过度训练大鼠肾组织热休克蛋白70的表达及山莨菪碱干预的研究

发布时间：2018-04-26 01:27

本文选题：热休克蛋白70（HSP70） + 过度训练　；参考：《河北医科大学》2012年硕士论文

【摘要】：目的：近年来对过度训练引起的急性肾损伤(overtraining-inducedacute kidney injury,OTIAKI)的研究越来越多，因为它已成为危害人类健康的重要疾病，但其发病机制和损伤方式尚不清楚，目前尚缺乏有效的防治方法。热休克蛋白(heat shock proteins, HSPs）又称应激蛋白，它是细胞应激反应的生物标志及内源性保护蛋白，其表达的强弱与组织细胞的损害程度有关。其中热休克蛋白70（heat shock protein70,HSP70）在生物细胞中含量最高，受到诱导最强，应激后生成最显著，具有多种生物学功能，对细胞有明确的内源性保护作用。本研究采取大鼠无负重单次游泳至力竭建立过度训练引起的急性肾损伤的模型，研究过度训练大鼠肾组织细胞HSP70的表达规律及其与肾组织细胞凋亡的关系，并观察山莨菪碱对HSP70表达的影响，探讨其作用机制。方法：(1)清洁级雄性SD大鼠48只，体重180－220g，按随机数字表随机分为三组：安静对照组(CN, n=8)、力竭运动组(ES, n=24)、山莨菪碱干预组(AD, n=16)。安静对照组（CN）未进行力竭运动，不注射任何药物；力竭组又根据力竭后恢复时间不同分为力竭后即刻组(ESI)、力竭后6小时组(ES6h)和力竭后24小时组(ES24h)三个亚组，且各亚组大鼠在运动前20min给予灭菌注射用水10mg/Kg一次性腹腔注射；山莨菪碱干预组也根据力竭后恢复时间不同分为山莨菪碱6h组(AD6h)和山莨菪碱24h组(AD24h)两个亚组，各亚组大鼠于运动前20min给予山莨菪碱注射液10mg/Kg一次性腹腔注射。每个时间点均为8只大鼠。采用大鼠无负重单次游泳至力竭建立过度训练引起急性肾损伤模型。(2)于各时间点，5%的水合氯醛（3ml/kg）腹腔注射麻醉，摘取两侧肾脏，将左侧肾脏放入EP管，迅速放入液氮中，-80℃冰冻保存，待用于Western blot技术检测肾组织HSP70的表达。右侧肾脏置于4℃4％多聚甲醛中固定，制作石蜡切片。(3)采用HE染色光镜下观察肾组织病理形态学变化。(4)采用免疫组化染色(PV法)观察肾组织HSP70的表达情况，并应用图像分析系统对免疫组化结果进行半定量分析。(5)应用Western blot技术检测肾组织HSP70表达的变化。(6)统计分析：所有实验数据均采用SPSS13.0软件包进行统计学处理，实验数据均以均数±标准差(X±S)表示。组间比较采用单因素方差分析(one-wayANOVA)分析，以P0.05认为有统计学意义。结果：(1)无负重单次游泳至力竭后大鼠表现为极度疲劳的神态，精神萎靡，对外界刺激反应迟钝，胸腹着地，无法站立，活动困难，严重者可有球结膜出血的现象。力竭运动组大鼠约2h后方可自由活动，进食水情况欠佳，而山莨菪碱干预组大鼠约20min后可自由活动，基本可正常进食。(2)正常对照组大鼠肾组织结构无明显变化。力竭后即刻组、力竭后6h组肾组织结构改变轻微，有部分肾小球囊、肾小管扩张，近端肾小管上皮细胞水肿，出现空泡变性；力竭后24h组大鼠肾组织结构改变明显，在皮髓质交界处及髓质可见大量细胞的细胞核深染及固缩现象，间质可见浸润细胞，肾小管上皮细胞的刷状缘出现不规整甚至脱落的改变。山莨菪碱6h组及山莨菪碱24h组较同期单纯力竭组比较大鼠肾组织结构改变明显减轻。(3)免疫组化染色检测肾组织HSP70的表达：阳性细胞多为肾小管上皮细胞，肾小球几乎无表达，且肾髓质中的细胞表达强于肾皮质。正常对照组大鼠肾组织中有较弱的HSP70的表达。力竭运动组大鼠随恢复时间的延长，肾组织HSP70蛋白的表达水平呈逐渐增高趋势，，且同正常对照组相比均有统计学意义（P＜0.05㖞。山莨菪碱干预组大鼠肾组织中HSP70的表达较同期力竭组比较阳性细胞数增加，且表达强度明显增强（P＜0.05㖞。(4)Western blot技术检测肾组织HSP70的表达：在内参GAPDH表达量基本一致的情况下，与正常对照组比较，随恢复时间的延长，力竭运动组肾组织HSP70表达逐渐增强（P＜0.05㖞。山莨菪碱6h组、山莨菪碱24h组较同期力竭组比较，肾组织HSP70表达增强（P＜0.05㖞。结论：(1)HSP70在过度训练大鼠肾组织中表达增强，且于力竭后即刻至力竭后24小时呈逐渐增高趋势，用免疫组化法观察肾髓质中的细胞表达强于肾皮质。(2)山莨菪碱干预组与同期力竭组比较，肾组织损伤减轻，肾组织HSP70的表达增强，山莨菪碱对OTIAKI的保护作用有可能与诱导HSP70表达增强有关。
[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.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R87

【参考文献】