本文選題：內(nèi)關(guān)　切入點(diǎn)：針刺　出處：《黑龍江中醫(yī)藥大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：目的：通過針刺內(nèi)關(guān)穴干預(yù)異丙腎上腺素致心肌肥厚模型小鼠,觀察其對心肌肥厚形態(tài)學(xué)、功能學(xué)及相關(guān)炎性因子的表達(dá)的影響,為針刺內(nèi)關(guān)穴逆轉(zhuǎn)心肌肥厚提供理論依據(jù)。方法：1.實(shí)驗(yàn)動(dòng)物及分組：選用體質(zhì)量為20-25g的清潔級雄性C5713L6野生型小鼠60只,隨機(jī)分為模型組(model)、針刺組(acupuncture)、非穴對照組(acupuncture-control)、藥物組(p-control)和空白對照組(control)各12只。2.造模方法：采用異丙腎上腺素(15mg/kg)連續(xù)8天每日1次皮下注射制作心肌肥厚小鼠模型。3.各組處理：模型組：背部三個(gè)部位鹽酸異丙腎上腺素注射液(15mg/kg)皮下注射,每日1次,連續(xù)8天。針刺組：小鼠麻醉后針刺雙側(cè)“內(nèi)關(guān)穴”,留針15min,針刺結(jié)束30min后注射造模藥物,造模方法同模型組,每日1次,連續(xù)8天。非穴組：非穴位對照組選取小鼠尾根部,每天麻醉后與針刺組同一時(shí)間針刺尾根,每次留針15min,30min后注射造模藥物,造模方法同模型組,每日1次,連續(xù)8天。藥物組：小鼠給予阿替洛爾(10mg/kg)灌胃,30min后注射造模藥物,造模方法同模型組,每日1次,連續(xù)8天�？瞻捉M：每天麻醉后30min給予生理鹽水1 ml/kg皮下注射,共8天。4.心電及心功能檢測：實(shí)驗(yàn)第9天,各組小鼠給予3%水合氯醛0.1 m1/10g小鼠體質(zhì)量腹腔注射麻醉后,描記小鼠標(biāo)準(zhǔn)12導(dǎo)聯(lián)心電圖,記錄各組小鼠心率。采用小動(dòng)物高頻彩色超聲儀采集各組小鼠左室前壁舒張末期厚度(LVAWD)、左室前壁收縮末期厚度(LVAWS)、左室后壁舒張末期厚度(LVPWD)、左室后壁收縮末期厚度(LVPWS)、射血分?jǐn)?shù)(EF%)并計(jì)算其左室短軸縮短率(FS%)。5.心臟重量／脛骨長度比值：實(shí)驗(yàn)第9天,稱取心臟質(zhì)量(HW),游標(biāo)卡尺測量脛骨長度,并計(jì)算心臟質(zhì)量／脛骨長度。6.小鼠死亡率：記錄造模過程中(實(shí)驗(yàn)第1-8天)各組小鼠死亡情況,分析各組小鼠死亡率差異。7.心肌組織形態(tài)學(xué)研究：分別采用HE染色觀察各組小鼠心肌細(xì)胞形態(tài)改變,采用masson染色觀察各組小鼠心肌纖維化程度,采用透射電鏡觀察各組小鼠心肌細(xì)胞超微結(jié)構(gòu)的改變。8.免疫熒光：免疫熒光法檢測各組小鼠心肌ANP及TNF-α表達(dá)的變化。9.免疫印跡法檢測各組小鼠心肌ANP和TNF-α表達(dá)的變化。結(jié)果：1.各組小鼠一般狀態(tài)：模型組與非穴組小鼠毛色及活躍度明顯較空白組降低,未見明顯呼吸費(fèi)力小鼠,針刺組與藥物組小鼠毛色及活躍狀態(tài)明顯好于模型組。2.各組小鼠死亡率分析：模型組和非穴組小鼠死亡率明顯高于空白組,而針刺和藥物治療均可降低各組小鼠死亡率。3.心率分析：與空白組相比,模型組與非穴組小鼠心率顯著加快,差異具有統(tǒng)計(jì)學(xué)意義(p0.01)；而與模型組相比,針刺組和藥物組均能顯著降低小鼠心率,差異具有統(tǒng)計(jì)學(xué)意義(p0.01)；而藥物組與針刺組小鼠心率相比,差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。4.超聲心動(dòng)圖分析：與空白組小鼠相比,模型組和非穴組小鼠在異丙腎上腺素皮下注射8天后表現(xiàn)出心肌肥厚和心功能減退的表現(xiàn)。其中以LVPWD、LVPWS、LVAWS增大為具有統(tǒng)計(jì)學(xué)意義(p0.05),而EF及FS值各組變化不明顯,未見顯著差異(p0.05)。與模型組小鼠相比,針刺組和藥物組表現(xiàn)出拮抗異丙腎上腺素所致心肌肥厚過程,其主要表現(xiàn)為LVPWD、LVPWS明顯低于模型組,差異顯著(p0.05)。5.心臟重量／脛骨長度比值分析：與空白組相比,模型組及非穴組小鼠心臟重量／脛骨長度比值明顯增加,差異顯著(p0.01)；.擦與模型組相比,針刺組、藥物組以及空白組小鼠心臟重量／脛骨長度比值明顯降低,差異具有統(tǒng)計(jì)學(xué)意義(p0.01)；而藥物組與針刺組心臟重量／脛骨長度比值差異不具有統(tǒng)計(jì)學(xué)意義(p0.05)。6.小鼠心肌組織形態(tài)學(xué)改變：HE染色及投射電鏡均可見針刺與藥物組均可改善異丙腎上腺素致心肌細(xì)胞及肌纖維的病變,masson染色可見與空白組相比,模型組與非穴組可見明濕心肌纖維纖維化。與模型組比較,針刺組與藥物組心肌纖維化程度明顯降低。7.小鼠心肌ANP的表達(dá)分析：經(jīng)免疫熒光及western blot分析均可見與模型組相比,針刺組和藥物組可顯著降低小鼠心肌ANP的表達(dá),差異顯著具有統(tǒng)計(jì)學(xué)意義,而假針刺組并不能降低小鼠心肌ANP的表達(dá),與模型組無顯著差異。8.心肌組織TNF-α表達(dá)分析：經(jīng)免疫熒光及Western blot研究發(fā)現(xiàn),與模型組相比,針刺組和藥物組可顯著降低小鼠心肌TNF-α的表達(dá),差異顯著具有統(tǒng)計(jì)學(xué)意義(p0.01),而假針刺組并不能降低小鼠心肌TNF-α的表達(dá),與模型組無顯著差異(p0.05)。結(jié)論：1.異丙腎上腺素連續(xù)8天皮下注射,可成功制作心肌肥厚小鼠模型。2.針刺內(nèi)關(guān)穴可顯著逆轉(zhuǎn)ISO誘導(dǎo)心肌肥厚小鼠心肌細(xì)胞形態(tài)學(xué)改變,改善其心肌細(xì)胞纖維化程度。3.針刺內(nèi)關(guān)穴可降低ISO皮下注射致小鼠心率增快,抑制心肌細(xì)胞炎癥因子TNF-α的表達(dá),進(jìn)而下調(diào)心肌肥厚標(biāo)志物ANP的表達(dá),逆轉(zhuǎn)小鼠心肌肥厚性重構(gòu)。
[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.

【學(xué)位授予單位】：黑龍江中醫(yī)藥大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R245

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4 李書霖;針刺內(nèi)關(guān)穴逆轉(zhuǎn)ISO誘導(dǎo)小鼠心肌肥厚的研究[D];黑龍江中醫(yī)藥大學(xué);2016年

5 邸若岷;雷帕霉素改善小鼠心肌梗死后心室重構(gòu)的分子機(jī)制研究[D];南京醫(yī)科大學(xué);2010年

6 程姝娟;Toll樣受體4在內(nèi)毒素誘導(dǎo)的小鼠心肌炎癥因子表達(dá)和左室功能不全中的作用[D];中國人民解放軍軍醫(yī)進(jìn)修學(xué)院;2004年

7 王振華;新生小鼠心肌再生機(jī)制的研究[D];北京協(xié)和醫(yī)學(xué)院;2013年

8 陳蓉;蛇床子素抑制異丙腎上腺素誘導(dǎo)小鼠心肌纖維化及其機(jī)制研究[D];蘇州大學(xué);2012年

9 王永梅;黃芩甙抗CVB_（3m）和調(diào)控CVB_（3m）感染小鼠心肌免疫損傷及細(xì)胞凋亡的實(shí)驗(yàn)研究[D];南京中醫(yī)藥大學(xué);2003年

10 周密;MiR-17-92簇在小鼠心肌缺血—再灌注中的作用[D];復(fù)旦大學(xué);2011年

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