本文選題：參草通脈顆粒 + klotho　； 參考：《遼寧中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:通過建立慢性心衰(CHF)大鼠模型,觀察參草通脈顆粒對慢性心衰大鼠心肌及腎組織klotho的影響以及心肌組織結構的改變。圍繞參草通脈顆粒對大鼠模型慢性心衰癥狀的作用機理和作用靶點,分別從基因、蛋白和組織形態(tài)學等渠道發(fā)現(xiàn)和探索將中醫(yī)藥防治作為治療慢性心衰重要手段的科學依據(jù)。材料與方法:1.通過對實驗大鼠采取結扎冠狀動脈前降支造成局部心梗,再對大鼠進行游泳及減食等方法造成慢性心衰模型,后觀察大鼠狀態(tài),且經(jīng)多普勒超聲心動圖檢測射血分數(shù)(EF)≤55%或CI≤180ml/min/kg,判定慢性心衰大鼠模型造模是否成功。2.將建立的慢性心衰大鼠模型分4組培養(yǎng)。首先,設立假手術組模型用于后期對比研究,該組模型不采取任何藥物干預,僅用常規(guī)等容積蒸餾水灌胃。其次,保留一組心衰大鼠模型為模型組,該組采取同假手術組培養(yǎng)方式。第三,設立西藥組采取灌服賴諾普利干預。最后,設立中藥組模型,灌服參草通脈顆粒,中藥組按照9.2g生藥/Kg/d灌服參草通脈顆粒,賴諾普利西藥組1.5mg生藥/Kg/d。預計培養(yǎng)4周時間,隨后經(jīng)大鼠尾靜脈采血后,麻醉處死全部大鼠,取其大鼠心臟心肌組織及腎組織。3.將所取各組樣本分別通過ELISA法、蛋白免疫印跡技術和實時熒光定量PCR技術收集相關數(shù)據(jù),主要對比不同組模型間BNP濃度,心肌及腎組織klotho的變化情況及電鏡觀察各組間心肌細胞組織形態(tài)學變化情況。結果:1.慢性心衰大鼠BNP濃度與假手術組比較,治療組(中藥組,西藥組)及模型組均升高,p0.05。其中假手術組BNP濃度為450±37.03pg/ml,模型組BNP濃度1017.55±98.6pg/ml,中藥組與西藥組BNP濃度分別為681.08±75.31pg/ml,686.53±74.86pg/ml,p0.05。2.慢性心衰大鼠模型組心肌klotho mRNA表達明顯降低,為0.0003±0.00004(與假手術組比較P0.01);西藥組樣本心肌klotho mRNA具體數(shù)值為0.0020±0.0009相較于中藥組(具體數(shù)值為0.0012±0.0006)p0.05,組間比較治療組與模型組P0.01與假手術組P0.01。3.慢性心衰大鼠模型組心肌klotho蛋白表達明顯下降,為0.0291±0.0025(與假手術組比較P0.01);西藥組樣本心肌klotho蛋白具體數(shù)值為0.2475±0.1407,相較于中藥組(具體數(shù)值為0.215±0.077)p0.05,組間比較治療組與模型組P0.01與假手術組P0.01。4.慢性心衰模型組腎組織klotho mRNA表達明顯降低,為0.0022±0.0007,(與假手術組比較P0.05);西藥組樣本腎klotho mRNA具體數(shù)值為0.0067±0.00209,相較于中藥組(具體數(shù)值為0.0063±0.0022)p0.05,組間比較治療組與模型組P0.01與假手術組P0.01。5.慢性心衰模型組腎組織klotho蛋白表達明顯降低,為0.0639±0.0203(與假手術組比較P0.01);西藥組樣本腎klotho蛋白具體數(shù)值為0.2150±0.0303,相較于中藥組(數(shù)值為0.2057±0.0457)p0.05,組間比較治療組與模型組P0.01與假手術組P0.01。6.假手術組,可見完整的心肌細胞,肌纖維整齊有序,呈均勻清晰的Z線排列,肌節(jié)可見明顯的周期性,心肌組織間質無水腫,大量橢圓形線粒體排列整齊于心肌肌纖維中。模型組,肌節(jié)結構不清或消失,即肌原纖維斷裂、溶解,肌節(jié)紊亂,心肌組織間質見明顯水腫,壞死區(qū)域面積大,壞死嚴重,線粒體形態(tài)不一,見腫脹、破裂或消失,空泡化嚴重。中藥組,肌節(jié)較清晰,仍有局部肌纖維斷裂、溶解,線粒體排列于肌絲之間,呈輕度水腫,尚有部分線粒體空化。西藥組,肌節(jié)較清晰,部分肌纖維斷裂、溶解,線粒體排列于肌絲之間,輕度腫脹,且空化線粒體較少。結論:1.通過對大鼠模型超聲心動圖檢測,并且輔以對比健康大鼠行為,證明利用結扎冠狀動脈前降支配合減食與力竭式游泳可成功造成大鼠心衰模型。2.通過4周參草通脈顆粒的治療后利用ELISA法檢測BNP濃度下降,通過實時熒光定量PCR、Western blot檢測大鼠心肌組織,發(fā)現(xiàn)參草通脈顆粒對心肌組織klotho mRNA與蛋白有明顯升高其表達的作用;并且通過電鏡觀察心肌組織證明參草通脈顆�？善鸬揭种苹蚰孓D心室重構的作用。3.通過4周參草通脈顆粒的治療后利用實時熒光定量PCR、Western blot檢測大鼠腎組織,發(fā)現(xiàn)參草通脈顆粒對腎組織klothomRNA與蛋白有明顯升高其表達的作用;參草通脈顆粒可起到抑制腎損傷的作用。
[Abstract]:Objective: by establishing a rat model of chronic heart failure (CHF), we observed the effect of Shen Cao Tongmai Granule on the Klotho of myocardium and kidney tissue in chronic heart failure rats and the changes of the structure of myocardium. The mechanism and target points of Shen Cao Tong Mai Granule on the symptoms of chronic heart failure in rat model were discussed from gene, protein and histomorphology. We found and explored the scientific basis of traditional Chinese medicine as an important means to treat chronic heart failure. Materials and methods: 1. by ligating the anterior descending branch of the coronary artery to cause local myocardial infarction in experimental rats, and then swimming and reducing food in rats, the model of chronic heart failure was created, and then the state of rats was observed, and the Doppler echocardiography was used to examine the rats. The score of ejection fraction (EF) was less than 55% or CI < 180ml/min/kg. To determine whether the model of chronic heart failure rat model was successful or not, the model of chronic heart failure in.2. would be divided into 4 groups. First, the model of sham operation group was set up for the later contrast study. The model group of heart failure rats was the model group, and the group adopted the same group as the sham operation group. Third, the western medicine group was set up to take the intervention of lisinopril. Finally, the Chinese medicine group was set up and filled with Shen Cao Tongmai granule. The Chinese medicine group was filled with Shen Cao Tong Mai Granule according to the 9.2g raw medicine /Kg/d, and the /Kg/d. of the 1.5mg medicine of lisinopixi drug group was expected to be cultivated for 4 weeks. After the rat tail vein was collected, all rats were killed, and the cardiac muscle tissue and.3. of the rats were taken to collect all the samples by ELISA, protein immunoblotting and real-time fluorescence quantitative PCR. The concentration of BNP and the changes of Klotho in the myocardium and kidney tissue were compared. Results: 1. the concentration of BNP in the chronic heart failure rats was higher than that in the sham group. The treatment group (Chinese medicine group, western medicine group) and the model group were all increased, and the concentration of BNP in the sham operation group was 450 + 37.03pg/ml, the concentration of BNP in the model group was 1017.55 + 98.6pg/ml, and the concentration of BNP in the Chinese medicine group and the western medicine group was respectively the concentration of BNP in the Chinese medicine group and the western medicine group. 681.08 + 75.31pg/ml, 686.53 + 74.86pg/ml, the expression of Klotho mRNA in the rat model group of p0.05.2. chronic heart failure was significantly reduced, 0.0003 + 0.00004 (compared with the sham operation group P0.01), and the specific value of Klotho mRNA in the western medicine group was 0.0020 + 0.0009 compared to the Chinese medicine group (with the body value of 0.0012 + 0.0006) P0.05, the comparison between the group and the model group was compared with the model group. The expression of Klotho protein in the myocardium of the model group P0.01 and the sham group P0.01.3. was 0.0291 + 0.0025 (compared with the sham operation group P0.01), and the specific value of Klotho protein in the western medicine group was 0.2475 + 0.1407, compared with the traditional Chinese medicine group (the specific value was 0.215 + 0.077) P0.05, and the comparative treatment group and the model group P0.01 were compared. The expression of Klotho mRNA in renal tissue of P0.01.4. chronic heart failure model group was significantly lower than that in sham group, which was 0.0022 + 0.0007 (compared with sham operation group P0.05), and the specific value of Klotho mRNA in the sample kidney of Western medicine group was 0.0067 + 0.00209, compared with the traditional Chinese Medicine group (the specific value was 0.0063 + 0.0022) P0.05, and the comparison group was compared with the model group P0.01 and the sham operation group P. In 0.01.5. chronic heart failure model group, the expression of Klotho protein in renal tissue decreased significantly, which was 0.0639 + 0.0203 (compared with sham operation group P0.01). The specific value of Klotho protein in the western medicine group was 0.2150 + 0.0303, compared with the traditional Chinese medicine group (0.2057 + 0.0457) P0.05, and the comparison treatment group and the model group P0.01 and the sham operation group P0.01.6. sham operation group, The complete myocardial cells were seen, the muscle fibers were orderly and orderly, the Z lines were arranged in a uniform and clear line, the myofibrils were obvious periodicity, the interstitial tissue of the myocardium was not edema, and a large number of oval mitochondria were arranged neatly in the muscle fibers. The model group, the myofibril structure was not clear or disappeared, that is, the myofibril rupture, dissolving, the myofibril disorder, and the interstitial tissue of the myocardium. Edema, necrotic area large area, necrosis serious, mitochondria form different, see swelling, rupture or disappearance, vacuolization serious. The Chinese medicine group, the myojoints are clear, there are still local muscle fiber fracture, dissolve, the mitochondria are arranged between the myes, with a mild edema, the western medicine group, the myojoints are clearer, some muscle fibers break and dissolve. The mitochondria were arranged between the muscle fibers, slightly swelling and the cavitation mitochondria were less. Conclusion: 1. through the echocardiographic examination of the rat model and the behavior of the healthy rats, it is proved that the model.2. of the rat heart failure model can be successfully made through the ligature of the anterior descending branch of the coronary artery and the exhaustive swimming of the rats through 4 weeks. After treatment, ELISA method was used to detect the decrease of BNP concentration. The myocardial tissue of rats was detected by real-time fluorescence quantitative PCR and Western blot. The effect of Shen Cao Tong Mai Granule on the expression of Klotho mRNA and protein was found in the myocardium, and the myocardial tissue was observed by electron microscope to prove that the ginseng Tongmai granule could inhibit or reverse the ventricular remodeling. The effect of.3. on renal tissue of rats was detected by real-time fluorescence quantitative PCR and Western blot after 4 weeks of ginseng Tongmai Granules. The effect of Shen Cao Tong Mai Granule on the expression of klothomRNA and protein in renal tissue was found, and the effect of Shen Cao Tongmai Granules could inhibit the renal injury.

【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】：2017
【分類號】：R285.5

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