【作者】 侯勇丽；

【导师】 郝敏；

【作者基本信息】 山西医科大学 ， 妇产科学， 2009， 硕士

【摘要】 研究背景:子宫内膜异位症(Endometriosis, EMs)简称内异症,发病率约占生育年龄妇女的15%,近年来有明显上升趋势。内异症所引起的痛经、下腹痛及不孕症,严重地影响了妇女的健康和生活质量,其病变广泛,形态多样,且有浸润性生长、广泛种植转移和易复发的恶性生物学行为,是一种常见且难治的妇产科疾病。目前诊断主要以微创的腹腔镜检查为金标准,但缺乏特异性的生化指标;治疗方法主要采用外科去势手术和激素治疗,这些方法虽对减轻症状,促进妊娠有一定帮助,但效果有限,副作用明显,且易复发,无法根治。影响内异症临床治疗效果的主要原因是本病的发病机制不清楚。因此,内异症发病机制的研究是当前妇产科基础研究的热点之一。己有的研究表明异位内膜的转移、种植、生长等类似肿瘤转移的生物学行为是内异症发病关键。核转录因子kappaB(NF-κB)是一种广泛存在的多效性的核转录因子,为Rel蛋白家族成员,由多肽P50和P65亚基形成同源或异源性二聚体。在静息细胞,NF-κB主要位于细胞浆,与具有抑制作用的IκB家族结合,其锚蛋白重复序列掩盖了NF-κB的DNA结合位点而干扰NF-κB的功能,在受到生理或有害刺激后,依靠信号传导系统由无活性状态转变为活性状态,激活后的NF-κB从NF-κB-IκBs复合物中解离,进入细胞核,与相应的靶基因结合,启动转录。其活化过程不需要合成新的蛋白,故能及时引起细胞反应早期的功能变化,继发引起其他因子的产生和活化。NF-кB可高效诱导多种前炎性细胞因子、白细胞粘附分子、单核细胞趋化因子等基因的表达,同时对参与炎症级联及放大效应的多种酶的基因转录也具有重要的调控作用。NF-кB的过度激活可引起一系列炎症相关性疾病,如哮喘、类风湿性关节炎、肠炎、癌症等,已引起人们的高度重视。上皮细胞来源的中性粒细胞活化肽-78(简称ENA-78)是一种重要的趋化因子及血管形成因子,其可趋化中性粒细胞到达病灶以及直接促进异位内膜细胞的增殖,近年来对其在内异症发病中的作用日益受到重视。研究表明,ENA-78是NF-кB的下游基因,是受到NF-кB调控的基因。但是NF-кB对ENA-78的调控在内异症中是否起作用尚不清楚。第一部分子宫内膜异位症体外细胞模型的建立研究对象正常妇女的子宫内膜细胞;子宫内膜异位症患者的在位及异位内膜细胞目的探索人正常、在位及异位子宫内膜细胞体外原代及传代培养的方法,建立子宫内膜异位症体外细胞模型方法收集子宫内膜异位症和正常妇女的在位及异位子宫内膜,采用胶原酶和铜网过筛法及差时贴壁等方法分离子宫内膜基质细胞和腺上皮细胞,进行单层培养。倒置显微镜观察细胞生长形态,免疫细胞化学进行细胞鉴定,MTT法绘制细胞生长曲线。结果子宫内膜腺上皮细胞(Endometrial epithelial cell ,EEC)和基质细胞(Endomet -rial stromal cel1 ,ESC)分别经角蛋白单抗和波形蛋白单抗组化染色为阳性,腺上皮可持续培养4-6周,传代2-3次;基质细胞可持续培养5-8周,传代4-6次。结论采用胶原酶消化、筛网二次过滤和贴壁纯化等方法,分离出高纯度的子宫内膜腺上皮细胞和基质细胞而没有细胞特征的改变。可为研究子宫内膜异位症相关病理生理机制提供满意的体外细胞平台。第二部分NF-KBp65蛋白、ENA-78在子宫内膜异位症发病中的作用研究目的检测核因子kappaBp65蛋白及ENA-78在内异症患者在位内膜、异位内膜和正常子宫内膜中的表达情况,探讨上述因子与内异症发病的关系。方法(1)体外传代培养至第3代的在位、异位及正常内膜细胞分别给予不同浓度的IL-1β及IL-1β+二硫氨基甲酸肽吡咯烷(PDTC)进行干预。用酶联免疫吸附双抗体夹心法(ELISA)检测三组子宫内膜培养上清液ENA-78浓度及IL-1β、IL-1β+PDTC对各组内膜基质细胞分泌ENA-78浓度的影响。(2)用免疫细胞化学方法检测三组子宫内膜细胞NF-KBp65蛋白的活化程度及IL-1β、IL-1β+PDTC干预下各组内膜基质细胞NF-KBp65蛋白的活化程度的改变。结果(1)在位及异位子宫内膜细胞培养上清液中ENA-78浓度明显高于正常子宫内膜细胞,差异有显著统计学意义(P<0.01);在位及异位子宫内膜细胞之间比较,异位子宫内膜细胞培养上清液中的ENA-78浓度高于在位子宫内膜细胞,差异有显著统计学意义(P<0.01),在分别给予IL-1β、IL-1β+PDTC干预后,正常子宫内膜细胞上清液中的ENA-78浓度无明显变化,差异无统计学意义(P>0.05);而在在位及异位内膜细胞的上清液中,ENA-78浓度在IL-1β组最高,明显高于对照组及IL-1β+PDTC组,差异有显著统计学意义(P<0.01)。(2)在子宫内膜细胞、在位及异位子宫内膜细胞这三组中,在位内膜细胞及异位内膜细胞的NF-KBp65蛋白的活化程度显著高于正常子宫内膜细胞,差异有统计学意义(P<0.05);在位及异位子宫内膜细胞NF-KBp65蛋白的活化程度IL-1β组最高,显著高于对照组及IL-1β+PDTC组,差异有统计学意义。IL-1β能显著诱导内异症异位及在位内膜间质细胞NF-кB的活化,这种作用能被PDTC所抑制。PDTC能明显抑制IL-1β诱导内异症异位及在位内膜基质细胞分泌ENA-78的作用。结论(1)NF-кBp65、ENA-78可能在内异症发病中起作用。(2)IL-1β可诱导在位及异位子宫内膜细胞NF-кBp65活化,使ENA-78的表达增高。PDTC可抑制此作用,说明NF-кBp65做为关键性的调控因子,可能在内异症的发病过程中起到“闸门”的作用,对ENA-78以及其它细胞因子起到中枢样调控作用。(3)在位内膜本质的先天性差异可能是内异症发病的关键。更多还原

【Abstract】 Background: EMs is short for endometriosis, whose morbility is 15% between women of reproductive ages. The number rises significantly in recent years. The dysmenorrheal, hypogastralgia and barrenness caused by EMs, severely affect women’s health and life quality. It spreads widely and changes variously. It has the biological behaviors of infiltrative growth, extensive implantation metastsis and easy recurrence, all of which are cacoethic. It is a common but incurable disease in the department of gynaecology and obstetrics. Abdominal speculum, which is the gold standard at present, is the main diagnosis method now, but it lacks specific biochemical indicator. At present, the treatment mainly depends on surgery and excitatory autacoid, which could relief symptoms and facilitate pregnancy, but its effect is limited and its side effect is obviou. Thus, it is not a radical cure. The main factor that influences the therapeutic efficacy is that its pathogenesis is not cleat till now. Therefore, the study on the pathogenesis of EMs is a hot field of the current elementary study of gynaecology and obstetrics. The existing study indicates that criticality of the EMs’onset is metastasis, implantation, growth and such biological behaviors of ectopic endometrium, which are similar to tumor metastasis.Nuclear factor kappaB(NF-KB) is a common existing and pleiotropy nuclear factor. It’s a member of the Rel protein family, whose homodimer or heterodimer is formed by polypeptide P50 and P65. In the resting cell, NF-KB is mainly located in cytolymph, which combines with IKB family who has depressant effect. Its ankyrin repeated sequence, by masking the binding site of NF-KB’s DNA, interferes the function of NF-KB’s. Stimulated by the physiological or harmful factors, it transforms from inactive phase into active phase through the signal transducting system. The activated NF-KB goes into the cell nucleus after its decollement from NF-KB-IKBs, and combines with the propotional target organ. The transcription starts at last. As the activated process needs no composition of new protein, it can timely provoke pristine functional change in the process of the cell reaction, and then provokes the creation and activation of other agents’. NF-KB can efficiently induce the gene expression of various ante-inflammatory cytokine, leucocyte adhesion molecule, monocyte chemotatic factor and so on. Meanwhile, it also has a significant controlling effect on the genetic transcription of many enzymes that participate in the inflammation-cascade and amplifying effectiveness. The excessive activation of NF-KB could provoke a series of inflammation related diseases, such as asthma, rheumatoid arthritis, enteritis, cancer, etc, which have already aroused high attention of human beings.The ENA-78, coming from epithelial cell, is a significant chemotactic factor as well as a angiogenesis factor. It can drive neutrophilic granulocyte to get to the focus of infection and facilitate the cell proliferation of ectopic endometrium. In recent years, more and more attention has been paid to its effect on the onset of EMs.According to various study, ENA-78 is NF-KB’s subtus gene, and is controlled by NF-KB. However, it is still not clear whether the NF-KB’s control on ENA-78 in the EMs is effective or not.Part ? Establishment of the exoteric cell model of EMs’Objective: To explore the method of exoteric primary cultivation and serial subcultivation of endometrial cells with normal, eutopic or ectopic states. To establish a exoteric cell model of EMs’.Methods: Collect the endometria of women with EMs, as well as eutopic and ectopic endometria of normal women. Use the collagenase and copper screen filtration to extract the endometrial epithelial cells and the endometrial stromal cells; adopt monolayer culture. Observe the growth morphous of the cells; identify cells by SABC; draw curves about the cell growth by MTT.Results: The EEC and ESC, stained by keratin single antibody and vimentin single antibody immunohistochemistry, appeare masculine. The EEC can be cultivated for 4 to 6 weeks and has a passage of 2 to 3 times; the ESC can be cultivated for 5 to 8 weeks and has a passage of 4 to 6 times.Conclusions: After the filter of collagenase and grit, highly purified EEC and ESC can be segregated without any change of the feature of the cells. It can offer a satisfying platform of exoteric cell for the study of the EMs-related pathomechanism and physiological mechanism. Part ?? The study of the effect of NF-KBp65 protein and ENA-78 in the onset of EndometriosisObjective: To detect the expressions of kappaBp65 and ENA-78 in the EMs’eutopic endometrium and ectopic endometrium as well as in the normal endometrium; to explore the relationship between the agents mentioned above and the onset of EMs.Methods: (1) The eutopic endometrium’s, ectopic endometrium’s and the normal endometrium’s cells, which are in third passage, were interfered by IL-1βand IL-1βadding PDTC with different concentration. Use ELISA to detect the concentrations of ENA-78 of those three groups, explore the effect of IL-1βand IL-1βadding PDTC to the concentration of ENA-78 of each group. (2) Use immunohistochemistry to detect the activating degree of NF-KBp65 of the endometrial cells in the three groups, to detect the change of NF-KBp65’s activating degrees after intervention of IL-1βand IL-1βadding PDTC.Results: (1) The normal endometrium’s concentration of the ENA-78 in the cells’culture media’s supernatant is remarkably thicker than the eutopic endometrium and the ectopic endometrium’s. There is a remarkable difference(P<0.01). The ectopic endometrium’s concentration of the ENA-78 in the cells’culture media’s supernatant is remarkably thicker than the eutopic endometrium’s. There is a remarkable difference(P<0.01). After the intervention, the concentration of the ENA-78 in the endometrial cells’culture media’s supernatant has no remarkable change. There is no remarkably difference(P>0.05). In the eutopic endometrium’s and the ectopic endometrium’s cells’culture media’s supernatant, the concentration of the ENA-78 in the group intervened with IL-1βwas the thickest. It is remarkably thicker than the control group and the group intervened by IL-1β+PDTC. There is a remarkably difference(P<0.01).(2) In three groups, the NF-KBp65’s activating degrees of eutopic and the ectopic endometrium are remarkably higher than the normal endometrium’s. The difference is statistically significant (P<0.05); for the NF-KBp65’s activating degrees in the eutopic endometrium’s and the ectopic endometrium’s cells, the IL-1βgroup’s is the highest, which is remarkably higher than the controlled group and the group intervened by IL-1β+PDTC. The difference is statistically significant) (P<0.05). IL-1βcan remarkably induce EMs’ectopia and the NF-KB’s activation of Leydig cell in eutopic endometrium. This induction can be inhibited by PDTC. IL-1βcan induce EMs’ectopia and the secretion of ENA-78 in the Leydig cell of eutopic endometrium. This induction can be remarkably inhibited by PDTC. Conclusions: (1) NF-KBp65 and ENA-78 may play a part in the onset of EMs. (2) IL-1βcan induce the activation of NF-KBp65 on eutopic and ectopic endometrium cells. It can strengthen the expression of ENA-78, while PDTC inhibits this effect. It suggests that as a key controlling agent, NF-KBp65 may play a penstock role in the onset of EMs, and it may have a controlling effect on ENA-78 and other cytokines. (3) The inborn differences of eutopic endometrium may be the criticality of the onset of EMs.更多还原