【作者】 王忠超；

【导师】 任立群；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2011， 博士

【摘要】 乳腺增生症是最常见的乳腺疾病,多见于30～50岁的中年妇女,占全部乳腺疾病的75%,在连续渐进的演变过程中可能发生癌变。近10年来,随着生活环境的变化,乳腺增生症发生率逐渐上升并趋向年轻化,以至于乳腺癌的发病率亦日趋增高,为此世界卫生组织在21世纪初提出了“提前干预乳腺增生症”的战略思想,已引起了医学界的广泛关注。乳腺增生症作为乳腺癌的风险因子之一,研究其发病机制,开发其治疗药物具有深远的现实意义。现代医学对于乳腺增生症的治疗尚无十分有效的方案,目前仍以内服激素和手术治疗为主,而民族医学对乳腺增生症的治疗具有丰富的经验,尤其是蒙药对乳腺增生症的治疗有独到之处。蒙药乳腺-Ⅰ号是内蒙古自治区的传统秘方,用其治疗符合蒙医“妇血症”又符合西医乳腺增生症标准的400例患者,总有效率为98%。但由于缺乏现代医学理论的支持,蒙药乳腺-Ⅰ号的开发力度及普及范围十分有限,仅作为院内制剂应用于临床。如果将现代医学的技术手段与蒙药乳腺-Ⅰ号研发相结合将会大大推动蒙药乳腺-Ⅰ号的开发与应用。如今在以功能基因组学和蛋白质组学为核心的后基因组时代,以高灵敏度和高通量检测为特点的药物蛋白质组学有望成为分析蒙药治疗机制的新方法。蛋白质组学研究技术的发展,为解析疾病的发病机制、寻找和发现药物治疗靶点创造了有利条件。运用蛋白质组学高通量地对比分析药物作用前后蛋白质表达谱或表达丰度的改变可筛选出与药物作用相关的蛋白靶点。近年来,蛋白质组学相关的实验技术不断涌现,如荧光差异双向凝胶电泳(2D-DIGE)技术,其与传统的双向电泳(2-DE)相比,提高了重复性,简化了比较模式,大大加宽了蛋白点检测的动态范围,已被广泛应用。目前关于乳腺癌的蛋白质组学研究报道较多,而乳腺增生症蛋白质组学的相关报道并不多见,通过实验动物建立乳腺增生动物模型,进而对蒙药乳腺-Ⅰ号治疗作用的蛋白质组学研究迄今未见报道。本研究通过采用雌激素联合孕激素肌内注射的方法复制大鼠乳腺增生模型,利用形态学、生物化学、免疫组织化学、Western blotting、RT-PCR等实验技术观察蒙药乳腺-Ⅰ号的治疗作用及蒙药乳腺-Ⅰ号对大鼠乳腺组织中ER,PR和AR在蛋白水平和基因水平的影响。利用2D-DIGE、MALDI-TOF/TOF-MS技术结合生物信息学方法,对蒙药乳腺-Ⅰ号治疗组及对照组大鼠乳腺组织的总蛋白进行双向电泳分离,得到高度重复性的双向凝胶电泳图谱,通过图象分析,得到蒙药乳腺-Ⅰ号治疗组和对照组大鼠乳腺组织中的差异蛋白质图谱,找到两者具有显著性差异的蛋白点,并对这些差异蛋白点进行质谱鉴定,从而为在蛋白水平研究蒙药乳腺-Ⅰ号的作用机制,深入研究与蒙药乳腺-Ⅰ号治疗相关的蛋白质和基因打下了坚实的基础,为今后应用现代科学的方法开发蒙药,提升蒙药研发的技术层次寻找出了一条新的途径。具体实验结果如下：1.通过给大鼠肌内注射苯甲酸雌二醇0.5 mg·kg-1·d-1 25d,继续注射孕酮4mg·kg-1·d-1 5d后,乳腺增生大鼠乳头直径较正常对照组大鼠乳头直径明显增宽(P<0.01),血清TC、TG、LDL-c、E2、LH及PRL水平较正常对照组显著增加(P<0.01或P<0.05),FSH水平有增高趋势,P、T水平较正常对照组显著降低(P<0.01或P<0.05),乳腺增生大鼠乳腺组织中ER、PR及AR的表达量较正常对照组显著升高(P<0.01),ER、AR的mRNA表达有显著的增加(P<0.01),PR的mRNA表达明显减少(P<0.01)。乳腺增生大鼠均出现典型的乳腺增生,表现为小叶小管及末梢导管不同程度扩张,上皮细胞增生,导管壁细胞层次增多,多数上皮细胞排列紊乱,向管腔内突出,管腔内有大量脱落的上皮和分泌物：乳腺小叶体积明显增大,分叶多,腺泡腔显著扩张：2.蒙药乳腺-Ⅰ号治疗组大鼠乳腺小叶小管、末梢导管扩张程度及上皮细胞增生程度较乳腺增生大鼠有所减轻,乳腺小叶体积、腺泡分叶均有所减少,腺泡腔可见轻微扩张.间质纤维组织增生减轻,部分可接近正常大鼠的乳腺腺泡结构。蒙药乳腺-Ⅰ号可显著降低乳腺增生大鼠血清中的TC、TG、LDL-c水平(P<0.01或P<0.05),显著降低乳腺增生大鼠血清中E2及LH水平,显著升高增生大鼠血清中P、T、FSH及PRL水平(P<0.01或P<0.05)：蒙药乳腺-Ⅰ号高剂量可显著降低乳腺增生大鼠乳腺组织中ER、PR及AR蛋白的表达(P<0.05或P<0.01).显著降低乳腺组织中ER、AR的nRNA的表达,升高PR mRNA的表达；3.对各组大鼠乳腺组织中的总蛋白进行双向电泳分离,得到高度重复性的双向凝胶电泳图谱,通过图象分析,找出正常对照组组与模型组之间差异大于1.3倍的蛋白点43个,其中受到蒙药乳腺-Ⅰ号调节的蛋白点27个；4.应用质谱和生物信息学技术,对27个蛋白点进行鉴定,其中17种蛋白质被成功鉴定。该17种蛋白质涉及到10类不同的生物学过程,包括代谢、信号转导、细胞骨架和细胞运动、氧化应激、免疫、细胞周期等。本研究得出以下结论：1.乳腺增生的发病机制与血清中内分泌激素紊乱、乳腺组织中激素受体的蛋白及基因表达增加密切相关；2.蒙药乳腺-Ⅰ号可以降低乳腺增生大鼠血清中雌激素水平,升高孕激素水平、改善乳腺增生大鼠体内性激素紊乱的状态；3.蒙药乳腺-Ⅰ号可调节乳腺增生大鼠乳腺组织ER,PR及AR蛋白的表达及各自mRNA的表达,减轻大鼠乳腺组织增生性损伤；4.通过注射外源性雌激素联合孕激素诱导的乳腺增生大鼠,其血清血脂代谢出现紊乱,蒙药乳腺-Ⅰ号可以降低实验性乳腺增生大鼠血清TC,TG和LDL-c,调节乳腺增生大鼠的血脂代谢紊乱；5.利用差异蛋白质组学技术对蒙药乳腺-Ⅰ号干预下的蛋白表达情况进行了研究,通过软件分析得到了27个表达发生显著变化蛋白点；应用质谱技术对蛋白点进行了鉴定,共鉴定出17个差异表达蛋白,这些蛋白分别涉及到代谢、信号转导、细胞骨架和细胞运动、氧化应激、免疫、细胞周期等多种生物学过程。本研究的创新点：1.采用给大鼠肌内注射雌激素联合孕激素的方法,建立乳腺增生动物模型,系统地研究了蒙药乳腺-Ⅰ号对大鼠乳腺增生的治疗作用,从内分泌激素和受体两方面初步探讨了其相关的作用机制；2.运用蛋白质组学技术初步建立了乳腺增生动物模型的血清和乳腺组织蛋白质组学研究技术平台；3.首次利用蛋白质组学技术,研究乳腺增生动物模型乳腺组织的蛋白质组变化,为乳腺增生症的发病机制的研究提供了新的研究思路和方法；4.首次采用蛋白质组学方法对蒙药复方制剂的药物机制进行研究,为研究蒙药作用靶点问题提供了崭新的思维模式。更多还原

【Abstract】 Benign lesions of the breast are far more frequent than malignant ones. Breast hyperplasia is one of these lesions and is also one of the risk factors for breast cancer.The World Health Organization advocates early prevention and detection in order to improve breast cancer outcome, and these measures remain the cornerstone of breast cancer control. However, patients with breast hyperplasia are reluctant to undergo tissue resection because it is painful, often allows recurrence, and does nothing to treat the underlying, causative endocrine imbalance. Instead, multiple applied estrogen or estrogen receptor antagonist agents, including tamoxifen, have been used. Although these drugs can relieve symptoms in patients, they have many side effects and complications, and their use also can lead to endocrine disorders Mongolian medicine plays an important role in Chinese ethnic medicine. But very little scientific evidence exist that support consumption of Mongolian medicine herbal preparations in traditional medicine. So RuXian-Ⅰis only in clinical use in the Affiliated Hospital of Inner Mongolia University for the Nationalities as a traditionally drug, lacks widespread application. RuXian-Ⅰtreatment of breast hyperplasia in 400 patients showed that the cure rate was 98%.But very little scientific evidence exist that support consumption of Mongolian medicine herbal preparations in traditional medicine. Two dimensional difference gel electrophoresis (2D-DIGE) can greatly reduce the inter-gel variation and excessive time/labor costs associated with standard 2-DE and can allow a more accurate qualitative and quantitative analysis. There have been several reports comparing the expression profiles of proteins in breast cancer tissues with those in nontumor breast tissues. However, expression profiles of proteins in breast hyperplasia have not been reported. In order to gain a better insight into the mechanism of RuXian-Ⅰ’s efficacy, diagnosis, therapy, outcomes, and prognosis in the breast hyperplasia, and the relation between breast hyperplasia and breast cancer, we used intramuscular estrogen and progesterone to induce rat breast hyperplasia and 2D-DIGE to study the proteins associated with RuXian-Ⅰtreatment. Differentially expressed spots were identified by MALDI-TOF/TOF-MS, and changes in the expression of the proteins of interest were further validated using immunohistochemical and biochemical detection methods.In this study, we tested the breast-protective effects of RuXian-Ⅰ,in rats with estradiol/progesterone induced breast hyperplasia. Rats were divided into 5 groups:a control group, a model group, a tamoxifen citrate (positive control) group, a RuXian-Ⅰhigh-dose group and a RuXian-Ⅰlow-dose group.As a first step toward the investigation of biomarkers associated with RuXian-Ⅰtreatment, a proteome-wide analysis of rat breast tissue was conducted in this study. First, rat breast hyperplasia was induced by injection with estradiol and progesterone. We used an approach that integrates size-based 2D DIGE, MALDI-TOF/TOF-MS,and bioinformatics to analyze data from the control group, the model group and the RuXian-Ⅰtreatment group.After 2D DIGE, the Cy2, Cy3, and Cy5 channels of each gel were individually imaged, and the images were analyzed using DeCyder 5.0 software.The main results of this study are as follows:1.Compared with the model group, the RuXian-Ⅰgroup experienced markedly limited increases in nipple diameter, reduced histopathological changes, and decreased serum estradiol (E2) and luteinizing hormone (LH) levels.RuXian-Ⅰwas increased significantly progesterone (P), testosterone (T) levels and significantly follicle stimulating hormone (FSH),but it did not affect prolactin (PRL) in breast hyperplasia rats at 30 d. RuXian-Ⅰdemonstrated the same treatment effect as tamoxifen citrate for nipple diameter size and histopathological change; however, tamoxifen citrate affected the regulation of serum hormones differently than RuXian-Ⅰ.2. The RuXian-Ⅰtreatment mechanism may involve decreased expression of estrogen receptor(ER), progesterone receptor (PR), androgen receptors(AR),ER mRNA and AR mRNA in breast hyperplasia tissue. The study showed that protein expression levels of ER, PR and AR in the model group increased relative to the control group (P<0.01).ER, PR and AR protein expression levels in RuXian-Ⅰgroups all decreased significantly compared with the model group (P<0.01)by immunohistochemistry and western blotting. RuXian-Ⅰregulated mRNA expression of ER, PR and AR of sex hormone receptor transcription. The results demonstrated that the mRNA expression of ER and AR increased (P<0.01) and PR mRNA expression decreased (P<0.01) significantly in the model group compared to the control group. RuXian-Ⅰhigh and low dose groups remarkably decreased ER and AR mRNA expression and increased PR mRNA expression. In conclusion, RuXian-Ⅰcould reduce increases in nipple diameter, decrease histopathological changes and adjust regulation of serum sex hormone. This protective effect may be partially mediated through affecting mRNA and protein expression of ER, PR and AR.3.17 spots corresponding to 16 different gene products were identified by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). There are two spots (corresponding to Tpil protein and myosin-4) that are significantly down-regulated in the disease model (ratio<1.3, p<0.01) but increased after treatment with RuXian-Ⅰ.The other 15 spots were significantly up-regulated (ratio>1.3, p<0.01) in the disease model group, but down-regulated after treatment with RuXian-Ⅰ.And two have the opposite change trend.4.Following protein identification, they were categorized using the PANTHER gene ontology database. The PANTHER classification system grouped the 17 proteins from the mammary glands into 10 major subcategories according to "biological processes",including the immune system process, metabolic process, response to stimulus, transport, et al.The conclusions of this study are as follows:1.The pathogenesis of breast hyperplasia is closely related to serum endocrine hormone disorders and hormone receptors gene and protein expression increase in breast tissue;2.RuXian-Ⅰcan reduce serum estrogen levels, increased progesterone levels, improve the hormone disorder state in the hyperplasia of mammary glands rats;3. RuXian-Ⅰcan adjust mammary glands ER, PR and AR protein expression and their respective mRNA expression, then treatment of rat mammary gland hyperplasia;4. Injection of exogenous estrogen and progesterone-induced hyperplasia of mammary glands in rats, serum lipid metabolism in disturbance;5. RuXian-Ⅰcan reduce serum TC,TG and LDL-c levels and regulate lipid metabolism in hyperplasia of mammary glands rats;6.RuXian-Ⅰtherapeutic effects of breast hyperplasia related to metabolism, signal transduction, cytoskeleton and cell motility, oxidative stress, immunity, cell cycle and other biological processes;The creative points of the present study are:1.Intramuscular injection to rats with estrogen and progesterone used to establish animal models of hyperplasia of mammary glands studied systematically of the RuXian-Ⅰ on the treatment of hyperplasia of mammary glands in rats, from both endocrine hormones and receptors discussed the related Mechanism of action;2.Initial establishment animal models of breast hyperplasia in serum and breast tissue proteomics technology platform by proteomics technology;3.The first use of proteomics technology to study animal models of hyperplasia of mammary glands proteome changes in breast tissue for breast hyperplasia in the pathogenesis of the study provides new ideas and methods;4. Proteomics approach was first introduced to study the Mongolian compound preparation mechanism for the study the problem of Mongolian medicine targets provides a new mode of thinking.更多还原