【作者】 宋文哲；

【导师】 宋燕；

【作者基本信息】 吉林大学 ， 外科学， 2004， 博士

【摘要】 乳腺癌是影响女性健康的一种常见恶性肿瘤，全世界每年约有120万妇女发生乳腺癌，有50万妇女死于乳腺癌。中国是发展中国家，乳腺癌的发病却与发达国家相似，呈不断上升趋势。有资料表明，近二十年来乳腺癌的发病率增长了37.6％，平均每年增长率达2.3％，到2000年我国乳腺癌的发病率已达28.8/10万，居女性肿瘤的第一位。现代的乳腺癌治疗是以手术治疗为主的综合治疗，乳腺癌内分泌治疗是综合治疗的重要组成部分。作为乳腺癌内分泌治疗的一线药物——三苯氧胺，应用已经有三十多年的历史。传统的观点认为三苯氧胺抑制乳腺癌细胞生长的机制是它与雌二醇竞争特异性受体部位，结合成活性较低的抗雌激素受体复合物，可以降低癌细胞活性作用，使肿瘤细胞停滞于G1期，减少S期细胞的比例。近年来学者发现TAM还可以通过诱导凋亡作用从而抑制乳腺癌细胞的生长。Survivin是由Altieri等在1997年发现的一种新型凋亡抑制因子，是凋亡抑制蛋白（inhibitor of apoptosis protein, IAP）家族中最小的成员。Survivin表达于胚胎、胎儿组织，及大多数常见的肿瘤组织，如肺、胃、结肠、胰腺、乳腺、肝脏、前列腺以及大约50％的非何杰金氏淋巴瘤，但不表达于终末分化的成人组织中。与其他IAP蛋白一样，survivin蛋白通过一种BIR依赖性的识别方式与caspase-3和caspase-7相结合，并抑制其活性，从而抑制凋亡。由于survivin的这种在肿瘤组织中特异性表达的特性，使其成为肿<WP=97>瘤治疗研究中的热点。研究发现，通过抑制肿瘤细胞内survivin基因的表达，可以增加肿瘤细胞对化疗药物的敏感性，而将survivin cDNA转入到肿瘤细胞内则促使肿瘤细胞对化疗药物产生耐受性，但对于TAM所诱导的乳腺癌细胞凋亡与survivin基因的关系，以及抑制survivin基因的表达能否增加乳腺癌细胞对TAM治疗的敏感性还未见报道。本实验的目的是研究TAM所诱导的乳腺癌细胞凋亡与survivin基因表达的关系。在这项实验中我们观察了TAM诱导MCF-7乳腺癌细胞凋亡、TAM对survivin基因作用情况，以及联合应用survivin mRNA反义寡核苷酸和TAM后，MCF-7乳腺癌细胞凋亡情况的变化。结果发现TAM所诱导的MCF-7细胞凋亡与survivin基因表达下调有关，survivin mRNA反义寡核苷酸可以增加MCF-7细胞对TAM的敏感性。三苯氧胺对survivin基因的作用及其机制研究 我们用不同浓度三苯氧胺（TAM）作用于MCF-7乳腺癌细胞株，在不同时间段提取标本。用流式细胞技术检测不同浓度TAM和作用时间后对MCF-7细胞凋亡率和细胞周期的影响；应用原位杂交技术和Western blotting方法检测不同浓度TAM和作用时间后MCF-7细胞survivin mRNA阳性率以及survivin蛋白表达的变化；应用免疫组织化学方法和比色法检测不同浓度TAM和作用时间后MCF-7细胞内caspase-3蛋白表达情况和caspase-3活性的变化。结果显示： （1）TAM所诱导的MCF-7细胞凋亡呈现出明显的时间依赖性，随着TAM作用时间的延长，MCF-7细胞凋亡率增加；同时TAM诱导凋亡作用亦呈现出剂量依赖性，随着TAM浓度的增加，细胞凋亡率增加。 （2）TAM可以影响MCF-7细胞株的细胞周期进程，TAM作用后细胞周期中G0/G1期细胞数量明显升高，相应的S期和G2/M期细胞数量明显降低，显示G0/G1期阻滞，且呈现出浓度、时间依赖性。 （3）当TAM浓度为10-8mol/L时，即出现明显的对survivin mRNA<WP=98>和survivin蛋白表达的抑制作用，而且这种抑制作用呈现出剂量依赖性，即随着TAM浓度的增加，survivin mRNA的阳性率以及survivin蛋白的表达逐渐降低。另外，TAM对survivin mRNA的抑制作用亦呈现出时间依赖性，随着TAM作用时间的延长，survivin mRNA阳性率以及survivin蛋白的表达逐渐降低。17β雌二醇（17β-E2）预处理能够阻断TAM对survivin的抑制作用，这说明TAM对survivin的抑制作用是雌激素受体依赖的。（4）不同浓度TAM和作用时间对MCF-7细胞中caspase-3蛋白表达无影响，但在TAM诱导MCF-7乳腺癌细胞凋亡的过程中，caspase-3的活性增高，而且具有量效关系，即随着TAM浓度的增加，caspase-3活性增加。而且survivin mRNA阳性率与caspase-3活性呈现明显负相关关系（P＜0.01）。总之，我们的实验结果表明，TAM能够以时间和剂量依赖的方式诱导MCF-7细胞凋亡，survivin基因参与了这一过程，其机制可能在于TAM通过干扰MCF-7乳腺癌细胞周期，减少S期和G2/M期的细胞数，使survivin蛋白表达降低，解除survivin蛋白对caspase-3活性的抑制作用，使caspase-3的活性增加，从而诱导乳腺癌细胞凋亡。靶向survivin的反义寡核苷酸协同三苯氧胺抗乳腺癌细胞的实验研究通过脂质体法将一条20-mer靶向survivin mRNA的反义寡核苷酸（ASODN）转染MCF-7乳腺癌细胞，实验分为空白对照组、TAM处理组、ASODN处理组、ASODN联合TAM处理组。应用MTT法检测各组别细胞生长抑制情况；用流式细胞技术检测各组对MCF-7细胞凋亡率和细胞周期的影响；应用原位杂交技术和Western blotting方法检测各组MCF-7细胞survivin mRNA阳性率以及survivin蛋白表达的变化；应用免疫组织化学方法和比色法检测转染后MCF-7细胞内caspase-3蛋白表达情<WP=99>况和caspase-3更多还原

【Abstract】 The breast cancer is a common malignant tumor which threaten to the health of women, every year there are new 1.2 million women that suffered from breast cancer all around the world, and five hundreds thousand women died of breast cancer. China is a developing country, but the morbidity of breast cancer is similar to developed countries, and there is a tendency of rise every year. Some data have demonstrated the morbidity of breast cancer have increased 37.6% in the recent two decades, the annual increasing rate have reached 2.3% averagely. By 2000, the morbidity of breast cancer has reached 28.8/100 000, which was the first one among all the women tumors. Modern management of breast cancer is integrated treatments that major in surgical treatment, and endocrine therapy is an important part of integrated treatment. Tamoxifen has been applied in clinical practice for over thirty years as a first line drug in endocrine therapy for breast cancer. Traditional opinion on the mechanism of TAM is believed to be combined to specific receptor site which competitive with estradiol, and form an anti-estrogen complex with low activity, downregulate the activities of cancer cell, arrest breast cancer cell in G1 phase, decrease the portion of S phase cells. Recently, some studies have demonstrated that TAM could inhibit the growth of breast cancer cells by apoptosis. Survivin is a novel anti-apoptotic factor found by Altieri et al in 1997, <WP=102>and it is the smallest member of IAP (inhibitor of apoptosis) family. Survivin was observed to express in fetal tissues and most common human cancers, including carcinomas of lung, stomach, colon, breast, and prostate as well as high-grade non-Hodgkin’s lymphomas, whereas no survivin transcripts were detected in normal, terminally differentiated adult tissues (except thymus gland and genital gland). Experimental data suggested that survivin functions similarly to other human IAPs, binding to active caspase-3 and -7 by a manner of BIR dependant, prevent apoptosis by inhibiting the activities of caspase-3 and -7. Because of the selective expression of survivin in cancer tissues, it has become the hot spot in many studies which focused on cancer treatment. Researches have demonstrated that tumor cells became more sensitive to chemotherapy after the expression of survivin was inhibited; on the other hand, after the survivin cDNA were transfected into tumor cells, these cells become resistant to chemotherapy. But until now, there are no reports on the relationship between survivin and apoptosis of breast cancer cells induced by TAM, and whether the sensitivity of breast cancer cells to TAM are increased by inhibiting the expression of survivin.The purpose of the research was to study the relationship between the expression of survivin and the apoptosis of breast cancer cells induced by TAM. In this experiment, we have observed the apoptosis of MCF-7 breast cancer cells induced by TAM, the roles of TAM on the changes of survivin gene, and the changes of apoptotic rate of MCF-7 cells after treated by the TAM which combined with antisense oligonucleotide targeting survivin mRNA. Our results have revealed that the apoptosis of MCF-7 cells induced by TAM were related to the downregulation of survivin expression, and <WP=103>antisense oligonucleotide targeting survivin mRNA could make MCF-7 cells more sensitive to TAM. Ⅰ. The role of TAM on survivin gene and its mechanismMCF-7 cell line was treated by various concentrations of TAM, and after various period of time, samples were extracted. The effects of TAM on the changes of apoptotic rate and distribution of cell cycle of MCF-7 cells were examined by flow cytometry; the positive rate of survivin mRNA and expression of survivin protein were confirmed by hybridization in situ and western blotting; the expression of caspase-3 protein and the activities of caspase-3 were evaluated by immune histochemistry and colorimetry. The results have demonstrated:(1) The apoptosis of MCF-7 cells induced by TAM was significantly time-dependa更多还原