顺铂和来曲唑对大鼠子宫内膜异位症的作用研究及临床问题研究

发布时间：2017-12-27 03:21

本文关键词：顺铂和来曲唑对大鼠子宫内膜异位症的作用研究及临床问题研究　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：研究背景子宫内膜异位症(简称内异症)是妇科临床的常见病、多发病,也是慢性病。其临床表现多样,发病机制复杂,被认为是遗传性疾病、炎症性疾病、免疫性疾病、出血引起的疾病、器官(子宫)依赖性疾病、激素(雌激素)依赖性疾病。现在更倾向于认为它是一种子宫内膜疾病、干细胞疾病、类肿瘤疾病。内异症极具侵袭性和容易复发,一直是妇科领域的研究热点和难点。由于受临床试验的限制,内异症动物模型研究一直是重点和热点,也是本文研究的出发点。在内异症的形成过程中,经血逆流种植应达到四个“必须”:a.经输卵管逆流入盆腔的经血中必须含有子宫内膜组织;b.内膜碎片中的腺上皮和间质细胞必须是“活的”;c.这些细胞必须有能力种植在盆腔组织器官上;d.盆腔内异症病灶的解剖分布与经输卵管播散的方式必须一致。而且,逆流之内膜需突破“三道防线”:腹水中的炎症因子、腹腔中的免疫细胞和腹膜的细胞外基质。经过多年的研究,有学者总结出了内异症形成“三步曲”:粘附、侵袭、血管形成,可将其称为“3A”程序(attachment-aggression-angiogenesis)。粘附是异位内膜“入侵”盆腹腔腹膜或其他脏器表面的第一步,继而突破细胞外基质,血管形成是其种植后生长的必要条件,亦即所谓“生根、生长、生病”的“三生”过程。基于上述理论,内异症模型的评价应该从以下方面来评价:异位病灶的外观表现及生长情况和内膜及血管生成的组织学检测,同时进行相关的免疫学标记物检测。既往的动物模型试验针对上述环节进行了众多的研究,而有关子宫内膜异位症的类肿瘤性质的相关研究却少见报道,这是本课题研究的出发点及创新点。内异症形成“三步曲”:粘附、侵袭、血管形成,同时内异症的复发和转移的特点,与肿瘤极其相似,因此,内异症被认为是“良性疾病,恶性行为”,这更体现了它的类肿瘤性质。在临床工作中,内异症恶变的情况也并不少见,恶变的病理类型多为子宫内膜样癌和透明细胞癌,属于子宫内膜癌或者卵巢癌的范畴,在这两类肿瘤的治疗中,腹腔灌注化疗占有重要位置,而在众多的腹腔灌注化疗方案中,顺铂是最常用而且疗效最为明显的一种。顺铂是细胞周期非特异性抗肿瘤药物,进入细胞后,与DNA发生反应,形成DNA内两点或两链的交叉连接,从而抑制DNA复制和转录,导致DNA断裂和错码,抑制细胞有丝分裂,达到抗肿瘤作用。芳香化酶是属于细胞色素P450的一种复合酶,可以催化雄烯二酮和睾酮转变成雌酮和雌二醇。动物试验研究表明,在内异症病灶中,芳香化酶相关蛋白表达水平增高。非留体类芳香化酶抑制剂来曲唑被研究认为有抑制大鼠内异症病灶生长的作用。另有研究证明,通过持续的高剂量的来曲唑灌胃试验,可以通过来曲唑的芳香化酶抑制剂作用诱导大鼠体内的高雄激素血症并进而引起卵巢的多囊性改变和排卵障碍,并且动情周期失去周期性。而在大鼠内异症模型治疗方案的探讨中,人们主要关注来曲唑灌胃治疗后的内异症病灶的变化,同时检测其动情周期及卵巢内分泌变化的研究未见报道。为弥补此类研究不足,本研究在研究来曲唑灌胃引起内异症病灶变化的同时检测大鼠动情周期、卵巢内分泌及排卵的变化情况,意在探讨来曲唑对内异症病灶的作用机制,究竟是直接抑制病灶中芳香化酶的表达为主要作用,还是由于来曲唑引起的大鼠内分泌的变化导致的体内低雌激素状态为主要作用。分子生物学、蛋白组学和动物模型的研究结果显示,在动物模型以及人类患者内异症病灶中MMP-2、TIMP-2和TGFb2mRNA表达水平升高。另有研究发现IL-1b、TNF-a、VEGF、MCP-1和PEDF在内异症动物模型发病中也有重要作用。众多的大鼠内异症模型研究,通过腹腔灌注治疗药物,并观察病灶变化和检测病灶中相关蛋白及因子的表达情况,来评价相应的药物疗效及探索可能的治疗方案。基于以上理论,本研究课题设计思路为通过构建动物模型探讨可能的新的内异症治疗方法。本实验选用的检测因子指标为:VEGF、P450arom、TGF-β和MMP-2。芳香化酶P450arom蛋白的作用如上所述,血管内皮细胞生长因子VEGF是内异症组织以及肿瘤组织在病灶生长以及转移灶生长过程中必不可少的生长因子,它表达水平的高低直接决定了病灶的活性。基质金属蛋白酶MMP-2是其家族的重要成员,基质金属蛋白酶及其抑制物(MMP/TIMP)是决定细胞外基质ECM降解的主要酶系,是内异症病灶和肿瘤组织向周围浸润植入和转移的关键因素。转化生长因子TGF-β也是重要的生长因子,对内异症病灶和肿瘤的生长起关键作用。本研究通过手术自体子宫内膜移植法建造大鼠内异症模型,评价动物模型成功后,随机分组进行试验,通过病灶外观观察及病灶体积测量,组织切片病理观察,以及异位内膜的指标检测,大鼠动情周期的动态观察,大鼠卵巢的形态学、组织学及免疫学变化,同时检测大鼠性激素的改变,通过和来曲唑灌胃治疗的比较,来探讨腹腔灌注顺铂治疗内异症的可能疗效以及来曲唑治疗内异症的更多机制,进一步从发病学及治疗学研究内异症,为子宫内膜异位症的临床诊断和治疗提供更多的实验依据。本研究共分为四个部分,阐述如下。研究目的第一部分:探讨内异症大鼠模型的制作过程、技术要点及模型评价,为应用大鼠模型进行内异症药物治疗研究提供理论依据。第二部分:研究应用顺铂腹腔灌注后,对子宫内膜异位症模型大鼠异位内膜病灶的作用。第三部分:研究在子宫内膜异位症大鼠模型中,给予来曲唑灌胃处理后,大鼠卵巢形态、体内性激素及卵巢功能的变化情况。第四部分:研究在子宫内膜异位症模型中,腹腔灌注顺铂和来曲唑灌胃,对模型大鼠的异位内膜病灶的作用,并且对二者的作用进行比较。方法与结果第一部分:45 只 SPF(specific-pathogen free)级雌性 SD(Spraue Dawley)大鼠。随机分为空白组、对照组、实验组,各15只。内异症模型应用子宫内膜自体移植法手术诱导,实验组移植物剥除浆膜层,对照组不剥除浆膜层,空白组仅开腹探查。空白组和实验组均每四日腹腔灌注生理盐水1ml一次;对照组不给予灌注。记录移植物的生长情况,测量并计算异位病灶的平均面积,及组织病理检查,来评价模型成功与否。整个试验过程,没有死亡和感染病例;内异症病灶在所有30只模型大鼠成功形成。在应用试验药物之前,对照组和实验组大鼠平均异位内膜病灶面积没有差别。在用药过程结束后,两组平均面积均明显增加,但是两组用药结束后的平均异位内膜病灶面积没有差别。组织病理学观察,对照组和实验组组织评分无明显差别;两组的异位组织病理评分与空白组大鼠在位内膜组织病理评分相比,差别均无统计学意义。第二部分:应用动物模型,36只SD大鼠在SPF设施内饲养。应用大鼠子宫内膜自体移植法诱导内异症模型。36只模型成功的大鼠随机分为三组,分别为对照组、低剂量来曲唑组和高剂量来曲唑组,每组12只。对照组12只大鼠不进行药物干预;低剂量顺铂组每四天行顺铂腹腔灌注一次,剂量为35mg/m2;高剂量顺铂组每四天行顺铂腹腔灌注一次,剂量为70mg/m2;所有大鼠用药疗程共24天。测量记录移植组织病灶的生长和病理评分。应用免疫组化、ELISA和Western blot 法检测病灶中 vascular endothelial growth factor(VEGF),aromatase P450(P450arom),transforming growth factor beta(TGF-β),和 matrix metalloprotein(MMP)-2蛋白的表达情况。结果显示,在顺铂用药疗程结束后,两顺铂组的平均病灶面积与对照组相比均明显降低,两组的病灶组织病理评分也都明显的低于对照组。与对照组相比,蛋白标记物VEGF,P450arom,TGF-β和MMP-2的表达在两个顺铂组均明显降低。同时,高剂量顺铂组上述检测指标的变化更加明显,表现出一定的剂量依赖性。另外,在高剂量顺铂组,部分大鼠表现出脱毛现象。第三部分:一项前瞻性的动物模型研究。大鼠在SPF设施内饲养。应用SD大鼠造模。内异症应用手术诱导法同上。对照组12只大鼠,每日应用1ml生理盐水灌胃;低剂量来曲唑组12只大鼠,每日以0.5 mg/kg来曲唑溶液灌胃;高剂量来曲唑组12只大鼠,每日以1mg/kg来曲唑溶液灌胃;每日行阴道涂片来观察大鼠的动情周期变化。应用化学发光法测量大鼠血清中性激素FSH,LH,E2,T和P的浓度;应用大体外观观察对比和HE染色研究大鼠内异症病灶和卵巢的组织病理变化。研究结果显示,与对照组相比,两来曲唑组大鼠在用药14天失去了规律的动情周期变化,而且大鼠血清FSH,LH和T水平明显升高,但是,来曲唑组的E2和P水平却明显低于对照组。两来曲唑组的卵巢也呈现多囊性改变,排卵征象罕见。两来曲唑组的大鼠异位内膜病灶面积和组织病理评分也明显的低于对照组。第四部分:应用动物模型进行前瞻性研究。实验动物在SPF设施内饲养,应用SD大鼠制造动物模型。通过大鼠内膜组织自体移植来手术诱发内异症模型。将30只模型大鼠随机分为三组,每组10只。第一组作为对照组不接受药物处理;第二组大鼠每日给予来曲唑灌胃,剂量标准为0.2 mg/kg;第三组大鼠每四天行腹腔灌注顺铂一次,剂量标准为35 mg/m2;所有的大鼠处理过程持续24天。对内膜移植物病灶的生长情况和组织学评分进行评价。应用免疫组化法和Western blotting法检测病灶中与组织和血管形成相关蛋白的表达;大鼠血清中的性激素水平用ELISA法检测。用药结束后,统计分析显示,来曲唑组和顺铂组大鼠内异症病灶的生长和组织评分均明显低于对照组;异位症病灶中VEGF,P450arom,TGF-β和MMP-2蛋白的表达水平,在来曲唑组和顺铂组均明显的低于对照组。另外,顺铂组中的P450arom蛋白水平明显的低于来曲唑组;来曲唑组中的TGF-β和MMP-2蛋白水平明显的低于顺铂组;来曲唑组大鼠血清中的T水平明显高于顺铂组,而E2水平低于顺铂组。结论1.大鼠子宫内膜异位症模型是一种易于建立和质量可靠的动物模型,在建造过程中要注意一些技术要点,传统的方法和改良的方法均可满足试验的要求,该模型对内异症的药物治疗研究有重要价值。2.顺铂可以导致内异症大鼠模型异位内膜病灶的萎缩退化,以及病灶中与组织增殖和血管生成相关的蛋白的表达水平降低,提示顺铂可能对内异症病人的临床药物治疗有作用。3.对子宫内膜异位症模型大鼠应用来曲唑后,大鼠在卵巢和内分泌方面表现出多囊卵巢综合征的状态,同时,也观察到大鼠异位内膜病灶体积缩小和组织学评分降低。提示来曲唑可能既可以在内异症病灶中通过抑制芳香化酶起直接的抑制雌激素生成的作用,也可以通过抑制卵巢的雌激素分泌间接的对内异症病灶起作用。4.顺铂和来曲唑可以在内异症模型大鼠异位内膜病灶中引起相似的组织萎缩退化的变化。但是,二者对内异症病灶中与组织增殖和血管生成相关的蛋白表达的影响表现出不同的模式;提示,顺铂和来曲唑可能通过不同的作用机制在内异症病灶中引起了相似的作用结果。研究背景输卵管切除术是妇产科临床的常见术式,广泛的应用于多种的妇科疾病的治疗。近些年来,随着手术技术的改进和提高,发展出许多种手术方式,包括经腹输卵管切除术、经阴道输卵管切除术及腹腔镜下输卵管切除术等。随着时代的发展,尤其是患者对术后生活质量和生殖医学要求的提高,输卵管切除术对卵巢血供和对卵巢功能的影响不容忽视,于是,如何采用更加微创的方法进行输卵管切除,在治疗输卵管疾病的同时,把手术对卵巢血供和对卵巢功能的影响降低到最小的程度,是妇科医生亟需解决的问题。目的本研究的目的是评价一种改良的、新型的及更加微创的输卵管切除方法,即腹腔镜下输卵管抽芯切除法,通过评价它的可行性,安全性,和有效性,来探讨该方法的潜在的临床价值和应用前景。材料和方法回顾性分析154例因“输卵管妊娠”行腹腔镜下输卵管切除术的患者的病历资料,其中76例患者施行腹腔镜下输卵管抽芯切除术(laparoscopic“core-pulling”salpingectomy,LCPS),78例患者施行传统的多孔腹腔镜下的输卵管切除术(conventional multi-port laparoscopic salpingectomy,MPLS),分析对比两组患者的临床一般特点、术中发现、术中和术后的情况及近期远期的并发症情况。结果所有患者共154例均成功施行腹腔镜手术,其中LCPS 76例,MPLS78例,没有中转开腹手术病例。两组患者的平均年龄分别为29.9±4.4岁(MPLS)和31.0±4.1 岁(LCPS),体重指数分别为 27.4±4.0 kg/m2(MPLS)和 27.1±3.3 kg/m2(LCPS),两组之间二者差别均无统计学意义(P0.05)。两组之间在病理特征方面,包括腹部手术病史及盆腔合并疾病如子宫内膜异位症和慢性盆腔炎症等,以及其他病理情况等,差别均无统计学意义(P0.05)。术中所见输卵管的病理情况包括输卵管管壁破裂情况及输卵管妊娠的位置等两组之间差别没有统计学意义(P0.05)。输卵管粘连的情况两组之间也相似,差别没有统计学意义(P0.05)。LCPS组和MPLS组在手术时间、估计手术出血量、术后发热的发生率以及术后住院时间方面,差别没有统计学意义(P0.05)。在术中及近期并发症方面,包括肠损伤、严重出血、休克、术中术后输血及术后切口感染方面,两组之间没有差别(P0.05)。在术后随访中,两组均没有发现与切口及创伤等相关的并发症。结论腹腔镜下输卵管抽芯切除术是安全的、可行的及有效的,在避免由于输卵管切除导致的卵巢血供受损及功能下降等不良副作用方面,有一定的应用价值和良好的前途,对该手术后卵巢功能及妊娠情况等方面,值得做深入的探索。
[Abstract]:Background endometriosis (endometriosis for short) is a common and frequently occurring disease in gynecologic clinic, and also a chronic disease. Its clinical manifestations are diverse, and its pathogenesis is complex. It is considered to be hereditary diseases, inflammatory diseases, immune diseases, hemorrhagic diseases, organ (uterine) dependent diseases and hormone (estrogen) dependent diseases. It is now more inclined to think of it as an endometrium disease, stem cell disease, and a tumor like disease. Endometriosis is very aggressive and easy to relapse. It has always been a hot spot and difficult point in the field of gynecology. Due to the limitation of clinical trials, the animal model of endometriosis has always been the focus and hot spot, and it is also the starting point of this study. In the process of the formation of endometriosis, retrograde menstruation should reach four "must": A. into the pelvic cavity through the fallopian tube countercurrent blood must contain endometrial tissue; B. fragments in endometrial glandular epithelial and stromal cells must be "live" C.; these cells must have the ability to grow in pelvic tissue organ; anatomic distribution mode D. pelvic endometriosis lesions and the tubal dissemination must be consistent. Moreover, the intima of countercurrent needs to break through the "three lines of defense": inflammatory factors in the ascites, immune cells in the abdominal cavity and extracellular matrix of the peritoneum. After many years of research, some scholars summed up the "three steps" of the formation of endometriosis: adhesion, invasion and angiogenesis, which can be called the "3A" program (attachment-aggression-angiogenesis). Adhesion is the first step to invade the peritoneum peritoneum or other organs by ectopic endometrium, and then break through the extracellular matrix. Angiogenesis is a necessary condition for its growth after planting, that is, the so-called "Sheng Sheng", "growth and illness". Based on the above theory, the evaluation of endometriosis models should be evaluated from the following aspects: appearance and growth of ectopic lesions, histological examination of intima and angiogenesis, and related immunological markers. Previous animal models have done many researches on these aspects. However, there are few reports about the nature of endometriosis, which is the starting point and innovation of this research. There are three steps in the formation of endometriosis: adhesion, invasion, angiogenesis, and the recurrence and metastasis of endometriosis, which are very similar to tumors. Therefore, endometriosis is considered as benign disease and malignant behavior, which shows its tumor like nature. In clinical work, endometriosis canceration situation is also not uncommon for pathological types of malignant endometrioid carcinoma and clear cell carcinoma, endometrial cancer or ovarian cancer belongs to the category of the two class, in the treatment of cancer, intraperitoneal chemotherapy occupies an important position in various programs of intraperitoneal chemotherapy cisplatin is the most commonly used, and the curative effect is one of the most obvious. Cisplatin is a cell cycle nonspecific anti-tumor drug. After entering the cell, it reacts with DNA to form a cross connection between two points or two chains in DNA, thus inhibiting DNA replication and transcription, resulting in DNA breakage and wrong code, inhibiting mitosis and achieving anti-tumor effect. Aromatase is a complex enzyme belongs to cytochrome P450, can catalyze androst-4-ene-3,17-dione in two and converted to estrone and estradiol testosterone. Animal experiments showed that the expression of aromatase related proteins increased in the lesion of endometriosis. The non retention of aromatase inhibitor, letrozole, is considered to inhibit the growth of the lesion in rats. Other studies have shown that continuous high dose letrozole gavage test can induce Kaohsiung steroid anemia in rats by letrozole aromatase inhibitor, and then cause ovarian polycystic changes and ovulation disorders, and estrous cycle is out of phase. In the treatment of rat models of endometriosis, people focused on the changes of endometriosis after letrozole treatment. Meanwhile, the estrous cycle and ovarian endocrine changes were not reported. In order to make up for the lack of such research, the study on the cause of endometriosis lesions change at the same time to detect the changes of estrous cycle and ovarian endocrine and ovulation in the study of letrozole, in order to explore the mechanism of letrozole to endometriosis, what is the expression of aromatase in the direct inhibition of lesions as the main role, or because in the low estrogen level leads to the change of letrozole in rats induced by endocrine as the main role. Molecular biology, proteomics and animal models showed that the expression levels of MMP-2, TIMP-2 and TGFb2mRNA increased in animal models and in patients with endometriosis. Other studies have found that IL-1b, TNF-a, VEGF, MCP-1 and PEDF also play an important role in the pathogenesis of the animal model of endometriosis. A large number of rat models of endometriosis were studied by intraperitoneal perfusion, observing the changes of lesions and detecting the expression of related proteins and factors in the lesions, so as to evaluate the corresponding drug efficacy and explore possible therapeutic options. Based on the above theory, this research project is designed to explore the possible new treatment of endometriosis through the construction of animal models. The test factors used in this experiment are VEGF, P450arom, TGF- beta and MMP-2. The role of aromatase P450arom protein is mentioned above. Vascular endothelial growth factor VEGF is an essential growth factor in the growth of endometriosis and tumor growth, and its expression level directly determines the activity of the lesion. Matrix metalloproteinase MMP-2 is an important member of its family. Matrix metalloproteinase and its inhibitor (MMP/TIMP) are the main enzymes that determine the ECM degradation of extracellular matrix. They are the key factors for the invasion and metastasis of endometriosis and tumor tissues. Transforming life
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R711.71

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