本文選題：熱休克蛋白70（HSP70） + 過度訓(xùn)練��； 參考：《河北醫(yī)科大學(xué)》2012年碩士論文

【摘要】：目的：近年來對過度訓(xùn)練引起的急性腎損傷(overtraining-inducedacute kidney injury,OTIAKI)的研究越來越多，因為它已成為危害人類健康的重要疾病，但其發(fā)病機制和損傷方式尚不清楚，目前尚缺乏有效的防治方法。熱休克蛋白(heat shock proteins, HSPs）又稱應(yīng)激蛋白，它是細(xì)胞應(yīng)激反應(yīng)的生物標(biāo)志及內(nèi)源性保護蛋白，其表達的強弱與組織細(xì)胞的損害程度有關(guān)。其中熱休克蛋白70（heat shock protein70,HSP70）在生物細(xì)胞中含量最高，受到誘導(dǎo)最強，應(yīng)激后生成最顯著，具有多種生物學(xué)功能，對細(xì)胞有明確的內(nèi)源性保護作用。本研究采取大鼠無負(fù)重單次游泳至力竭建立過度訓(xùn)練引起的急性腎損傷的模型，研究過度訓(xùn)練大鼠腎組織細(xì)胞HSP70的表達規(guī)律及其與腎組織細(xì)胞凋亡的關(guān)系，并觀察山莨菪堿對HSP70表達的影響，探討其作用機制。 方法：(1)清潔級雄性SD大鼠48只，體重180－220g，按隨機數(shù)字表隨機分為三組：安靜對照組(CN, n=8)、力竭運動組(ES, n=24)、山莨菪堿干預(yù)組(AD, n=16)。安靜對照組（CN）未進行力竭運動，不注射任何藥物；力竭組又根據(jù)力竭后恢復(fù)時間不同分為力竭后即刻組(ESI)、力竭后6小時組(ES6h)和力竭后24小時組(ES24h)三個亞組，且各亞組大鼠在運動前20min給予滅菌注射用水10mg/Kg一次性腹腔注射；山莨菪堿干預(yù)組也根據(jù)力竭后恢復(fù)時間不同分為山莨菪堿6h組(AD6h)和山莨菪堿24h組(AD24h)兩個亞組，各亞組大鼠于運動前20min給予山莨菪堿注射液10mg/Kg一次性腹腔注射。每個時間點均為8只大鼠。采用大鼠無負(fù)重單次游泳至力竭建立過度訓(xùn)練引起急性腎損傷模型。(2)于各時間點，5%的水合氯醛（3ml/kg）腹腔注射麻醉，摘取兩側(cè)腎臟，將左側(cè)腎臟放入EP管，迅速放入液氮中，-80℃冰凍保存，待用于Western blot技術(shù)檢測腎組織HSP70的表達。右側(cè)腎臟置于4℃4％多聚甲醛中固定，制作石蠟切片。(3)采用HE染色光鏡下觀察腎組織病理形態(tài)學(xué)變化。(4)采用免疫組化染色(PV法)觀察腎組織HSP70的表達情況，并應(yīng)用圖像分析系統(tǒng)對免疫組化結(jié)果進行半定量分析。(5)應(yīng)用Western blot技術(shù)檢測腎組織HSP70表達的變化。(6)統(tǒng)計分析：所有實驗數(shù)據(jù)均采用SPSS13.0軟件包進行統(tǒng)計學(xué)處理，實驗數(shù)據(jù)均以均數(shù)±標(biāo)準(zhǔn)差(X±S)表示。組間比較采用單因素方差分析(one-wayANOVA)分析，以P0.05認(rèn)為有統(tǒng)計學(xué)意義。 結(jié)果：(1)無負(fù)重單次游泳至力竭后大鼠表現(xiàn)為極度疲勞的神態(tài)，精神萎靡，對外界刺激反應(yīng)遲鈍，胸腹著地，無法站立，活動困難，嚴(yán)重者可有球結(jié)膜出血的現(xiàn)象。力竭運動組大鼠約2h后方可自由活動，進食水情況欠佳，而山莨菪堿干預(yù)組大鼠約20min后可自由活動，基本可正常進食。(2)正常對照組大鼠腎組織結(jié)構(gòu)無明顯變化。力竭后即刻組、力竭后6h組腎組織結(jié)構(gòu)改變輕微，有部分腎小球囊、腎小管擴張，近端腎小管上皮細(xì)胞水腫，出現(xiàn)空泡變性；力竭后24h組大鼠腎組織結(jié)構(gòu)改變明顯，在皮髓質(zhì)交界處及髓質(zhì)可見大量細(xì)胞的細(xì)胞核深染及固縮現(xiàn)象，間質(zhì)可見浸潤細(xì)胞，腎小管上皮細(xì)胞的刷狀緣出現(xiàn)不規(guī)整甚至脫落的改變。山莨菪堿6h組及山莨菪堿24h組較同期單純力竭組比較大鼠腎組織結(jié)構(gòu)改變明顯減輕。(3)免疫組化染色檢測腎組織HSP70的表達：陽性細(xì)胞多為腎小管上皮細(xì)胞，腎小球幾乎無表達，且腎髓質(zhì)中的細(xì)胞表達強于腎皮質(zhì)。正常對照組大鼠腎組織中有較弱的HSP70的表達。力竭運動組大鼠隨恢復(fù)時間的延長，腎組織HSP70蛋白的表達水平呈逐漸增高趨勢，，且同正常對照組相比均有統(tǒng)計學(xué)意義（P＜0.05㖞。山莨菪堿干預(yù)組大鼠腎組織中HSP70的表達較同期力竭組比較陽性細(xì)胞數(shù)增加，且表達強度明顯增強（P＜0.05㖞。(4)Western blot技術(shù)檢測腎組織HSP70的表達：在內(nèi)參GAPDH表達量基本一致的情況下，與正常對照組比較，隨恢復(fù)時間的延長，力竭運動組腎組織HSP70表達逐漸增強（P＜0.05㖞。山莨菪堿6h組、山莨菪堿24h組較同期力竭組比較，腎組織HSP70表達增強（P＜0.05㖞。 結(jié)論：(1)HSP70在過度訓(xùn)練大鼠腎組織中表達增強，且于力竭后即刻至力竭后24小時呈逐漸增高趨勢，用免疫組化法觀察腎髓質(zhì)中的細(xì)胞表達強于腎皮質(zhì)。(2)山莨菪堿干預(yù)組與同期力竭組比較，腎組織損傷減輕，腎組織HSP70的表達增強，山莨菪堿對OTIAKI的保護作用有可能與誘導(dǎo)HSP70表達增強有關(guān)。
[Abstract]:Objective: in recent years, there are more and more studies on overtraining-inducedacute kidney injury (OTIAKI) caused by overtraining, because it has become an important disease that endangers human health, but its pathogenesis and damage methods are not yet clear, and there is still a lack of effective methods of prevention and control. Heat shock protein (heat shock proteins) HSPs, also known as stress protein, is a biomarker and endogenous protective protein of cell stress reaction. The intensity of its expression is related to the damage of tissue cells. Heat shock protein 70 (heat shock protein70, HSP70) has the highest content in biological cells, is most strongly induced, and is most significant after stress, with a variety of biological functions. In this study, the model of acute renal injury induced by excessive training in rats without weight negative single swimming to exhaustive training was used to study the expression of HSP70 in renal tissue cells in overtraining rats and the relationship with apoptosis of renal tissue cells, and to observe the effect of anisodamine on the expression of HSP70. Mechanism of action.
Methods: (1) 48 clean male SD rats, weighing 180 - 220g, were randomly divided into three groups according to the random number table: the quiet control group (CN, n=8), the exhaustive exercise group (ES, n=24), the anisodamine intervention group (AD, n=16). The quiet control group (CN) did not exercise exhaustive exercise, and no drugs were injected; the exhaustive group was divided into force according to the recovery time after exhaustion. Immediately after exhaustion group (ESI), 6 hours after exhaustion (ES6h) and 24 hours after exhaustion (ES24h) three subgroups, and the rats in each subgroup were given a one-time intraperitoneal injection of Sterile Water for Injection 10mg/Kg before exercise; and the anisodamine intervention group was also divided into two groups of anisodamine 6h group (AD6h) and anisodamine 24h group (AD24h), according to the recovery time after exhaustion. Subgroup, the rats in each subgroup were given an intraperitoneal injection of anisodamine injection 10mg/Kg at 20min before exercise. Each time point was 8 rats. The rat model of acute renal injury was induced by unloaded single swimming to exhaustion training in rats. (2) 5% of chloral chloral (3ml/kg) was injected into the abdominal cavity at each time point, and the kidney was extracted from both sides. The left kidney was put into the EP tube and stored in liquid nitrogen and frozen at -80 C. The expression of HSP70 in renal tissue was detected by Western blot. The right kidney was fixed in 4% polyformaldehyde at 4 degrees C and made of paraffin section. (3) the pathological changes of renal tissue were observed under the HE staining light microscope. (4) immunohistochemical staining (PV method) was used to observe the kidney. The expression of HSP70 was organized and the results of immunohistochemistry were semi quantitative analyzed with image analysis system. (5) Western blot technique was used to detect the changes in the expression of HSP70 in renal tissue. (6) statistical analysis: all experimental data were statistically treated with SPSS13.0 software package, and the experimental data were expressed with mean mean standard deviation (X + S). A single factor analysis of variance (one-wayANOVA) was used to compare the results, and P0.05 was considered to be statistically significant.
Results: (1) the rats without a single swim to the exhaustion were extremely tired, the spirit was depressed, the irritation of the outside world was slow, the chest and abdomen could not stand, the activity was difficult, and the serious person could have the hemorrhage of the conjunctiva. The rats in the exhaustive exercise group were free to move about 2h, and the water situation was not good, and anisodamine intervention group was large. Rats were free to move after 20min. (2) there was no obvious change in the renal tissue structure in the normal control group. After exhaustion, the renal tissue structure changed slightly in the 6h group after exhaustion. Some small renal balloon, renal tubules dilated, the proximal renal tubular epithelial cells edema, and vacuolated degeneration, and the renal tissue structure of group 24h rats after exhaustion. In the medullary junction and medulla, the cell nucleus was deeply dyed and the cell nucleus was sequestrated. The infiltration cells were seen in the interstitium, and the brush like margin of the tubular epithelial cells of the renal tubule appeared irregular or even shedding. The changes of the renal tissue structure of the anisodamine group 6h and the Anisodamine 24h group were significantly lower than those in the simple exhaustion group. 3) the expression of HSP70 in renal tissue was detected by immunohistochemical staining: the positive cells were mostly renal tubular epithelial cells, and the glomeruli were almost no expression, and the expression of cells in the renal medulla was stronger than that of the renal cortex. There was a weak expression of HSP70 in the renal tissue of the normal control rats. The expression of HSP70 protein in the rats of the exhaustive exercise group with the prolongation of the time of recovery and the expression of the renal tissue protein. The level increased gradually, and was statistically significant compared with the normal control group (P < 0.05?. the expression of HSP70 in the renal tissue of the rats in the anisodamine intervention group was increased and the expression intensity was significantly enhanced (P < 0.05? (4) Western blot technique to detect the expression of HSP70 in the renal tissue: the expression of GAPDH in the internal parameter of the renal tissue. " Compared with the normal control group, the expression of HSP70 in the renal tissue of the exhaustive exercise group increased gradually with the time of recovery. (P < 0.05?. anisodamine 6h group. The HSP70 expression in the renal tissue was enhanced compared with the group 24h (P < 0.05?).
Conclusion: (1) the expression of HSP70 was enhanced in the renal tissue of overtrained rats and increased gradually at 24 hours after exhaustion. The expression of cells in the renal medulla was stronger than that in the renal cortex by immunohistochemistry. (2) the renal tissue injury was lightened and the expression of renal tissue HSP70 increased in the anisodamine intervention group. The protective effect of scopolamine on OTIAKI may be related to the enhanced expression of HSP70.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R87

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