晚期妊娠维持和抑制早产中cAMP信号活化PR的作用机制研究

Premature birth is the most important cause of neonatal morbidity and mortality. Despite intensive research, preterm labour remains the most important cause of premature birth, primarily because there is a lack of understanding of the underlying mechanisms responsible. Although in clinics, progesterone treatment has been shown to reduce the risk of preterm labour in high-risk populations, there is no difference between the circulating concentration of progesterone in normal labour and preterm labour groups. In addition, in human being, progesterone level is maintained at a high and increasing concentration throughout parturition, which leads to the notion that a "functional progesterone withdrawal" occurs before parturition. It is agreed that the factors responsible for preterm labour adjust the balance between uterine quiescence and contractility, to prematurely promote the onset of labour. Increasing evidence supports the concept that cAMP promotes the relaxation of the myometrium. Based on recent data that cAMP could act together with progesterone to promote decidualisation in endometrial stromal cells, and it is also involved in the interations between PR and its co-regulators.Furthermore, our preliminary laboratory data has shown that the combination of progesterone and cAMP can inhibit the expression of the genes related preterm labour, decrease the ratio of PR-A/B and increase the PRE activity of myometrial cells. Since there is extensive crosstalk between second messenger systems and nuclear steroid receptors in a variety of settings, it is possible that progesterone and cAMP could act together to prolong pregnancy. In our project, the myometrial cells were isolated from myometrial biopsies at the time of caesarean section at about 28-37 weeks gestations. Then, the changes of PR isoforms ratio, PR response genes and PRE activity will be detected under the stimulus of specific cAMP effectors agonists, antagonists, shRNA or overexpression. Subsequently, in order to explore the potential molecular mechanism of cAMP regulate PR function via its co-modulators, PRE or nuclear transcription factors, co- immunoprecipitation and reverse chromatin immunoprecipitation assay could be employed for this part of the research work. Finally, we would like to elucidate the effectiveness of tocolytic agents which may contribute to maintain or even increase PR activity, and these agents can be combined with progesterone and tested in vivo or vitro model. Thus, this research would further illustrate the theory of "functional progesterone withdrawal" from a new perspective and provide new potential strategies for prevention of preterm labour.

晚期妊娠维持具体机制尚未完全阐明，严重影响早产防治。本项目拟以"功能性孕激素撤退学说"为理论基础，基于PR在妊娠维持中的关键作用，以我们预实验发现cAMP可协同孕激素抑制子宫平滑肌细胞早产相关基因表达、降低PR-A/B比例、提高PRE活性为线索，结合文献我们提出假设"cAMP提高PR活性维持晚期妊娠"。以来自早产的子宫平滑肌细胞为研究对象，先采用不同拮抗剂、shRNA等技术干扰cAMP及下游信号，观察细胞亚型、调控基因、PRE等的表达变化，确定cAMP信号对PR活性的影响及关键作用分子；着重利用免疫共沉淀法和反向ChIP技术研究cAMP信号刺激时与PR、PR启动子、PRE结合的辅助因子或核转录因子，阐明cAMP信号活化PR的分子调控机制；最后在子宫平滑肌细胞、组织牵拉实验和小鼠早产模型以验证cAMP信号提高PR活性的作用机制。力图从新视角阐释"功能性孕激素撤退学说"，为早产防治提供新策略。

早产是造成新生儿残疾或死亡的主要原因之一，但晚期妊娠维持机制尚未阐明严重影响早产防治，目前广泛接受的假说是“功能性孕激素撤退学说”，孕激素受体(PR)活性降低是引起孕激素功能性撤退的重要原因。体内3',5'-环腺苷酸(cAMP)水平在正常妊娠过程中从孕32周开始持续下降直至分娩发动，是影响子宫平滑肌细胞PR活性的关键因素之一。故推测在晚期妊娠过程中当cAMP浓度减少至一定水平可导致PR活性降低，引起孕激素功能性撤退，从而分娩发动或早产发生。.为了阐明子宫平滑肌细胞内cAMP在PR活性维持中的作用与机制，本项目：①系列观察了cAMP对PR活性的影响；②筛选出cAMP影响PR活性的cAMP下游信号通路；③研究cAMP下游信号通路活化PR的分子调控机制；④完成在体内外模型验证cAMP信号活化PR的作用机制。.本课题的重要实验结果、发现及科学意义如下：.①通过forskolin刺激培养子宫平滑肌实验，发现cAMP对OTR、CX-43、COX-2等早产相关基因产生明显的影响，cAMP通过提高PRB的表达，加强PRB与PRE的结合，从而提高PRE的活性。.②利用cAMP下游信号通路PKA、Epac、AMPK等效应物的激动剂、抑制剂和shRNA刺激子宫平滑肌细胞的系列实验显示，cAMP通过PKA调控PRB的表达，这为妊娠维持和早产发生的机制研究提供重要实验依据。探讨了cAMP抑制子宫平滑肌细胞OTR表达的作用机制，发现cAMP是通过下游信号通路PKA降低子宫平滑肌细胞OTR表达，为妊娠维持机制研究提供新方向。.③孕激素与PR结合后通过募集辅助调节子与PRE启动子相结合而启动转录。系列实验研究了参与PR与PRE相互作用的NCoR、SMRT、CREB、SRC-1等不同辅助调节子，发现cAMP明显减少NCoR与PR的相互作用，cAMP刺激显著降低NCoR的表达，下调NCoR可加强cAMP对MPA/PR-B驱动PRE的激活，表明NCoR在cAMP影响PR活性中发挥重要作用，为阻断早产发生提供新靶点。.④在体外牵拉实验模型和IL-1β诱导炎症模型验证了cAMP可提高子宫平滑肌细胞PRB的表达，cAMP联合孕激素通过提高PRB的表达以活化PR。在LPS诱导小鼠早产模型体内验证了cAMP激动剂与孕激素联合可更有效抑制早产的发生。以上结果表明cAMP激动剂与孕激素联合有望成为早产防治的新策略。

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