【作者】 吕丽；

【导师】 王莉芬；

【作者基本信息】 大连医科大学 ， 病理学与病理生理学， 2015， 博士

【摘要】 背景:乳腺癌是女性最常见的恶性肿瘤之一,严重影响患者的健康及生存。寻找乳腺癌预后危险因素及潜在的治疗靶点,是当前乳腺癌研究热点之一。乳腺癌属激素依赖性肿瘤,体内雌激素水平是调控乳腺癌细胞增殖的重要因素,雌激素主要通过雌激素受体(Estrogen receptor,ER)发挥作用。雌激素与ER形成复合体后转移至细胞核,作为一种转录因子,通过调节下游靶基因的转录发挥作用。这种机制称为雌激素的基因组作用模式或核启动的雌激素应答。但近年来研究已经证实,雌激素还存在非基因组作用模式,也称为膜启动的雌激素应答,在这种模式中,雌激素直接与分布在细胞膜/细胞质内的雌激素结合蛋白结合,快速激活细胞内信号传导通路,主要包括ERK/MAPK信号通路、Ras-PI3K通路、JNK通路及第二信使系统等,使细胞产生快速反应,促进细胞发生增殖及恶性转化。关于分布在癌细胞质/膜中的雌激素结合蛋白究竟为何种成分,目前尚无定论。目前在临床上广泛使用免疫组化法检测乳腺癌细胞的ER状态,普遍认为ERα蛋白定位于细胞核内,而质/膜ERα由于其成分尚无定论,临床意义不明,目前尚未引起广泛关注。本课题组在前期工作中发现,部分乳腺癌病例ERα免疫组化染色可见不同程度、不同比例的细胞质/膜着色,目前尚不清楚这些定位于细胞膜和/或细胞质中的蛋白究竟是何种成分以及这种蛋白具有何种临床意义。我们推测该蛋白可能为ERα66的剪切变异体:ERα36。目前,该蛋白在乳腺癌发生、进展中的作用及其对相关治疗药物疗效的影响及机制尚未明确。目的:1、研究ERα蛋白在人乳腺癌组织中的定位,确定是否存在细胞质/膜定位,探讨细胞质/膜ERα表达的临床病理意义及其对患者预后的影响。2、研究乳腺癌病例中质/膜ERα表达与ERα36表达及定位之间的关系,探讨质/膜ERα究竟为何种蛋白。3、研究ERα36高表达后,MCF-7细胞系中ERα蛋白细胞内定位情况以及HER-2、RARα、ki-67蛋白表达量的变化,探讨ERα36促进乳腺癌细胞转移可能的作用机制。方法:1、免疫组化染色法检测ERα蛋白表达量及细胞内定位,以>1%肿瘤细胞出现确切的细胞核和/或细胞质/膜不同强度的黄色细颗粒状着色计为ER核阳性或质/膜阳性。统计分析质/膜ERα蛋白与患者的月经状态、家族史、乳头大导管受累、乳腺癌组织学分级、临床分期、T分期、N分期、M分期、复发、HER-2表达等临床病理特征之间的相关性。2、应用q-PCR方法检测36例人乳腺癌新鲜标本中ERα36、ERα66 m RNA表达量,分析二者与ERα核定位及质/膜定位的相关性;3、应用核蛋白-胞浆蛋白抽提试剂盒分别提取癌细胞的核蛋白和质/膜蛋白,得到高纯度的核蛋白和质/膜蛋白,用Western-blot方法分别在核蛋白样本及质/膜蛋白样本中检测ERα36和ERα66蛋白的表达情况,并对Western蛋白条带扫描,进行灰度分析和计算,分析ERα36和ERα66在细胞内的定位情况。4、构建p IRES2-EGFP-ERα36基因过表达载体,瞬时转染至人乳腺癌细胞系MCF-7中,提取m RNA和蛋白质,分别应用q-PCR和Western-blot方法鉴定ERα36和ERα66表达情况。5、收集转染后MCF-7细胞,制作细胞蜡块,免疫组化方法检测ERα、HER-2及ki-67的表达。6、收集转染后MCF-7细胞,提取总蛋白,Western-blot方法检测HER-2、RARα蛋白表达。结果:1、在1164个病例中,ERα免疫组化染色阳性定位主要位于细胞核,部分病例可观察到细胞质/膜部位着色,质/膜着色可以与核着色同时出现(即核、质/膜均阳性),也可以单独出现(质/膜阳性,核阴性)。ERα质/膜着色与较高的N分期、较高的HER-2评分及复发转移有关,ERα质/膜阳性患者无进展生存期较短。2、36例样本中共有18例(占50.0%)检测到ERα66和/或ERα36 m RNA,其中10例检测到ERα66m RNA,阳性百分比为27.8%;14例检测到ERα36 m RNA,阳性百分比为38.9%。ERα66与ERα36共同表达的样本共6例,占所有样本的百分比为16.7%。ERα66 m RNA阳性的病例均出现免疫组化核着色,ERα36 m RNA阳性病例除1例以外免疫组化染色均出现ERα细胞质/膜着色。ERα66、ERα36 m RNA表达量分别与ERα免疫组化核定位及质膜定位有显著的相关性(p<0.05)。3、免疫组化ERα核及质/膜均阳性者Western blot显示ERα36和ERα66蛋白均有表达;ERα核阳性者多数为ERα66蛋白表达,少数为ERα36表达;ERα质/膜阳性者仅有ERα36蛋白表达;ERα核及质/膜均阴性者ERα36和ERα66蛋白均未见表达。4、p IRES2-EGFP-ERα36真核表达载体成功转染MCF-7细胞,转染72小时后,转染效率约为70%。q PCR检测结果显示,ERα36基因真核表达载体转染MCF-7细胞后,ERα36基因过表达129.23倍;western-blot检测到特异性ERα36条带,两种结果均证实转染成功。5、转染后MCF-7细胞ERα着色部位变化,除核表达之外,细胞质/膜中也可见阳性着色,进一步证实ERα36主要定位于细胞质或细胞膜;HER-2蛋白表达增加,部分肿瘤细胞胞膜出现阳性着色(1+~2+);ki-67指数增高。6、转染72小时后,p IRES2-EGFP-ERα36与p IRES2-EGFP-NC相比,HER-2蛋白表达量增加,而RARα蛋白表达减少。结论:1、人乳腺癌雌激素受体α(ERα)蛋白表达定位于癌细胞核和/或细胞质/膜中。质/膜ERα主要为ERα36,核ERα主要为ERα66,少部分为ERα36。2、乳腺癌细胞质/膜ERα表达量与癌细胞HER-2表达量呈等级正相关,提示二者之间存在调控关系。3、乳腺癌质/膜ERα阳性组患者淋巴结转移率较高、无进展生存期较短,乳腺癌质/膜ERα表达是一种独立的预后不良因素。4、ERα36促进HER-2蛋白表达,抑制RARα蛋白表达;ERα36可能通过下调RARα促进HER-2蛋白表达增加。更多还原

【Abstract】 Background: Breast cancer as one of the most common malignant tumors in women seriously affect patients’ health and survival.To looking for the prognostic risk factors and potential therapeutic targets of breast cancer is one of the hot topics of breast cancer research at present. Breast cancer belongs to hormone-dependent tumor, and estrogen levels in body is the important factor in regulation of breast cancer cell proliferation, and estrogen plays a role mainly through estrogen receptor(ER).Estrogen as a transcription factor transfers to the nucleus after forming complex with ER to make function by adjusting the transcription of downstream target genes.And this mechanism is called the action mode of estrogen genome or nuclear launch of estrogen response. But in recent years, studies have shown that estrogen also exists the non-genome function model which is also known as the membrane start of estrogen response.In this mode, the estrogen combines with the estrogen binding protein distributing in cytoplasm and membrane directly to activate the intracellular signaling pathways rapidly.And these intracellular signaling pathways mainly include ERK/MAPK signaling pathways, Ras- PI3 K pathway, JNK pathway and the second messenger system and so on. Besides, they can make the cells to produce rapid response and promote cell proliferation and malignant transformation.About the composition of estrogen binding protein distributing in the membrane and plasma of cancer cells, it has not been determined. Currently immunohistochemical method is widely used to detect the state of ER of breast cancer cells in clinical. It is widely believed that the ER alpha protein is Located in the nucleus, however the ER in cytoplasm and membrane has little clinical significance and has not been caused widespread attention due to its uncertain content. In the early study,our group discoverd that we can see different degree and different proportion of cytoplasm/membrane coloring by ER alpha immunohistochemical stainning among part of the breast cancer cases.However the composition of these proteins located in cell membrane and/or cytoplasm is unclear and whether the protein has clinical significance is also unclear currently.We hypothesized that the protein may be ER alpha 36, an shear variants of ER alpha 66. At present, the protein’s role in occurrence and progress of breast cancer and its influence on curative effect of related drug have not been clear.Objective:1、To research the chromatic way of ER alpha protein in human breast cancer tissue, determine whether there is a cytoplasmic/membrane positioning and explore the clinical pathology of cytoplasm /membrane ER alpha expressing and its effect on the prognosis of patients.2、To research the relationship between the expression and localization of ER alpha and ER alpha 36 among breast cancer cases, and explore the kind protein of ER alpha in plasma and membrane. 3、To research the intracellular localization of ER alpha protein and the protein expression changes of HER-2、RARα and ki-67 in MCF- 7 cell line after high expression of ER alpha 36 and discusses its possible mechanisms of ERα36 which promotes breast cancer cells to transfer.Method: 1、Immunohistochemical staining method to detect the intracellular expression and localization of ER alpha protein,if different intensity yellow fine granular staining in nucleus or cytoplasm / membrane occurs in smore than one-percent tumor cells,we see it as ER positive nuclear or positive cytoplasm / membrane. Analyze and gather the correlation between ER alpha in cytoplasm / membrane and the patient’s clinicopathologic feature,such as menstruation state, family history, the involvement of nipple big duct, histological grade of breast cancer, clinical stages,T-staging, N-staging, M-staging, recrudesce, HER-2 expression and so on.2、q-PCR to detect the expression quantity of ERα36 m RNA and ERα66 m RNA,analyze the correlation between the two and nuclear localization and cytoplasm/membrane localization of ERα.3、Using nucleoprotein- cytoplasm protein extraction kit from Thermo Fisher to extract nucleoprotein and cytoplasm/ membrane protein respectively,western-blot to detect the expression of ERα36 and ERα66 proteins after getting highly purified nucleoprotein and cytoplasm/membrane protein,then western protein stripe scanning, gray analysis and calculation are needed. 4、Structure p IRES2-EGFP- ERα36 gene overexpression vector,transient transfection to human breast cancer cell line MCF- 7, extract m RNA and protein,detect the expression of ERα36 and ERα66 by q-PCR and western-blot.5、Collect MCF 7 cells after transfection, make cell wax, immunohistochemical method to detect the expression of ERα、HER-2 and ki-67.6、Collect MCF 7 cells after transfection, extract total protein, western-blot method to detect the expression of HER-2 and RARα.Outcome:1 、 Among the 1164 cases, ER alpha positive orientation through immunohistochemical staining was mainly in the nucleus,and some cases could be observed in cytoplasm/membrane. Cytoplasm/ membrane staining could be observed with nuclear staining in the same time(the nuclear, cytoplasm/membrane are both positive).The two could also be observed respectively(positive cytoplasm/membrane,negative nuclear).The cytoplasm/membrane coloring of ERα was related with high N-staging, the score of HER-2, recurrence and metastasis. Furthermore, the patient with positive cytoplasm/membrane of ERα had short progression-free surial.2、There were 18 cases(accounting for 50%) that ERα66 m RNAand/or ERα36 m RNA were detected among 36 samples.In the results,ERα66 m RNA was detected in 10 cases and its positive percentage was 27.8%;ERα36 m RNA was detected in 14 cases and its positive percentage was 38.9%.There were 6 cases in which ERα36 and ERα66 expressed meanwhile, and its percentage of all samples was 16.7%. Cases with ERα66 m RNA positive appeared nuclear coloring by immunohistochemical staining. In the same way,cases with ERα36 m RNA positive appeared cytoplasm/ membrane coloring except one case. There was a significant correlation between the expression of ERα36 m RNA and ERα66 m RNA and immunohistochemical cell subcellular localization(p<0.05). 3、ERα36 and ERα66 were both detected in cases with positive nuclear and positive cytoplasm/membrane by western blot method.However, cases with ERα nuclear positive only expressed ERα66,and cases with ERα cytoplasm/membrane positive only expressed ERα36,and cases with ERαnuclear and cytoplasm/membrane negative expressed no ERα36 and ERα66. 4 、 p IRES2-EGFP-ERα36 eukaryotic expression vector transfered into MCF-7 cells successfully.Its transfection efficiency was about 70% after transfection 72 hours.The results of q PCR detection showed that ERα36 gene overexpressed 129.23 times after ERα36 gene eukaryotic expression vector transfered into MCF-7 cell line;western-blot detected specific ERα36 stripe.And both two kinds of results confirm successful transfection. 5、About the changes of coloring location after transfection in MCF-7 cells, cell cytoplasm and membrane also appeared positive coloring besides nucleus expression. It confirmed that ERα36 expressed primarily in cell cytoplasm or cell membrane; The expression of HER-2 increased, and part of the tumor cell membrane appeared positive staining(1+~2+);Ki- 67 index increased slightly.6、In p IRES2-EGFP- ERα36 expression vector,the expression of HER-2 increased and expression of RARα decreased compared with p IRES2-EGFP- NC after transfection 72 hours.conclusion:1、Human breast cancer estrogen receptor alpha(ERα) protein expressed in nuclear and/or cytoplasm/membrane.Cytoplasm/membrane ERα was mainly for ERα36,and nuclear ERα was mainly for ERα66,with a few of ERα36.2 、 The expression of cytoplasm/membrane ERα in breast cancer cells was correlated to the expression of HER-2 in cancer cells, and it revealed that there was a regulating association between the two.3、The patient with positive breast cancer plasma /membrane ER alpha had high lymph node metastasis rate and short progression-free surial. The expression of cytoplasm/membrane ERα in breast cancer was an independent poor prognosis factor.4、ERα36 promoted the expression of HER-2, while inhibited RARα protein; ERα36 may promote HER-2 expression through down-regulation of RARα protein.更多还原