基于平滑肌细胞肌球蛋白轻链激酶（MLCK）信号传导通路的脾虚证胃肠动力障碍以及四君子汤干预的分子机制研究

The project would plan to establish the rat model of"dysfunction of the spleenin transportation"(namely the syndrome of spleen deficiency and gastrointestinal dynamic disfunction)；apply nuclide solid empty method to observe the gastrointestinal evacuation of the deficiency of spleen rat；apply pathologic and electron microscopy to observe the ultrastructure of gastrointestinal SMC of the normal and the" dysfunction of the spleen in transportation " rat；isolate and culture the SMC of gastric antrum and jejunum from the normal and the" dysfunction of the spleen in transportation " rat；apply electron microscopy to observe SMC length；apply the fluorescence detecting method to test the dynamic change of intracellular Ca2+ concentration；apply immunohistochemistry to observe the expression of CaM and MLCK in SMC；apply electrophoresis of glycerol or urea to test myosin phosphorylation; apply Malachitegreen to test the changes of myosin Mg2+-ATPase activity. We would explore the mechanism of "dysfunction of the spleen in transportation" in accordance with the gastrointestinal smooth muscle cell Ca2+－CaM－MLCK signal transduction pathway.The project would apply the regulation theory of gastrointestinal motion contraction to research the splenasthenic syndrome, to explain the mechanism of "dysfunction of the spleen in transportation" from a totally new point of view, and would open up a new thinking and method for the contemporary research for the spleen and stomach doctrine of Traditional Chinese Medicine.

制造脾虚证胃肠动力障碍大鼠模型，应用核素固体胃肠排空法观察模型大鼠胃排空和小肠推进运动，应用图像分析系统观察正常和模型大鼠胃肠平滑肌细胞（smooth muscle cell，SMC）超微结构；分离、培养正常和模型大鼠胃窦和小肠SMC后并分别应用电子显微镜观察SMC长度，应用荧光探测法检测SMC胞浆游离Ca2+浓度变化，应用免疫组化法检测SMC中钙调素（calmodulin，CaM）和肌球蛋白轻链激酶（myosin light chain kinase，MLCK）的表达，孔雀绿法检测myosin Mg2+-ATPase活性以及四君子汤干预后以上指标的变化。从胃肠平滑肌细胞MLCK上下游细胞信号传导通路（Ca2+-CaM-MLCK）探讨脾虚证胃肠动力障碍以及健脾益气方干预的分子机制，为研究脾虚证胃肠动力障碍疾病、开发调节胃肠运动中药提供新思路、新方法和新靶点，为中医药走上国际打下坚实的基础。

本研究从体内和体外实验两部分，对胃肠平滑肌细胞CaM-MLCK上下游细胞信号传导通路（Ca2+-CaM-MLCK）进行研究，探讨了脾虚证胃肠动力障碍以及健脾益气方干预的分子机制。本研究首先对脾虚证胃肠动力障碍动物模型的造模方法进行研究，分别尝试了传统“破气耗气法联合劳倦、饥饱失常法”和国内最新“碘乙酰胺、劳倦联合水环境小平台站立”法，实验结果表明“碘乙酰胺、劳倦联合水环境小平台站立”效果最佳，可作为脾虚证胃肠感觉-动力异常障碍模型。在体实验部分，课题组运用了半固体胃排空和小肠推进实验、电镜观察SMC超微结构实验、Western Blot、免疫组化等实验，观察了脾虚证胃肠动力障碍大鼠及健脾益气方干预后CaM-MLCK信号通路上下游的改变。结果表明脾虚证胃肠动力障碍大鼠存在体重减轻，胃肠动力减弱，胃肠平滑肌CaM、MLCK蛋白和基因水平升高等改变，运用四君子汤干预后相关指标能一定程度上恢复至正常水平，但平滑肌组织电镜超微结构未见明显差异。在体外实验部分，我们探索、优化方法，成功从幼年期SD大鼠分离胃肠SMC原代细胞，对细胞进行了鉴定，运用Fluo-4 AM探针法、Western Blot法、RT-PCR法观察健脾益气中药四君子汤对胞浆游离Ca2+浓度、CaM-MLCK上下游蛋白和基因改变情况，结果与在体实验趋势基本一致。同时，我们对与脾虚证胃肠感觉-动力相关的其他信号通路，如动力相关通路CNP-NPRB-cGMP信号通路、痛觉相关通路TRPV1信号通路也进行了部分研究，结果表明脾虚证感觉-动力异常大鼠存在相关信号通路异常，四君子汤能调节该通路恢复至正常水平，丰富了脾虚证胃肠动力障碍和健脾益气中药干预分子机制的内容。本项目从胃肠平滑肌Ca2+-CaM-MLCK信号通路上下游以及CNP-NPR-B -cGMP信号通路、TRPV1信号通路探讨“脾虚失运”的发生机制，以全新的角度阐明“脾虚失运”证的发生机制，为中医脾胃研究和中医药治疗脾虚证胃肠感觉-动力紊乱疾病研究开辟新思路、新方法和新靶点。