标本配穴电针对缺血性心肌细胞凋亡基因及其相关信号通路调控的机制研究

Ischemic Heart Disease is closely related to the process of apoptosis in vivo,with points based on the early stage of research resultson about Acupuncture of Combined Biao and Ben Acupoints on chronic myocardial ischemia,inte- nded to chronic myocardial ischemia model in rats for the study,selected the Acupuncture of Combined Biao and Ben Acupoints,with a selection of sample points to a single acupuncture as control,from the detection indicators of the ultrasound cardiac function, myocardial enzymes and ultrastructure of myocardial apoptosis,observing the Acupuncture of Combined Biao and Ben Acupoints of synergy to improve the effect of myocardial ischemia.Simultaneously Combined with gene chip,Western blot,RT-PCR techniques to observe the affect of apoptosis related genes protein expression of Acupuncture of Combined Biao and Ben Acupoints on myocardial ischemia,while using inhibitor LY294002 to block the PI3K/Akt signaling pathway as a control sample,observed the effect of Acupuncture of Combined Biao and Ben Acupoints on related signaling pathways,to clarify the synergistic improvement effect of the Acupuncture of Combined Biao and Ben Acupoints on ischemic myocardium damaging and the regulatory mechanisms of apoptosis-related genes,then to prove that scientific and practical of the Acupuncture of Combined Biao and Ben Acupoints theory from the genetic level,while to provide a scientific basis for clinical acupoints compatibility.

缺血性心脏病与体内细胞凋亡过程密切相关，基于前期标本配穴防治慢性心肌缺血研究成果，拟以慢性心肌缺血模型大鼠为研究对象，选用标本配穴，以单穴针刺为对照，从心脏功能彩超、心肌酶谱及心肌凋亡超微结构等检测指标，观测标本配穴对心肌缺血的协同改善效应。同时结合基因芯片、Western blot、RT-PCR等技术，观察标本配穴对心肌缺血大鼠细胞凋亡相关基因和蛋白表达的影响，同时采用抑制剂LY294002阻断PI3K/Akt信号通路作为对照，观察标本配穴对相关信号通路激活途径的影响，以阐明标本配穴在心肌缺血损伤中的协同改善作用及对凋亡相关基因的调控机制，进而从基因水平验证标本配穴理论的科学性和实用性，为临床腧穴配伍提供科学依据。

选用心肌缺血模型大鼠为研究对象，在中医针灸“治病求本”思想指导下，采用“标本配穴”电针干预，并以单取“内关”穴作为对照，通过观察心电图、心肌病理形态学、心肌梗死面积、细胞凋亡指数及线粒体超微结构的变化，并通过对 PI3K/Akt 通路的激活和抑制，观察 PI3K/Akt 通路及凋亡相关基因、蛋白等指标的变化，系统整理和分析“标本配穴”电针对慢性心肌缺血大鼠PI3K/Akt信号通路及相关基因、蛋白表达的调控作用机制；另一方面，以细胞凋亡为切入点，运用miRNA基因芯片筛选、实时荧光定量PCR（qRT-PCR）及蛋白质印迹（Western blot）等技术，观察心肌细胞凋亡相关miRNA及其靶基因表达的影响及调控关系，探讨“标本配穴”针刺干预心肌缺血的miRNA基因调控机制，以期为针刺防治心肌缺血疾病提供理论支撑和科学依据。结果表明，“标本配穴"电针治疗通过PI3K/Akt信号通路可有效调控Bcl-2和Bax蛋白的表达活性，抑制细胞凋亡，从而起到对慢性心肌缺血大鼠的保护作用。而miRNA基因及其靶基因是影响心肌缺血细胞凋亡的主要途径，其中在“标本配穴”电针防治心肌缺血的miRNA基因信号调控通路中，miRNA-133-5p与Caspase-3、Aifm2之间的基因调控信号是主要途径。Caspase-3和Aifm2因子是激活细胞凋亡通道的主要执行因子，“标本配穴”电针可有效防治心肌缺血，其保护机制可能与上调miRNA-133的表达，抑制心肌miRNA-1、miRNA-208、miRNA-499表达的双重调控作用有关。