The Study Of The Effect Of NF-KBp65 Protein And ENA-78 In The Onset Of Endometriosis

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.