptosis interim: nuclear chromatin condensation, margination crescent-shaped nuclear pore disappear, the nuclear membrane was corrugated shrinkage; apoptosis late the: karyopyknosis, membrane budding to form vesicles, apoptotic bodies. The 3. Medication group and cisplatin group, GnRHa NS group, the combined group NFkb downregulated caspase-3 upregulation. Compared with the control group, the differences were statistically significant (P lt; 0.05). The data are shown in Table 3. Reduced joint the drug group NFkb expression than cisplatin group, the difference was statistically significant (P lt; 0.05). The conclusions the GnRHa not affect the anti-proliferative effects of cisplatin on ovarian cancer; alone GnRHa and cisplatin-induced apoptosis of ovarian cancer in nude mice tumor cells. Cut NFkb expression raised Caspase-3 in one of the molecular mechanisms of its induction of apoptosis.

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