【作者】 李静怡；

【导师】 栾杰；

【作者基本信息】 北京协和医学院 ， 外科学， 2012， 博士

【摘要】 一.研究背景：乳房不但做为女性的第二性征和哺乳器官满足生理的需要,而且它也是形成人体曲线美和体现女性魅力的重要结构。乳房的过度肥大会给女性带来肉体和心理的双重痛苦。虽然治疗乳房肥大的手术逐渐的成熟和完善,但对其发病原因的研究相对较少,发病机制仍不明了。乳房的发生和发育受多种激素和体液因子的调节,而雌激素做为女性的主要性激素,在其中起着重要的作用。作为生长因子,雌激素促进正常乳腺细胞和癌细胞增殖。人们对乳房肥大病因的研究大多数都集中在雌激素和雌激素受体(Estrogen Receptor, ER)上面,结果不一,观点各异。研究发现：在绝大多数的乳房肥大症患者中血清的雌激素水平是正常的；乳房肥大的发生可能与ER含量升高有关,小乳房则与ER含量或敏感性降低有关。但有学者认为乳房肥大与ER含量没有直接关系。ER基因调控一系列基因表达及介导细胞内信号转导,ER基因的多态性可能改变基因的转录调控水平从而影响雌激素的生理功能。人的ER包括a和β两种亚型。各亚型在乳腺组织细胞中均有不同的表达,发挥不尽相同的作用,大量研究证明ERa基因更多的承担着雌激素介导作用。有报道：ERa基因多态性可能通过影响雌激素的敏感性与乳腺癌的发生有关,ERa基因Xba Ⅰ基因多态性与乳腺增生症的发病有关,而ERa基因Pvu Ⅱ基因多态性则与其无明显相关。因此,ERa基因是否是乳房肥大和小乳房发生的关键因素、其生物学机制如何?还需进一步的研究。二.目的：探讨国人乳房肥大患者的ERa基因与相关基因的表达差异以及乳房肥大与ERa基因多态性的关系,以期为乳房肥大症的诊断、治疗和预防提供理论依据。三.方法：1)选取2011年8月～10月我院收住的3名女性乳房肥大患者与行乳房纤维腺瘤切除术的3名女性患者作为研究对象。手术中取乳房肥大患者废弃的乳腺组织和乳腺纤维腺瘤患者距离病灶3cm的位置的乳腺组织,分别作为实验组和对照组,应用Illumina芯片来进行全基因组扫描而找到差异基因,分析ERa基因与下丘脑-垂体-性腺轴的促性腺激素释放激素(Gonadotropin-releasing hormone, GnRH)、黄体生成素(Luteinizing hormone, LH)、卵泡雌激素(Follicle stimulating hormone,FSH)等相关基因的表达差异。2)选取2009年12月-2011年6月我院收住的年龄18-45岁的乳腺发育异常的女性患者和乳房大小形态正常的健康女性作为研究对象。分为三组：(1)乳房肥大组(28例)：左右两侧每侧乳腺切除的重量≥350g的患者；(2)小乳组(70例)：经乳晕切口、腋窝切口行隆乳术患者,经术前测量乳房组织总量在200g以下；(3)正常对照组(69例)：乳房大小及形态正常的健康女性。收集入组对象外周全血样本,应用PCR-限制性酶切方法对三组进行基因组DNA ERα基因的Xba Ⅰ和Pvu Ⅱ酶切多态性位点研究,对结果进行了相应的统计学分析并采用限制性片段长度多态性(RFLP)分析。3)利用报告基因技术,将上述乳房肥大组、小乳组和正常乳房组的人群中的第一内含子中具有增强子、启动子作用序列克隆入报告基因载体pGL3-Basic,重组报告基因载体质粒与内参照质粒pRL-TK一起转染入乳腺癌细胞(MCF-7),化学发光仪检测转染后细胞荧光素酶的相对活性,分析三组人群第一内含子多态性对雌激素受体基因转录水平的差异。并利用双荧光素酶报告基因技术对基因突变进行转录调节能力研究。四.结果：1.乳房肥大患者的ERa基因与相关基因的差异性表达本研究共筛选分析得到2808个差异表达的mRNA,其中上调的1815个,下调的993个和223个差异表达的miRNA基因,其中有123个(占55%)差异表达基因为上调]miRNA基因,100个(占45%)基因为下调miRNA基因。其中ERa基因在乳房肥大组是上调的,而与下丘脑-垂体-性腺轴相关的GnRH、LH、FSH基因的表达水平均无上调或下调,提示ERa基因和乳房肥大的发生有关。通过进行两类基因对应基因组序列富集分析,发现上调表达基因主要富集在GnRH信号传导通路、趋化因子信号传导通路,Fc7受体介导的吞噬作用,内皮细胞介导的白细胞迁移途径及血管平滑肌收缩作用等途径中；而下调表达基因主要富集在氧化磷酸化、蛋白酶体、心肌收缩作用、柠檬酸盐循环(TCA循环)、mRNA介导的信号传导通路及泛素介导的蛋白酶解等途径中。2. ERαPvuⅡ和Xba Ⅰ多态性与乳房肥大和小乳房的关系1)ERa基因上Pvu Ⅱ酶切位点分析结果为：正常对照组：PP型、Pp与PP型分别是10.14%、68.12%和21.74%；乳房肥大组：PP型、Pp与PP型分别是25%、57.14%和17.86%；两组比较差异无统计学意义(p>0.05)。小乳组：pp型40%,与正常对照组比较差异有统计学意义(p<0.0001)；Pp型47.14%,与正常对照组比较差异有统计学意义(p=0.0124)2)ERa基因Xba Ⅰ的酶切多态性分析结果为：正常对照组：xx, Xx与XX分别为63%、10%和26%。乳房肥大组：xx, Xx与XX分别为25%、64%和1O%,与正常对照组等位基因构成比较差异有统计学意义(p<0.0001)。小乳组：xx, Xx与XX分别为77%、21%和0.10%,与正常对照组等位基因构成组比较差异也有统计学意义(p<0.0001)。3)PvuⅡ酶切多态性相对易感度分析：小乳组：P和p两等位基因的OR值为0.4540(95%CI：0.2093～0.8326),即可能p基因是小乳症的易感基因；隐性基因型PP与pp携带者的OR值为0.15(95%CI：2.3622～14.7604),说明小乳症与pp基因型有关联。4)Xba Ⅰ酶切多态性相对易感度分析：乳房肥大组：X和x两等位基因的OR值为1.6570(95%CI：1.8735～3.1431),提示X基因是该疾病的易感基因；隐性基因型xx与X携带者的OR值为0.1894(95%CI：0.0706～0.5078),杂合子与纯合子基因型的OR值为15.9429((95%CI：5.3110～47.8580),提示乳房肥大症发生与Xx基因型有关联。小乳组：X和x两等位基因的OR值为0.305(95%CI：0.1639～0.5688),即可能x基因是该疾病的易感基因；隐性基因型xx与X携带者的OR值为1.9176,(95%CI：1.9121～4.0317)提示小乳症与xx基因型有关联。3.双荧光素酶报告基因活性测定结果：正常对照组、乳房肥大组和小乳组荧光素酶的相对活性分别为2.66±1.41、3.13±1.17和1.97±0.79。乳房肥大组荧光素酶的相对活性较正常对照组明显升高,差异有统计学意义(p<0.05),小乳组荧光素酶的相对活性较正常对照组显著降低,差异有统计学意义(p<0.01)。各组标本XbaI多态性与荧光素酶的相对活性对比分析：xx基因型的ERa基因启动转录表达活性显著低于xX和XX基因型,小乳组中xx基因型的ERa基因启动转录表达活性显著低于正常对照组中xx基因型。乳房肥大组中XX基因型的ERa基因启动转录表达活性显著高于对照组XX基因型个体,但xX和xx的ERa基因启动转录表达活性与对照组差异无统计学意义。结合第二部分结果,乳房肥大组中XX基因型个体仅占10.1%(3/28)。推测XX基因型的ERa基因启动转录表达水平升高可能不是巨乳形成的主要原因。正常组和小乳组中pp基因型的雌激素受体基因启动转录表达活性显著低于pP和PP基因型；小乳组中pp基因型的ERa基因启动转录表达活性显著低于正常组中pp基因型；乳房肥大组中PP基因型的ERa基因启动转录表达活性显著高于正常组中PP基因型,提示肥大组中PP基因型的ERa基因表达上调；肥大组中pp和pP基因型的ERa基因启动转录表达活性与正常组中pp和pP基因型无显著性差异。结合第二部分结果,乳房肥大组中PP基因型占17%(5/28),提示尽管ERa基因Pvu Ⅱ PP基因型与ERa基因启动转录表达水平上调有关,但可能不是巨乳形成的主要原因。五.结论：1)应用基因芯片技术对乳房肥大组和正常对照组进行的全基因组的扫描结果结合上调基因主要富集的通路分析发现：乳房肥大患者组中ERa基因表达是上调的,而与乳房的发育有着密切的关系的下丘脑调节垂体-性腺-雌激素轴相关的GnRH基因、LH基因、FSH基因的表达水平均无上调或下调,提示ERa基因和乳房肥大的发生有关。从而从基因层面上支持了ERa基因表达的上调是导致乳房肥大的发生的因素之一。同时还提示,其他基因的表达也存在着明显的差异,说明乳房肥大的发生可能是一个由多种基因和多种通路参与的复杂的生物学网络。2)乳房肥大的发生与杂合基因型xx有关,而小乳症的发生与隐性基因型pp及xx有关,乳房大小和形态的差异并非由某个基因差异决定的。3)ERal号内含子多态性可导致雌激素受体基因转录表达的启动水平的差异；乳房肥大组人群ERal号内含子的增强子和启动子的调节序列,对ERa基因转录表达的启动水平,显著高于正常对照组人群,而小乳组人群,则显著低于正常对照组人群。结合对乳房肥大和小乳症ERa基因多态性的研究,推测xx和pp这两个基因型的低水平启动转录表达可能是小乳症形成的部分原因。尽管ERa基因Pvu Ⅱ PP基因型与ERa启动转录表达水平上调有关,但可能不是巨乳形成的主要原因。更多还原

【Abstract】 Background:Breast in females does not only involve in the physiological functions like development of secondary sexual characteristics and involves in the feeding mechanism (breast feeding), but it is also a forming embody feminity, formation of body curves. Commonly the physical and psychological stress and pain is expressed in females regarding excess development and dysplasia of breast. Although the surgical treatment of breast dysplasia gradually improve, the causes of the disease are few studied and the pathogenesis is still not clear. Various hormones and humoral factors involve in the breast development, and estrogen is the main sexual hormone in female and play an important role. Most of the research on hypermastia are concentrated in estrogen and estrogen receptor(ER), but the results and opinions are varying.The variation of estrogen and its receptors will cause the malformations and abnormal development of breast. Estrogen as the growth factor promotes the breast cells and cancer cell proliferations by means of ER. The levels of estrogen are found normal in vast number of patients with gigantomastia. Gigantomastia may associated with increased ER, but micromastia may associated with depressed ER or reduced sensitivity. Some scholars believe that gigantomastia has not a directly association with ER. ER regulates a series of genes expression and mediate intracellular signal transduction. ER gene polymorphisms may alter gene transcriptional regulation, and then impact the physiological function of estrogen.ER gene includes two subtypes, they are ERa and ERβ.They have different expression and play different role in breast tissue. Many studies have demonstrated that the Eragene play the more important role. One report demonstrated that ER gene polymorphisms may effecting sensibility of estrogen, that may be related to breast cancer, ER a gene XbaI polymorphism is related to cyclomastopathy, however, ER a gene PvuII gene polymorphism is not related to it. Therefore, it need to further investigate the biological mechanism of ER and whether ER play a key role in gigantomastia and micromastia.Objective:To investigate the relationship between polymorphism of ERaand gigantomastia, and the relevance of gene differential expression in Chinese, in order to supply theory foundation for the diagnosis, therapy and prevention of gigantomastia and hypogenetic micromastia.Methods:1) Select the patients admitted in August to October2011,3female patients with breast hypertrophy, and3female patients after breast fibroadenoma excision for the study. Surgery to take the distance of the breast tissue and fibroadenoma of the breast in patients with breast hypertrophy patients abandoned the lesions3cm in breast tissue, as the experimental group and control group, the application Illumina chips to carry out genome-wide scan to find differentially expressed genes. Furthermore, analyzing the level of ERa gene, Gonadotropin-releasing hormone(GnRH), Luteinizing hormone(LH), Follicle stimulating hormone(FSH) in hypothalamus-pituitary-sex gland axle and other related genes expression.2.) A control case study was conducted in our hospital during2009.12-2011.6. The women admitted to us for abnormal galactophore dysplasia and gigantomastia, and the healthy check-up, who aged18to45yeas old. They were divided into3groupes.(1)Macromastia group (28cases):patients with the weight of each paredmastectomy>350g;(2)Micromastia group (70cases):The breast augmentation by incision of areola and armpit. The preoperative measurment of the total weight of breast tissue<200g;(3)Normal group(69cases):Healthy women whose breast size and form are normal.Collected peripheral blood samples from the objectives, then extract genomic DNA of3groups and investigate the polymorphism of ERa of XbaI and PvuII by PCR-reconstruction enzyme studies. The analysis were done by statical mean and (RELP)analysis.3) Using reporter gene:the sequences which have enhancer and promoter function in the first intron in the above three groups were cloned into the pGL3-Basic reporter vector, and restructuring the reporter gene plasmid within the reference plasmid and pRL-TK was transfected into breast cancer cells (MCF-7) cells relative luciferase activity after trarisfection was detected by chemiluminescence analyzer, analysis of the first intron polymorphism of the three groups by differences in the levels of estrogen gene transcription. Using dual-luciferase reporter gene technology to regulate transcription of the gene mutation.Results:1) Differentially expressed genes in hypermastia.This study analyzed and selected2808differentially expressed mRNA include1815up-regulated mRNA and993down-regulated mRNA,223differentially expressed miRNAs.123(55%) of differentially expressed genes were up-regulated miRNAs, and other100(45%) miRNAs were down-regulated. Further study found that these genes may play an important role in the occurrence of breast hypertrophy. By means of enrichment analysis of two types of genes corresponding gene ontology term, found that up-regulated genes enriched in chemokine signaling pathway, Fc gamma R-mediated phagocytosis, GnRH signaling pathway, leukocyte transendothelial migration and vascular smooth muscle contraction and so on. However, the down-regulated genes enriched in oxidative phosphorylation, proteasome. cardiac muscle contraction,citrate cycle (TCA cycle). mRNA-mediated pathway and ubiquitin mediated proteolysis pathway.2) The relationship between ERaPvu Ⅱ, Xba Ⅰ polymorphism and hypermastia and micromastiaThe analytical results of ERa gene at Pvu Ⅱ restriction sites as follows:breast hypertrophy group:the incidence of polymorphisms were25%,57.14%and17.86%of PP, Pp and PP-type Respectively; Normal control group:type of PP, Pp and PP-type10.14%,68.12%and21.74%respectively. The difference was not statistically significant (P>0.05). Micromastia group:pp-type40%,10.14%of the normal control group, the difference was statistically significant (P<0.0001); Pp, 47.14%,68.12%of the normal control group, the difference was statistically significant (P=0.0124).The analytical results of ERa gene Xba I restriction enzyme polymorphism as follows:the results of breast hypertrophy group:xx, Xx and XX were25%,64%and10%; normal control group:xx, Xx and XX were63%,10%and26%,in the two groups the difference was statistically significant (P<0.0001). Micromastia group:xx, Xx and XX were77%,21%and0.10%respectively; Normal control group:xx, Xx and XX respectively63%,10%and26%, further analysis of the alleles constitute of the two groups, the difference was statistically significant (P<0.0001).The relative susceptibility analysis of Pvu II restriction enzyme polymorphism: Micromastia group:P/p-two allele of the OR value is0.4540(95%CI:.2093～.8326), which may be p gene is a small breast disease susceptibility gene. Recessive genotype pp and P carriers OR value is5.9048(95%CI:2.3622-14.7604), which accounted for the relationship between Small breast disease and the pp genotype.The relative susceptibility analysis of Xba I restriction enzyme polymorphism: breast hypertrophy group:X/x alleles OR value is1.6570(95%CI:0.095～1.2634), which may be the X gene is the disease easy to sense gene; Recessive genotype xx and X carriers OR value is0.1894(95%CI:0.0706～0.5078), heterozygous and pure and sub-genotype OR value is15.9429((95%CI:5.3110～47.8580), breast hypertrophy associated with Xx genotype. Micromastia group:X/x two-allele OR value is0.3054(95%CI:.1639～0.5688),which means that x gene may be susceptibility gene of the disease; Recessive genotypes xx and X carriers, the OR value is1.9176(95%CI:.9121～4.0317) suggesting that small breast disease associated with the xx genotype.3) The dual luciferase reporter gene activity assay.The relative luciferase activity of normal control group, hypermastia and micromastia were2.66±1.41,3.13±1.17and1.97±0.79respectively. Compared With normal control group, the relative activity luciferase enzyme of hypermastia group was significantly elevated (P<0.05), and the relative activity luciferase enzyme of micromastia group was significantly depressed(P<0.01). Through analyzing the relative luciferase activity and XbaI polymorphism in each group, we find that the transcriptional expression activity of ER gene that showing xx genetype was significant lower than xX and XX genetypes, whereas, between the micromastia and normal control, the individual that showing xx genetype, its transcription expression activity of ER gene significant lower in the former. In hypermastia, the transcription expression activity of ER a gene that showing XX genetype was high relative to the normal control, and there has no significant in the xX genetype and xx genetype compared with normal control. Combined with the second part of the results, the XX genetype individuals account for10.1%(3/28) in hypermastia. It supposed that the reason of hypermastia was not the transcriptional expression activity of ER a gene with XX genetype.Between normal control and micromastia groups, the transcriptional expression activity of ER gene was lower in pp genetype polymorphism than other polymorphisms(pP or PP). In PP polymorphism, the activity was high in hypermastia but in normal control. It demonstrate that the expression of ERa gene was up-regulated in PP polymorphism in hypermastia. In pp and pP polymorphisms of ER a gene, the activity was no significant compared to the normal control. Combined with the second part, PP polymorphisms account for17%(5/28) in hypermastia. It suggested that although PP genetype of ER gene Pvu Ⅱ have a relationship with the up-regulated transcriptional expression activity of ER a gene, it may not the main cause of the hypermastia formation.Conclusion:1) The scan results of gene chip technology in hypermastia group and normal control group show that ER a gene was up-regulated in hypermastia and enriched in GnRH signaling pathway which was related in the pituitary gland-gonad-estrogen axis. This axis is closely related to breast development. These indicate that up-regulate of ER a gene is the one reason of hypermastia formation. Meanwhile, there have some other genes different expressed, so it demonstrated that hypermastia may be a complex biological network involved multiple genes and multiple pathways.2) The occurrence of micromastia was related to recessive genotype pp and xx, whereas hypermastia was associated with heterozygous genotype Xx. The differences in breast size and shape are not determined by differential expression of one gene.3) The polymorphism of the first intron in ER a gene may result in different transcriptional of ER gene. The transcriptional expression activity of ER in hypermastia group was significant higher than normal control, whereas, the micromastia was lower than normal control. Combined the polymorphism of ER a gene in hypermastia and micromastia, it supposed that the low level transcriptional expression of the genetype xx and pp may be a part reason of the micromastia. Although PP genetype has a relationship with the transcriptional expression level of ER a gene, it may not the main reason of the hypermastia formation.However, the cases that we investigated are less, it need to increase more samples and in-depth studies to get a more definite conclusion.更多还原