本文選題：高血壓前期 + 腎臟細胞外基質沉積��； 參考：《遼寧中醫(yī)藥大學》2017年碩士論文

【摘要】：目的:本實驗采用實時定量PCR及蛋白免疫印跡法(Western boltting)的方法,檢測鹽敏感大鼠建立的高血壓前期模型中,腎臟組織中轉化生長因子-β1(transforming growth factor-beta1,TGF-β1)介導的p38MAPK信號傳導通路相關基因和蛋白的表達,主要有p38絲裂素活化蛋白激酶(p38mitogen-activated protein kinase,p38MAPK)、c AMP反應元件結合蛋白(c AMP-responsive element binding protein,CREB),及α-平滑肌肌動蛋白(α-smooth muscle actin,α-SMA)、纖維黏連蛋白(fibronectin,FN)基因的表達,探討針刺對高血壓前期大鼠延緩腎臟細胞外基質沉積的作用原理,從更深層次上理解高血壓病的發(fā)生發(fā)展過程。材料與方法:本次實驗按照實驗目的將實驗動物分為四組,分別為正常對照組,模型組,針刺組以及非經非穴組。全部實驗動物為40只健康雄性大鼠,其中有30只為Dahl鹽敏感大鼠(Dahl salt sensitive rats,DS),給予高鹽飼料喂養(yǎng)后血壓升高。將它們按照隨機數字表法平均分為三組,每組10只,分別記為模型、針刺及非經非穴組。另外10只為Dahl鹽抵抗大鼠(Dahl salt resistant rats,DR),給予高鹽飼料喂養(yǎng)后血壓基本穩(wěn)定不變。將每組大鼠分籠飼養(yǎng),適應性喂養(yǎng)7天,無異常后給予高鹽飼料喂養(yǎng)并每天檢測血壓變化,待鹽敏感大鼠血壓升高10-20mm Hg后停止高鹽喂養(yǎng)改為普通飼料喂養(yǎng),即造模成功并開始治療。其中模型組大鼠僅每日捆綁20min靜置;針刺組針刺雙側“足三里”“曲池”穴3-5mm,接通電針20min,采用疏密波,頻率以針柄輕微震顫為度,每日1次,每周6次,治療五周;非經非穴組針刺大鼠雙側髂嵴上15mm、后正中線旁開20mm這一固定對照點,同樣采用疏密波,接通電針20min,頻率以針柄輕微震顫為度,治療日期與針刺組相同。治療時每周檢測大鼠血壓一次并記錄。停止針刺治療結束。取材時,捆綁固定大鼠后注射10%水合氯醛(每100g大鼠給藥0.35ml),待大鼠完全麻醉后,腹部正中切口,充分暴露腎臟后切除雙腎,去包膜,沿最大冠狀切面縱行切開,取其中一部分腎組織做勻漿液,提取腎臟組織總RNA,采用實時定量PCR方法檢測腎臟中TGF-β1、p38MAPK、CREB、FN及α-SMAm RNA的表達;用蛋白免疫印跡法檢測腎臟組織中p38MAPK、CREB的磷酸化蛋白表達量。結果:1.電針對Dahl鹽敏感大鼠腎組織TGF-β1基因表達的影響模型組大鼠與正常組大鼠相比,腎組織中TGF-β1基因表達升高(P0.05)。經過五周治療后,針刺組及非經非穴組大鼠與模型組相比,TGF-β1基因表達量均有所降低(P0.05),與非經非穴組相比,針刺組降低明顯,具有統(tǒng)計學意義(P0.05)。2.電針對Dahl鹽敏感大鼠腎組織p38MAPK信號通路的影響模型組大鼠與正常組大鼠相比,腎組織中p38MAPK、CREB基因表達及其磷酸化蛋白表達升高(P0.05)。經過五周治療后,針刺組及非經非穴組大鼠與模型組相比,p38MAPK、CREB基因表達及其磷酸化蛋白表達量均有所降低(P0.05),與非經非穴組相比,針刺組降低明顯,具有統(tǒng)計學意義(P0.05)。3.電針對Dahl鹽敏感大鼠腎組織α-SMA、FN基因表達的影響模型組大鼠與正常組大鼠相比,腎組織中α-SMA、FN基因表達均升高(P0.05)。經過五周治療后,針刺組及非經非穴組大鼠與模型組相比,α-SMA、FN基因表達均有所降低(P0.05),與非經非穴組相比,針刺組降低明顯,具有統(tǒng)計學意義(P0.05)。結論:1.電針雙側“足三里”“曲池”穴有延緩高血壓前期大鼠腎臟細胞外基質沉積的作用。2.延緩的作用機制可能與抑制TGF-β1介導的p38MAPK信號傳導通路上的相關基因及磷酸化蛋白的表達有關。3.其中TGF-β1、p38MAPK、CREB、FN及α-SMA是其關鍵的作用點。
[Abstract]:Objective: to detect the expression of genes and proteins related to p38MAPK signal transduction pathway mediated by transforming growth factor - beta 1 (transforming growth factor-beta1, TGF- beta 1) in the prehypertensive rat model of salt sensitive rats by real-time quantitative PCR and Western boltting. 8 p38mitogen-activated protein kinase (p38MAPK), C AMP reactive element binding protein (C AMP-responsive element binding protein, CREB), and the expression of alpha smooth muscle actin, fibronectin gene, and the effect of acupuncture on prehypertensive rats The mechanism of the inhibition of extracellular matrix deposition in the kidney and understanding the process of the occurrence and development of hypertension from a deeper level. Materials and methods: the experimental animals were divided into four groups according to the purpose of the experiment, which were the normal control group, the model group, the acupuncture group and the non non acupoint group. All the experimental animals were 40 healthy male rats, including 3 of the healthy male rats. 0 Dahl salt sensitive rats (Dahl salt sensitive rats, DS) were given high salt diet after feeding their blood pressure. They were divided into three groups according to the random number table method. 10 rats in each group were recorded as model, acupuncture and non non acupoint group. The other 10 were Dahl salt resistant rats (Dahl salt resistant rats, DR), and given high salt feed after feeding blood. The pressure was stable and constant. The rats in each group were kept in cage and fed for 7 days, and the high salt feed was fed without abnormality and the blood pressure was measured every day. After the blood pressure of the salt sensitive rats was raised 10-20mm Hg, the high salt feeding was stopped and the diet was changed to the ordinary feed. The acupuncture group was treated with the acupuncture of "Zusanli" "Zusanli" "Qu Chi" (3-5mm) and electroacupuncture (20min). The needle was used as an electric needle for a slight tremor of the needle handle, 1 times a day, 6 times a week for five weeks, and five weeks in the treatment of non acupoint acupuncture rats on the bilateral iliac crest on the bilateral iliac crest, and the 20mm fixed control point beside the middle line, and the same density wave was used to connect the electroacupuncture 20min, frequency. The treatment date was the same as that in the acupuncture group with the slight tremor of the needle handle. The blood pressure of the rats was detected once a week and the end of the acupuncture treatment was stopped. When the material was taken, 10% chloral chloral hydrate (0.35ml) was injected into the rats. After the rats were completely anaesthetized, the abdominal median incision was fully exposed and the kidneys were fully exposed and the double kidneys were excised after the kidney was excised and went to the bag. The membrane was cut along the maximum coronal section, and a part of the kidney tissue was taken as homogenate to extract the total RNA of kidney tissue. The expression of TGF- beta 1, p38MAPK, CREB, FN and alpha -SMAm RNA in kidney was detected by real-time quantitative PCR. The expression of p38MAPK and CREB phosphorylated protein in renal tissue was detected by protein immunoblotting. Results: 1. electroacupuncture was used for Dahl. The expression of TGF- beta 1 gene in renal tissue of salt sensitive rats was affected by the increase of TGF- beta 1 gene expression in the renal tissue (P0.05). After five weeks of treatment, the expression of TGF- beta 1 in the acupuncture group and the non non acupoint group was lower than that in the model group (P0.05). Compared with the non non acupoint group, the acupuncture group decreased the brightness. The effect of (P0.05).2. electroacupuncture on the p38MAPK signaling pathway of renal tissue in Dahl salt sensitive rats model group, compared with normal rats, the p38MAPK, CREB gene expression and phosphorylated protein expression increased (P0.05) in the renal tissue. After five weeks of treatment, the acupuncture group and the non non acupoint group were compared with the model group, p38MAPK, The expression of CREB gene and the expression of phosphorylated protein decreased (P0.05). Compared with the non acupoint group, the acupuncture group decreased obviously and had statistically significant (P0.05).3. electroacupuncture on the renal tissue of the Dahl salt sensitive rat renal tissue alpha -SMA, and the expression of FN gene in the model group was higher than that in the normal group, and the expression of alpha -SMA and FN gene in the renal tissue increased (P0). .05) after five weeks of treatment, the expression of alpha -SMA and FN genes in the acupuncture group and the non acupoint group were lower than that in the model group (P0.05). Compared with the non acupoint group, the acupuncture group decreased obviously and had statistical significance (P0.05). Conclusion: 1. electroacupuncture bilateral "foot three li" "Qu Chi" point postpones the renal extracellular base of prehypertensive rats. The mechanism of.2. delayed action may be related to the inhibition of the related genes and the expression of phosphorylated protein on the p38MAPK signaling pathway mediated by TGF- beta 1, and.3. in which TGF- beta 1, p38MAPK, CREB, FN and alpha -SMA are the key points of action.
【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】：R245

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