本文選題：標(biāo)本配穴 + 針刺�。� 參考：《湖北中醫(yī)藥大學(xué)》2016年博士論文

【摘要】：目的心血管疾病是當(dāng)今威脅人類健康和生命的主要疾病之一,心肌缺血是心血管疾病的主要因素。針刺用于防治心肌缺血已有數(shù)千年的歷史,具有操作簡(jiǎn)便快捷、療效確切的特點(diǎn),但其作用機(jī)制尤其是分子生物學(xué)機(jī)制目前尚不完全清晰,在一定程度影響了針灸在心肌缺血臨床的應(yīng)用及推廣。相關(guān)研究表明,在心肌缺血的過(guò)程中存在普遍心肌細(xì)胞凋亡表現(xiàn),而細(xì)胞凋亡是受基因嚴(yán)格控制的,其中Micro RNA(miRNA)的調(diào)控具有關(guān)鍵作用。針刺是否是通過(guò)miRNA基因調(diào)控途徑干預(yù)心肌細(xì)胞凋亡?主要是通過(guò)什么miRNA基因調(diào)控的?主要的miRNA基因是通過(guò)什么信號(hào)調(diào)控途徑發(fā)揮作用的?這些都是需用研究的問(wèn)題。有據(jù)于此,本課題選用心肌缺血模型大鼠為研究對(duì)象,在中醫(yī)針灸“治未病”理論為指導(dǎo)下,采用“標(biāo)本配穴”電針干預(yù)方法,以細(xì)胞凋亡為切入點(diǎn),運(yùn)用miRNA基因芯片篩選、實(shí)時(shí)熒光定量PCR(q RT-PCR)及蛋白質(zhì)印跡(Western blot)等技術(shù),觀察心肌細(xì)胞凋亡相關(guān)miRNA及其靶基因表達(dá)的影響及調(diào)控關(guān)系,探討“標(biāo)本配穴”針刺干預(yù)心肌缺血的miRNA基因調(diào)控機(jī)制,以期為針刺防治心肌缺血疾病提供理論支撐和科學(xué)依據(jù)。方法1.選用SPF級(jí)雄性Wistar大鼠40只,體重180-220g,隨機(jī)分為正常組、模型組、內(nèi)關(guān)電針組(內(nèi)關(guān)組)、標(biāo)本配穴電針組(標(biāo)配組),每組10只。模型組、內(nèi)關(guān)組、標(biāo)配組大鼠予異丙腎上腺素(ISO)2mg/(kg·d)腹部皮下注射,連續(xù)14天,后用BL-420生物機(jī)能系統(tǒng)檢測(cè)模型組大鼠心電圖,以QRS波、QT波持續(xù)時(shí)間延長(zhǎng)(QRS0.1s)、T波形態(tài)改變作為造模成功標(biāo)志。正常組大鼠給予等量0.9%生理鹽水腹部皮下注射,連續(xù)14天。正常組、模型組大鼠只抓取固定,不針刺治療。內(nèi)關(guān)組針刺雙側(cè)“內(nèi)關(guān)”(大鼠腕橫紋正中上5 mm,針刺深約0.5 cm),標(biāo)配組針刺雙側(cè)“內(nèi)關(guān)”、“關(guān)元”(大鼠臍下約25 mm,針刺深約0.5 cm)、雙側(cè)“足三里”(大鼠膝關(guān)節(jié)后外側(cè)、腓骨小頭下約5mm,針刺深約0.8 cm),標(biāo)配組同側(cè)穴位組成一對(duì)電極,內(nèi)關(guān)組及標(biāo)配組中內(nèi)關(guān)穴在穴位右旁開(kāi)0.5cm處另皮下淺刺一針作輔助電極,后連接HANS LH202H電針治療儀,連續(xù)波,頻率2 Hz,強(qiáng)度1m A,以大鼠肢體有震顫感為適宜強(qiáng)度,通電10min。每日治療1次,共21天。大鼠于第22天行超聲心動(dòng)圖機(jī)檢測(cè)大鼠左室舒張末期內(nèi)徑(LVEDd)、左室收縮末期內(nèi)徑(LVESD)、左室射血分?jǐn)?shù)(EF)和左室短軸縮短率(FS);TUNEL法檢測(cè)大鼠心肌細(xì)胞凋亡指數(shù)(AI);ELISA法檢測(cè)血清肌酸激酶同工酶(CK-MB)、血管內(nèi)皮細(xì)胞粘附分子1(VCAM-1)、內(nèi)皮素1(ET-1);實(shí)時(shí)熒光定量PCR法檢測(cè)大鼠心肌組織miRNA-133-3p、miRNA-133-5p、miRNA-1-3p、miRNA-486表達(dá)及靶基因Nol3、Caspase-3、Aifm2、Api5、RGD1564319、Aatk的表達(dá)。后對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)分析,并對(duì)miRNA與其靶基因進(jìn)行相關(guān)性驗(yàn)證。2.SPF級(jí)雄性Wistar大鼠12只,每組3只,分組、治療、取材方法同上。用微列陣基因芯片技術(shù)檢測(cè)各組大鼠心肌miRNA表達(dá)譜狀況。3.SPF級(jí)雄性Wistar大鼠70只,隨機(jī)分為正常組、模型組、內(nèi)關(guān)組、標(biāo)配組、miRNA-133a-5p抑制劑組(抑制劑組)、miRNA-133a-5p激動(dòng)劑組(激動(dòng)劑組)、miRNA-133a-5p抑制劑+“標(biāo)本配穴”電針組(抑制劑+標(biāo)配組),每組10只。模型組、內(nèi)關(guān)組、標(biāo)配組、抑制劑組、激動(dòng)劑組及抑制劑+標(biāo)配組造模方法與上(方法1)相同。抑制劑組、抑制劑+標(biāo)配組腹部皮下按10mg/kg于實(shí)驗(yàn)第1、7、14天進(jìn)行miRNA-133a-5p抑制(antagomir)劑注射,共3次;激動(dòng)劑組注射miRNA-133a-5p激動(dòng)(agomir)劑,劑量、方法同抑制劑組相同。正常組方法與上(方法1)相同。正常組、模型組、抑制劑組、激動(dòng)劑組大鼠只抓取固定,不針刺治療。內(nèi)關(guān)組、標(biāo)配組、抑制劑+標(biāo)配組進(jìn)行電針治療,抑制劑+標(biāo)配組與標(biāo)配組治療方法相同,電針治療方法與上(方法1)相同。實(shí)驗(yàn)第22天心臟取材,用實(shí)時(shí)熒光定量PCR法檢測(cè)各組大鼠心肌組織miRNA-133-5p及其靶基因Caspase-3、Aifm2的表達(dá);Western blot法檢測(cè)各組靶基因Caspase-3、Aifm2的表達(dá),并對(duì)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)及相關(guān)性分析。結(jié)果1.模型組大鼠心臟LVEDd、LVESD高于正常組(均為P0.01),而EF、FS值均低于正常組(均為P0.01);內(nèi)關(guān)組、標(biāo)配組LVEDd、LVESD均低于模型組(均為P0.01),標(biāo)配組低于內(nèi)關(guān)組(分別為P0.01,P0.05);內(nèi)關(guān)組、標(biāo)配組中EF、FS值均高于模型組(均為P0.01),標(biāo)配組高于內(nèi)關(guān)組(P0.05)。2.正常組大鼠心肌AI值低于模型組(P0.01);內(nèi)關(guān)組、標(biāo)配組均低于模型組(均為P0.01);標(biāo)配組低于內(nèi)關(guān)組(P0.05)。3.模型組大鼠血清CK-MB、VCAM-1、ET-1值均高于正常組(均為P0.01);內(nèi)關(guān)組、標(biāo)配組均低于模型組(均為P0.01);標(biāo)配組均低于內(nèi)關(guān)組(均為P0.05)。4.運(yùn)用微陳列芯片技術(shù)共篩選出758個(gè)miRNA基因信號(hào),有20個(gè)具有差異化意義的miRNA基因信息:12個(gè)表達(dá)上調(diào),8個(gè)表達(dá)下調(diào);其中miR-133a-5p和miR-133a-3p上調(diào)趨勢(shì)、miR-1-3p和miR-486下調(diào)趨勢(shì)明顯優(yōu)于其它miRNA基因。通過(guò)Target Scan、miRanda和Pic Tar3種生物信息數(shù)據(jù)庫(kù)交叉對(duì)上述4個(gè)miRNA基因與凋亡相關(guān)的靶基因進(jìn)行預(yù)測(cè):miR-133a-3p的靶基因?yàn)镹ol3,miR-133a-3p的靶基因?yàn)镃aspase-3和Aifm2,miR-1-3p的靶基因?yàn)锳pi5,miR-486的靶基因?yàn)镽GD1564319和Aatk。5.(1)模型組大鼠心肌miRNA-133-3p、miRNA-133-5p表達(dá)均低于正常組(均為P0.01),miRNA-1-3p、miRNA-486表達(dá)均高于正常組(均為P0.01);內(nèi)關(guān)組、標(biāo)配組miRNA-133-3p、miRNA-133-5p表達(dá)均高于模型組(分別為P0.05,P0.01,P0.01,P0.01),標(biāo)配組均高于內(nèi)關(guān)組(分別為P0.05,P0.01);內(nèi)關(guān)組、標(biāo)配組miRNA-1-3p、miRNA-486表達(dá)均低于模型組(分別為P0.05,P0.05,P0.01,P0.01),標(biāo)配組均低于內(nèi)關(guān)組(均為P0.05)。(2)模型組心肌Nol3表達(dá)高于正常組(P0.01),模型組與內(nèi)關(guān)組、標(biāo)配組沒(méi)有顯著差異(均為P0.05);模型組Caspase-3、Aifm2表達(dá)均高于正常組(均為P0.01),內(nèi)關(guān)組、標(biāo)配組均低于模型組(均為P0.01),標(biāo)配組均低于內(nèi)關(guān)組(均為P0.01);模型組Api5表達(dá)低于正常組(P0.01),內(nèi)關(guān)組與模型組無(wú)顯著差異(P0.05),標(biāo)配組低于模型組(P0.05);正常組RGD1564319表達(dá)與模型組無(wú)顯著差異(P0.05);模型組Aatk表達(dá)高于正常組(P0.01),內(nèi)關(guān)組與模型組無(wú)顯著差異(P0.05),標(biāo)配組高于模型組(P0.05)。(3)整體組間相關(guān)性及相關(guān)系數(shù)強(qiáng)度分析:miRNA-133a-3p與Nol3、miRNA-1-3p與Api5為中度負(fù)相關(guān);miRNA-486與Aatk為中度正相關(guān);miRNA-486與RGD1564319為輕度負(fù)相關(guān);miRNA-133a-5p與Caspase-3、Aifm2均為高度負(fù)相關(guān)。6.(1)模型組大鼠心肌miRNA-133a-5p表達(dá)低于正常組(P0.01);內(nèi)關(guān)組、標(biāo)配組、激動(dòng)劑組、抑制劑+標(biāo)配組均高于模型組(均為P0.01),抑制劑組低于模型組(P0.01);標(biāo)配組、激動(dòng)劑組、抑制劑+標(biāo)配組均高于內(nèi)關(guān)組(分別為P0.01,P0.01,P0.05),抑制劑組低于內(nèi)關(guān)組(P0.01);激動(dòng)劑組高于標(biāo)配組(P0.01),抑制劑組低于標(biāo)配組(P0.01),抑制劑+標(biāo)配組與標(biāo)配組無(wú)顯著差異(P0.05)。(2)模型組大鼠心肌Caspase-3、Aifm2表達(dá)(q RT-PCR檢測(cè))均高于正常組(均為P0.01);內(nèi)關(guān)組、標(biāo)配組、激動(dòng)劑組、抑制劑+標(biāo)配組均低于于模型組(均為P0.01),抑制劑組高于模型組(分別為P0.01,P0.05);標(biāo)配組、激動(dòng)劑組均低于于內(nèi)關(guān)組(均為P0.01),抑制劑組高于內(nèi)關(guān)組(均為P0.01),抑制劑+標(biāo)配組與內(nèi)關(guān)組無(wú)顯著差異(P0.05);抑制劑組高于標(biāo)配組(均為P0.01),激動(dòng)劑組、抑制劑+標(biāo)配組與標(biāo)配組無(wú)顯著差異(均為P0.05)。(3)模型組大鼠心肌Caspase-3、Aifm2表達(dá)(Western-blot檢測(cè))均高于正常組(均為P0.01);內(nèi)關(guān)組、標(biāo)配組、激動(dòng)劑組、抑制劑+標(biāo)配組均低于于模型組(均為P0.01),抑制劑組高于模型組(均為P0.05);標(biāo)配組、激動(dòng)劑組均低于內(nèi)關(guān)組(均為P0.01),抑制劑組高于內(nèi)關(guān)組(均為P0.01),抑制劑+標(biāo)配組(Caspase-3)與內(nèi)關(guān)組無(wú)顯著差異(P0.05),抑制劑+標(biāo)配組(Aifm2)低于內(nèi)關(guān)組(P0.01);抑制劑組高于標(biāo)配組(均為P0.01),激動(dòng)劑組、抑制劑+標(biāo)配組與標(biāo)配組無(wú)顯著差異(均為P0.05)。(4)整體組間相關(guān)性及相關(guān)系數(shù)強(qiáng)度分析:miRNA-133a-5p分別與Caspase-3、Aifm2(q RT-PCR、Western-blot檢測(cè))均為較強(qiáng)負(fù)相關(guān)。結(jié)論1.電針能夠通過(guò)降低心肌缺血模型大鼠的LVEDd、LVESD值、提升EF、FS值,有效改善心肌缺血細(xì)胞凋亡導(dǎo)致的代償性心臟擴(kuò)張、心功能低下,降低心肌細(xì)胞凋亡程度,且“標(biāo)本配穴”電針?lè)ū葐渭儍?nèi)關(guān)電針?lè)ň哂懈玫男?yīng)。2.對(duì)于心肌缺血細(xì)胞凋亡后引發(fā)的心肌酶等指標(biāo)改變,“標(biāo)本配穴”電針和內(nèi)關(guān)電針?lè)ň芡ㄟ^(guò)有效減少實(shí)驗(yàn)大鼠血清CK-MB、VCAM-1及ET-1的活性表達(dá)發(fā)揮抗心肌缺血作用的,且“標(biāo)本配穴”電針?lè)ū葐渭儍?nèi)關(guān)電針?lè)ň哂懈玫男?yīng)。3.多miRNA基因參與了異丙腎上腺所致的大鼠心肌缺血細(xì)胞凋亡過(guò)程,心肌缺血過(guò)程中,主要表現(xiàn)在miRNA-133-3p、miRNA-133-5p表達(dá)下降,miRNA-1-3p、miRNA-486上升;“標(biāo)本配穴”電針?lè)軌蛲ㄟ^(guò)有效地提升miRNA-133-3p、miRNA-133-5p的表達(dá),降低miRNA-1-3p、miRNA-486的表達(dá),其保護(hù)作用是通過(guò)調(diào)控這4個(gè)miRNA基因的方式抑制心肌細(xì)胞凋亡。4.miRNA基因及其靶基因是影響心肌缺血細(xì)胞凋亡的主要途徑;“標(biāo)本配穴”電針?lè)乐涡募∪毖膍iRNA基因信號(hào)調(diào)控通路中,miRNA-133-5p與Caspase-3、Aifm2之間的基因調(diào)控信號(hào)是主要途徑。5.Caspase-3和Aifm2因子是激活細(xì)胞凋亡通道的主要執(zhí)行因子,”標(biāo)本配穴”電針抑制細(xì)胞凋亡、保護(hù)心肌缺血的作用主要是通過(guò)提升miRNA-133-5p的表達(dá),進(jìn)而減少Caspase-3、Aifm2的表達(dá)的雙通道調(diào)控途徑實(shí)現(xiàn)的。
[Abstract]:Objective cardiovascular disease is one of the major diseases that threaten human health and life today. Myocardial ischemia is the main factor of cardiovascular disease. Acupuncture has been used to prevent and cure myocardial ischemia for thousands of years. It has the characteristics of simple and quick operation and accurate curative effect. But the mechanism of its action, especially the molecular biological mechanism, is not completely clear at present. It has influenced the clinical application and popularization of acupuncture and moxibustion in myocardial ischemia to a certain extent. Related studies have shown that there is a general expression of cardiomyocyte apoptosis in the process of myocardial ischemia, and the apoptosis is strictly controlled by genes, in which the regulation of Micro RNA (miRNA) is the key to be used. Whether acupuncture is the intervention of miRNA gene regulation pathway Cardiomyocyte apoptosis? What is the main miRNA gene regulated by miRNA gene? Are the main miRNA genes play the role of the signaling pathway? These are the problems that need to be studied. Acupoint electroacupuncture intervention method, taking cell apoptosis as the breakthrough point, using miRNA gene chip screening, real-time fluorescence quantitative PCR (Q RT-PCR) and Western blot (Western blot) techniques to observe the influence of apoptosis related miRNA and its target gene expression in cardiac myocyte apoptosis and its regulatory relationship, and explore the miRNA gene of "specimen matching point" acupuncture intervention on myocardial ischemia Regulation mechanism, in order to provide theoretical support and scientific basis for the prevention and treatment of myocardial ischemia by acupuncture. Method 1. 40 SPF male Wistar rats, weighing 180-220g, were randomly divided into normal group, model group, Neiguan electroacupuncture group (Neiguan group), group acupoint electroacupuncture group (marking group), 10 in each group. Model group, Neiguan group, and standard group rats were given the isopropionic kidney. Adenin (ISO) 2mg/ (kg / D) was injected subcutaneously for 14 days. Then BL-420 biological function system was used to detect the electrocardiogram of rats in the model group. QRS wave, QT wave duration extended (QRS0.1s), T wave morphologic change was used as a successful model. The normal group rats were given the same amount of 0.9% raw saline under the abdominal subcutaneous injection for 14 days. Normal group, model group rats The Neiguan group needled bilateral "inner customs" (5 mm in the middle of the wrists in the middle of the rat's wrist and 0.5 cm deep in the needle), and the marking group was puncturing bilateral "inner customs", "Guan yuan" (about 25 mm under the umbilicus of the rat, 0.5 cm in deep acupuncture), bilateral "Zusanli" (the posterior lateral of the knee of the rat, the small head of the fibula about 5mm, and the needling depth about 0.8 cm). Group the same side acupoints composed of a pair of electrodes, Neiguan group and the mark group Neiguan point at the right side of the acupoint on the right side of the acupoint at 0.5cm, another needle as an auxiliary electrode, then HANS LH202H electroacupuncture treatment instrument, continuous wave, 2 Hz, the intensity 1m A, with the limbs of the rats have a feeling of tremor as suitable strength, 1 times a day, a total of 21 days. Rats on the twenty-second day. The left ventricular end diastolic diameter (LVEDd), left ventricular end systolic diameter (LVESD), left ventricular ejection fraction (EF) and shortening rate of left ventricular short axis (FS) were detected by echocardiography. Myocardial apoptosis index (AI) was detected by TUNEL, serum creatine kinase isoenzyme (CK-MB), vascular endothelial cell adhesion molecule 1 (VCAM-1) and endothelin 1 (ET-1) were detected by ELISA method. The expression of miRNA-133-3p, miRNA-133-5p, miRNA-1-3p, miRNA-486 and target gene Nol3, Caspase-3, Aifm2, Api5, RGD1564319, Aatk were detected by real time fluorescence quantitative PCR, and the data were analyzed, and 12 male rats in each group were 3 rats in each group. 70.3.SPF male Wistar rats were detected by microarray gene chip technology, and 70 male rats were randomly divided into normal group, model group, Neiguan group, marker group, miRNA-133a-5p inhibitor group (inhibitor group), miRNA-133a-5p irritant group (agonist group), miRNA-133a-5p inhibitor + "specimen matching acupoint" "Electroacupuncture group (inhibitor + labeling group), each group of 10. Model group, Neiguan group, marker group, inhibitor group, agonist group and inhibitor + marking group model method and (method 1) the same. Inhibitor group, inhibitor + labeling group under the abdominal subcutaneous 10mg/kg on the first 1,7,14 day of the experiment, miRNA-133a-5p inhibition (antagomir) injection, a total of 3 times; agonist group Injection of miRNA-133a-5p agonist (agomir), dose, method is the same as the inhibitor group. Normal group method and (method 1) the same. Normal group, model group, inhibitor group, excitant group rats only take fixed, no acupuncture treatment. Neiguan group, marking group, inhibitor + marking group for electroacupuncture treatment, inhibitor + marking group and the matching group treatment method is the same The method of electroacupuncture treatment was the same as that of the upper (method 1). The heart samples were obtained on the twenty-second day of the experiment. The expression of miRNA-133-5p and its target gene Caspase-3 and Aifm2 were detected by real-time fluorescence quantitative PCR. The expression of Caspase-3 and Aifm2 of the target genes of each group was detected by Western blot method, and the data were statistically analyzed and the correlation analysis was carried out. The results of the 1. model group were in the 1. model group. The LVEDd, LVESD of the rat heart was higher than that of the normal group (all P0.01), while the value of EF and FS were lower than that of the normal group (all P0.01), and the Neiguan group, LVEDd and LVESD were lower than the model group (all P0.01), and the marking group was lower than the Neiguan group (P0.01, P0.05), and the Neiguan group was higher than the model group (all respectively), and the marking group was higher than the Neiguan group. The AI value of the normal group was lower than that of the model group (P0.01), and the Neiguan group was lower than the model group (all P0.01), and the serum CK-MB, VCAM-1, ET-1 value of the labeled group were lower than that of the normal group (P0.05) in the.3. model group (all P0.01), and the Neiguan group was lower than the model group (all P0.01), and the standard group were all lower than the Neiguan group (all P0.05). .4. microarray technology was used to screen 758 miRNA gene signals, and there were 20 miRNA gene information with differential significance: 12 expression up-regulated and 8 down-regulation, of which miR-133a-5p and miR-133a-3p were up-regulated, and the downward trend of miR-1-3p and miR-486 was obviously better than that of other miRNA genes. The target genes related to apoptosis of the 4 miRNA genes are predicted by the material information database. The target base of miR-133a-3p is because of Nol3, the target of miR-133a-3p is Caspase-3 and Aifm2, the target gene of miR-1-3p is Api5, and the target gene of miR-486 is RGD1564319 and Aatk.5. (1) model group of rat myocardium miRNA-133-3p. The expression of miRNA-1-3p and miRNA-486 in the normal group (all P0.01) was higher than that in the normal group (all P0.01), and the expression of miRNA-133-3p and miRNA-133-5p in the Neiguan group was higher than that in the model group (P0.05, P0.01, P0.01, P0.01), and the markers were all higher than those in the Neiguan group (P0.05, P0.01), and the Neiguan group was lower than the model group. P0.05, P0.05, P0.01, P0.01) were all lower than the Neiguan group (all P0.05). (2) the expression of Nol3 in the model group was higher than that of the normal group (P0.01). There was no significant difference between the model group and the Neiguan group (all P0.05), and the expression of Caspase-3 in the model group was higher than that of the normal group (all P0.01), and the Neiguan group and the marking group were all lower than the model group (all P0.0). 1), the standard group was lower than the Neiguan group (all P0.01), and the expression of Api5 in the model group was lower than that of the normal group (P0.01). There was no significant difference between the Neiguan group and the model group (P0.05), the marking group was lower than the model group (P0.05), and the RGD1564319 expression in the normal group was not significantly different from the model group (P0.05), and the expression of Aatk in the model group was higher than that in the normal group (P0.01), and there was no significant difference between the Neiguan group and the model group. (P0.05), the marker group was higher than the model group (P0.05). (3) the correlation between the whole group and the correlation coefficient intensity analysis: miRNA-133a-3p and Nol3, miRNA-1-3p and Api5 are moderate negative correlation; miRNA-486 and Aatk are moderate positive correlation; miRNA-486 and RGD1564319 are mild negative correlation; miRNA-133a-5p and Caspase-3, both are highly negative correlation (1) model group. The expression of miRNA-133a-5p in rat myocardium was lower than that of the normal group (P0.01), and the Neiguan group, the marker group, the agonist group and the inhibitor + marker group were all higher than the model group (all P0.01), and the inhibitor group was lower than the model group (P0.01). The marker group, the agonist group, the inhibitor + marking group were all higher than the Neiguan group (P0.01, P0.01, P0.05), and the inhibitor group was lower than the Neiguan group (P0.01); the inhibitor group was lower than the Neiguan group (P0.01); the inhibitor group was lower than the Neiguan group (P0.01). The activity agent group was higher than the marking group (P0.01), the inhibitor group was lower than the marking group (P0.01), the inhibitor + marker group had no significant difference (P0.05). (2) the myocardium of the rat model group was Caspase-3, and the Aifm2 expression (Q RT-PCR detection) was higher than that of the normal group (all P0.01), and the Neiguan group, the marker group, the agonist group, and the inhibitor + labeling group were all lower than the model group (all P0.01). The inhibitor group was higher than the model group (P0.01, P0.05), and the activator group was lower than the Neiguan group (all P0.01), the inhibitor group was higher than the Neiguan group (all P0.01), the inhibitor + marker group and the Neiguan group had no significant difference (P0.05), the inhibitor group was higher than the marking group (all P0.01), the agonist group, the inhibitor + marker group and the marking group were not significant. The difference (all P0.05). (3) the myocardium of rats in the model group was higher than that of the normal group (all P0.01) in Caspase-3 and Aifm2 expression (all P0.01), and in the Neiguan group, the marker group, the agonist group and the inhibitor + marker group were all lower than the model group (all P0.01), and the inhibitor group was higher than the model group (all P0.05); the group of the markers were all lower than the Neiguan group (all P0.01). The inhibitor group was higher than the Neiguan group (all P0.01), and there was no significant difference between the inhibitor + marker group (Caspase-3) and the Neiguan group (P0.05), the inhibitor + marker group (Aifm2) was lower than the Neiguan group (P0.01), the inhibitor group was higher than the marking group (P0.01), the agonist group, the inhibitor + marker group and the marking group had no significant difference (all P0.05). (4) the correlation and phase of the whole group. Relationship strength analysis: miRNA-133a-5p and Caspase-3, Aifm2 (Q RT-PCR, Western-blot detection) were both strong negative correlation. Conclusion 1. electroacupuncture can reduce LVEDd, LVESD value, EF, FS value of myocardial ischemia model rats, effectively improve the compensatory cardiac dilatation caused by myocardial ischemia cell apoptosis, heart dysfunction, and reduce myocardial fine. The degree of apoptosis, and the Electroacupuncture of "sample acupoint" electroacupuncture is better than the simple Neiguan electroacupuncture method..2. can change the index of myocardial enzymes caused by myocardial ischemia cell apoptosis. "Specimen acupoint" electroacupuncture and Neiguan electroacupuncture can effectively reduce the activity of CK-MB, VCAM-1 and ET-1 in experimental rats to play anti myocardial deficiency. Blood effect, and the "specimen matching point" electroacupuncture method has a better effect than pure Neiguan electroacupuncture..3. multiple miRNA gene participates in the apoptosis process of myocardial ischemia cells in rats induced by the adrenal gland. During the process of myocardial ischemia, the main manifestations are miRNA-133-3p, miRNA-133-5p, miRNA-1-3p, miRNA-486 rise, and "specimen with acupoint" electricity. The needle method can effectively enhance the expression of miRNA-133-3p and miRNA-133-5p and reduce the expression of miRNA-1-3p and miRNA-486. The protective effect is to inhibit the apoptosis of myocardial cells by controlling these 4 miRNA genes and the target gene is the main way to affect the apoptosis of myocardial ischemia cells. "Specimen matching point" electroacupuncture is used to prevent and cure myocardium. In the miRNA signaling pathway of ischemia, the gene regulation signal between miRNA-133-5p and Caspase-3 and Aifm2 is the main way of activating.5.Caspase-3 and Aifm2 factor, which is the main executive factor to activate the apoptotic pathway. "Specimen matching" electroacupuncture inhibits apoptosis and protects the heart from the function of miRNA-133-5p. Thus, the dual channel regulation of Caspase-3 and Aifm2 expression can be reduced.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R245

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