Rac1高表达激活CaMKⅡ影响Ca2+通道重构在早产发病机制中的作用研究

Preterm birth is the main leading cause of perinatal morbidity and mortality，the rate of preterm births has been increasing. However, the mechanism of preterm birth remains elusive. It is reported that intracellular calcium concentration play a central role in uterine contraction, the remodeling of calcium channels in the uterine smooth muscle cells depends regulation of signal protein pathway. Research shows that CaMKII transducts calcium signal, moreover, Rac1 modulates smooth muscle contraction via up-regulating the expression of CaMKII. It was demonstrated that Western-Blot not only showed the expression of Rac1 and CaMKII protein in uterine smooth muscle but also the expression of Rac1 and CaMKII protein levels were higher in preterm birth group compared with the control group. Taken all these together, we hypothesize that Rac1 overexpression or activation can up-regulate CaMKII signal pathway, which results in calcium channels remodeling in uterine smooth muscle cells and uterine contraction, leading preterm birth. On the basis of previous works, the project intends to observe the effect of Rac1 on preterm birth via isometric tension recordings and animal model. Furthermore, the inhibitor of Rac1 was add in vivo and in vitro , uterine smooth muscle cell was isolated to investigate the pathway for the regulation of Rac1/ CaMKII on calcium channels by using Patch-clamp technique. The project will explore the mechanism for Rac1 on preterm birth and provide a new clinical idea for preterm birth treatment.

早产是新生儿死亡和长期发病的主要原因，但目前早产的具体发生机制并不清楚。子宫平滑肌的收缩张力取决于胞浆内Ca2+的浓度，平滑肌细胞胞内钙重构的过程依赖于蛋白信号通路调节。相关研究表明Rac1能促进CaMKII表达，传递Ca2+信号，参与平滑肌收缩过程。我们前期研究证实子宫平滑肌细胞上Rac1和CaMKII蛋白的表达，且早产子宫平滑肌Rac1和CaMKII蛋白含量高于非早产。我们假设Rac1的过表达，上调CaMKII信号通路，改变子宫平滑肌细胞膜上钙通道特性，导致钙离子重构，使平滑肌发生异常收缩导致早产。本课题在前期研究基础上，通过早产模型研究Rac1在早产中的表达变化。以子宫平滑肌细胞为研究对象，采用膜片钳技术记录钙通道电流，明确不同亚型Ca2+通道在早产中的作用。结合体内外实验，给予Rac1拮抗剂证明Rac1/ CaMKII对钙通道的调控，从而为临床防治早产提供新的思路。

-早产是导致围产期新生儿发病和死亡的主要原因，而且增加早产新生儿罹患远期慢性病的风险，影响其远期预后。早产是多因素导致的一种综合征，目前研究认为感染相关的炎症激活是早产最主要的发病机制。Rac1（Ras-related C3 botulinum toxin substrate 1）属于Rho蛋白家族，是细胞内重要的信号转导分子，调节细胞收缩、迁移等，但涉及Rac1与早产子宫炎症的分子机制研究很少。本课题提出科学假设Rac1激活CaMKⅡ信号通路，可调控子宫平滑肌细胞钙电流，调节子宫平滑肌炎症反应，进一步调节子宫收缩相关蛋白引起子宫平滑肌异常收缩，从而导致早产发生。.本课题主要研究内容及结果如下：.(1)孕小鼠注射LPS建立早产模型,通过HE、Western-Blot、离体肌张力测定、Pull-Down等手段检测发现模型组小鼠早产发生率高、子宫炎症评分增加、离体子宫收缩张力增加，Rac1-Total和Rac1-GTP表达显著上升。.(2)模型小鼠体内给与Rac1抑制剂(NSC23766)后发现NSC23766降低Rac1活性含量、延缓模型小鼠发生早产的时间、抑制离体子宫收缩张力、降低子宫组织炎症因子（IL-1β、IL-6、TNF-α）和子宫收缩相关蛋白（COX-2、Connexin43）的表达程度。.(3) 急性分离小鼠子宫平滑肌细胞，采用膜片钳技术检测平滑肌细胞钙电流，采用尼莫地平后能阻滞该电流。.(4) 原代培养子宫平滑肌，体外细胞实验采用腺病毒感染敲低Rac1表达和过表达Rac1，证实敲低和过表达Rac1分别能下调和上调细胞炎症反应和子宫收缩相关蛋白，同时影响子宫平滑肌细胞收缩功能。.(5) Rescue实验，体外细胞实验采用CaMKII抑制剂KN-93和过表达Rac1，证实KN-93能减弱过表达Rac1后引起的子宫收缩相关蛋白。.通过上述结果明确Rac1是调节子宫平滑肌炎症应答和子宫收缩活化的关键调控因子，最终为临床人为防治早产提供可能的解决途径。