【作者】 王强；

【导师】 李荷莲；

【作者基本信息】 吉林大学 ， 妇产科学， 2004， 博士

【摘要】 子宫内膜异位症(Endometriosis，EMs)是指有活动功能的子宫内膜出现在子宫内壁以外的部位并生长。其在生育年龄妇女中发病可达20%,复发率达40%。症状体征明显，如痛经，子宫异常出血，性交痛，盆腔肿块；临床治疗较为困难，不管是手术还是药物治疗均易复发。目前缺乏简便、有效、经济和不良反应小的治疗手段。内异症是雌激素依赖性疾病。因此对该病的药物治疗其目的主要是抑制下丘脑-垂体-卵巢轴，减少卵巢激素的分泌或对抗雌激素的作用，使异位生长的内膜组织萎缩乃至消失。近年来GnRH-a在治疗内异症上得到了越来越广泛的应用，GnRH-a是天然促性腺激素释放激素（GnRH）的结构类似物，与GnRH受体的亲和力较天然GnRH高100-200倍，故活性明显升高，且半衰期延长。短期应用可刺激垂体功能，GnRH-a通过占据垂体大部分GnRH-a受体，首先使垂体分泌FSH、LH增加。长期应用对垂体起降调节作用，使GnRH-a受体脱敏，数量减少。因此称为“暂时药物性卵巢切除”，引起一系列类似绝经期的表现，称“药物性卵巢切除”可用来治疗多种雌激素依赖性疾病。已有大量研究证实，GnRH-a对异位内膜具有很好的治疗效果，那法瑞林是常用的GnRH-a之一。它是由D-萘基丙氨酸取代了天然GnRH第6位上的甘氨酸得到的，其活力约为天然GnRH的200倍。那法瑞林（长期大剂量应用）具有强效、低毒、易为病人接受的优点；其作用主要通过抑制下丘脑-垂体-卵巢轴，造成体内的低雌激素状态，引起异位内膜萎缩。本研究采用那法瑞林作为研究药物疗效的对照。LHRH类似物可作用于垂体卵巢轴，通过抑制生成卵巢激素而诱导闭经，使体内雌激素水平降到绝经期或卵巢切除后的水平，从而治疗子宫内膜异位症及雌激素依赖性疾病。LHRH－PE40是用基因工程的方法将<WP=78>LHRH基因和一部分铜绿假单孢菌外毒素基因在体外重组后所表达出的一种融合蛋白，包括LHRH蛋白和一部分铜绿假单孢菌外毒素蛋白。LHRH-PE40是由军需大学军事兽医研究所合成的治疗雌激素依赖性疾病的新药。LHRH-PE40产品的理化性质已达到国家基因工程药物规程标准，目前正进行药效药理学实验。对此药用于动物模型子宫内膜癌的治疗已有报道，此药对子宫内膜癌治疗有效。国内外尚无LHRH-PE40用于子宫内膜异位症治疗的报道。本实验探讨药物LHRH-PE40对大鼠子宫内膜异位症的治疗效果。子宫内膜异位症是一种良性妇科疾病，但它的某些生物学行为却与恶性肿瘤类似，尤其是同样具有组织侵袭和血管形成能力。尽管EM发病机理仍众说纷纭，但异位灶在腹腔种植成功后，其进一步的发展则必须要有新生血管提供血液，显然血管形成在EMs的发病中具有重要作用。VEGF是血管形成最关键的因子，可直接特异性地作用于血管内皮细胞，导致新生血管形成。且研究发现：异位子宫内膜组织中VEGF的阳性表达率为72%，而非异位症的子宫内膜组织中无VEGF表达。多数学者认可Sampson提出的经血逆流学说，研究发现经血逆流在生育期妇女中非常普遍，其发生率可高达90%，而Ems的患病率仅占20%左右，说明经血逆流只是诱因，因此，逆流入腹腔的内膜组织必须降解基底膜和其他的细胞外基质（ECM）成分才能植入内膜，使异位内膜能在卵巢，子宫肌层，腹膜等处种植生长。基质金属蛋白酶是一组水解细胞外基质中多种成分（如胶原，纤维连接蛋白，蛋白多糖等）的蛋白酶，由于其活性的产生依赖于Zn2+，Ca2+，Mg2+等金属离子，因此称为金属蛋白酶。MMPs在肿瘤侵袭，生长，转移中发挥重要作用。MMPs是一种蛋白分解酶，可分解所有细胞外基质成分。细胞外基质（extracelluar mtrix，ECM）是维持细胞生理活动的外环境，不仅在细胞与细胞之间起机械支撑和连接作用，而且还是细胞与细胞之间信号转导的桥梁，参与细胞的多种生理和病理过程。其中MMP-2不仅具有降解明胶蛋白和Ⅳ，Ⅴ型胶原的作用，参与形成早期的<WP=79>EMS，而且能降解BM周围的毛细血管基底膜，形成血管，为EMS病灶提供血供，促进腺体的形成，进而促进EMS的生长和增殖。VEGF主要定位于内膜腺上皮，基质细胞不表达。空白组，假手术组VEGF蛋白表达较低，主要定位于内膜腺上皮。模型组VEGF蛋白表达高。用药后，LHRH-PE40组和那法瑞林组较模型组VEGF蛋白表达降低。 MMP-2主要定位于子宫内膜组织的上皮细胞及间质细胞中，在异位内膜组织的腺上皮中染色较强，在间质细胞中也呈阳性表达。空白组，假手术组MMP-2蛋白表达较低，主要定位于内膜腺上皮。模型组MMP-2蛋白表达高，主要定位于子宫内膜组织的上皮细胞及间质细胞中。用药后，LHRH-PE40组和那法瑞林组较模型组MMP-2蛋白表达降低。EMS与E2和P密切关联，EMS多发生于育龄期妇女，绝经后病灶渐消退，E2水平高者常患EMS，子宫平滑肌瘤，子宫内膜癌等疾病，子宫内膜不管在位或异位均受卵巢性激素的调节，并随着血中的E2水平的消长而增殖或萎缩。本实验对80只Wister大鼠进行研究。随机选取对照组11只大鼠做空白对照组。随机选取假手术组取11只，行开腹手术，只用镊子轻轻牵拉子宫，不进行内膜移植。对其余58只大鼠采用外科诱导法行建模手术。在术后的第4、6和8周动态观察病灶的生长情况，运用光镜进行组织学观察，病理确定?更多还原

【Abstract】 Endometriosis(EMS), the present of endometrium outside of the uterine cavity, is a common disease, causing abdominal pain,dysmenorrhea, dyspareunia, and infertility in 10-15% of menstruating women. Endometriosis is considered to be an estrogen-dependent disease. A variety of hormonal treatments for endometriosis have been used, including drugs such as danazol, progestogens, gestrinone, prostaglandin synthetase inhibitors and GnRH analogues in the treatment of endometriosis comes from comparative with danazol. Many studies have investigated the effect of GnRH analogued in comparison with danazol. Studies show that GnRH-a is the most effective and the most promising medicine for treating endometriosis. It is estimated that approximately one third of the women who complain to a physician of pelvic pain or infertility have endometriosis and that its incidence is incerasing. Endometriosis remains an enigma among women’s diseases and presents many problems regarding its treatment and prognosis due partly to the great variation in the severity of the disease. Although EMS is a benign gynecological disease，its biological action is similar to malignant tumor .The formation of new from existing blood vessels is essential for tumor growth, invasion and metastasis. VEGF is one of the vasual inner epithelial cells leading to new vascular formation.Matrix metalloproteinases(MMPs) play an important role in degradation of extracell matrix and basement membrane components . MMPs are highly homologous protelytic zinc enzymes responsible for degradation of extracellular matrix components，such as collagen，proteolycans，fibrotectin and laminin. MMP-2，which degrades type IV collagen，may play a role in tumor metastasis.Materials and Methods: A total of 80 animals underwent the procedure for induction of endometrisis. We chose 11 rats as the case-control group <WP=83>randomly. We chose 11 rats as the operation group randomly and draw the uterine not making endometrium transplant. The rest of 58 animals underwent the surgery operation. Then three pieces of 5×5mm fragments of endometrial tissue were transplanted to different places；We observe and measure the length，width and height of the implantion and count each volume of them and compare the volume at the forth, sixth and eighth week after the operation.We use the light telescope to observe the histology of ectopic endometrium to make sure that the transplant is endometrium. Among them there are 34 rats，which are successful in endometrium transplant.Rats with experimental endometriosis were randomly divided into 3 groups:1 Group LHRH-PE40 :12 rats，15ug/kg /d，im.continuly for one month.2. Group Naf : 11 rats，5ug/kg /d，im.continuly for one month.3.Model group:11 rats，not giving any medicine and raising them normally. To kill all the rats by pulling the heads and they were laparotomized. To compare the size and structure of the transplant endometrium by naked eyes and lightelescope.Measuring the level of the E2 ，FSH，LH and PRL by radioimmunoassay. VEGF and MMP-2 in eupotic and ecpotic endometrium were measured by immunohistochemitry method. We use RT-PCR to compare the effects of Nafarelin and LHRH- PE40 on endometriosis respectively.Because cyclic estrogen and progesterone are the primary stimuli for endometrial growth and remodeling, medical therapies for endometriosis have included attempts to suppress steroid action. Gonadotropin-releasing hormone agonists that cause a reversible block of ovarian cyclicity have been used in the mangement of endometriosis. Results: 1.There are 34 rats in the 58 rats which are successful in creating models and successful rate is 59%. Eutopic endometrium is becoming larger at first and then becoming smaller with the time of the transplant. The volume in uterin ligament is much larger than in the abdominal wall at the same time <WP=84>and the difference is significant（P<0.05）.There is no significantly difference on the eutopic endometrium volume between the abdominal wall and the crossing site of the uterine（P>0.05更多还原