t-ICC损伤致子宫内膜异位症中输卵管性不孕的分子机制

Tubal motility abnormalities are one of important factors for infertility patient suffered with endometriosis. Cajal-like type of tubal interstitial cells (t-ICC) generate slow waves within oviduct smooth muscles and has been deemed to be oviduct pacemaker cells. Ano1 is a Ca2+-activated Cl- channel expressed in t-ICC, which was encoded by Tmem16a in myosalpinx. Ano1 contribute to regulator of proliferation of t-ICC and initiation of membrane potential and oviduct slow waves. As we know, genital infections cause loss of pacemaker activity of t-ICC and inhibit oocyte transport within oviduct. We have confirmed in our preliminary results that t-ICC network were damaged in endometriosis and tubal ectopic pregnancy. However, little is known regarding the adverse effects of endometriosis on altered expression of Tmem16a transcriptional variants, Ano1 channel protein and subsequent pacemaker activity of t-ICC and associated smooth muscle contractions. The central hypothesis is that disturbances of pelvic micro-environments (abnormal immune status, unbalanced oxidative stress, elevated local estrogen levels, local tissue adhesion and invasion) which were induced by endometriosis will inevitably cause damage to Tmem16a, Ano1 and t-ICC, leading to disruption of pacemaker activity and loss of associated muscle contractions, finally cause tubal factor infertility. .To test this hypothesis, in this application, our work will focus on the newly discovered protein Ano1 in t-ICC in animal model of endometriosis in SD rat: firstly evaluate damage of t-ICC network (nerve fibers- t-ICC- smooth muscle). Then investigate altered expression of Tmem16a transcriptional variants, Ano1 protein expression, followed with proliferation status and slow wave activity changes of t-ICC. Finally evaluate damage of t-ICC network (nerve fibers- t-ICC- smooth muscle). We will use a combination of innovative techniques including Animal model, Cell cultures, Immunohistochemistry, Western blots, RT-PCR, siRNA Transfection, Patch clamp techniques, Electrophysiological experiments, Flow cytometry, Immunofluorescence. .Successful completion of this application will provide both basic and clinical significance. Unveil molecular mechanisms of t-ICC down-regulation and disruption of oviduct pacemaker activity and tubal muscle contraction, and resultant increased risk for tubal factor infertility or ectopic pregnancy. In addition, we will use GnRH-a and heme oxygenase-1 to try to protect damage of Tmem16a encoded Ano1 in t-ICC. With the aim to provide potential target for clinicians on use of Tmem16a and Ano1 to earlier assess damage of t-ICC in tubal motility disorders and provide targets for future pharmaceutical and genetic therapeutic interventions.

输卵管蠕动障碍是内异症不孕的重要病因。输卵管Cajal样间质细胞(t-ICC)产生起搏慢波并激发肌层运动。而Tmem16a编码的钙激活氯通道(Ano1)是t-ICC增殖调控及慢波的重要离子基础，但尚无Tmem16a异常表达致t-ICC起搏活性及肌层运动异常的报道。本课题组前期实验已证实内异症抑制t-ICC网络。故设想，内异症盆腔局部异常的微环境（免疫环境，氧化应激，高雌激素，局部粘连浸润），可能损伤Ano1介导的t-ICC起搏活性。遂拟t-ICC为切入点，首次探讨SD大鼠腹腔内异症中Tmem16a表达亚型、Ano1蛋白及起搏活性、t-ICC增殖状态与"神经-t-ICC-平滑肌"的改变情况。且尝试"GnRH-a与血红素加氧酶-1"保护t-ICC。旨在以Tmem16a/Ano1为靶点，阐明内异症中t-ICC病变的分子机制，为临床使用Ano1早期评估、治疗t-ICC损伤的输卵管肌层病变提供依据。

经典理论认为，子宫内膜异位症引起不孕的机制复杂，涉及机械、排卵、受精及胚胎着床等诸多因素。Telocytes（TC）是近年发现的一种新型间质细胞（超微结构显著不同于既往的cajal间质细胞）。目前TC的研究多在正常组织中，尚缺少病理状态时TC的改变及意义。而对输卵管中TC的研究，以及其在生殖过程中的角色鲜有报道。. 首先我们在正常SD大鼠中，观察到TC的免疫表型为CD34 (+)/ vimentin (+)/ c-kit (-)，分布在输卵管肌层及固有层，胞体呈细长的梨形/纺锤/三角形，有2-3条极其细长的念珠状突起（telopode），由膨大（podom）与细小部分（podomer）相间构成，且TC细胞之间，以及与免疫、平滑肌、干细胞等诸多类型细胞间形成连接结构，环绕干细胞并构成干细胞巢。这些结构特征提示，TC参与输卵管组织内的细胞间信号传递（输卵管肌层运动的神经体液调节，免疫监视/调节）；干细胞介导的组织损伤后修复，发生，血管再生，重塑与组织内稳定；间质组织的三维结构支持。TC对输卵管生殖功能的结构学基础的维持有重要意义，为生殖功能障碍的研究提供了崭新的视角。. 其后在SD大鼠子宫内膜异位症、急性输卵管炎模型中，首次观察到受影响的输卵管组织中，TC数量显著减少，超微结构损伤：胞质溶解、空泡样变性，细胞器数量减少，线粒体明显肿胀，糙面内质网扩张；TC的同种或异型细胞间连接出现模糊或消失。失去正常TC的典型超微结构特征，且伴随组织内炎性白细胞显著增高、炎症因子（iNOS，COX2）氧化应激源（LPO）与Estrogen的过量生成。提示输卵管局部炎症因子增高，缺氧与氧化应激反应，是导致TC损伤的分子机制；而高水平雌激素亦会导致TC功能异常。. 我们认为，TC损伤与丢失势必导致：细胞能量供应不足及合成功能下降，输卵管结构与生殖功能的紊乱，包括细胞间信号传递与旁分泌的异常，输卵管肌层运动的神经体液调节的异常，组织修复与内稳定的异常，间质组织三维支持结构的异常。进一步并发输卵管粘连/阻塞，最终致输卵管生殖功能异常或减低，与输卵管性不孕或异位妊娠有密切关系。可见，TC在输卵管疾病发生、发展、转归中扮演一定的角色。本研究具源头创新，提出了输卵管性不孕与异位妊娠的新机制、新的治疗靶点，在目前教科书的经典理论中尚未见述及。这一层面上相对应的工作在国内外尚未见报道。