促性腺激素释放激素激动剂以及联合顺铂对卵巢癌裸鼠移植瘤化疗效果的影响及分子机制的研究

1、促性腺激素释放激素激动剂以及联合顺铂对卵巢癌裸鼠移植瘤化疗效果的影响及分子机制的研究         11-05-21 16:12:00     编辑：studa20                   作者：翁慧男, 王沂峰,林琼燕  【摘要】  目的 探讨单独应用促

2、性腺激素释放激素激动剂(gonadotropin-releasing hormone agonist,GnRHa)及与顺铂联合应用对卵巢癌裸鼠移植瘤化疗效果的影响及其分子机制。方法 将24只裸鼠腹腔种植低分化卵巢浆液性囊腺癌细胞株OVCAR-3，成瘤后随机分为4组，即对照组(NS)、顺铂组、GnRHa组(GnRHa+NS)、联合用药组(GnRHa+顺铂)。剥取肿瘤组织称重，透射电镜检测各组肿瘤组织中细胞凋亡情况，免疫组织化学法检测肿瘤组织中ki67、NFkb及caspase-3表达水平。结果 对照组中未见到明显凋亡细胞及凋亡小体;顺铂组以核边集固缩的早期凋亡细胞为主;GnRHa+NS组可见凋亡

3、细胞及坏死溶解细胞;联合用药组以中晚期凋亡细胞为主。肿瘤重量及ki67评分：对照组分别为(0.149±0.072)g、(9.533±2.380);顺铂组(0.034±0.010)g、(6.283±0.491);GnRHa组(0.116±0.039)g、(8.733±0.531);联合用药组(0.026±0.024)g、(7.70±0.745)。统计结果，顺铂组和联合用药组肿瘤重量及ki67分别与对照组相比，差异有显著性(P<0.05);而GnRHa组与对照组肿瘤重量及ki67比较、顺铂组和联合用药组肿瘤重量及

4、ki67比较差异均无显著性(P>0.05)。顺铂组与联合用药组肿瘤重量及ki67均明显低于其余两组，差异有显著性(P<0.05);与对照组比较，顺铂组、GnRHa组、联合用药组NFkb表达减弱，caspase-3表达增强，差异均有显著性(P<0.05)。联合用药组NFkb表达量比顺铂组减少，差异具有显著性(P<0.05)。结论 单独应用GnRHa不影响肿瘤细胞的增殖;联合应用GnRHa及顺铂不影响顺铂对卵巢癌细胞的抗增殖作用;单独应用GnRHa、顺铂及GnRHa与顺铂联合应用均能诱导卵巢癌裸鼠移植瘤肿瘤细胞的凋亡。下调NFkb表达、上调caspase-3表达可能为诱导肿

5、瘤细胞凋亡的分子机制之一。 【关键词】  卵巢肿瘤;促性腺激素释放激素激动剂;顺铂;细胞凋亡Abstract objective To explore the anti-proliferation and apoptosis inducing effects of GnRHa ,cisplatin or both on the ovarian cancer model of athymic mice and their related molecular mechanisms.Methods Twenty-four nu/nu athymic mice were injected i

6、ntraperitoneally with human ovarian serous cystadenocarcinoma cells of the line OVCAR-3 and then randomly divided into 4 groups:control group (n=6,used as controls),CDDP group (n=6,receiving NS 0.2ml after inoculation, CDDP 4 weeks after 2 weeks of the injection of NS),GnRHa group(n=6,receiving GnRH

7、a + NS) and GnRHa + CDDP group(n=6,receiving GnRHa + CDDP). At the end of the experiment,mice were killed,and the tumor tissue was excised and histochemical analysis.Transmissino electron microscopy detect the apoptosis of the tumor tissue . The protein levels of ki67,NFkb and caspase-3 in the tumor

8、 tissue was detected by immunohistochemistry.Results There was no apoptosis cell or apoptosis body in control group.The viable apoptotic cells were observed in the cisplatin group.Apotosis cells and necrosis dissolve cells were seen in GnRH a+NS group.In combined group there were mid and non-viable

9、apoptotic cells. The immunohistochemistryscore of ki67 and the weight of the ovarian cancer of the CDDP group and the combination group were lower than the control group and the GnRHa group,but there were no significantly difference between the CDDP group and the combination group(P>0.05).Compare

10、d 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 group of cisplatin,which had significantly difference (P<0.05).Conclusion GnRHa combined with CDDP

11、in the nu/nu athymic mice ovarian cancer model do not influence the anti-proliferation of CDDP to the ovarian cancer.GnRHa ,cisplatin or both of them could also induce the apoptosis through up-regulating caspase-3 and down-regulating expression of NFkb on the ovarian cancer model of athymic mice,whi

12、ch probably one of its molecular mechanisms.Key words ovarian cancer;gonadotropin-releasing hormone agonist;cisplatin;anti-proliferation apoptosis卵巢癌目前仍为女性恶性肿瘤死亡率最高的疾病，且发病年龄日趋年轻化。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。目前有报道在肿瘤化疗过程中，使用GnRHa有卵巢功能保护的作用。而顺铂是卵巢癌标准化疗方案的用药之一。目前尚未有研究联合使用GnRHa及顺铂对肿瘤细胞化

13、疗的相关影响。在体外实验方面，有研究证实GnRHa能直接抑制卵巢癌细胞增殖1，并呈时间剂量依赖性2，3。而在凋亡方面，GnRHa是否能诱导肿瘤细胞凋亡，与顺铂联合用药是否有协同作用尚未清楚。本研究拟建立卵巢癌裸鼠移植瘤模型，探讨体内单独应用GnRHa、GnRHa与顺铂联合应用对卵巢癌裸鼠移植瘤模型肿瘤细胞凋亡及增殖的影响及可能的分子机制。现报道如下。1 材料与方法1.1 细胞株和裸鼠 人卵巢浆液性乳头状囊腺癌细胞株OVCAR-3购自中国科学院细胞库，46周龄成年雌性BALB/c-nu裸鼠24只，体重1416g。购自广东省医学实验动物研究中心动物许可证号：SYXK(粤)2005-0058，中山大

14、学眼科中心SPF实验动物部饲养，恒温(25±2)，恒湿45%50%，饲养于无菌实验室的净化空气层流架中。顺铂(CDDP)为齐鲁制药厂产品;醋酸曲普瑞林(达菲林)为博福-益普生(天津)制药有限公司产品;高糖型DMEM培养基、胰蛋白酶、胎牛血清为美国GIBCO公司产品;免疫组化试剂(北京中杉金桥)一抗为NFkb抗体及caspase-3抗体(广州杰特伟生物科技有限公司)。选择即用型免疫组化(IHC)二步法染色试剂盒(北京中杉金桥);透射电镜型号为日本电子株式会社(JELO100CX)。1.2 细胞培养 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

15、消防立管化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。1.3 动物接种及实验分组 在SPF环境超净工作台内，以75%酒精消毒裸鼠腹部皮肤，腹腔接种0.2ml细胞悬液，观察动物成瘤情况，接种后第10天可观察到裸鼠腹部稍膨隆，活动减少，腹腔进针处可见米粒样大小结节。腹腔肿瘤原代培养细胞形态与OVCAR-3细胞形态一致。遂将24只裸鼠随机分为4组每组6只：即对照组腹腔注射生理盐水(NS)0.2ml/w×5w、顺铂组(第一天腹腔注射NS 0.2ml,2周后腹腔注射顺铂5mg/w×4w)、GnRHa+NS组(GnRHa 0.3mg皮下注射，2周后NS 0.2ml/w腹腔注射×4w)、联合用药组(GnRHa 0.3mg皮下注射，2周后顺铂腹腔注射5mg/w×4w)。1.4 观察指标1.5 统计学处理 数据以(x±s)表示，运用SPSS