【作者】 丁晓艳；

【导师】 郑伟；

【作者基本信息】 浙江大学 ， 临床医学， 2010， 博士

【摘要】 子宫内膜异位症(endometriosis, EMs)是生育年龄妇女常见的良性疾病,它又可浸润病灶周围的正常组织,治疗后易于复发,具有一定的“恶性”行为,近年来发病率有逐年升高趋势,成为妇科疾病中的热点问题。内异症病因与发病机制至今未明,经血逆流、种植以及腹膜或间皮细胞化生是两种被广泛接受的学说,其中以Sampson的经血逆流种植学说为主导理论。经血逆流是妇女月经期常见的现象,可发生于80%-90%的月经周期,但临床上仅10%-15%育龄妇女罹患内异症,这是Sampson学说无法解释之处。新近的研究焦点聚焦于子宫在位内膜,有谓“在位内膜决定论”。有研究也表明内异症患者在位内膜的一些生物因子表达异常,有别于正常子宫内膜,使其具有更强的粘附和侵袭能力。约有30%的子宫内膜异位症患者同时合并子宫腺肌病(adenomyosis),子宫腺肌病也是发生于女性生殖系统常见的良性病变,随着诊断水平的提高,其发病率也呈上升趋势。子宫腺肌病的发病机理尚未达成一致,其中被普遍接受的假说是：子宫腺肌病起源于子宫内膜基底层,由于某些特异性酶的作用,子宫肌层的平滑肌纤维束间失去紧密的连接,同时基底层内陷,高水平雌孕激素刺激也可能是促进子宫内膜向肌层生长的原因之一。可见,在位内膜的侵袭性增强和肌层薄弱是子宫腺肌病形成的两大重要要素。约15%的子宫腺肌病同时合并子宫内膜异位症,以往认为子宫腺肌病是内在性的子宫内膜异位症,但研究发现其发病机制和病理特点有别于外在性的子宫内膜异位症,故有的学者建议将子宫腺肌病作为一种独立性的疾病进行研究。子宫内膜异位症和子宫腺肌病的发病机制至今不明,诊断的金标准仍然依赖手术或病理检查,因此临床诊断困难,但其临床症状和并发症却对女性健康和生育却有着重大的影响。探讨子宫内膜异位症和子宫腺肌病的发病机制,寻找微创、灵敏、特异的标志物成为基础研究向应用研究过渡的热点之一线粒体不仅是机体的能量代谢中心,而且还参与多种重要的细胞病理过程。线粒体也有可能成为癌症和其他疾病早期筛查、风险评估和诊断的新标志物,同时为寻找新的治疗靶点和疾病干涉手段提供基础。以往的研究中,子宫内膜异位症和子宫腺肌病的线粒体功能变化研究甚少,仅有少量研究表明,氧化应激和线粒体DNA突变可能参与了内异症的发生和发展,而子宫内膜异位症和子宫腺肌病线粒体蛋白组学水平的研究,至今鲜有报道。蛋白质而并非核酸才是生命活动的具体执行者和体现者,因此深入研究子宫内膜异位症和子宫腺肌病的发病机制,寻找微创、特异、灵敏的标志物用于早期诊断和随访监测,客观上要求在蛋白质组的水平进行进一步探索。近年来开展的表面增强激光解吸-电离-飞行时间质谱(SELDI-TOF-MS)技术为这一探索提供了优良的技术平台。这一方法最大的优势在于高通量、高灵敏度的实现了质谱技术对临床样本的直接检测。本课题旨在应用SELDI-TOF-MS和生物信息学技术从子宫在位内膜中筛选子宫内膜异位症和子宫腺肌病的差异表达线粒体蛋白并构建诊断模型；并将得到的线粒体差异蛋白与血清和内膜总蛋白标志物进行对比,以期找到适合子宫内膜异位症和子宫腺肌病早期诊断、监测的候选标志物,为其分离、纯化、解码等进一步研究提供资源和基础,为探索子宫内膜异位症和子宫腺肌病的发病机制和治疗靶点提供新思路。第一部分应用SELDI-TOF-MS筛选子宫内膜异位症在位内膜线粒体差异蛋白的研究目的：筛选子宫内膜异位症在位内膜线粒体差异蛋白,并建立检测模型。方法：采用SELDI-TOF-MS技术检测24例内异症在位内膜和29例对照患者正常内膜组织中线粒体蛋白,并用支持向量机方法建立判别模型,用留一法评估模型的预测效果；采用卡方检验检验各类模型间特异度和灵敏度的差异,t检验检验不同临床分期间蛋白质峰值表达差异。结果：1.内异组在位内膜／对照组正常内膜组织间共检测到线粒体蛋白质峰78个,有统计学意义的差异蛋白质峰10个(P<0.05),其中5,574m／z和9,378 m／z两个蛋白质峰在内异组中的表达低于对照组,其余8个蛋白质峰在内异组呈现高表达；5,574、7,573和7,965m／z三个蛋白质峰出现在增殖期,其它7个蛋白质峰均出现在分泌期。2.3个线粒体蛋白质峰(15,334,15,128和16,069 m／z)组合所构建的诊断模型(模型1)鉴别内异症患者的特异度为86.2%,灵敏度为87.5%。3.分泌期内异组在位内膜／对照组正常内膜组织间共检测到线粒体蛋白质峰69个,有统计学意义的差异蛋白质峰14个(P<0.05),其中5,367、5,432、9,526和9,378 m／z 4个蛋白质峰在内异组中的表达低于对照组,其余10个蛋白质峰在内异组呈现高表达。4.3个分泌期线粒体蛋白质峰(7,615,5,432和15,868 m／z)组合所构建的诊断模型(模型2)鉴别内异症患者的特异度为76.9%,灵敏度为80.0%,与模型1相比,无统计学差异。5.增殖期内异组在位内膜／对照组正常内膜组织间共检测到线粒体蛋白质峰64个,均无统计学意义(P>0.05)。6.2个增殖期线粒体蛋白质峰(3,456 m／z和15,128 m／z)组合所构建的诊断模型(模型3)鉴别内异患者的特异度为87.5%,灵敏度为85.7%。7.内异组在位内膜／对照组正常内膜组织间检测到的有统计学意义的10个线粒体差异蛋白质峰中,处于Ⅲ、Ⅳ期的内异症患者15,334、15,128、7,573、15,868和7,982 m／z五个蛋白质峰值的表达水平显著高于临床分期Ⅰ、Ⅱ的患者(P<0.05),5,574和9,378 m／z两个峰值在Ⅲ、Ⅳ期的内异症患者的表达水平显著低于临床分期Ⅰ、Ⅱ的患者(P<0.05)。另外3个蛋白质峰值在不同的临床分期中表达无统计学差异(P>0.05)。第二部分应用SELDI-TOF-MS筛选子宫腺肌病在位内膜线粒体差异蛋白的研究目的：筛选子宫腺肌病在位内膜线粒体差异蛋白,建立检测模型,并将得到的线粒体差异蛋白与第一部分得到的子宫内膜异位症线粒体差异蛋白进行对比。方法：采用SELDI-TOF-MS技术检测13例子宫腺肌病在位内膜和29例对照患者正常内膜组织中线粒体蛋白,并用支持向量机方法建立判别模型,用留一法评估模型的预测效果。结果：1.腺肌病组在位内膜／对照组正常内膜组织间共检测到线粒体蛋白质峰82个,两组间有统计学意义的差异蛋白质峰1个。2.3个线粒体蛋白质峰(7,614,14,978和6,370 m／z)组合所构建的诊断模型鉴别子宫腺肌病患者的特异度为93.1%,灵敏度为84.6%。3.14个线粒体蛋白质峰同时出现在内异组和腺肌病组,其中3,499m／z的峰在两组中有统计学差异(P=0.029),其余13个峰值在两组中的表达无统计学差异(P>0.05)；3,384,3,499,4,237和5,388m／z的蛋白质峰在内异组和腺肌病组有相反的表达趋势,其余10个峰值在两组中有相同的表达趋势。结论：1.子宫内膜异位症和子宫腺肌病患者在位内膜与对照组正常内膜相比有差异表达的线粒体蛋白,在位子宫内膜线粒体蛋白的变化可能与内异症和子宫腺肌病的发生有关。2.内异症患者在位内膜与对照组正常内膜相比大部分差异表达的线粒体蛋白质峰出现在分泌期,分泌期内膜线粒体蛋白可能在内异症形成过程中较增殖期内膜线粒体蛋白发挥了更大的作用。3.在内异症中有差异表达的7个线粒体蛋白质峰与内异症临床分期有相关性,提示在位子宫内膜线粒体蛋白的变化可能与内异症的发展、预后有关。4.内异症在位内膜线粒体差异蛋白与内异症血清蛋白标志物相比,无吻合的蛋白质峰,可能是蛋白质从细胞内到血液循环的过程中经过了修饰、剪接等过程,进而造成了分子量的改变。5.内异症在位内膜线粒体差异蛋白与内异症在位内膜总蛋白标志物相比较,有3个吻合的蛋白质峰,对这些蛋白质峰进行分离、纯化和解码是下一步的重要工作。6.组合线粒体蛋白标志物(15,334,15,128和16,069 m／z)所构建的模型能达到鉴别内异症的最佳效果。7.组合线粒体蛋白标志物(7,614,14,978和6,370 m／z)所构建的模型对子宫腺肌病具有较好的诊断价值。8.子宫腺肌病与内异症在位内膜有14个表达相同的线粒体蛋白质峰,且大部分蛋白质峰(13／14)在腺肌病组和内异组的表达无差异,大部分蛋白质峰(10／14)在两组中有相同的表达趋势,二者可能在线粒体相关的功能异常方面有类似也有不同的改变。更多还原

【Abstract】 Endometriosis is a chronic condition characterized by the presence of endometnal tissue outside of the uterus in lesions of varying sizes and appearance containing endometrial glands and stroma. The pathogenesis of this enigmatic disorder still remains controversial despite multiple theories regarding the etiology exist. Among them, the implantation hypothesis of retrograde menstruation suggested by Sampson is the most widely accepted one. However, the implantation theory doesn’t explain that adhesion and persistence of endometriotic lesions in the pelvis only happen in approximately 10%-15% of women in reproductive age while the retrogradation of endometrial tissues via fallopian tubes during menstruation happens in approximately 80%-90% menstrual cycles. It is assumed that additional factors favor the adhesion and growth of endometrial cells that spill into the peritoneal cavity. Some studies indicate that eutopic endometrium of women affected by endometriosis is abnormal compared with that of healthy women. Although the morphology of eutopic endometrium from women with endometriosis is similar to the normal, its physiology and biochemistry are different.Adenomyosis is characterized by the presence of endometrial glands and stroma within the myometrium. The cause(s) and mechanisms of development of intramyometrial endometrium are not clear. At present, three theories have been proposed, but unanimous agreement has yet to be reached. The most popular hypothesis is that adenomyosis originates from the deep part (basalis) of the endometrial mucosa. The latter would invaginate between bundles of smooth muscle fibres of the myometrium, possibly due to loss of tissue cohesion caused by specific enzymes. About 30% of patients with endometriosis also have adenomyosis, while about 15% of patients with adenomyosis also have endometriosis. Some believed that adenomyosis is generally considered a variant of endometriosis widely referred to as internal endometriosis. While some believed that adenomyosis is a distinct disease from endometriosis requiring different therapeutic approaches.Despite of significant improvement of the diseases the pathogenesis of endometriosis and adenomyosis is still unclear. Opration approaches remain the gold standard for the diagnosis of endometriosis and adenomyosis. It has inherent limitations, and the patients suffer from the symptoms and complications a lot. Therefor it is urgent to get a better understanding of disease and identify groups of proteins involved in its pathogenesis and find sensitive, specific and convenient new biomarkers.To the best of our knowledge, the cellular function of mitochondria, however, is not limited to bioenergetics. They play crucial roles in the metabolism of amino acids and lipids, the biosynthesis of heme and iron-sulfur clusters, cell signaling, and apoptosis. Mitochondria might be new markers for the early detection, risk assessment, and diagnosis of cancer and other diseases, and for the identification of new targets for therapeutic prevention and intervention. Few reports were about the dysfunctions of mitochondria in eutopic endometria of endometriosis and adenomyosis. It was reported that oxidative stress and mitochondrial DNA mutations might be anticipated in the initiation or progression of endometriosis.Protein but not nuclear acid is the material executant and embodiment of life. So the studies on proteomics are preferred to approach to the pathogenesis of endometriosis and adenomyosis and screen sensitive and specific biomarkers. A novel technology, surface-enhanced laser desorption/ionization time-off light mass spectrometry (SELDI-TOF-MS) carved out such a new path to proteomics research on endometriosis and adenomyosis. It can directly obtain high-throughput protein profilings from clinical samples with high sensitive and this is the main advantage of this technology. The present study focused on the discrepant mitochondrial proteins of eutopic endometria from patients with endometriosis, adenomyosis and controls, using SELDI-TOF-MS and bioinformatics tools; establishing diagnostic models of endometriosis and adenomyosis; comparing the discrepant mitochondrial proteins screened with those in serum and eutopic endometrial. The purposes of the study are to screen novel biomarkers of endometriosis and adenomyosis that are fit for diagnosis and monitoring of endometriosis and adenomyosis, to provide materials and elements for the further studies such as the separation, purification and decoding of biomarkers and to provide new ideas of pathogenesis and therapeutic approaches of endometriosis and adenomyosis.Part one Detection of discrepant mitochondrial proteins in eutopic endometrium of endometriosis using SELDI-TOF-MSObjective:To detect specific mitochondrial proteins in eutopic endometrial samples from women with and without endometriosis and build diagnostic models.Methods:Eutopic endometrial samples from women with endometriosis (excluding adenomyosis) (n=24) and women with benign indications as control (n=29) were studied, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip technology. After finding the biomarkers, the diagnostic model was evaluated and validated by leave-one cross validation.χ2 test was used to evaluate the sensitivity and specificity of different diagnostic models. T-test was used to evaluate the protein peaks in different rAFS stages of endometriosis.Results:1.78 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with and without endometriosis and 10 of them had a significant difference (P＜0.05). The 5,574 m/z and 9,378 m/z peaks were expressed at a lower level in endometriosis than in controls; the other 8 peaks were expressed in the opposite way.5,574,7,573 and 7,965m/z peaks were from proliferation phase and the rest 7 peaks were from secretory phase.2. Three combined potential biomarkers, with m/z of 15,334,15,128 and 16,069, were found and the diagnostic system (pattern 1) distinguished endometriosis from control samples with a specificity of 86.2% and a sensitivity of 87.5%.3.69 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with endometriosis and controls in their secretory phase and 14 of them had a significant difference (P＜0.05). The 5,367,5,432,9,526 and 9,378 m/z peaks were expressed at a lower level in endometriosis than in controls; the other 10 peaks were expressed in the opposite way.4. Three combined potential biomarkers in secretory phase, with m/z of 7,615, 5,432 and 15,867, were found and the diagnostic system (pattern 2) distinguished endometriosis from control samples with a specificity of 76.9% and a sensitivity of 80.0%. The specificity and sensitivity had no significant difference from those of pattern 1.5.64 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with endometriosis and controls in their proliferation phase and none of them have a significant difference (P＞0.05).6. Two combined potential biomarkers in secretory phase, with m/z of 3,456 and 15,128, were found and the diagnostic system (pattern 3) distinguished endometriosis from control samples with a specificity of 87.5% and a sensitivity of 85.7%.7. Among the 10 discrepant mitochondrial protein peaks in eutopic endometrium from women with and without endometriosis which had a significant difference (P＜0.05), peaks with m/z of 15,334,15,128,7,573,15,868 and 7,982 were expressed at a higher level in endometriosis of stageⅢ&Ⅳthan of stageⅠ&Ⅱ.5,574 and 9,378 m/z peaks were expressed at an opposite way.Part two Detection of discrepant mitochondrial proteins in eutopic endometrium of adenomyosis using SELDI-TOF-MSObjective:To detect specific mitochondrial proteins in eutopic endometrial samples from women with and without adenomyosis and build diagnostic models, and comparie the discrepant mitochondrial proteins screened with those in part one.Methods:Eutopic endometrial samples from women with adenomyosis (n=13) and women with benign indications as control (n=29) were studied, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein chip technology. After finding the biomarkers, the diagnostic model was evaluated and validated by leave-one cross validation.Results:1.82 qualified mitochondrial protein peaks were detected in eutopic endometrium from women with and without adenomyosis and 1 of them had a significant difference (P＜0.05).2. Three combined potential biomarkers, with m/z of 7,614,14,978 and 6,370, were found and the diagnostic systemdistinguished adenomyosis from control samples with a specificity of 93.1% and a sensitivity of 84.6%.3.14 eutopic mitochondrial protein peaks were detected in both endometriosis and adenomyosis patients. The 3,499m/z peak had a significant difference (P=0.029).3,384, 3,499,4,237 and 5,388m/z peaks had the same trend in the two groups.Conclusions:1. Discrepant mitochondrial proteins of eutopic endometria from patients with endometriosis and adenomyosis were detected, which might play a role in the pathogenesis of the two diseases.2. Most of the discrepant eutopic mitochondrial proteins were found in the secretory phase. It was indicated that mitochondrial proteins at secretory phase had a high relationship to the characteristics of endometriosis.3. There were 7 peaks which had a correlation with the rAFS stages of endometriosis. They might play a role in the development of endometriosis.4. There were no similar peaks detected in eutopic mitochondrial proteins and serum from women with endometriosis. Mass/Charge of the proteins might change after being modified and sheared from cells to the circulation system.5. Three eutopic peaks were detected in both mitochondrial proteins and total proteins from women with endometriosis. Purification and identificatoin of the proteins will be an important part of our future study.6. The model composed by 15,334,15,128 and 16,069 m/z could do the best in division of endometriosis and controls. 7. The model composed by 7,614,14,978 and 6,370 m/z could do the best in division of adenomyosis and controls.8. 14 eutopic mitochondrial protein peaks were detected in both endometriosis and adenomyosis patients. Mitochondrial functions might have similar changes in the two diseases, but not totally the same.更多还原