| Endometriosis is a common, benign, oestrogendependent, chronic gynaecological disorder. The prevalence of pelvic endometriosis approaches6-10%in the general female population; in women with pain, infertility, or both, the frequency is35-50%. Symptoms of endometriosis include reduced fertility and several types of pain such as severe dysmenorrhea, deep dyspareunia, dyschezia, and chronic pelvic pain. Most commonly used gonadotropin-releasing hormone(GnRH) agnonists seem generally efficient for alleviating the painful symptoms because endometriotic lesions are hormone dependent, but the time for use of these agents is limited, and for most serious cases the painful symptoms may reappear even under medical treatment.TRPV1(Transient Receptor Potential Vanilloid Type1, TRPV1) is a receptors controlled non-selective cation channel expressed in nociception, is a member of TRP(transient receptor potential, TRP) family. The prominent role of TRPV1in nociception has been illustrated in studies with TRPV1null mutant mice. In these studies, TRPV1-/-mice don’t show thermal hyperalgesia after mustard oil or complete Freund’s adjuvant-induced inflammation.The role of TRPV1in occurrence,conductance and sensitization of pain in central nervous system(CNS) has been conducted massively, but it hasn’t been reported in ectopic endometrial stromal cells(ESC). Considering the inflammatory feature of endometriosis, we conduct this study to investigate the possible reason of endometriosis associated pain from its origin. Part I The expression of TRPVl in endometriosisObjectives:To investigate the expression of TRPV1in endometriosis and to determine its correlation with severity of pain.Methods:Ectopic endomemetrium from68Patients with endometriosis and endometrium from24women without endometriosis were included for this study when patients underwent surgery at Shanghai OB/GYN Hospital, Fudan University Shanghai Medical College from2007to2009. The expression of TRPV1in ectopic and normal endometrium was assessed with immunohistochemistry and TRPV1immunoreactive nerve fibres in ectopic endometium was assessed with immunofluorescence.Results:1.The TRPV1expression was significantly higher in ectopic endometrium of endometriosis than normal endometrium (p<0.05). The TRPV1expression was significantly different among women complained of pain and no pain in ectopic endometrium(p<0.05). The TRPV1expression in ectopic endometrium was positively correlated with severity of pain(p<0.05).2. The positive rate of TRPV1immunoreactive nerve fibres in ectopic endometrium was higher in women with endometriosis who had pain symptoms (53.1%) than those who had no pain symptoms(26.3%); The mean density (x±D/mm2) of TRPV1-immunoactive nerve fibres in ectopic endometrium in women with and without pain was0.2±0.12and0.4±0.27, respectively; both are correlated with severity of pain.Consusions:The TRPV1immunoreactivity and TRPV1immunoreactive nerve fibres in ectopic endometrium are both correlated with severity of pain. Part Ⅱ The effect of PGE2and TNF-a on the expression of TRPVl in primarily cultured ectopic ESCObjectives:Endometriosis is an inflammatory disease.women with endometriosis have an increased concentration and activation of peritoneal macrophages and polymorphonuclear leukocytes, which can mediate the release of prostaglandins(PGs) and cytokines. And correlations have been found between pain severity and the proinflammatory cytokines,prostaglandins, chemokines,NO,etc. It had been proved that PGE2and TNF-a concentrations were increased in peritoneal or ovarian endometrioma fluid of women with endometriosis. In the first part, we found the TRPV1expression was higher in ectopic endometrium of endometriosis than normal endometrium, so in this part we will evaluate the impact of proinflammatory factors PGE2and TNF-a on the expression of TRPV1in prmary cultured ectopic ESC.Methods:The ectopic endometrial stromal cells were isolated and cultured in vitro, then exposed to PGE2(10-8M) or TNF-α(1uM). And the expression of TRPV1was ananlyzed by Realtime PCR and Western Blot.Results:The expression of mRNA and protein of TRPV1in ectopic ESC were significantly increased after the treatment of PGE2or TNF-a.Consusions:Proinflammatory factors of PGE2and TNF-a could increase the expression of TRPV1in ectopic ESC. Part Ⅲ The electrophysiological characteristics and function of TRPV1in ESCObjectives:It have been massively conducted for the electrophysiological characteristics and function of TRPV1in neurons, but it hasn’t been reported in ESC. So in this part we studied the basic electrophysiological characteristics and pain related function of TRPV1in membrane of ectopic ESC.Methods:The ectopic and normal endometrial stromal cells were isolated and cultured in vitro. Capsaincin(CAP), a specific agonist of TRPV1, induced membrane currents were recorded with whole-cell membrane patch-clamp. The effects of CAP on intracellular Ca2+level([Ca2+]) were measured by calcium imaging, and also the influence of PGE2on the effect of CAP. The effect of CAP on the secretion of pain related factors, i.e. NO and IL-1β in the culture medium of ectopic ESC was determined by NO detection kit and enzyme-linked immunosorbent assay(ELISA) of IL-1β.Results:1. Applcation of capsaicin(1uM) for3sec could evoke an outward current by using whole-cell patch clamp technique, which could be blocked by capzepine(10uM), a specific antagonist of TRPV1, and this current did not show significant desensitization.2. Application of1uM capsaicin(CAP) via bath superfusion significantly decreased the intracellular [Ca2+]in ESC, and this effect could be washed out by balanced salt solution(BSS). Ectopic ESC showed greater capacity of Ca2+outflux compared with normal ESC when treated with CAP. Pretreated with PGE2could increase the Ca2+outflux induced by CAP.(n=10,p<0.05)3. Treatment of ectopic ESC with CAP could increase the production of NO and IL-1β in the culture medium(n=10, p all<0.05).Consusions:1. The TRPV1in ectopic ESC is functional, it could produce an outflux of Ca2+after activation, which could be sensitized by PGE2.2. TRPV1could increase the ectopic ESC to produce pain related factors, i.e. NO and IL-1β. |
PDF Full Text Request