【作者】 林琼燕；

【导师】 王沂峰；

【作者基本信息】 广州医学院 ， 妇产科学， 2010， 硕士

【摘要】 背景卵巢癌目前仍是妇科恶性肿瘤中妇女首要的死亡原因。目前卵巢癌的发病日趋年轻化,且多数确诊时已到晚期。传统手术治疗、放、化疗及生物治疗等治疗措施疗效有限,不良反应大,5年生存率徘徊在30%左右。顺铂为目前治疗卵巢肿瘤的一线化疗药物。但顺铂不仅对肿瘤细胞有杀灭作用,同时也对正常的卵巢组织,特别是卵母细胞造成伤害,导致卵巢功能下降甚至早衰(premature ovarian failure)。文献报道80%卵巢癌细胞表达GnRH受体,体外实验证实,GnRH类似物通过与GnRH受体结合发挥直接抑制肿瘤生长和抗增殖作用、诱导凋亡作用。有实验研究促性腺激素释放激素激动剂(gonadotropin-releasing hormone agonist GnRHa)可能抑制下丘脑-垂体-性腺轴,阻止原始卵泡的募集及进一步发育成熟,在肿瘤化疗过程中有卵巢功能保护的作用。目前甚少有研究关于卵巢肿瘤化疗过程中应用GnRHa对化疗效果的影响。故通过动物体内试验研究顺铂化疗过程中应用GnRHa对肿瘤细胞增殖及凋亡的影响及是否有卵巢功能保护作用,以寻求既提高卵巢癌患者的生存质量又不影响肿瘤化疗效果的治疗方法,具有重要的临床指导意义。目的通过建立裸鼠上皮性卵巢癌移植瘤动物模型,并应用顺铂化疗及联合应用GnRHa(曲普瑞林),探讨GnRHa对顺铂化疗效果的影响及对卵巢功能保护作用。(一)构建动物模型及探讨GnRHa的卵巢功能保护作用方法1.人卵巢癌细胞株OVCAR-3在高糖型DMEM培养基中培养至对数生长期,0.25%胰蛋白酶消化3min,终止反应,制备成单细胞悬液,细胞计数,调整细胞密度为2×107/ml。2.4只裸鼠进行成瘤预实验。在SPF环境超净工作台内,以75%酒精消毒腹部皮肤,腹腔接种0.2ml细胞悬液,观察两组动物成瘤情况。3.观察1月后取肿瘤组织进行HE染色病理学检查及腹腔肿瘤原代培养,确定病理结果为低分化卵巢浆液性囊腺癌及原代细胞培养与OVCAR-3一致。再取24只裸鼠随机分为4组按预实验方法进行动物接种,成瘤后遂将24只裸鼠随机分为4组:即对照组[腹腔注射生理盐水(NS)0.2ml/w×6w]、顺铂组(第一天腹腔注射NS0.2ml,2周后腹腔注射顺铂5mg/w×4w)、GnRHa+NS组(GnRHa0.3mg皮下注射,2周后NS0.2ml/w腹腔注射×4w)、联合用药组(GnRHa0.3mg皮下注射,2周后顺铂腹腔注射5mg/w×4w);每组6只。每周称裸鼠体重。4.卵巢组织形态学观察及计数具有正常结构的原始卵泡及生长卵泡数。5.小鼠血清抗苗勒管激素(AMH)ELISA检测。结果裸鼠腹腔接种后第10天可观察到腹部稍膨隆,活动减少,腹腔进针处可见米粒样大小结节,各组体重增长曲线见附图;打开腹腔,可见肿瘤结节分布于肠管、腹膜表面、肠系膜为主,直径约3-15mm(见附图)。仅有对照组中1只裸鼠尸解时发现有淡黄色腹水约0.4ml。总成瘤率为96%。腹腔肿瘤原代培养细胞形态与OVCAR-3细胞形态一致。病理学检查结果:为低分化卵巢浆液性囊腺癌。肿瘤组织呈团块状,侵犯脂肪组织及横纹肌组织;细胞核核浆比例增大,细胞浆透明空泡状,部分红染;细胞核呈卵圆或不规则形,见有核仁,并可见散在凋亡细胞(图2、3)。用药后对照组裸鼠卵巢形态正常,顺铂腹腔注射给药后卵巢组织肉眼观卵巢组织体积缩小,表面凸凹不平。4组双侧卵巢重量、卵巢原始卵泡数及生长卵泡数见表1。顺铂组卵巢重量明显降低,与其它三组比较差异具有统计学意义(P<0.05);联合用药组与对照组、GnRHa组两两比较,差异无统计学意义(P>0.05)。4组裸鼠卵巢原始卵泡数、生长卵泡数分别比较差异具有统计学意义(P<0.05)。顺铂组原始卵泡数、生长卵泡数明显少于其余三组,差异具有统计学意义(P<0.05)。但联合用药组分别与对照组、GnRHa组两两比较,差异无统计学意义(P>0.05)。用药后,4组裸鼠血清中AMH水平见表1,各组比较差异具有统计学意义(P<0.05)。顺铂组明显低于其余三组,差异具有统计学意义(P<0.05);但联合用药组分别与对照组、GnRHa组两两比较,差异无统计学意义(P>0.05)。结论应用人卵巢癌细胞株OVCAR-3构建裸鼠卵巢癌腹腔移植瘤的方法简单易行, 4×106/0.2ml即能成瘤,能够较完整保持了人卵巢浆液性囊腺癌的特点;顺铂对裸鼠卵巢功能具有损害作用;GnRHa不仅对顺铂引起的裸鼠卵巢功能损害具有保护作用,在化疗早期还能明显减少化疗引起体重下降的副作用。(二)探讨GnRHa对顺铂化疗效果的影响方法1.各用药组裸鼠肿瘤组织重量及增殖抗原ki67免疫组化评分2.透射电镜标本制备并观察肿瘤组织细胞凋亡情况3.肿瘤组织半胱胺酸蛋白酶蛋白-3(Caspase-3)及NFkb检测结果1.4组裸鼠肿瘤组织重量及增殖抗原ki67免疫组化评分见表2,4组裸鼠肿瘤组织重量分别比较差异具有统计学意义(P<0.05),顺铂组与联合用药组肿瘤重量明显低于对照组及GnRHa组,差异具有统计学意义(P<0.05);顺铂组与联合用药组比较差异无统计学意义(P>0.05);对照组与GnRHa组比较,差异无统计学意义(P>0.05)。2.对照组未发生凋亡的卵巢癌细胞细胞膜,线粒体、细胞核等细胞器正常;而在经顺铂、GnRHa、GnRHa+顺铂处理后的三组用药组呈细胞凋亡的形态改变,胞质浓缩,细胞核深染,染色质浓缩成块并边集,其中以早、中、晚期凋亡为最具特征性改变。细胞凋亡早期:细胞核染色体发生边集,核形不规整,核膜表面凹凸;凋亡中期:核内染色质凝聚,趋边呈月牙状,核膜孔消失,核膜呈波纹状皱缩;凋亡晚期:核固缩,细胞膜出芽形成小泡,可见凋亡小体。3.三组用药组,顺铂组、GnRHa+NS组、联合用药组NFkb表达下调,Caspase-3表达上调。与对照组比较,差异均有统计学意义(P<0.05)。数据见表3。联合用药组NFkb表达量比顺铂组减少,差异具有统计学意义(P<0.05)。结论GnRHa不影响顺铂对卵巢癌细胞的抗增殖作用;单独应用GnRHa及与顺铂联合应用能诱导卵巢癌裸鼠移植瘤肿瘤细胞凋亡。下调NFkb表达、上调Caspase-3表达可能为其诱导凋亡的分子机制之一。更多还原

【Abstract】 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.更多还原