The Study Of Peritoneal Mesothelial Cells And TGF On The Pathogenesis Of Endometriosis Associated Adhesion

Abdomino-pelvic adhesions represent the common and the typical symptom in endometriosis. Such adhesions can cause complications such as pelvic pain, infertility, and cause an increase repeat surgery risk and more complexity for future surgery. The characteristics of adhesion formation in endometriosis include that:1.Endometriosis has the high rate of adhesions, more than80%endometriosis have adhesions formation preoperatively, and nearly100%recurrent endometriosis have adhesions formation.2. Endometriosis has stricky adhesion, which makes the anatomical structure of pelvic organs unclear, and leads to increase in the potential for further functional injury to organs within the peritoneal cavity.3. Adhesions cause the variation of pelvic structure, which could leads to pelvic pain, fertility, and encapsulated fluid.4. Postoperative adhesions reformation could be increased in endometriosis, which also cause complicated repeat surgery. However, the mechanisms of adhesions formation in endometriosis are not really clear. Therefore, the study of development and various associated factors in endometriosis adhesions formation, could represent a significant clinical advance in further understanding of the pathogenesis of endometriosis, and in the search for therapeutic intervention to prevent adhesion formation.The peritoneum is lined by mesothelial cells loosely attached to the basement membrane. Injury to the peritoneum, leads to the disruption of mesothelial cells, and exposure of underlying extracelluar matrix(ECM), and then, a local inflammatory reaction and increased chemoattractant in blood vessels supplying the damaged area, attract normal mesothelial cells around to the damage area and develop new mesothelium from islands of mesothelial cells, which later peoliferate into sheets of cells. The process of peritoneal healing concludes different biochemical processes, such as inflammation, coagulation, revascularization, fibrin and ECM organization, remesothelialization. The proliferation of mesothelial cells play a vital role in peritoneal healing. However, if the normal healing process is impaired, the adhesion will develop.Peritoneal micro-environment, composed of peritoneal mesothelial cells(PMCs), peritoneal fluid(PF), and various of cells and cytokines, play a role in the development of endometriosis and associated symptoms such as infertility and pain. Investigators have proposed that patients with endometriosis have an altered peritoneal micro-environment. It appears that a composite theory of retrograde menstruation with implantation of endometrial fragments in conjunction with peritoneal factors to stimulate cell growth is the most widely accepted explanation for peritoneal endometriosis. Evidence of the key role of PMCs in peritoneal healing and adhesiogenesis falls into explored. Mesothelial cells are capable of regulating, producing, and secreting several enzymes, and are essential in the local peritoneal regulation and production of components from the fibrinolytic system. The mechanisms of peritoneal adhesions formation in endometriosis have been unexplored.Transforming growth factor(TGF)-β acts as chemoattractant of inflammatory cells, and stimulates angiogenesis and regulates the expression of various components of extracellular matrix, directs collagen and fibronectin biosynthesis, is normally found in macrophages, endometrial cells. There are three isoforms of TGF-β:TGF-β1, TGF-β2, and TGF-β3, found in mammals. After activation, the TGF-βs bind with high affinity to TGF-β receptor (TbR)Ⅱ that phosphorylates TbRⅠ, and then, propagate a downstream signaling cascade through phosphorylation of receptor associated Smads, lead to fibrosis. TGF-β1is a mainly potent inflammatory cytokine, by acting as a chemoattractant and stimulatory factor for monocytes and macrophages, and immunosuppressor agent, while profoundly inhibiting T and B lymphocyte activation and natural killer cell proliferation. Oosterlynck et al approved that the peritoneal fluid of women with endometriosis have demonstrated increased TGF-β1activity. It is suggested that increased TGF-β1activity in PF was related to the decreased NK activity of PF in women with endometriosis.In recent years, with the leadership of professor Lang, a systematic study had been focused on the role of eutopic endometrium in the pathogenesis of endometriosis, and had made much breakthrough, which systematically demonstrated the differences between the eutopic endometrium of endometriosis and the normal endometrium, in ultrastructure, gene expressions, and functional status, and differences in adhesion, invasion, angiogenesis. With the impact of ectopic endometrial cells on PMCs, leads to abnormality of the peritonieum and mechanisms of peritonieum repair, and leads to peritoneal adhesions formation. In our study, we examined whether the PMCs of endometriosis is different from the normal or not, what the effect of TGF-β1on PMCs, what the effect of the ovarian steroid on TGF-β1, what the effect of TGF-β1neutralizing antibody on the development of EMs and peritoneal adhesions formation in the nude model of endometriosis.Objective1. Demonstrate the difference of PMCs between endometriosis and controls2. Demonstrate the effect of TGF-β1on cell viability and celluar cycles of PMCs3. Demonstrate the effect of estrogen and progesterone on the expression of TGF-β1,Smad3,Smad7of PMCs4. Demonstrate the effect of TGF-β1neutralizing antibody on the development of endometriosis and peritoneal adhesion formation in the nude model of endometriosisMaterials and Methods1. Isolated and cultured human peritoneal mesothelial cells (HPMCs) from the lateral pelvic peritoneum2. Detected the effect of TGF-β1on the viability of PMCs by MTT3. Detected the effect of TGF-β1on the proliferation of PMCs by flow cytometry4. Detected the effect of ovarian steroid on the TGF-β1and Smad3,7of PMCs by immunochemistry, Real time-PCR, and Western blot5. Detected the effect of TGF-β1neutralizing antibody on the development of EMs lesions and the peritoneal adhesions formation in the nude model of endometriosisResults1. Human peritoneal mesothelial cells (HPMCs) from the lateral pelvic peritoneum in endometriosis were succefully isolated and cultured in vitro, the same part of HPMCs in non-endometriosis were hardly cultured in vitro2. TGF-β1decreased the viability of HPMCs, especially when the concentration of TGF-β1was less than2.5ng/ml3. TGF-β1inhibited the proliferation of HPMCs, especially when the concentration of TGF-β1was less than2.5ng/ml4. The TGF-β1was regulated by the ovarian steroids,17β-estradiol promoted the expression of TGF-β1in PMCs5. Progesterone inhibited the expression of TGF-β1in HMrSV5, and promoted the expression of TGF-β1in HPMCs in endometriosis, and promoted the expression of Smad7in peritoneal mesothelial cell.6. The nude model of endometriosis was established successfully; The neutralizing TGF-β1antibody decreased the development of EMs lesions and associated adhesions in nude model, had no effect on the extent of adhesions formationConclusions:1. The HPMCs in macroscopically normal peritoneum of endometriosis is more easily cultured in vitro than the normal HPMCs, suggesting that changes of HPMCs in EM might be associated with high adaptability to the enviroment2. TGF-β1has the effect on the viability and proliferation of HPMCs, which further verifies the effect of cytokines in peritoneal cavity on the HPMCs, leads to the abnormality of HPMCs which could be an important process to peritoneal adhesion formation3. The expression of TGF-β/Smad in HPMCs is regulated by the ovarian steroids, it implies that peritoneal adhesion formation might be related with the abnormal inter-action between the adhesion cytokines and local estrogen4. The neutralizing TGF-(31antibody could decrease the development of EMs associated adhesion in nude model, had no effect on the extent of adhesion formation, suggesting that TGF-β1play a role in the process of the earlier EMs development, the neutralizing TGF-(31antibody could decrease the occurrence of adhesion by blocking the inhibited effect of TGF-β1on HPMCs, TGF-β1could be a target in the future of preventing and treatment of EMs associated adhesions, it is deserved to be further explored...