【作者】 哈春芳；

【导师】 刘培淑；

【作者基本信息】 山东大学 ， 妇产科学， 2011， 博士

【摘要】 研究背景：子宫内膜异位症(Endometriosis, EMS)是育龄期妇女的多发病,是引起80%患者慢性盆腔疼痛,30%妇科手术的和20%不孕患者的主要原因。尽管基础和临床研究显示EMS属于一种始于细胞水平,止于盆腔疼痛、不孕和盆腔包块为特点的激素依赖性的连续性病变,但目前发病机制不清,治疗对策有限,仍然是临床上亟待解决的难点问题之一。手术治疗是内异症首选的治疗方法,但手术常难以彻底清除所有病灶,术后复发率高,手术结合术后用药可抑制残余病灶、推迟内异症复发的时间,因此药物治疗仍居重要地位,抗粘附、抗侵袭、抗血管生成药物将是下一步治疗内异症的靶点。因此进一步探索内异症细胞异位粘附、侵袭的机制、通路上有效的通路信息分子,将有助于研究有效的药物治疗靶点,为从机制上进行有效的药物干预、指导临床治疗和随访提供依据。骨桥蛋白(osteopontin, OPN)是一种重要的磷酸化基质蛋白,与其受体整合素和CD44结合通过多种信号途径介导细胞侵袭过程、细胞外基(Extracellular Matrix, ECM)的降解和重塑、细胞迁移、宿主免疫细胞的逃逸和新生血管的形成；朱耀奎、马彩虹等均研究表明OPN蛋白出现在人EMS患者在位和异位内膜中而且表达明显升高；金属基质蛋白酶9(MatrixmetalloProteinase-9, MMP9)是金属基质蛋白酶(MatrixmetalloProteinase, MMPS)家族中分子量最大的明胶酶,来源于结缔组织细胞、巨噬细胞、中性白细胞等,在子宫内膜细胞的异位黏附、种植和生长的过程中发挥重要作用。肿瘤学研究已经证实OPN通过诱导细胞内MMP-9的和其他细胞因子的表达发挥异位黏附、侵蚀和转移作用。内异症虽为良性疾病,但具有明显的侵袭、转移及复发等恶性生物学行为,根据在位内膜决定学说即在位内膜本身的基因与蛋白表达紊乱是决定脱落内膜是否能异地粘附、种植的根本原因,OPN/MMP9作为肿瘤细胞粘附和侵蚀不可缺少的因子,在脱落的内膜细胞种植中是否发挥同等重要的作用。关于此方面的研究国内外从未有相关报道。实验证实,OPN基因5’上游侧区有孕激素调控元件,孕激素及其受体水平可调节OPN的水平；而MMP9上游区的雌激素结合区域,可通过雌激素或旁通路因子调节MMP9的水平。我们既往的研究也显示OPN和MMP9在正常和内异症患者在位、异位内膜中均有表达,而且异位内膜中的表达明显高于正常对照组,提示二者在内异症的发生发展中发挥重要作用,而且受卵巢雌孕激素的调节。由此我们推断雌孕激素是否是通过调节OPN和MMP9表达发挥细胞粘附作用?丙氨瑞林(GnRH-A)、高效孕激素制剂孕三烯酮及其受体拮抗剂米非司酮是目前临床治疗子宫内膜异位症的一线药物,研究显示上述药物通过调节和抑制下丘脑-垂体-卵巢轴的功能、竞争性抑制局部受体的结合、降低病变局部的雌激素浓度导致异位病症的萎缩；同时也可以通过直接作用于病灶局部,改变病变局部的分子生物学环境,抑制细胞的凋亡和提高细胞的凋亡能力,但是确切机制不清。本课题拟选取丙氨瑞林、孕三烯酮等激素相关类药物治疗EMS时异位/在位内膜中OPN/MMP9表达的变化为研究目的,通过建立和评估大鼠内异症模型,借助RT-PCR等分子生物学实验方法,探讨药物干预前后组织中OPN/MMP9蛋白和mRNA的变化,进一步揭示内异症中OPN与子宫内膜异位症发生发展的关系、药物作用的可能机制,为临床合理选择药物治疗提供可靠的依据和更多的预后判定指标。第一部分大鼠子宫内膜异位症模型的建立与评估[研究目的]应用传统法和开放法两种方法建立大鼠子宫内膜异位症模型,探讨两种方法建立大鼠子宫内膜异位症模型的特征、判定指标及建模成功率,为进一步研究药物治疗EMS的作用机制奠定基础。[研究方法]开腹取同一只大鼠的子宫,左侧宫腔采用开放法将子宫内膜与骶韧带或大网膜贴近建模,右侧采用移植法将1.0cm的子宫内膜移植于侧腹膜上建模,4周后二次开腹判定成模的特征、成模率,免疫组化测定骨桥蛋白(OPN)及金属蛋白酶-9(MMP-9)的表达。[研究结果]1.雌激素在建模中的作用及两种方法成模率比较：给药组子宫直径明显较未给药组径线大(P左-左=0.007,P右-右=0.006),二次手术发现给药组与未给药组成模率相比无明显差异(X2开放=0.01,P>0.05；x2移植=0.193 P>0.05))；开放法与移植法成模率比较两组无差异(x2=0.031,P>0.05)。2.两种方法主要大体观及并发症比较：移植法以透亮囊肿为主,占64.80%,其次为紫蓝色结节(10.81%),开放法主要以紫蓝色结节为主,占35.13%,其次为透亮囊肿(24.3%)；术后盆腹腔粘连开放法器官粘连率为64.86%,明显高于移植法的18.9%(P<0.01)；肠梗阻及感染等并发症发生率两组无明显差异(P>0.05)；3.病灶大体观与镜下观分析：大体观见到的各个病灶中,72.70%(24/33)透亮囊肿中镜下可以见到典型的腺上皮、腺体等,不典型腺体或间质细胞仅占21.2%(7/33)；紫蓝色结节中29.41%(5/17)结节中可见到腺体或腺上皮细胞,而火焰状病灶中68.42%(13/22)的标本中可见到镜下典型的腺体或腺上皮细胞；干酪样表现的脓肿镜下未见腺体细胞,仅为大量的淋巴细胞及毛细血管的增生。[结论]1.开放法和移植法均有较高的成模率；2.观察开放法成模的大体观更类似于人类的子宫内膜异位症改变,且具有成模面积大、简单、易行等特点；3.开放法建模病灶弥散,镜下腺体少见,不利于实验性药物干预。第二部分OPN与MMP9在大鼠子宫内膜异位症中的表达及意义[研究目的]检测OPN与MMP9在内异症大鼠在位、异位病灶中蛋白、基因表达的差异,探讨OPN与MMP9在子宫内膜中表达的特点、内异症中OPN与MMP9表达相关关系。[研究方法]建模成功的37只大鼠,建模28天后二次手术开腹,大体观合并组织病理学检查确定建模成功后,取大鼠的在位(在位组)、异位内膜组织(异位组)和未建模的正常大鼠子宫内膜组织(对照组),通过RT-PCR、免疫组化法测定内膜中OPN与MMP9 mRNA、蛋白表达。[研究结果]1. OPN、MMP9在大鼠子宫内膜异位症内膜中的表达：OPN主要高表达与腺上皮细胞的细胞膜上,间质内少量表达,而MMP9主要表达于腺上皮细胞的胞浆内,核内无表达；2. OPN、MMP9蛋白在内异症在位、异位内膜中表达的差异：免疫组化显示异位内膜中OPN表达的IOD值为53735.4±4896,明显高于在位内膜组的33733.8±5322和对照组的37291.2±4588(t在位-异位=17.010；P=0.000；t对照-异位=9.122；P=0.000),对照组和在位组的IOD值无明显差异(t=1.973,P=,0.52)；MMP9表达在异位内膜组为32645±9967,高于在位内膜组和对照组((t在位-异位=8.559；P=0.000；t对照-异位=5.386；P=0.000),而在位组和对照组比较无明显差异(t=0.198,P=,0.844)。3. OPN、MMP9 mRNA在内异症在位、异位内膜中的表达：异位组内膜中OPN、MMP9 mRNA表达明显高于对照组和在位组,而对照组和在位内膜组OPN、MMP9 mRNA表达无明显差异。4.在位、异位内膜组织中OPN、MMP9 mRNA表达的相关性分析：OPN、MMP9 mRNA的表达呈现明显的正相关关系(r在位=0.511,P=0.036；r异位=0.600,P=0.015)。[结论]1.内异症大鼠异位内膜中高表达的OPN与MMP9蛋白和mRNA与内异症细胞的异位粘附、侵袭密切相关。2.大鼠内异症子宫内膜中OPN表达与MMP9表达存在正相关关系,二者通过相应的信息通路途径,共同促进异位细胞的异位粘附和侵袭。第三部分药物干预对内异症大鼠内膜中OPN/MMP9表达的影[研究目的]检测雌孕激素相关药物干预前后内异症大鼠内膜中OPN/MMP9表达的变化,探讨药物与OPN/MMP9表达的关系以及临床药物治疗病灶局部的可能机制,为临床药物个体化治疗提供依据。[研究方法]1.分组并药物干预：取建模成功的大鼠26只随机分为四组：第一组丙氨瑞林组(GnRH组)6只：丙氨瑞林7.5ug/250g皮下注射每天一次连用一月；第二组：米非司酮组7只：0.62mg/250g每天一次连用一月；第三组：孕三烯酮组7只：0.062mg/次,一周两次连用一月；第四组对照组6只：酵母片半片/天连用一月；2.实验方法：药物干预后一月处死大鼠,取在位、异位内膜,RT-PCR法和免疫组化法测定OPN与MMP9蛋白、基因表达。[研究结果]1.药物干预前、后异位病灶囊肿大小的变化：药物干预后各组异位囊肿的直径均比干预前缩小,但统计学处理无差异；干预前各组囊肿大小组间比较无差异；药物干预后各组囊肿大小组间比较无差异。2.药物干预前后在位、异位内膜中OPN表达2.1干预前后在位、异位内膜中OPN蛋白表达：干预前各组在位内膜OPN表达均低于异位内膜组(P<0.05),药物干预后,米非司酮组、孕三烯酮组及GnRH组中在位内膜和异位内膜中OPN表达无明显差异(P>0.05),没有药物干预的对照组在位内膜中OPN的表达低于异位内膜组织表达(P<0.05)；干预后无论是在位内膜还是异位内膜,OPN表达均较干预前明显降低(P<0.01)；而没有药物干预的对照组中在位、异位内膜中OPN干预后亦低于干预前(P<0.05)。2.2药物干预前后各组间OPN蛋白表达变化：干预前在位、异位内膜中各组间两两比较无明显差异(P>0.05)；干预后,在位内膜对照组高于米非司酮组及GnRH组,而与孕三烯酮组无差异；异位内膜中对照组高于其他三组,而米非司酮组与孕三烯酮组及GnRH组比较无明显差异(P>0.05)；孕三烯酮组与GnRH组比较有显著性差异(P<0.05)3.药物干预前后在位、异位内膜中MMP9蛋白表达3.1干预前后各组在位内膜、异位内膜中MMP9表达：药物干预前各组在位内膜中MMP9表达明显低于异位内膜(P=0.000)；药物干预后干预组在位与异位内膜MMP9表达无差异(P>O.05),对照组本身异位内膜中MMP9表达仍高于在位内膜的表达(P<0.05)；3.2药物干预前后在位、异位内膜中MMP9蛋白表达差异：干预前各组间两两比较MMP9表达在位、异位内膜中均无差异(F在位=2.827,P=0.062；F异位=2.790 P=0.064)；干预后米非司酮组、孕三烯酮组及GnRH组在位、异位内膜中MMP9表达均较对照组明显降低；米非司酮组在位、异位内膜中MMP9表达均低于孕三烯酮组、高于GnRH组在位内膜但低于异位内膜(P<0.001)；在位内膜中孕三烯酮组与GnRH组比较无差异；异位内膜中GnRH组明显低于孕三烯酮组和米非司酮组(P<0.001)。4.药物干预前后在位、异位内膜中OPNmRNA、MMP9mRNA表达4.1药物干预前后在位、异位内膜OPN mRNA表达：干预前在位内膜中OPN mRNA表达低于异位内膜(P<0.001),但各组间两两比较在位、异位内膜中OPNmRNA表达无差异(F在位=0.970,P=0.428；F异位=1.947,P=0.156)；干预后对照组在位、异位中OPN mRNA表达高于其他三组在位与异位内膜中表达(F在位=9.365, P=0.001; F异位=25.249, P=0.000)米非司酮组、孕三烯酮组及GnRH组两两比较均无差异；4.2药物干预前后在位、异位内膜MMP9 mRNA表达：干预前在位内膜中MMP9mRNA表达低于异位内膜(P<0.001),但各组间两两比较在位、异位内膜中MMP9mRNA表达无差异；干预后MMP9 mRNA在位、异位内膜中表达对照组明显高于各干预组(p<0.001)；组间两两比较在位无差异而异位中孕三烯酮组低于米非司酮组和GnRH组(P<0.001)。[结论]1.药物干预治疗改变了病变局部的分子生物学环境,但对异位囊肿的大小影响不大。2.药物干预治疗通过改变病灶局部OPN和MMP9表达水平,调节异位细胞的粘附和侵袭而发挥疗效。3.药物抑制病灶局部OPN/MMP9表达上,GnRH-a组优于米非司酮组和孕三烯酮组,异位内膜中的效果优于在位内膜中。更多还原

【Abstract】 BackgroundThe endometrosis (EMS) is a common disease in reproductive age women, followed by clinical signes of 80% chronical pelvical pain,30% of gynecological operations and 20% of infertility.Although studys from theory and clinics show that EMS is successive homonal depended pathological changes, beginning from cells to the pelvic pain, infertility and pelvical mass in clinics. However, it’s a difficult question in clinics with unclear mechanism and limited treatment. Operation is the first choise for its treatment. But it is differcult to remove all the signs and it is not superise to be recurrance. Operation combined medicine treatment can inhabbit the rest of the disease, delay the reocurance, and it shows that the medicine for anti-adherisve, anti-invasive, ant-angiogenesis is the next target for the endometriosis. Therefore, further study for the mechanism of ectope adhersion and invasion as well as effective signal molecule will be useful to research the medicine target and latter medicine treatment,and instruction the cilincal treatment and follow-up.Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two cell adhesion molecules avb3-integrin and CD44, is an acidic hydrophili glycophosphoprotein. Potential steps that may involve OPN include tumor cell attachment to basement membrane through cell-surface adhesion molecules, proteolytic degradation of the extracellular matrix (ECM) and tumor cell migration through the ECM. Studys from Zhu Yaokui and Ma Chaihong has identified that OPN protein appeared higher in ectopic and uctopic endometrium in patients with endometriosis;Matrix metalloproteinase-9(MMP-9), the largest enzyme for degradation of extra cellular matrix, is playing an important role involved in adherision,implantation and growth of ectopic endometrium cells. Investigators has showed that MMP-9 plays a direct role in OPN-induced cell migration, invasion and tumor growth and that demonstrates(see the film attached). Although endometrosis is a benige desiease, it has clear malignant behavior such as invasion, transfer and recurance. However, it is nover to be reported about OPN and MMP-9 in endometriosis.Experiments confirmed:OPN gene 5’upstream side of the area pregnant hormone regulatory elements. Progesterone receptor levels can be adjusted OPN. The MMP-9 upstream of estrogen binding region. Or side by estrogen pathway factor regulating the level of MMP-9. Our previous study also showed that OPN and MMP9 were expressed in patients with endometriosis and normal eutopic and ectopic endometrium. And ectopic endometrium was significantly higher than the normal control group. Prompted the development of both play an important role in the EMS. But also by the regulation of ovarian estrogen and progesterone. Thus, we infer whether estrogen and progesterone by regulating the expression of OPN and MMP9 play a role in cell adhesion?GnRH-A, high progesterone preparations gestrinone and its receptor antagonist mifepristone are the clinical treatment of endometriosis in first-line drugs.Studies have shown that these drugs through the regulation and inhibition of the hypothalamus - pituitary - ovarian axis function competitive inhibition of the local receptor binding reduce the concentration of estrogen in the local lesions and cause ectopic disease atrophy. These drugs can also directy effect on the local lesions, transforming the molecular biology environment of Pathological changes inhibiting cell apoptosis and increaseing cell apoptosis ability.But the exact mechanism is unclearAnd the different mechanisms of drug action.In particular, whether the change in drug treatment in the molecular mechanism of membrane is not clear.The topic to be selected the changed expression of OPN/MMP9 when leuprolide, gestrinone other hormone-related drugs treating ectopic and eutopic for research purposes. Through the establishment and evaluation of rat endometriosis model. Using experimental methods such as RT-PCR. Discussion of changes in drug intervention organization OPN/MMP9 protein and mRNA. Further revealed endometriosis in the OPN and the relationship between the development of EMS. The possible mechanism of drug action,The drug of choice for clinical treatment of a reasonable and reliable basis and more indicators of prognosis. Part OneEstablishment and Estimate of an Endometriosis Model by Openning- method in RatsObjectiveTo research the reliability, estimate index and achievement ratio of an endometriosis model by openning-method in rats compared with transfering-method..MethodFemale virginal rats were operated with openning-method (lefe uterus) and transfering-method (right uterus). In left side of the uterine, endometrum was stick to the uterosacro ligament or omentum majus while right side of the uterine was taken and the endometrium was implanted to the right abdominal wall. Second operation was taken 4 weeks after implantation. Samples from the rats were examined by OPN antibody and MMP9 antibody.Results1.The achievement ratio in both mothed model:The achievement ratio of openning-mothed model is 81.08%, lower than that of 83.78% in transpering-method model without statistical significiance.2.The different signs in both mothed model:Clear vesicle is main gross appearance with 64.8% of all rats, the dark bule nodus are the second appearance with 10.81% in transfer-method model.However, the dark bule nodus is the main in openning-method model with 35.13% while Clear vesicle is the second with 24.3%. Extensive adherent rate in transfering-method model is 18.9%, significient lower that of 64.86% in openning-method model (P<0.01).There is no different in both intestinal obstruction and infection rate between two groups (P>0.05).3.The comparion between clinical manifestation and microscope features:the typical and atypical gland and endometrial cells could be found in 72,7% nodules or vesicles, and 68.4% flame signes with a strong staining of OPN and MMP9 in the ectopic endometrium.Conclusion The appearance of animal endometriosis in openning-method model is more similar to the human endometriosis in appearance with feature of reliable, large eara, convenient and less complication. Part Two: The Expression and Significiance of OPN &MMP9 in Endometriosis of RatsObjectiveAnalysis of differences between OPN and MMP9 endometriosis eutopic and ectopic lesions in the protein, gene in rats. Discussion of OPN and MMP9 expression in ectopic endometrium features and the relationship between OPN expression and MMP9 in endometriosis.MethodSuccess of the 37 pre-built model rats, the second surgery laparotomy after the model 28 days, the rat’s reign (reigned group), ectopic endometrial tissue (ectopic group) and eutopic endometrium former model (control group), determination of the expression of intima OPN and MMP9mRNA, protein.Results1.OPN mainly highly expressed in the plasma membrane of epithelial cellsand a small amount of expression of interstitial.But MMP9 was mainly expressed in the cytoplasm of glandular epithelial cells and no expression in the nucleus;2. Immunohistochemistry showed that OPN expression in ectopic endometrium IOD of 53735.4±4896. Group was significantly higher than that of eutopic 33733.8±5322 and a control group of 37291.2±4588. (T reign-ectopic=17.010; P=0.000; t control-ectopic=9.122; P=0.000). Control group and the reign of group no significant difference in IOD (t=1.973, P=,0.52);3. The IOD of MMP9 expressed in ectopic endometrium group was 32645±9967. Higher than that of eutopic endometrium and control groups(T reign-ectopic= 8.559; P=0.000; t control-ectopic=5.386; P=0.000), while in office and control groups showed no significant difference (t=0.198, P=,0.844).4. The expression of OPN and MMP9 significant positive normal relationship. (R reign=0.511, P=0.036; r ectopic=0.600, P=0.015).Conclusion 1.The expression of OPN and MMP9 in eutopic and ectopic endometrium in rats are highly;2.The expression of OPN and MMP9 correlated with positive relationship. They may exist in the regulation of cell information between cascade regulation. Part ThreeThe Impact of Estrogen-related Drugs on the Expression of OPN/MMP9 in Rat Endometrium of EndometriosisObjectiveAnalysis of the relationship between estrogen and progesterone-related drugs and the expression of OPN/MMP9 in rat endometrium of endometriosis. Explore the possible mechanism of the relationship between Estrogen and progesterone and OPN/MMP9 and clinical drug treatment. Individual drugs for clinical therapy.Methods1.24 cases of successful model rats were randomly divided into four groups.There are 6 cases in groupⅠof Wagner Ruil:The dose of 7.5ug/250g was done by subcutaneous injection for every day till one month. There are 7 cases in groupⅡ:The mifepristone group:0.62mg/250g/day for a month. There are 7 cases in groupⅢ:gestrinone group:0.062mg/250g/day twice a week for one month. Control group, There are 6 cases in groupⅣ:yeast tablets.2..Rats were killed after one month, take eutopic and ectopic endometrium, RT-PCR, and immunohistochemical determination of OPN and MMP9 protein, gene expression.Results1. The diameter in each group after drug:The diameter of the cysts in each group after drug intervention reduced than before. But there was no difference between treatment.2. The OPN protein expression before and after drug intervention in eutopic and ectopic endometrium:Before the intervention, OPN expression in eutopic endometrium in each group were lower than in ectopic endometrium (P<0.05). The OPN express in drug intervention group eutopic and ectopic endometrium difference disappeared. However, the expression of the control group remained below eutopic endometriotic tissues (P<0.05). OPN expression of each group after the intervention and control group at eutopic and ectopic endometrium significantly lower than before intervention. (P intervention<0.01, P were<0.05). Before the intervention, eutopic and ectopic endometrium in each group showed no significant difference between two(F reign=0.161, P=0.921, F ectopic=2.550, P=0.082). After the intervention, eutopic endometrium (F=3.45 P=0.034) higher than the control group compared mifepristone group and GnRH groups. Ectopic endometrium in the control group was higher than the other three groups. Between GnRH group and Gestrinone mifepristone group and the group no significant difference.3. The MMP9 express in eutopic and ectopic endometrium after and before drug intervention:Each group before drug intervention MMP9 expression in eutopic endometrium was significantly lower than in ectopic endometrium (P=0.000). Drug intervention group, MMP9 expression in eutopic and ectopic endometrium was no difference (P> 0.05). Ectopic endometrium in the control group, the expression of MMP9 expression is still higher than in eutopic endometrium (P<0.05). MMP9 expression in each group before the intervention in place, there were no differences in ectopic endometrium. (F reign=2.827, P=0.062; F ectopic=2.790 P=0.064) Mifepristone group, gestrinone group and GnRH after the interventiond eutopic and ectopic endometrium in MMP9 expression was significantly lower than control group. MMP9 in mifepristone group eutopic and ectopic endometrium was ower than the Gestrinone group. Gestrinone group compared with the GnRH group was no difference in the eutopic endometrium. However, MMP9 in ectopic endometrium was 13291±2914, higher than the GnRH group,3951±265 (P=0.00).4.The expression differences at before and after intervention in eutopic and ectopic endometrium4. OPNmRNA, MMP9mRNA expression in endometrium:4.1 OPNmRNA expression:Before the intervention, OPNmRNA expression in eutopic endometrium wa significantly lower than ectopic endometrium. (P<0.001). However, compared with the reign and the reign of each group、eutopic and ectopic no difference (F reign=9.365, P=0.001; F ectopic=25.249, P=0.000); After the intervention of any two control groups in eutopic and ectopic expression in OPNmRNA higher than the other three groups.However the mifepristone group, gestrinone group, GnRH group had no difference in the three groups. 4.2 MMP9mRNA expression:Before the intervention, eutopic and ectopic endometrium compared P<0.001. the expression of MMP9 mRNA in the intervention group eutopic and ectopic endometrium was no difference. The control group significantly (p<0.001).After the intervention, MMP9mRNA expressed in eutopic and ectopic endometrium were lower than the control group. In gestrinone group ware lower than in the mifepristone group and the GnRH group (P<0.001) and no differences among other groups.Conclusion1.Drug intervention changed the environment for local lesion of the molecular biology, however, the size of the cysts had little effect.2.By changing the focus of drug intervention of local expression of OPN and MMP9. Play the treatment by adjusting adhesion and invasion of the ectopic cell.3.In the drugs inhibit the expression of OPN/MMP9 in the local lesions, GnRH-a group better than the mifepristone group and gestrinone group, the ectopic endometrium better than the eutopic endometrium.更多还原