针刺内关穴逆转ISO诱导小鼠心肌肥厚的研究

发布时间：2018-03-16 01:09

本文选题：内关　切入点：针刺　出处：《黑龙江中医药大学》2016年博士论文　论文类型：学位论文

【摘要】：目的：通过针刺内关穴干预异丙肾上腺素致心肌肥厚模型小鼠,观察其对心肌肥厚形态学、功能学及相关炎性因子的表达的影响,为针刺内关穴逆转心肌肥厚提供理论依据。方法：1.实验动物及分组：选用体质量为20-25g的清洁级雄性C5713L6野生型小鼠60只,随机分为模型组(model)、针刺组(acupuncture)、非穴对照组(acupuncture-control)、药物组(p-control)和空白对照组(control)各12只。2.造模方法：采用异丙肾上腺素(15mg/kg)连续8天每日1次皮下注射制作心肌肥厚小鼠模型。3.各组处理：模型组：背部三个部位盐酸异丙肾上腺素注射液(15mg/kg)皮下注射,每日1次,连续8天。针刺组：小鼠麻醉后针刺双侧“内关穴”,留针15min,针刺结束30min后注射造模药物,造模方法同模型组,每日1次,连续8天。非穴组：非穴位对照组选取小鼠尾根部,每天麻醉后与针刺组同一时间针刺尾根,每次留针15min,30min后注射造模药物,造模方法同模型组,每日1次,连续8天。药物组：小鼠给予阿替洛尔(10mg/kg)灌胃,30min后注射造模药物,造模方法同模型组,每日1次,连续8天。空白组：每天麻醉后30min给予生理盐水1 ml/kg皮下注射,共8天。4.心电及心功能检测：实验第9天,各组小鼠给予3%水合氯醛0.1 m1/10g小鼠体质量腹腔注射麻醉后,描记小鼠标准12导联心电图,记录各组小鼠心率。采用小动物高频彩色超声仪采集各组小鼠左室前壁舒张末期厚度(LVAWD)、左室前壁收缩末期厚度(LVAWS)、左室后壁舒张末期厚度(LVPWD)、左室后壁收缩末期厚度(LVPWS)、射血分数(EF%)并计算其左室短轴缩短率(FS%)。5.心脏重量／胫骨长度比值：实验第9天,称取心脏质量(HW),游标卡尺测量胫骨长度,并计算心脏质量／胫骨长度。6.小鼠死亡率：记录造模过程中(实验第1-8天)各组小鼠死亡情况,分析各组小鼠死亡率差异。7.心肌组织形态学研究：分别采用HE染色观察各组小鼠心肌细胞形态改变,采用masson染色观察各组小鼠心肌纤维化程度,采用透射电镜观察各组小鼠心肌细胞超微结构的改变。8.免疫荧光：免疫荧光法检测各组小鼠心肌ANP及TNF-α表达的变化。9.免疫印迹法检测各组小鼠心肌ANP和TNF-α表达的变化。结果：1.各组小鼠一般状态：模型组与非穴组小鼠毛色及活跃度明显较空白组降低,未见明显呼吸费力小鼠,针刺组与药物组小鼠毛色及活跃状态明显好于模型组。2.各组小鼠死亡率分析：模型组和非穴组小鼠死亡率明显高于空白组,而针刺和药物治疗均可降低各组小鼠死亡率。3.心率分析：与空白组相比,模型组与非穴组小鼠心率显著加快,差异具有统计学意义(p0.01)；而与模型组相比,针刺组和药物组均能显著降低小鼠心率,差异具有统计学意义(p0.01)；而药物组与针刺组小鼠心率相比,差异不具有统计学意义(p0.05)。4.超声心动图分析：与空白组小鼠相比,模型组和非穴组小鼠在异丙肾上腺素皮下注射8天后表现出心肌肥厚和心功能减退的表现。其中以LVPWD、LVPWS、LVAWS增大为具有统计学意义(p0.05),而EF及FS值各组变化不明显,未见显著差异(p0.05)。与模型组小鼠相比,针刺组和药物组表现出拮抗异丙肾上腺素所致心肌肥厚过程,其主要表现为LVPWD、LVPWS明显低于模型组,差异显著(p0.05)。5.心脏重量／胫骨长度比值分析：与空白组相比,模型组及非穴组小鼠心脏重量／胫骨长度比值明显增加,差异显著(p0.01)；.擦与模型组相比,针刺组、药物组以及空白组小鼠心脏重量／胫骨长度比值明显降低,差异具有统计学意义(p0.01)；而药物组与针刺组心脏重量／胫骨长度比值差异不具有统计学意义(p0.05)。6.小鼠心肌组织形态学改变：HE染色及投射电镜均可见针刺与药物组均可改善异丙肾上腺素致心肌细胞及肌纤维的病变,masson染色可见与空白组相比,模型组与非穴组可见明湿心肌纤维纤维化。与模型组比较,针刺组与药物组心肌纤维化程度明显降低。7.小鼠心肌ANP的表达分析：经免疫荧光及western blot分析均可见与模型组相比,针刺组和药物组可显著降低小鼠心肌ANP的表达,差异显著具有统计学意义,而假针刺组并不能降低小鼠心肌ANP的表达,与模型组无显著差异。8.心肌组织TNF-α表达分析：经免疫荧光及Western blot研究发现,与模型组相比,针刺组和药物组可显著降低小鼠心肌TNF-α的表达,差异显著具有统计学意义(p0.01),而假针刺组并不能降低小鼠心肌TNF-α的表达,与模型组无显著差异(p0.05)。结论：1.异丙肾上腺素连续8天皮下注射,可成功制作心肌肥厚小鼠模型。2.针刺内关穴可显著逆转ISO诱导心肌肥厚小鼠心肌细胞形态学改变,改善其心肌细胞纤维化程度。3.针刺内关穴可降低ISO皮下注射致小鼠心率增快,抑制心肌细胞炎症因子TNF-α的表达,进而下调心肌肥厚标志物ANP的表达,逆转小鼠心肌肥厚性重构。
[Abstract]:Objective: through the acupuncture intervention in isoproterenol induced myocardial hypertrophy in mice, observe the myocardial hypertrophy morphology, function and related inflammatory cytokines expression, and provide a theoretical basis for acupuncture and reversal of cardiac hypertrophy. Methods: 1. experimental animal and group: the body mass of male C5713L6 wild type of mouse 20-25g 60 rats were randomly divided into model group (model), acupuncture group (acupuncture), non acupoint control group (acupuncture-control), drug group (P-control) and control group (control) of the 12.2. modeling methods: using isoproterenol (15mg/kg) for 8 consecutive days, 1 times daily subcutaneous injection production myocardial hypertrophy mice model.3. group treatment: Model group: three sites on the back of Isoprenaline Hydrochloride Injection (15mg/kg) subcutaneous injection, 1 times daily for 8 days. The acupuncture group: the mice were anesthetized with acupuncture" Neiguan, acupuncture needle 15min, 30min after injecting drugs, the modeling method with the model group, 1 times daily for 8 consecutive days. Non acupoint group: non acupoint control group selected mice tail root day after anesthesia and acupuncture acupuncture group at the same time the root of the tail, each time for 15min, 30min after injection modeling of drug modeling method with the model group, 1 times daily for 8 days. The drug group: mice were given intragastric administration of atenolol (10mg/kg), 30min after injecting drugs, the modeling method with the model group, 1 times daily for 8 consecutive days. Control group: every day after anesthesia given saline 30min 1 ml/kg subcutaneous injection, a total of 8 days of.4. ECG and heart function detection: the ninth day of the experiment, mice were anesthetized by 3% chloral hydrate 0.1 m1/10g the weight of the mice after intraperitoneal injection, mice were recorded on standard 12 lead ECG, heart rate were recorded in mice. Using a small animal ultrasound high frequency acquisition of mice The left ventricular anterior wall end diastolic thickness (LVAWD), left ventricular end systolic anterior wall thickness (LVAWS), left ventricular posterior wall end diastolic thickness (LVPWD), left ventricular posterior wall systolic thickness (LVPWS), ejection fraction (EF%) and calculate the left ventricular fractional shortening (FS%).5. heart weight / tibia length ratio: the ninth day of the experiment, take heart mass (HW), vernier caliper to measure the length of tibia, and calculate the heart quality / tibia length:.6. mice mortality recorded during (the 1-8 day) of mice death, morphological study of.7. myocardial tissue differences in mortality of mice in each group were stained to observe the morphology of myocardial cells of mice were changed by HE, Masson staining was used to observe the myocardial fibrosis in mice, observe the changes of.8. immunofluorescence of Mice Myocardial Ultrastructure by transmission electron microscopy, immunofluorescence method to detect myocardial AN in mice The expression of P and TNF- expression of.9. alpha in mice were detected by Western blot of myocardial ANP and TNF- alpha. Results: 1. the general state of mice in each model group and non acupoint group mice coat color and activity was significantly lower than those in the control group, there was no obvious respiratory effort in mice, acupuncture group and drug group mouse hair color and the active state is significantly better than the model group.2. mice mortality analysis: mortality in model group and non acupoint group were significantly higher than the control group, and acupuncture and drug treatment can reduce the mortality of mice in each group of.3. heart rate: compared with the blank group, model group and non acupoint group of mice heart rate increased significantly, the difference was statistically significant (P0.01); compared with the model group, acupuncture group and drug group were significantly decreased in mice heart rate, the difference was statistically significant (P0.01); compared with drug group and acupuncture group, the heart rate of mice, the difference was not statistically The significance of.4. (P0.05) analysis of echocardiography: compared with the blank group, model group and non acupoint group mice showed myocardial hypertrophy and cardiac dysfunction in isoproterenol injection for 8 days. The LVPWD, LVPWS, LVAWS increased with statistical significance (P0.05), and EF and FS groups the change is not obvious, but there were no significant difference (P0.05). Compared with the model group, acupuncture group and drug group showed antagonism of isoprenaline induced myocardial hypertrophy, the main performance for LVPWD, LVPWS was significantly lower than that of model group, significant difference (P0.05) analysis of.5. heart weight / tibia length ratio: compared with blank control group. The model group and non acupoint group mice heart weight / tibia length ratio was significantly increased, significant difference (P0.01); compared with the model group. Wipe, acupuncture group, drug group and control group of mice heart weight / tibia length ratio decreased significantly, The difference was statistically significant (P0.01); and the drug group and acupuncture group, the heart weight / tibia length ratio difference was not statistically significant (P0.05) changes of.6. in myocardial tissue morphology: HE staining and transmission electron microscopy showed the acupuncture and medicine group can improve the isoprenaline induced myocardial cells and muscle fiber lesions, Masson staining compared with the blank group, model group and non acupoint group showed myocardial fibrosis. The wet fiber compared with the model group, acupuncture group and drug group significantly reduced the degree of myocardial fibrosis and expression of myocardial ANP in.7. mice by immunofluorescence and Western blot analysis were compared with the model group, acupuncture group and drug group significantly decreased the expression of myocardial ANP in mice, the difference was statistically significant, while the sham acupuncture group did not decrease the expression of myocardial ANP in mice, and the model group had no significant difference.8. TNF- myocardial tissue Expression analysis: the study of immunofluorescence and Western blot found that, compared with the model group, acupuncture group and drug group can significantly reduce the expression of myocardium TNF- alpha, significant difference was statistically significant (P0.01), and sham acupuncture group did not reduce the expression of mouse cardiac TNF- alpha, had no significant difference compared with the model group (P0.05 1.). Conclusion: isoproterenol subcutaneous injection for 8 consecutive days, can be successfully established cardiac hypertrophy mouse model of.2. acupuncture significantly reversed the ISO induced cardiac hypertrophy in mice cardiomyocytes morphology change, improve the degree of myocardial fibrosis in.3. acupuncture Neiguan can reduce the subcutaneous injection of ISO mice induced by increased heart rate, inhibit the expression of myocardial cell inflammation factor TNF- alpha, and down regulate the expression of cardiac hypertrophy marker ANP, reverse remodeling of cardiac hypertrophy in mice.

【学位授予单位】：黑龙江中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R245

【相似文献】