本文選題：心肌缺血 + 內(nèi)關��； 參考：《遼寧中醫(yī)藥大學》2016年碩士論文

【摘要】：目的:實驗一從分子水平研究電針內(nèi)關穴對心肌缺血小鼠心肌細胞Gi、Gs蛋白表達的影響,實驗二用慢性應激刺激,結(jié)合情志因素來加重心肌缺血的損傷,觀察電針內(nèi)關穴對炎癥因子及心肌細胞Gi、Gs蛋白表達的影響。探討內(nèi)關穴治療心肌缺血的機理和內(nèi)關穴治療慢性應激抑郁合并心肌缺血的機理,為內(nèi)關穴與心以及經(jīng)絡與臟腑之間關聯(lián),及情志因素在心肌缺血的發(fā)病與治療的相關性之中提供理論依據(jù)。材料與方法:在研究電針內(nèi)關穴對心肌缺血小鼠心肌細胞Gi、Gs蛋白表達的影響的實驗中,選取24只C57BL/6(SPF級)小鼠和21只同品系酸敏感離子通道3(SPF級)基因敲除小鼠,隨機分為5組,為正常組、模型組、內(nèi)關組、列缺組和非經(jīng)非穴組,給予多點皮下注射異丙腎的方法建立心肌缺血模型,觀察電針內(nèi)關組與電針列缺組、電針非經(jīng)非穴組比較心電圖ST段值以及T波值的改變;用Western-blot法分析Gi、Gs蛋白表達,光鏡觀察心肌組織形態(tài)學的變化。在研究電針內(nèi)關穴對慢性應激抑郁合并心肌缺血大鼠炎癥因子IL-1、IL-2、IL-8及心肌細胞Gi、Gs蛋白表達的影響實驗中,選取50只SD大鼠隨機分為5組,為正常組、心肌缺血模型組、心肌缺血合并慢性應激抑郁模型組、內(nèi)關治療1組、內(nèi)關治療2組,給予多點皮下注射異丙腎上腺素和強制束縛加游泳的方法建立心肌缺血合并慢性應激抑郁模型,經(jīng)電針內(nèi)關治療1組與內(nèi)關治療2組后,采用Western-blot法檢測心肌細胞Gi、Gs蛋白表達的影響,采用ELISA法檢測血漿中IL-1、IL-2、IL-8的含量。結(jié)果:1.C57BL/6和ASIC3~(-/-)兩種小鼠造模后,除正常組外,各組小鼠ST段值與T波值都有不同程度抬高,差異有統(tǒng)計學意義(P0.05),表明心肌缺血模型成功建立。經(jīng)過7天電針干預后,內(nèi)關組、列缺組和非經(jīng)非穴組ST段均有降低,各組與本組造模后比較差異有統(tǒng)計學意義(P0.05),與模型組7天電針干預后比較有差異(P0.05)。內(nèi)關組ST段降低更加明顯,與列缺組和非經(jīng)非穴組比較差異有統(tǒng)計學意義(P0.05)。但未降低到正常水平。提示:電針內(nèi)關穴,可明顯改善小鼠急性心肌缺血損傷后的心電圖ST段值與T波值的異常抬高,促進缺血心肌心電圖ST段值與T波值的恢復,列缺穴和非經(jīng)非穴組干預效果差于內(nèi)關穴組。C57BL/6小鼠內(nèi)關組與ASIC3~(-/-)小鼠內(nèi)關組比較,C57BL/6小鼠組心電圖ST段值與T波值降低更顯著。2.通過光鏡檢測心肌缺血心肌組織形態(tài)改變,電針內(nèi)關穴、列缺穴和非經(jīng)非穴對心肌缺血的兩種小鼠心肌細胞均有一定的作用,但是電針內(nèi)關穴對C57BL/6與ASIC3~(-/-)小鼠的心肌保護作用更明顯。3.C57BL/6和ASIC3~(-/-)兩種小鼠造模后與正常組比較,模型組、內(nèi)關組、列缺組和非經(jīng)非穴組C57BL/6小鼠Gs蛋白表達均有所降低,Gi蛋白表達均有所升高,且具有統(tǒng)計學意義(P0.05);經(jīng)電針干預后與模型組比較,內(nèi)關組、列缺組Gs蛋白表達均有所上升,Gi蛋白表達均有所下降,且具有統(tǒng)計學意義(P0.05);內(nèi)關組與列缺組比較,內(nèi)關組Gs蛋白表達上升更明顯,Gi蛋白表達下降更明顯,且具有統(tǒng)計學意義(P0.05)。提示:電針內(nèi)關穴可明顯增加C57BL/6和ASIC3~(-/-)兩種小鼠Gs蛋白表達,降低兩種小鼠Gi蛋白表達,但沒能恢復到正常水平,列缺組沒內(nèi)關組效果明顯,且具有統(tǒng)計學意義(P0.05),非經(jīng)非穴組效過不明顯。C57BL/6小鼠內(nèi)關組與ASIC3~(-/-)小鼠內(nèi)關組比較,C57BL/6小鼠組Gi、Gs蛋白表達更明顯。4.與正常組比較,心肌缺血模型組、心肌缺血合并慢性應激抑郁模型組,血清CK-MB、LDH都有所升高,且具有統(tǒng)計學意義(P0.05)。提示心肌缺血造模成功。與正常組、心肌缺血模型組以及內(nèi)關治療1組比較,心肌缺血合并慢性應激抑郁組和內(nèi)關治療2組大鼠體重增加減慢,蔗糖攝入量減少,差異具有統(tǒng)計學意義(P0.05),提示造模方法可誘發(fā)大鼠慢性應激抑郁的發(fā)生。與正常組比較,心肌缺血模型組與心肌缺血合并慢性應激抑郁模型組血清的IL-1、IL-2、IL-8含量升高,Gi蛋白表達升高、Gs蛋白表達降低,且具有統(tǒng)計學意義(P0.05);與心肌缺血模型組比較,心肌缺血合并慢性應激抑郁模型組的血清IL-1、IL-2、IL-8含量升高更明顯、Gi蛋白表達升高更明顯、Gs蛋白表達降低更明顯,且具有統(tǒng)計學意義(P0.05),說明慢性應激抑郁能加重炎癥因子含量、加重Gi蛋白表達升高、加重Gs蛋白表達下降。電針內(nèi)關穴后,與心肌缺血模型組及心肌缺血合并慢性應激抑郁模型組比較,內(nèi)關治療1組與內(nèi)關治療2組的血清IL-1、IL-2、IL-8含量降低,Gi蛋白表達降低、Gs蛋白表達上升,且具有統(tǒng)計學意義(P0.05);與內(nèi)關治療2組比較,內(nèi)關治療1組IL-1、IL-2、IL-8含量降低更明顯,Gi蛋白表達降低更明顯、Gs蛋白表達上升更明顯,且具有統(tǒng)計學意義(P0.05)。提示:電針內(nèi)關穴可明顯改善心肌缺血合并慢性應激抑郁大鼠血清中IL-1、IL-2、IL-8含量,改善Gi、Gs蛋白異常表達,但是電針內(nèi)關穴對單純心肌缺血大鼠各項指標的改善較心肌缺血合并慢性應激抑郁大鼠的效果更明顯。結(jié)論:1.電針內(nèi)關穴通過降低Gi蛋白表達及升高Gs蛋白表達,達到保護心肌細胞,從心電圖的改善、組織形態(tài)學的改善中體現(xiàn)。2.針刺對C57BL/6小鼠治療作用明顯優(yōu)于對ASIC3~(-/-)小鼠,可能的機制是ASIC3參與針刺作用的外周感受器傳入神經(jīng)機制,因此針刺內(nèi)關穴可以激活ASIC3受體離子通道,從而抑制交感神經(jīng),緩解因異丙腎上腺素興奮交感神經(jīng)所引起的心肌缺血的癥狀。3.慢性應激抑郁加重了心肌缺血損傷,說明情志因素可以通過炎癥因子改變影響心肌缺血的發(fā)生發(fā)展。電針內(nèi)關穴可通過調(diào)節(jié)炎癥因子表達和心肌細胞Gi、Gs蛋白來減輕心肌缺血合并慢性應激抑郁大鼠的心肌損傷,從而同時治療情志病及心系病,說明內(nèi)關穴是情志與心系病相互關聯(lián)的經(jīng)絡基礎。
[Abstract]:Objective: To study the effect of electroacupuncture Neiguan point on the expression of Gi and Gs protein in myocardial ischemia mice from electroacupuncture at molecular level. Experiment two with chronic stress stimulation, combined with emotional factors to aggravate myocardial ischemia injury, and observe the effect of electroacupuncture Neiguan point on the expression of inflammatory factors and Gi and Gs protein in cardiac myocytes. The mechanism of ischemia and the mechanism of Neiguan Acupoint in the treatment of chronic stress depression combined with myocardial ischemia provide a theoretical basis for the association between Neiguan acupoint and heart, the relationship between the meridian and the viscera, and the relationship between the emotional factors and the correlation between the pathogenesis and treatment of myocardial ischemia. Materials and methods: the study on the Gi, Gs egg of the myocardial ischemia mice by the electroacupuncture Neiguan point In the experiment of the effect of white expression, 24 C57BL/6 (SPF grade) mice and 21 same line acid sensitive ion channel 3 (SPF grade) gene knockout mice were randomly divided into 5 groups, which were divided into normal group, model group, Neiguan group, column deficiency group and non non acupoint group, and the myocardial ischemia model was established by multiple subcutaneous injection of isopropyl kidney. The ECG ST segment value and the change of T wave value were compared by Electroacupuncture without non acupoint group. The changes of Gi, Gs protein expression and myocardial histomorphology were observed by Western-blot method. The expression of IL-1, IL-2, IL-8 and myocardium cells Gi and Gs protein were studied in the electroacupuncture Neiguan point for the chronic stress depression with myocardial ischemia rats, IL-1, IL-2, IL-8 and cardiac myocytes. In the experiment, 50 SD rats were randomly divided into 5 groups, which were normal group, myocardial ischemia model group, myocardial ischemia combined with chronic stress depression model group, 1 groups of Neiguan treatment, 2 groups of Neiguan treatment, multi point subcutaneous injection of isoproterenol and compulsory shackle and swimming. After 1 groups and 2 groups of Neiguan treatment, the effect of Western-blot method was used to detect the expression of Gi and Gs protein in cardiac myocytes. The content of IL-1, IL-2 and IL-8 in plasma was detected by ELISA. Results: after two kinds of mice model of 1.C57BL/6 and ASIC3~ (- / -), the ST segment value and T wave value of all the mice were elevated in different degrees, and the difference was statistically significant. Learning significance (P0.05) showed that the model of myocardial ischemia was successfully established. After 7 days of electroacupuncture, the ST segment of Neiguan group, lacked group and non acupoint group decreased, and there was a significant difference between the groups and the group (P0.05), and the prognosis was different from that of the model group at 7 days (P0.05). The decrease of the ST segment in the Neiguan group was more obvious, and the group and the absence of the group. The difference between non acupoint group and non acupoint group was statistically significant (P0.05), but it did not decrease to normal level. It suggested that electroacupuncture Neiguan acupoint can obviously improve the abnormal elevation of ST segment value and T wave value of ECG after acute myocardial ischemia injury in mice, promote the recovery of ST segment value and T wave value of ischemic myocardium, and the intervention effect of column deficiency and non acupoint group is worse than that of the non acupoint group. Compared with the ASIC3~ (- / -) Neiguan group of Neiguan group.C57BL/6 mice and Neiguan group of ASIC3~ (- / -), the electrocardiogram of C57BL/6 mice and T wave value decreased more significantly by.2. to detect the changes of myocardial tissue morphology by light microscope, and the electroacupuncture Neiguan point, the lacing points and non non acupoints had a certain effect on the two kinds of myocardial cells of myocardial ischemia. The protective effect of electroacupuncture at Neiguan point on the myocardium of C57BL/6 and ASIC3~ (- / -) mice was more obvious than that of the two mice of.3.C57BL/6 and ASIC3~ (- / -). The expression of Gs protein in the model group, the Neiguan group, the absence group and the non non acupoint group of C57BL/6 mice decreased, and the expression of Gi protein increased, and was statistically significant (P0.05). Compared with the model group, the expression of Gs protein in the Neiguan group and the absence group were all increased, and the expression of Gi protein decreased and was statistically significant (P0.05). In Neiguan group, the expression of Gs protein in Neiguan group increased more obviously, and the expression of Gi protein decreased more clearly, and was statistically significant (P0.05). It suggested that the electroacupuncture Neiguan point was clear. Increase the expression of C57BL/6 and ASIC3~ (- / -) two mice Gs protein and reduce the expression of Gi protein in two mice, but did not recover to the normal level. The effect of the group without the Neiguan group was significant and had statistical significance (P0.05). The non acupoint group and the.C57BL/6 mice Neiguan group were compared with the ASIC3~ (- / -) mouse Neiguan group, and C57BL/6 mice group Gi, Gs. The expression of.4. was more obvious than that in the normal group. The myocardial ischemia model group, the myocardial ischemia combined with the chronic stress depression model group, the serum CK-MB, LDH all increased, and had statistical significance (P0.05). It suggested that the model of myocardial ischemia was successful. Compared with the normal group, the myocardial ischemia model group and the Neiguan treatment group, the myocardial ischemia combined with the chronic stress in 1 groups. In the 2 groups of depression group and Neiguan treatment, the weight gain decreased and the sucrose intake decreased, the difference was statistically significant (P0.05), suggesting that the modeling method could induce chronic stress depression in rats. Compared with the normal group, the serum IL-1, IL-2, and IL-8 levels in the myocardial ischemia model group and the myocardial ischemia combined with chronic irritable depression model group were increased, Gi The expression of protein expression increased, Gs protein expression decreased and had statistical significance (P0.05). Compared with myocardial ischemia model group, the levels of serum IL-1, IL-2, IL-8 in the group of myocardial ischemia combined with chronic stress depression were more obvious, the increase of Gi protein expression was more obvious, the decrease of Gs protein expression was more obvious, and there was statistical significance (P0.05), indicating chronic (P0.05). Stress and depression could aggravate the content of inflammatory factors, increase the expression of Gi protein and increase the expression of Gs protein. After electroacupuncture Neiguan point, compared with myocardial ischemia model group and myocardial ischemia combined with chronic stress depression model group, the content of serum IL-1, IL-2, IL-8 in Neiguan treatment group and Neiguan treatment group 2 were decreased, the expression of Gi protein was reduced, and the expression of Gs protein was expressed. The increase was statistically significant (P0.05). Compared with the 2 groups in the Neiguan treatment group, the content of IL-1, IL-2, IL-8 decreased more obviously in Neiguan treatment, the decrease of Gi protein expression was more obvious, the expression of Gs protein increased more obviously, and it had statistical significance (P0.05). It suggested that the electroacupuncture Neiguan point could obviously improve the serum IL in the myocardial ischemia combined with chronic stress depression rats. -1, IL-2, IL-8 content, improve the abnormal expression of Gi, Gs protein, but the effect of electroacupuncture Neiguan point on myocardial ischemia rats is more obvious than that of myocardial ischemia combined with chronic stress depression rats. Conclusion: 1. electroacupuncture Neiguan point can protect cardiomyocytes by reducing the expression of Gi protein and increasing the expression of Gs protein, and change the electrocardiogram from electrocardiogram. The improvement of the histomorphology shows that the therapeutic effect of.2. acupuncture on C57BL/6 mice is obviously better than that of ASIC3~ (- / -) mice. The possible mechanism is that ASIC3 participates in the peripheral receptor of the peripheral receptor of the acupuncture effect. Therefore, the acupuncture Neiguan point can activate the ASIC3 receptor ion channel, thus inhibiting the sympathetic nerve and relieving the isoprenaline. .3. chronic stress depression aggravates myocardial ischemia injury, indicating that emotional factors can affect the development of myocardial ischemia through the change of inflammatory factors. The electroacupuncture Neiguan point can reduce myocardial ischemia and chronic stress depression by regulating the expression of inflammatory factors and myocardial cells Gi, Gs egg white. Myocardial injury in rats, so as to treat emotional diseases and heart diseases at the same time, shows that Neiguan is the basis of meridians and collaterals, which is related to emotion and heart disease.
【學位授予單位】：遼寧中醫(yī)藥大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：R245

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