基于P13K/Akt与PLC-γ信号通路对Cajal间质细胞的调控探讨枳术丸纠正结肠慢波的机制研究

Slow transit constipation (STC) seriously affects the quality of life of modern people.The decrease in the number of ICC and abnormal structure and function caused by abnormal colon smooth muscle contraction weakened, slow electric wave is one of the important mechanisms on STC , the C-kit/SCF pathway is a key of ICC's occurrence, development and phenotype maintenance. The PI3K/ATK and PLC-γdownstream pathway of C-kit/SCF is vital for calcium slow wave potentials. The expression of ICC and the signal transmission of signaling pathways downstream is damaged, the slow wave potential and nervous excitement of generation and transmission loss of control can cause on STC. Definite pills can invigorating spleen and moving bowels. The single herbs or compatibility of Pill can promote the gastric emptying and intestinal propulsion, improving the ICC colon morphology and increase the number of ICC and the expression of C-kit. So we speculated that the definite technique pill may through regulating the PI3K/ATK and PLC-γdownstream pathway of C-kit/SCF to influence the expression of Cajal interstitial cells and achieve the effect of purging and strong the spleen. This topic proposed by Spleen deficiency syndrome STC mice Including vivo and invitro experiments discuss the molecular mechanism of system on STC generation and definite the intervention effect of intraoperative pill in detail from the perspective of morphology, cytology and molecular biology to open up new areas in the clinical application of the classic party, providing new theoretical basis.

慢传输型便秘（STC）严重影响现代人的生活质量。ICC数量减少、结构及功能异常致结肠平滑肌收缩减弱、电慢波异常是STC发病的重要机制，C-kit/SCF通路是ICC发生、发育及表型维持的关键。C-kit/SCF下游的PI3K-ATK及PLC-γ通路对于钙离子产生慢波电位至关重要。ICC的表达及下游信号通路的信号传递受损，慢波电位及神经兴奋的产生及传递失去调控可致STC。枳术丸具有健脾运脾，理气导滞通便之功，能促进胃排空和肠推进，改善结肠ICC形态并提高ICC数量及C-kit的表达。我们推测枳术丸可能通过调控C-kit/SCF下游的PI3K/AKT及PLC-γ信号通路来影响Cajal间质细胞的表达达到健脾通便之效。本课题以脾虚证STC小鼠为研究对象，包括在体和体外实验，从形态学、细胞学和分子生物学角度，探讨STC发生的分子机制以及枳术丸的干预作用，从而为该经典方在临床的应用提供新的理论依据。

本研究基于Cajal间质细胞是慢传输型便秘的关键靶细胞，Cajal间质细胞数量、形态及功能的改变是STC 发生、发展的关键机制之一，C-kit/SCF参与Cajal间质细胞表型的调控，以C-kit/SCF下游的PI3K/Akt及PLC-γ两条信号通路为切入点，运用功能评价方法和免疫组化、WB、RT-PCR等现代分子生物学技术，从行为学、分子生物学和细胞学角度，开展在体、体外PI3K/Akt及PLC-γ信号通路对于Cajal间质细胞调控机制的探索研究，以及以健脾理气，导滞通便立法的枳术丸干预研究。. 整体动物实验结果显示，枳术丸可改善模型小鼠的一般状态，增加体质量、调节脾脏功能；枳术丸通过提高肠道推进率，调节结肠慢波紊乱，促进肠道蠕动治疗便秘。枳术丸改善脾虚证慢传输型便秘小鼠的便秘症状，提高Cajal间质细胞的数量可能与上调结肠黏膜PLC-γ1、PLC-γ2、PI3K、AKT蛋白和基因表达有关，且以枳术丸中剂量组作用疗效最佳。. 体外细胞实验结果显示，枳术丸、枳实、白术在一定浓度下均能通过增加XIAP、PCNA蛋白表达促进Cajal间质细胞的增殖，对其凋亡无明显影响；枳术丸促进Cajal 间质细胞增殖的效果优于单药枳实和白术；枳术丸汤剂含药血清能提高Cajal间质细胞的数量，并能提高PLC-γ1、PLC-γ2、、PI3K、AKT的蛋白和基因表达。. 以上研究结果表明，C-kit/SCF 下游的 PI3K/Akt及PLC-γ信号通路在结肠Cajal 间质细胞的发育过程中起重要作用，枳术丸汤剂对Cajal间质细胞的干预作用可能与调控PI3K/Akt及PLC-γ信号通路有关，枳术丸汤剂可能调控PI3K/Akt及PLC-γ信号通路提高结肠Cajal间质细胞的数量促进胃肠运动。本研究为明确枳术丸调节胃肠运动的作用靶点，揭示慢传输型便秘的病因病机提供了新的理论依据。