【作者】 郭君兰；

【导师】 王林波；

【作者基本信息】 浙江大学 ， 肿瘤学， 2011， 硕士

【摘要】 乳腺癌是女性最为常见的恶性肿瘤之一。美国癌症协会研究表明,乳腺癌已成为女性发病率最高的恶性肿瘤,死亡率仅次于肺癌居第二位。随着我国经济的不断发展,近些年来乳腺癌发病率已位居女性恶性肿瘤首位。如何有效提高乳腺癌综合治疗效果,降低发病率和死亡率,已成为当前乳腺癌研究重点之一乳腺癌组织中雌激素受体(Estrogen receptor, ER)的状态被广泛认为是预测患者对内分泌治疗反应性和预后的关键指标之一。雌激素受体主要有两个亚型,即ERa和ER6。由于剪切异构体的存在,ER66尚有三个亚型,即ERa66, ERa46和ERa36,其中ERα66即为通常所说的ERα,也简称为ER66。大量临床试验和回顾性研究都表明ER66阳性的乳腺癌患者对化疗的反应性不如阴性患者敏感,ER66可能影响乳腺癌患者化疗的有效性。紫杉醇是乳腺癌化疗最有效的药物之一,近些年来大量的临床试验表明ER66可能影响乳腺癌患者对紫杉醇的敏感性。本课题研究了转染ER66 (BC-ER)和转染空质粒(BC-V)的乳腺癌细胞对紫杉醇敏感性的差异及可能机制。具体内容如下：1.乳腺癌细胞的转染我们将pcDNA3.1-ER66和pcDNA3.1空载体转入ER66-的Bcap37乳腺癌细胞,进行RT-PCR和Western-blot鉴定,建立稳定转染细胞株,即转染ER66的BC-ER细胞和转染空载体的BC-V细胞。2. BC-ER和BC-V乳腺癌细胞对紫杉醇敏感性差异的研究以建立的同源细胞模型(BC-ER和BC-V细胞)为基础来研究ER66的表达对紫杉醇敏感性的影响。用MTT法和流式PI染死细胞法检测BC-ER(转染ER66)和BC-V(转染空质粒)细胞对紫杉醇的敏感性。实验结果表明在E2作用下BC-ER细胞较BC-V细胞对紫杉醇耐药。3. BC-ER和BC-V乳腺癌细胞对紫杉醇敏感性差异的机制研究(1)细胞增殖速度在BC-ER和BC-V乳腺癌细胞对紫杉醇敏感性差异中的作用研究化疗敏感性和细胞增殖速度有一定关系,增殖慢的细胞对化疗耐药。MTT结果提示E2可以抑制BC-ER细胞的增殖活力,我们进一步检测了BC-ER和BC-V在E2作用下细胞周期和周期蛋白D1的表达变化,结果提示,在BC-ER细胞,E2作用下G2/S期细胞减少,周期蛋白D1表达下调,提示E2作用可抑制BC-ER细胞的增殖速度。推测BC-ER细胞在E2作用下细胞增殖速度的减慢可能对紫杉醇耐药有一定的影响。(2)细胞凋亡通路在BC-ER和BC-V乳腺癌细胞对紫杉醇耐药差异中的作用研究BC-ER和BC-V乳腺癌细胞对紫杉醇敏感性差异的研究表明Bcap37细胞表达ER66后可降低其对紫杉醇的敏感性,接下来我们检测了ER66在紫杉醇诱导乳腺癌细胞凋亡中的可能作用。用Western-blot检测两种细胞在紫杉醇作用下凋亡蛋白caspase3的表达差异,结果表明在BC-ER细胞E2作用下可以抑制紫杉醇诱导的caspase3酶原活化裂解,而转染空载体的BC-V细胞没有此变化,也就是说在BC-ER细胞ER66可以通过caspase3途径抑制紫杉醇的促凋亡作用。在具体机制上,我们用Western-blot检测了凋亡信号通路蛋白NF-κB/Iκ-Bα, JNK和AKT的表达及磷酸化状态的变化。结果提示,紫杉醇作用下可以引起BC-ER和BC-V细胞IK-Ba磷酸化降解。而在转染ER66的BC-ER细胞,E2可抑制紫杉醇诱导的IK-Ba磷酸化降解,在转染空载的BC-V细胞没有发现E2对Iκ-Ba有明显影响。推测NF-κB/Iκ-Bα通路可能在BC-ER细胞对紫杉醇耐药上起一定作用。JNK和AKT通路我们只是初步的研究,对于它们在ER66介导紫杉醇诱导凋亡中的具体作用仍需要深入研究。更多还原

【Abstract】 Breast cancer is the most commonly diagnosed cancer type in women. American Cancer Sociaty estimates, breast cancer has been the highest incidence of femal malignant tumor and ranks the second in female cancer deaths. With the development of economy, in our country, breast cancer has ranked first in female cancer incidences. So more understandings about breast cancer are needed for the improvement of early detection and comprehensive treatment.The expression status of estrogen receptor (ER) has been widely accepted as a prognostic marker and a predictor for endocrine therapy response. There are two main subtypes of ER, ERa and ERβ. Due to the existence of splice variants, ERa has three subtypes, namely ERa66, ERa46 and ERa36, ERa66 is simply called ER66. Data from clinical trials or retrospective analyses suggested that ER66 status might also affect the efficacy of chemotherapy, Specifically, it has been observed that some chemotherapeutic agents may be less effective in patients with ER66+breast tumors than those with ER66-breast tumors. Taxanes (paclitaxel and docetaxel) is an active agent used in breast cancer chemotherapy. Evidence is accumulating that improvements in taxane-based adjuvant chemotherapy disproportionately benefit patients with ER66-breast tumors. In the present study, we investigate paclitaxel susceptibility difference in BC-ER and BC-V cells and its underlying mechanism. The details are listed below:1. Stable transfection of breast cancer cellsWe established several isogenic ER66 cell lines by stable transfection of ER66 expression vectors and empty vector into ER66-Bcap37 breast cancer cells, The expression vector was successfully transfected into Bcap37 breast cancer cells and the expression of ER66 protein was confirmed by RT-PCR and Western-blot, and established Bcap37 transfected with pIRES-ER66 expression vector (BC-ER), or empty vector (BC-V) cells.2. To investigate paclitaxel susceptibility difference in BC-ER and BC-V cellsTo investigate the possible influence of ER66 on the therapeutic efficacy of paclitaxel in BC-ER and BC-V cells. We did MTT assays and Flow cytometric analyses to determine whether the expression of ER66 would affect the sensitivity of Bcap37 cells to paclitaxel. We showed that E2 significantly reduces the overall cytotoxicity of paclitaxel in Bcap37-expressing ER66 but has no influence on the ER66-parental cells.3. To investigate the mechanisms underlying paclitaxel susceptibility difference in BC-ER and BC-V cells(1) Cell proliferation rate was examined to investigate the paclitaxel susceptibility difference in BC-ER and BC-V cellsChemotherapy susceptibility is associated with cell proliferation rate, High growth rate tumors are sensitivity to chemotherapy. MTT assays show E2 would affect the vitality of BC-ER cells, next, we examine the possible effect of E2 on Cell cycle and cyclinD1 protein, the results show E2 decreases the population of cells at G2/S phase and the protein levels of cyclin D1, so E2 can restrain proliferation rate of BC-ER cell.To infer cell proliferation rate decreasesing via E2 in BC-ER cells is likely to strongly influence variation the efficacy of paclitaxel therapy;(2) Apoptosis-associated proteins were examined to investigate the paclitaxel susceptibility difference in BC-ER and BC-V cellsThe data mentioned above reveal that E2 significantly reduces the overall cytotoxicity of paclitaxel in Bcap37-expressing ER66, next, we examined the possible inhibitory effect of ER66 on the ability of paclitaxel to induce breast cancer cell apoptosis. Western-blot exames paclitaxel induce caspase3 protein expressing difference in BC-ER and BC-V cells. The result shows, in BC-ER cells, pretreatment with E2 attenuated the paclitaxel-induced caspase3 down-regulation, it indicates inhibitory effect of ER66 on the ability of paclitaxel to induce apoptosis through caspase3 apoptosis pathway. To investigate the possible molecular mechanisms, we analyzed the expression and possible alterations of apoptosis-associated proteins including NF-KB/IK-Ba, JNK和AKT. The results reveal paclitaxel causes degradation of Iκ-Ba in BC-ER and BC-V cells. However, pretreatment with E2 attenuated the paclitaxel-induced IK-Ba down-regulation in BC-ER cells, although it has no effect in BC-V cells. Our studies have suggested that activation of the NF-κB/Iκ-Bαsignaling pathway maybe play an active role in mediating paclitaxel-induced apoptosis in BC-ER cells. We just preliminary studies JNK and AKT signaling pathway, the roles they play in ER66 mediating paclitaxel-induced apoptosis need further research.更多还原