| BackgroudOvarian cancer is still the primary cause of death in women of the gynecologic malignancy, Whose incidence is increasingly younger, and most is a late stage when diagnosed. Traditional surgical treatment, chemotherapy and biological therapy treatments have limited efficacy and large adverse reactions. The five-year survival rate remained at about 30%.Cisplatinum is the first-line treatment of ovarian cancer chemotherapy drugs in current. However, cisplatin not only kills the tumor cells, but also the normal ovarian tissue,especially the oocytes, which causes premature ovarian failure(POF).Gonadotropin-releasing hormone agonist(GnRHa) can inhibit the hypothalamus-pituitary-gonadal axis to prevent primordial follicle recruitment and further development to hamper the chemotherapy drug-induced damage of ovarian function. 80% of ovarian cancer cell expression of GnRH receptors,in vitro experiments confirmed that GnRH analogues binding to GnRH receptors plays a direct inhibition of tumor growth and anti-proliferation effects.There were few research which explore that influence of GnRHa on the effect of chemotherapy .Therefore,it has important clinical significance to explore the influence of gonadotropin-releasing hormone agonist on the effects of chemotherapy upon ovarian cancer and the prevention of chemotherapy-induced ovarian damage in vivo experiment,which can improve the quality of life of ovarian cancer patients and not affect the chemotherapy effect.PurposeThrough the establishment of the epithelial ovarian cancer animal model in nu/nu athymic mice,we explore the influence of the gonadotropin-releasing hormone agonist(GnRHa) on the effects of chemotherapy upon ovarian cancer and the prevention of chemotherapy-induced ovarian damage .Establish the animal model in nu/nu athymic mice and explore the role of GnRHa in the prevention of chemotherapy- induced ovarian damage.Methods1. Human ovarian cancer cell line OVCAR-3 in high-glucose-based DMEM culture medium to logarithmic phase, prepared into single cell suspension, adjust the cell density of 2×107/ml .2. Four nude mice for tumorigenic pre-test. In the SPF environment, 75% alcohol disinfection of the abdomen skin, inoculated intraperitoneally with 0.2ml cell suspension ,observe the tumorigenic of the animals.3. Observations after one month, HE staining in tumor tissue for pathological examination and intra-abdominal tumor primary culture to determine the pathological results of poorly differentiated ovarian serous adenocarcinoma and primary cell culture coherenting with OVCAR-3 line. And then take 24 nude mice were randomly divided into four groups according to pre-experimental method for animal inoculation: the control group [intraperitoneal injection of normal saline (NS) 0.2ml / w×5w], CDDP group (the first day of intraperitoneal injection of NS0.2ml, 2 weeks after intraperitoneal injection of cisplatin 5mg / w ×4w), GnRHa + NS group (GnRHa0.3mg subcutaneous injection, 2 weeks after NS0.2ml / w intraperitoneal injection of×4w), the combination group (GnRHa0.3mg subcutaneously 2 weeks after intraperitoneal injection of cisplatin 5mg / w×4w); 6 mice in each group.4. Morphological observation and counting the number of primordial follicles and growth follicles .5. Mouse serum anti-Mullerian hormone (AMH) ELISA detectionResultNude mice bulging abdomen, can be observed were after inoculated 10 days. activities reduced, nodules could be seen in the injection position. Body weight growth curve of each group shows in Figure 1.Opening the abdominal cavity, we could seen the distribution of tumor nodules in the intestine, peritoneal surface, mesentery mainly a diameter of about 3-15mm. There is about 0.4ml of yellow ascites in one mouse of the control group. Assembly tumor rate was 96%. Intraperitoneal tumor cells in primary culture morphology and OVCAR-3 cells is coincident. Pathological examination is poorly differentiated ovarian serous cystadenocarcinoma (Figure 2,3).Ovarian morphology of Control group is normal. But ovarian tissue volume reduced, the surface rugosity in CDDP group. Data of the bilateral ovarian weight, ovarian primordial follicle number and growth of the number of follicles of the 4 groups shows in Table 1. Ovarian weight of CDDP group decreased significantly.comparing with the other three groups ,there is statistically significant difference (P <0.05).combination group and control group, GnRHa group pairwise comparison, the difference was not statistically significant (P> 0.05). The number of ovarian primordial follicles and growth follicles in 4 groups has statistically significance (P <0.05). the original number of primordial and growth follicles in CDDP group is less than the other three groups ,which has significantly difference(P <0.05). However, the combination group, control group and GnRHa group pairwise comparison, the difference was not statistically significant (P> 0.05). After treatment, four groups of nude mice in serum AMH levels in Table 1. CDDP group was significantly lower than the other three groups, the difference was statistically significant (P <0.05); However, the combination group,control group and GnRHa group pairwise comparison, the difference was not statistically significant (P> 0.05).ConclusionApplication of human ovarian cancer cell line OVCAR-3 to establish nude mice ovarian cancer model is successful, 4×106/0.2ml is the most optimal density. It can maintain the characteristics of human ovarian serous cystadenocarcinoma. cisplatin has the effect of ovarian function damage in nude mice .GnRHa can prevent the ovarian damage which caused by CDDP.Explore the influence of GnRHa on the effects of chemotherapy upon ovarian cancer animal model1. Measured the weight of the tumor weight and the ki67 of the tumor tissue was analyzed by immunohistochemistry.2. TEM sample preparation and observation of tumor cell apoptosis.3. Cysteine protease protein -3 (Caspase-3) and NFkb detectionResult1. Tumor weight and ki67 immunohistochemical score of 4 groups in Table 2. CDDP group and combination group were significantly lower than the two other groups, the difference was statistically significant (P <0.05); while There was not statistically significant difference between the CDDP group and the combined group (P >0.05); control group and GnRHa group has not statistically significant difference (P> 0.05).2.TEM observation of tumor cell apoptosis: There was not apoptosis of ovarian cancer cells in the control group, cell membrane, mitochondria, nucleus and other organelles was normal ; while the CDDP, GnRHa, combination groups showed morphological changes of apoptosis, cytoplasmic condensation, nuclei deeply stained, chromatin condensed condensation and edge sets. Apoptosis early state: nuclear chromosome occurrence edge set, the nuclear shape is not structured, the nuclear membrane surface, uneven; Apoptosis in the middle state: the nucleus chromatin condensation, margination was crescent-shaped, nuclear membrane pore disappearance ,nuclear membrane was crinkle; Apoptosis in the late state :cell membrane budding to form vesicles, apoptotic bodies can be seen.3. Compared with the control group,the protein level of NFkb was decreased while Caspase-3 expression was increased in the other three groups(P>0.05).the level of NFkb in combined group was lower than the one of the cisplatin,which had significantly difference between them(P<0.05).ConclusionGnRHa alone could not inhibit tumor cell proliferation; Combined with GnRHa does not affect the anti-proliferative effect of cisplatin in ovarian cancer; Alone GnRHa, cisplatin, and the two combined can induced apoptosis of ovarian cancer xenograft in nude mice . it probably one of its molecular mechanisms to up-regulate Caspase-3 and down-regulate expression of NFkb on the ovarian cancer model of athymic mice.
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