艾灸对结肠炎相关性癌变的ATP-P2X7R的作用及调控机制研究

P2X7R，β-catenin abnormal expressions are key factors to colitis-associated cancer (CAC), this project focus the effect on purinergic receptor P2X7R in the immune system. The application of modern science and technology may be used, and combine gene knockout methods. The relation ship between ATP-P2X7R inflammation pathway and Wnt/β-catenin pathway may be co-targeted transcription factor NF-κB. So, we explore crosstalk in inflammation related tumor transformation interaction between ATP-P2X7R/NF-κB and Wnt/β-catenin/N F-κB, and the expressions of downstream tumor immune factor IL-6, C-myc, etc. In order to further clarify the role of P2X7R in regulating physiological and pathological states, P2X7R knockout mice used for in-depth study. Moreover, to confirm moxibustion that may activate ATP-P2X7R, inhibit Wnt/β-catenin pathway and interact NF-κB, the intervention mechanisms of P2X7R are researched under normal and disease states by different moxibustions. Inhibition of colitis inflammation in the intestinal epithelial cells may prevent intestinal epithelial cell differentiation and proliferation, and to expect to provide the scientific evidence for mechanism of acupuncture on therapy in CAC.

P2X7R和β-catenin表达异常是导致结肠炎相关癌变的关键环节之一，本项目拟从嘌呤受体P2X7R的免疫调节机制为切入点，应用现代科学技术，结合基因敲除的方法，从ATP-P2X7R炎症、Wnt/β-catenin癌变通路均靶向转录因子NF-κB的角度，探讨ATP-P2X7R/NF-κB 及Wnt/β-catenin/NF-κB两个通路异常激活在炎症向肿瘤转化中的交互串扰，转录调控下游肿瘤免疫因子IL-6、C-myc等的表达，为进一步明确P2X7R在生理病理状态的调节作用；采用P2X7R基因敲除小鼠深入研究其免疫炎症与肿瘤的相关性。并研究不同灸法在正常与疾病状态下对P2X7R的干预机制，拟证实艾灸可能是通过激活ATP-P2X7R、抑制Wnt/β-catenin通路，交互作用NF-κB，由此达到抑制结肠炎的肠上皮细胞的炎症，阻止肠上皮细胞的分化与增殖，有效防治结肠炎相关癌变之目的。

结肠炎相关CAC与慢性炎症和肠上皮细胞异型增生密切相关，ATP-P2X7/NF-κB和Wnt/β-catenin/NF-κB通路异常是导致结肠炎相关癌变的关键环节之一，针灸治疗UC具有一定的疗效，本项目从嘌呤受体P2X7R的免疫调节机制为切入点，采用Western Blot、免疫组化、免疫荧光、RT-qPCR等技术与方法，结合P2rx7-KO基因敲除小鼠多角度、多层次的深入研究ATP-P2X7R/NF-κB, Wnt/β-catenin/NF-κB通路导致结肠炎及其相关癌变机制，以及灸法对其的干预作用。1）UC大鼠研究结果提示，艾灸可下调结肠组织P2X7R、NF-κBp65、TNF-α蛋白表达，降低结肠组织TNF-α、IL-9 mRNA表达，降低血清IL-6、TNF-α的蛋白浓度；上调结肠组织TGF-β1的蛋白表达。2）CAC大鼠研究结果提示，艾灸可上调结肠组织P2X7R的蛋白表达，下调结肠组织NF-κBp65、β-catenin、Wnt1、Wnt9a、Wnt5a的蛋白表达，降低结肠组织C-myc、GSK-3β、β-catenin、Wnt1、Wnt9a的mRNA表达，降低血清CEA、VEGF、CysC、Frat1、Wnt9b的蛋白浓度。3）UC小鼠研究结果提示，P2rx7-KO基因敲除后，UC小鼠结肠组织NF-κBp65、CD3、CD8、CD39的蛋白表达下调，Ccl5、Cxcl10的mRNA表达下调；4）CAC小鼠研究结果提示，P2rx7-KO基因敲除后，CAC小鼠肿瘤组织Wnt9b、Wnt5a、β-catenin、C-myc、GSK-3β的蛋白表达下调。综上所述，揭示了艾灸能通过调节P2X7R表达，调控NF-κB的活性，抑制促炎症因子TNF-α和促癌基因β-catenin异常表达，由此达到抑制结肠炎的肠上皮细胞的炎症，阻止肠上皮细胞异型增生的分化与增殖的目的，为针灸防治结肠炎及相关CAC效应机制的阐明提供了科学的实验资料。