AP-1和NF-κB在NMBR启动分娩中交互作用及机制研究

Based on the prior studies,GST pull-down assay and immunoprecipitation technologies were performed to identify the interaction between NF-κB and AP-1 in the NMBR induced uterine smooth muscle contraction in vitro and vivo,while the positioning of actived NF-κB and AP-1 were determined using multiple immunofluorescence labeling and laserconfocal microscopy technology. Then the interaction mode on NF-κB and AP-1 was revealed when the specific agonists of AP-1 and NF-κB were exerted on the pregnant mice model with NMBR silence and primary cultured pregnant mice uterine smooth muscle cells,respectively,combination of NMBR, AP-1 and NF-κB gene silencing and overexpression techniques.Moreover,the mechanism on interaction between NF-κB and AP-1 was determined using jointly the transcription factor activity analysis, GST pull-down technology ,JNK and IKK activity assay, active change of different truncaed fragments of AP-1 and NF-κB , regulating to the downstream target genes expression of MMP-9, IL-6 and COX-2 in vitro and vivo .It can be demonstrated the interaction mode and mechnisim of NF-κB and AP-1 in NMBR induced labor while it can be porovided the strong theoretical basis on the NMBR as a novel tocolytic drug target.

在前期研究的基础上，首先应用蛋白质体外结合实验（GST pull-down）和免疫共沉淀技术，确定NMBR诱发子宫平滑肌收缩时NF-κB和AP-1存在相互作用；应用多重免疫荧光标记和激光共聚焦显微镜技术确定其在细胞中活化、定位信息；进而利用AP-1和NF-κB特异的激动剂作用于NMBR基因敲除小鼠模型及原代培养的孕鼠子宫平滑肌细胞，结合NMBR、AP-1及NF-κB基因联合沉默和过表达等技术，确定NF-κB和AP-1相互作用的方式；并通过AP-1和NF-κB结构域中关键性转录部位、不同截短型的构建及转录活性的分析、对下游基因MMP-9、IL-6和COX-2表达的调节，确定AP-1和NF-κB相互作用的机制。为阐明AP-1和NF-κB在NMBR启动分娩中相互作用的方式及机制、NMBR能否作为宫缩抑制药物的靶点提供实验依据。

本课题在前期研究“NF-κB介导NMBR在分娩启动中作用及作用机制研究”的基础上首先采用质谱分析、免疫共沉淀及细胞免疫荧光技术，确定在NMBR诱发子宫平滑肌收缩时NF-κB和AP-1之间存在相互作用；然后应用Western Blotting、多重免疫荧光标记技术确定NF-κB和AP-1在原代培养子宫平滑肌细胞中活化、核内易位等变化；进而利用AP-1和NF-κB特异性的激动剂/抑制剂作用于孕鼠模型，及利用原代培养的人妊娠晚期子宫平滑肌细胞，结合NMBR、AP-1及NF-κB基因分级和联合沉默/过表达等技术，确定在NMBR诱发子宫平滑肌收缩时，AP-1和NF-κB之间存在相互协同作用；进而通过检测AP-1和NF-κB信号通路中关键性蛋白的表达、活化及其对下游基因MMP-9、IL-6和COX-2表达的调节，确定AP-1和NF-κB相互作用的机制。为阐明AP-1和NF-κB在 NMBR启动分娩中相互作用的方式及机制、NMBR能否作为宫缩抑制药物的靶点提供实验依据。