【作者】 庞晓静；

【导师】 高福禄； 牛嗣云；

【作者基本信息】 承德医学院 ， 人体解剖与组织胚胎学， 2009， 硕士

【摘要】 衰老是一种随着年龄增长、组织细胞老化、器官功能下降引起的不可避免的生理过程。中医学认为肾脏为主导人的生长发育和衰老的主轴器官,它的重要作用表现在对生命活动过程的各个环节的调节与控制,包括胚胎形成、个体发育、生殖与衰老等生理过程。中医学中有肾虚致衰之说,认为肾气亏损则五脏之气血津液生化乏源,各种衰老病症日益显露出来,且多以补肾中药对抗衰老和促进机体功能的恢复。本研究旨在观察何首乌饮（HY）延缓肾脏衰老过程中的各种变化,探讨何首乌饮的作用机制,为进一步研究中药抗衰老的作用机理奠定理论和实验基础。目的:本研究采用光镜、免疫组织化学和Western Blotting方法,通过观察各实验组肾脏组织形态学的变化,转化生长因子-β₁（TGF-β₁）和组织金属蛋白酶抑制物-1（TIMP-1）的表达,以及P16和pRB含量的变化,从而探讨何首乌饮延缓大鼠肾脏组织衰老的作用机制。方法:1.实验动物的选择:选用8周龄SD雄性大鼠100只,体重180-220g。2.衰老模型的建立:随机选取大鼠14只作为正常组（N）（腹腔注射等量的生理盐水）,其余大鼠腹腔注射6%D-半乳糖溶液（用生理盐水稀释）,按300mg/kg/d给药,连续注射60d,建立亚急性衰老大鼠模型。在造模的过程中观察大鼠体重、毛色、行为、精神等外观特征的变化情况,测定衰老大鼠的外周血血清超氧化物歧化酶（SOD）、丙二醛（MDA）含量的变化,并进行Morris水迷宫行为测试,以确定模型是否建立成功。3.动物分组:选用造模成功的衰老大鼠,随机分为:模型组（M）、何首乌饮低（HY-LD）、中（HY-MD）、高剂量组（HY-HD）和何首乌丸组（HW）,每组14只。模型组和正常组灌胃等量的生理盐水,其它各组分别灌胃何首乌饮（低、中、高剂量）和何首乌丸,每天一次,连续60d。4.标本处理:（1）每组选用8只大鼠经4%多聚甲醛灌注固定,取双侧肾,常规石蜡包埋、切片,片厚5μm。HE染色观察肾脏形态结构的变化,显微镜下计数肾小球数目,测量肾小囊囊腔宽和肾小球直径;SP免疫组织化学方法检测肾脏中TGF-β₁和TIMP-1的表达。（2）其余大鼠断头处死,取双侧肾,液氮中保存,采用Western Blotring方法检测大鼠肾脏组织pRB和P16含量。结果:1.外观的变化:实验开始前,模型大鼠与正常大鼠体重无差别,动物毛色白,有光泽,精神状态良好。随着实验的进展,各组大鼠体重逐渐增加,但模型大鼠体重增加比例低于正常大鼠。随着造模的时间延长,模型大鼠与正常组比较,毛色逐渐发黄,无光泽,并且出现掉毛的情况;精神萎靡、畏寒怕冷、体形臃肿和行动迟缓等症状。2.外周血血清SOD、MDA含量的变化:衰老大鼠外周血血清SOD含量明显降低,MDA含量明显升高,与正常大鼠相比有显著性差异（P＜0.01）,表明衰老模型建立成功。3.Morris水迷宫定位航行测试结果前2d正常组和模型组成绩无显著性差异,随学习时间的延长各组潜伏期均逐渐缩短,连续5d的定位航行实验发现,模型组的潜伏期与正常组比较明显延长（P＜0.01）,表明模型建立成功。4.肾脏组织形态学变化:模型组较正常组肾脏组织血管球不规则而松散,体积相应缩小,缩向血管极一侧,可见散在的空泡样肾小球。肾小管多数管腔扩大,上皮细胞低矮变薄或有体积明显增大。肾小球数目显著低于正常组（P＜0.01）,肾小囊囊腔宽和肾小球直径高于正常组（P＜0.01）。何首乌饮与何首乌丸组肾脏组织的形态结构较模型组有所改善,其中何首乌饮高剂量组肾脏组织的形态结构较模型组大鼠明显恢复,且肾小球数目、肾小囊囊腔宽和肾小球直径与模型组差异显著（P＜0.01）。5.TGF-β₁免疫组化染色结果:免疫阳性产物表达在细胞质,呈棕黄色或棕褐色,分布在肾小球毛细血管管壁,肾小球壁层细胞,肾小管间质及间质血管管壁;模型组除分布在上述部位外,肾小管上皮细胞也见表达。阳性率明显高于正常组（P＜0.01）。何首乌饮各组随剂量的增高阳性率呈下降趋势,高剂量组和模型组有显著差异（P＜0.01）,并低于何首乌丸组（P＜0.01）。6.TIMP-1免疫组化染色结果:免疫阳性产物表达在细胞质,呈棕黄色或棕褐色,位于肾小球球内系膜细胞、肾小管间质和肾小管上皮细胞,肾小球毛细血管管壁见少量表达。模型组TIMP-1的阳性率明显高于正常组（P＜0.01）,何首乌饮低、中剂量组和模型组接近无明显差别（P＞0.05）,高剂量组和模型组有显著差异（P＜0.01）,且好于何首乌丸组（P＜0.01）。7.P16的含量变化模型组大鼠肾脏组织P16含量明显高于正常组（P＜0.01）,何首乌饮各组大鼠肾脏组织P16含量均低于模型组,且呈剂量依赖性,尤以高剂量组与模型组差别显著（P＜0.01）,且好于何首乌丸组。8.pRB的含量变化模型组大鼠肾脏组织pRB含量明显降低,与正常组相比有显著性差异（P＜0.01）,而何首乌饮各组大鼠肾脏组织pRB含量均高于模型组,并且以高剂量组大鼠肾脏组织pRB含量水平最高,与模型组差别显著（P＜0.01）,且好于何首乌丸组。结论:衰老大鼠肾脏肾小球数目明显减少,肾小囊囊腔宽和肾小球直径明显增大;TGF-β₁和TIMP-1和P16含量明显增高,而pRB含量明显降低。何首乌饮可提高大鼠肾小球数目,减小肾小囊囊腔宽和肾小球直径;降低肾脏TGF-β₁、TIMP-1和P16的表达量;提高pRB在大鼠肾脏组织的表达。因此,本研究证明何首乌饮具有良好的延缓大鼠肾脏衰老的作用。更多还原

【Abstract】 Senescence is an unavoidable physiological process that is caused by aging of tissue and cells,declining of organ functions along with the increasing of age.Kidney,as the main organ dominating human growth and aging,its important role is to adjust and control every aspect in vital activities, including formation of embryos,individual development,reproductive and aging.In traditional Chinese medical science,there is the theory that renal deficiency causes debility,that is,renal deficiency causes poor sources for internal organs’ chemical and biological fluid,then a variety of diseases of aging will appear.Many Chinese traditional medicines that reinforce kidney were used to delay aging and restore body functions.This study is designed to observe the changes in the process of anti-aging effect of HESHOUWUYIN（HY）,to explore its mechanism,thus to establish theoretical and experimental basis for clinical using of Chinese traditional medicine.Objective:To observe the conformation change,the expression of TGF-β₁（transforming growth factor-β₁） and TIMP-1（tissue type inhibitors of metalloproteinase-1）,the content change of P16 and pRB in kidney of rat by HE-staining,immunohistochemistry and western blotting methods,thus to discuss the anti-aging mechanism of HY on the kidney of rat.Methods:1 Selection of experimental animals:100 male SD rats（8-weeks） were used in this study,the rats weight was 180-220g.2 Establishment of the aging model:14 rats were selected at random as normal group（N）（injected the same dose of physiological saline）,and the remains were injected 6%D-gal solution through abdominal cavity（diluted by physiological saline） continually by 300mg/kg/d for 60 days,to construct the sub-acutely aging rat model.Observe the change of weight,hair color, behavior and spirit,and measure the content of SOD（Superoxide Dismutase） and MDA（Malonaldehyde Biscdimethy） in serum,and make Morris water maze behavior test,all these to make sure whether the model was successfully establishment.3 Animal groups:The aging model rats were randomly divided into five groups:model group（M）,HY low dose group（HY-LD）,middle dose group（HY-MD）,high dose group（HY-HD） and HESHOUWUWAN group（HW）（14 rats each group）.Model and normal groups rats were poured the same dose physiological saline,the others were respectively poured HY（low,middle and high dose） and HW continually for 60 days,once a day.4 Sample disposal:（1）The testes were taken after the rats being infused with 4%polyformaldehyde,embedded in paraffin and sectioned as usual.The thickness was 5-7μm.HE-staining was used to observed the conformation changes of kidney;the number and diameter of renal corpuscle and the width of renal capsular space were measured under microscope;the expression of TGF-β₁ and TIMP-1 were observed by SP immunohistochemistry.（2） The rats were killed by amputating the head and the kidneys were taken.Western blotting was used to analyze the P16 and pRB content of the kidneys.Results:1 The appearance change:Rats in normal and model groups were no discrepancy in aspects of weight,hair color and spirit before test.All rats weight increased with the test proceeding,but in model rats it increased lower than in normal rats and then some aging signs appeared.2 The level changes of SOD and MDA in serum:SOD in serum was lower and MDA was higher significantly in model rats than those in normal rats （P＜0.01）,and this suggests the model have been made successfully.3 Morris water maze behavior test:the first two days the result of each group didn’t show significant difference and each group latency all shortenned gradually with study time extension,the locating sea trial of the continuous 5 days discover,the latency result of model group obviously prolong than normal group（P＜0.01）,and express that the model builds up successfuly. 4 The confonnation changes of kidney:the number of renal corpuscle decreased,the width of renal capsular space and the diameter of renal corpuscle augment in model rats,and had a statistical significance with normal rats（P＜0.01）.HY-HD and HW could restore the number and diameter of renal corpuscle and the width of renal capsular space（P＜0.01）.5 The expressive result of TGF-β₁:The positive productions were brown and located at the cytoplasm of endothelial cells of blood vessel and Leydig cells,and was also found in epithelial cells of renal tubule in model group.The positive rate in model rats was much higher than in normal rats（P＜0.01）.The positive rate in HY group decreased with the increasing dose,and HY-HD group was much less than model group（P＜0.01）,and less than HW group.6 The expressive result of TIMP-1:The positive productions were brown and located at the cytoplasm of mesangial cell,Leydig cells and epithelial cells of renal tubule.The positive rate in model rats was much higher than in normal rats（P＜0.01）.HY-LD group and HY-MD group rats were nearly the same as model rats（P＞0.05）.HY-HD group was less than model group significantly（P＜0.01）,and less than HW group.7 The content change of P16:The content of P16 in model group was much higher than in normal group（P＜0.01）.Each HY group was lower than model group,especially HY-HD group has statistical significance（P＜0.01）,and lower than HW group.8 The content change of pRB:The content of pRB in model group was much lower than normal group（P＜0.01）.Each HY group was higher than model group,especially HY-HD group has statistical significance（P＜0.01）, and higher than HW group.Conclusion:In model rats,the number of renal corpuscle decreased,the width of renal capsular space and the diameter of renal corpuscle enlarged markedly;the content of TGF-β₁,TIMP-1 and P16 increased distinctively, whereas the content of pRB depressed significantly.HY can improve the number of renal corpuscle,decrease the width of renal capsular space,and the diameter of renal corpuscle;impair the expression of TGF-β₁,TIMP-1 and P16,and increase the content of pRB.So this experimental study indicate that HY has definite anti-aging effect on the kidney of rat.更多还原