“标本配穴”电针对心肌缺血大鼠细胞凋亡miRNA的影响及调控机制研究

发布时间：2018-07-02 12:23

本文选题：标本配穴 + 针刺　；参考：《湖北中医药大学》2016年博士论文

【摘要】：目的心血管疾病是当今威胁人类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。针刺用于防治心肌缺血已有数千年的历史,具有操作简便快捷、疗效确切的特点,但其作用机制尤其是分子生物学机制目前尚不完全清晰,在一定程度影响了针灸在心肌缺血临床的应用及推广。相关研究表明,在心肌缺血的过程中存在普遍心肌细胞凋亡表现,而细胞凋亡是受基因严格控制的,其中Micro RNA(miRNA)的调控具有关键作用。针刺是否是通过miRNA基因调控途径干预心肌细胞凋亡?主要是通过什么miRNA基因调控的?主要的miRNA基因是通过什么信号调控途径发挥作用的?这些都是需用研究的问题。有据于此,本课题选用心肌缺血模型大鼠为研究对象,在中医针灸“治未病”理论为指导下,采用“标本配穴”电针干预方法,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(q RT-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。方法1.选用SPF级雄性Wistar大鼠40只,体重180-220g,随机分为正常组、模型组、内关电针组(内关组)、标本配穴电针组(标配组),每组10只。模型组、内关组、标配组大鼠予异丙肾上腺素(ISO)2mg/(kg·d)腹部皮下注射,连续14天,后用BL-420生物机能系统检测模型组大鼠心电图,以QRS波、QT波持续时间延长(QRS0.1s)、T波形态改变作为造模成功标志。正常组大鼠给予等量0.9%生理盐水腹部皮下注射,连续14天。正常组、模型组大鼠只抓取固定,不针刺治疗。内关组针刺双侧“内关”(大鼠腕横纹正中上5 mm,针刺深约0.5 cm),标配组针刺双侧“内关”、“关元”(大鼠脐下约25 mm,针刺深约0.5 cm)、双侧“足三里”(大鼠膝关节后外侧、腓骨小头下约5mm,针刺深约0.8 cm),标配组同侧穴位组成一对电极,内关组及标配组中内关穴在穴位右旁开0.5cm处另皮下浅刺一针作辅助电极,后连接HANS LH202H电针治疗仪,连续波,频率2 Hz,强度1m A,以大鼠肢体有震颤感为适宜强度,通电10min。每日治疗1次,共21天。大鼠于第22天行超声心动图机检测大鼠左室舒张末期内径(LVEDd)、左室收缩末期内径(LVESD)、左室射血分数(EF)和左室短轴缩短率(FS);TUNEL法检测大鼠心肌细胞凋亡指数(AI);ELISA法检测血清肌酸激酶同工酶(CK-MB)、血管内皮细胞粘附分子1(VCAM-1)、内皮素1(ET-1);实时荧光定量PCR法检测大鼠心肌组织miRNA-133-3p、miRNA-133-5p、miRNA-1-3p、miRNA-486表达及靶基因Nol3、Caspase-3、Aifm2、Api5、RGD1564319、Aatk的表达。后对数据进行统计分析,并对miRNA与其靶基因进行相关性验证。2.SPF级雄性Wistar大鼠12只,每组3只,分组、治疗、取材方法同上。用微列阵基因芯片技术检测各组大鼠心肌miRNA表达谱状况。3.SPF级雄性Wistar大鼠70只,随机分为正常组、模型组、内关组、标配组、miRNA-133a-5p抑制剂组(抑制剂组)、miRNA-133a-5p激动剂组(激动剂组)、miRNA-133a-5p抑制剂+“标本配穴”电针组(抑制剂+标配组),每组10只。模型组、内关组、标配组、抑制剂组、激动剂组及抑制剂+标配组造模方法与上(方法1)相同。抑制剂组、抑制剂+标配组腹部皮下按10mg/kg于实验第1、7、14天进行miRNA-133a-5p抑制(antagomir)剂注射,共3次;激动剂组注射miRNA-133a-5p激动(agomir)剂,剂量、方法同抑制剂组相同。正常组方法与上(方法1)相同。正常组、模型组、抑制剂组、激动剂组大鼠只抓取固定,不针刺治疗。内关组、标配组、抑制剂+标配组进行电针治疗,抑制剂+标配组与标配组治疗方法相同,电针治疗方法与上(方法1)相同。实验第22天心脏取材,用实时荧光定量PCR法检测各组大鼠心肌组织miRNA-133-5p及其靶基因Caspase-3、Aifm2的表达;Western blot法检测各组靶基因Caspase-3、Aifm2的表达,并对数据进行统计及相关性分析。结果1.模型组大鼠心脏LVEDd、LVESD高于正常组(均为P0.01),而EF、FS值均低于正常组(均为P0.01);内关组、标配组LVEDd、LVESD均低于模型组(均为P0.01),标配组低于内关组(分别为P0.01,P0.05);内关组、标配组中EF、FS值均高于模型组(均为P0.01),标配组高于内关组(P0.05)。2.正常组大鼠心肌AI值低于模型组(P0.01);内关组、标配组均低于模型组(均为P0.01);标配组低于内关组(P0.05)。3.模型组大鼠血清CK-MB、VCAM-1、ET-1值均高于正常组(均为P0.01);内关组、标配组均低于模型组(均为P0.01);标配组均低于内关组(均为P0.05)。4.运用微陈列芯片技术共筛选出758个miRNA基因信号,有20个具有差异化意义的miRNA基因信息:12个表达上调,8个表达下调;其中miR-133a-5p和miR-133a-3p上调趋势、miR-1-3p和miR-486下调趋势明显优于其它miRNA基因。通过Target Scan、miRanda和Pic Tar3种生物信息数据库交叉对上述4个miRNA基因与凋亡相关的靶基因进行预测:miR-133a-3p的靶基因为Nol3,miR-133a-3p的靶基因为Caspase-3和Aifm2,miR-1-3p的靶基因为Api5,miR-486的靶基因为RGD1564319和Aatk。5.(1)模型组大鼠心肌miRNA-133-3p、miRNA-133-5p表达均低于正常组(均为P0.01),miRNA-1-3p、miRNA-486表达均高于正常组(均为P0.01);内关组、标配组miRNA-133-3p、miRNA-133-5p表达均高于模型组(分别为P0.05,P0.01,P0.01,P0.01),标配组均高于内关组(分别为P0.05,P0.01);内关组、标配组miRNA-1-3p、miRNA-486表达均低于模型组(分别为P0.05,P0.05,P0.01,P0.01),标配组均低于内关组(均为P0.05)。(2)模型组心肌Nol3表达高于正常组(P0.01),模型组与内关组、标配组没有显著差异(均为P0.05);模型组Caspase-3、Aifm2表达均高于正常组(均为P0.01),内关组、标配组均低于模型组(均为P0.01),标配组均低于内关组(均为P0.01);模型组Api5表达低于正常组(P0.01),内关组与模型组无显著差异(P0.05),标配组低于模型组(P0.05);正常组RGD1564319表达与模型组无显著差异(P0.05);模型组Aatk表达高于正常组(P0.01),内关组与模型组无显著差异(P0.05),标配组高于模型组(P0.05)。(3)整体组间相关性及相关系数强度分析:miRNA-133a-3p与Nol3、miRNA-1-3p与Api5为中度负相关;miRNA-486与Aatk为中度正相关;miRNA-486与RGD1564319为轻度负相关;miRNA-133a-5p与Caspase-3、Aifm2均为高度负相关。6.(1)模型组大鼠心肌miRNA-133a-5p表达低于正常组(P0.01);内关组、标配组、激动剂组、抑制剂+标配组均高于模型组(均为P0.01),抑制剂组低于模型组(P0.01);标配组、激动剂组、抑制剂+标配组均高于内关组(分别为P0.01,P0.01,P0.05),抑制剂组低于内关组(P0.01);激动剂组高于标配组(P0.01),抑制剂组低于标配组(P0.01),抑制剂+标配组与标配组无显著差异(P0.05)。(2)模型组大鼠心肌Caspase-3、Aifm2表达(q RT-PCR检测)均高于正常组(均为P0.01);内关组、标配组、激动剂组、抑制剂+标配组均低于于模型组(均为P0.01),抑制剂组高于模型组(分别为P0.01,P0.05);标配组、激动剂组均低于于内关组(均为P0.01),抑制剂组高于内关组(均为P0.01),抑制剂+标配组与内关组无显著差异(P0.05);抑制剂组高于标配组(均为P0.01),激动剂组、抑制剂+标配组与标配组无显著差异(均为P0.05)。(3)模型组大鼠心肌Caspase-3、Aifm2表达(Western-blot检测)均高于正常组(均为P0.01);内关组、标配组、激动剂组、抑制剂+标配组均低于于模型组(均为P0.01),抑制剂组高于模型组(均为P0.05);标配组、激动剂组均低于内关组(均为P0.01),抑制剂组高于内关组(均为P0.01),抑制剂+标配组(Caspase-3)与内关组无显著差异(P0.05),抑制剂+标配组(Aifm2)低于内关组(P0.01);抑制剂组高于标配组(均为P0.01),激动剂组、抑制剂+标配组与标配组无显著差异(均为P0.05)。(4)整体组间相关性及相关系数强度分析:miRNA-133a-5p分别与Caspase-3、Aifm2(q RT-PCR、Western-blot检测)均为较强负相关。结论1.电针能够通过降低心肌缺血模型大鼠的LVEDd、LVESD值、提升EF、FS值,有效改善心肌缺血细胞凋亡导致的代偿性心脏扩张、心功能低下,降低心肌细胞凋亡程度,且“标本配穴”电针法比单纯内关电针法具有更好的效应。2.对于心肌缺血细胞凋亡后引发的心肌酶等指标改变,“标本配穴”电针和内关电针法均能通过有效减少实验大鼠血清CK-MB、VCAM-1及ET-1的活性表达发挥抗心肌缺血作用的,且“标本配穴”电针法比单纯内关电针法具有更好的效应。3.多miRNA基因参与了异丙肾上腺所致的大鼠心肌缺血细胞凋亡过程,心肌缺血过程中,主要表现在miRNA-133-3p、miRNA-133-5p表达下降,miRNA-1-3p、miRNA-486上升;“标本配穴”电针法能够通过有效地提升miRNA-133-3p、miRNA-133-5p的表达,降低miRNA-1-3p、miRNA-486的表达,其保护作用是通过调控这4个miRNA基因的方式抑制心肌细胞凋亡。4.miRNA基因及其靶基因是影响心肌缺血细胞凋亡的主要途径;“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。5.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”电针抑制细胞凋亡、保护心肌缺血的作用主要是通过提升miRNA-133-5p的表达,进而减少Caspase-3、Aifm2的表达的双通道调控途径实现的。
[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.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

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