【作者】 黄文玲；

【导师】 金哲；

【作者基本信息】 北京中医药大学 ， 中西医结合妇科学， 2006， 博士

【摘要】 目的:子宫内膜癌又称子宫体癌,是女性生殖道系统常见的三大恶性肿瘤之一,高发年龄为58～61岁,约占女性癌症总数的7%,已占到女性生殖系统恶性肿瘤的20%～30%。子宫内膜癌是一种起源于子宫内膜的雌激素依赖性恶性肿瘤。近年来,随着激素替代治疗(HRT)广泛应用,子宫内膜癌在我国发病率逐年上升,与宫颈癌比较,已趋于接近甚至超过,受到研究者高度重视。虽然有多家研究机构致力于子宫内膜癌的研究工作,但对其发病机制仍不十分明确。更深入的研究工作仍需要进一步开展。为此,本实验以子宫内膜癌细胞系Ishikawa细胞为研究对象,研究子宫内膜癌的主要发病机制,首次探讨葛根提取物对Ishikawa细胞的影响,以及葛根提取物抑制Ishikawa细胞体外增殖作用机制,为中医药治疗子宫内膜癌提供理论基础和体外实验的资料。方法:1、MTT法分别地检测葛根提取物对Ishikawa细胞体外增殖的抑制作用:不同浓度葛根提取物处理Ishikawa细胞48小时后,测定光吸收值,计算抑制率,获得量效数据。取ICB50B浓度葛根提取物处理Ishikawa细胞24、48、72、96、120小时后,测定光吸收值,计算抑制率,获得时效数据。2、流式细胞术分别测定葛根提取物对Ishikawa细胞周期及凋亡的作用:不同浓度葛根提取物处理Ishikawa细胞48小时后,固定细胞,RNAase消化半小时,PI避光染色1小时,上机检测,Modi Fit软件进行细胞周期分析并绘制DNA分布图。3、RT-PCR法检测葛根提取物、雌二醇、孕激素对Ishikawa细胞ERRαmRNA表达的调节作用:不同浓度葛根提取物、雌二醇、孕激素处理Ishikawa细胞48小时后,提取总RNA,进行RT-PCR反应后,电泳、凝胶图像分析仪器采集图像,分析灰度值。结果:1、葛根提取物对Ishikawa细胞体外增殖作用有明显的抑制作用,并随着药物浓度增加其抑制作用更加明显,在一定浓度范围内均呈剂量依赖性。葛根提取物对Ishikawa细胞体外增殖抑制最显著的时间是加药后48小时,加药后72小时的抑制率虽然有所上升,更多还原

【Abstract】 Objective: endometrial cancer（EC）,carcinoma of corpusuteri,is one of the malignant tumor in the most frequent female genital tract disease. It occurs easily in 58-61 years old. It is about 7% in the female tumor and 20-30% in the tumor of the female reproductive system. EC is one of the estrogen dependent malignant tumor causing by endometia. Recently, the disease incidence is increasing year by year, with hormone replacement therapy （HRT） generally being used. It get close, even exceed, to cancer of the cervix. Although there are many research institute going in for rsch of EC, The pathogenesy is inadequacy identified. It’s necessary to study more deeply. For this reason, this study’s object is endometrial cancer cell line Ishikawa. We want to identify the mechanisms of EC, investigate the mechanisms of inhibiting proliferous effect of endometrial cancer cell line Ishikawa treated with Genistein in vitro and offer rationale and experimental data for treating EC by TCM.METHODS:1、To detect the effect of inhibiting proliferous to Ishikawa cells treated with Genistein by MTT respectively: After different density Genistein treating to Ishikawa cells 48 hours, we evaluated photoabsorption value, calculating inhibition ratio and getting quantity and effect data. After treating Ishikawa cells by IC₅₀ BGenistein 24 hours、48hours、72hours、96hours、120hours, we evaluated photoabsorption value, calculating inhibition ratio and getting seasoning data.2、To detect the effect of cell cycle and apoptosis to Ishikawa cells treated with Genistein by flow cytometry. After different density Genistein treating Ishikawa cells 48 hours, fixing cells, digesting half an hour by RNAase, dyeing 1 hour by PI away from light, we detected it, analyzeed cell cycle by ModiFit software and draw a DNA profile.3、To detect the effect of Bregulating to ERαBmRNA of Ishikawa cells treated with Genistein, E₂ and P₄ by RT-PCR method: After different density Genistein, E₂ and P₄ treating Ishikawa cells 48 hours, we extracted RNA, did RT-PCR reaction, collected image by electrophoresis and gel image analytical apparatus and analyzed gray scale.Result:1、Genistein markedly inhibited proliferation of Ishikawa cell in vitro. Its更多还原