【作者】 吴克芬；

【导师】 胡予；

【作者基本信息】 复旦大学 ， 内科学， 2013， 硕士

【摘要】 第一部分衰老大鼠模型的建立[背景]社会日趋老年化,与衰老相关的各种老年疾病及如何延缓衰老成为老龄化社会关注的重点与热点。研究衰老需要衰老动物模型。理论上,以自然衰老动物为模型进行研究最理想,但自然衰老动物存在饲养周期长、价格昂贵、来源稀缺同源性差等缺点,不能被广泛使用。因此,学者开始探讨、研究建立衰老动物模型,来满足科研的需要。到目前为止,常用的人工动物衰老模型主要有：D-半乳糖(D-galactose, D-gal)注射、β-淀粉样蛋白注射、快速老化小鼠、辐照Y射线、臭氧损伤、去除胸腺等方法建立衰老模型。这些衰老模型各有利弊。其中,多项实验证实,D-gal可使许多组织出现类似衰老的退行性改变,如与自然衰老相似的生化改变、免疫功能低下、基因表达与调控异常、细胞生长繁殖能力下降及细胞退行性改变。且D-gal皮下注射这一方法简便易行、价格低廉、结果稳定。因此,实验性衰老模型——D-gal模型已成为国内外学者研究老年病常用的病理模型。[目的]本实验采用国内最常用的建立衰老动物模型方法,即通过对SD大鼠采取D-半乳糖连续皮下注射的方法建立衰老大鼠模型,并对其相关的衰老指标进行初步分析与评价,来观察大鼠在衰老过程中体重、衰老指标如超氧化物歧化酶(supemxide dismutate, SOD)、丙二醛(malondiadehycle, MDA)等方面的变化。同时利用该衰老大鼠模型,对后续研究提供衰老实验动物。[材料和方法](1)选取3月龄SD大鼠10只(A组)、12月龄SD大鼠10只(B组)、24月龄衰老模型SD大鼠10只(C组),其中24月龄衰老模型大鼠由3月龄SD大鼠予D-gal按0.125g·kg-1·d1剂量皮下连续注射40d的方法建立。各组均常规摄食和饮水饲养40d,饲养期间每10天测量各组大鼠体重变化,饲养40d后测量各组大鼠血清SOD活力、MDA含量；取大鼠胸腺、脾脏称重,计算胸腺、脾脏指数。(2)比较3月龄组(A组)、12月龄组(B组)和24月龄衰老模型组(C组)间体重、血清SOD活力、MDA含量、胸腺指数、脾脏指数的差异。[结果](1)常规饮食饲养40d后,24月龄衰老模型鼠(C组)、12月龄大鼠(B组)与3月龄大鼠(A组)相比,体重增长均减慢[24月龄(41.3±11.1)g,12月龄(85.6±10.9)g,3月龄(125.3±15.2)g,P<0.05],且24月龄衰老模型鼠体重增长较12月龄亦减慢(P<0.05)。(2)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)与3月龄大鼠(A组)相比,血清SOD活力下降[24月龄(50.48±10.12)U/m1,12月龄(58.40±5.57)U/ml,3月龄(66.38±5.39)U/m1,P<0.05],且24月龄衰老模型鼠血清SOD活力较12月龄亦下降(P<0.05)。(3)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)与3月龄大鼠(A组)相比,血清MDA含量增加[24月龄(11.83±3.13)nmol/ml,12月龄(8.41± 2.04)nmol/ml,3月龄(4.81±1.37)nmol/ml,P<0.05],且24月龄衰老模型鼠血清MDA含量较12月龄亦增加(P<0.05)。(4)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)与3月龄大鼠(A组)相比,胸腺指数、脾脏指数均降低[胸腺指数：24月龄(0.089±0.006)g/100g,12月龄(0.107±0.005)g/100g,3月龄(0.230±0.011)g/100g,P<0.05；脾脏指数：24月龄(0.147±0.011)g/100g,12月龄(0.138±0.007)g/100g,3月龄(0.305±0.017)g/100g,P<0.05],且24月龄衰老模型鼠胸腺指数较12月龄亦降低(P<0.05)。[结论]用D-gal按0.125g·kg-1·d-1剂量皮下连续注射40d的方法能成功建立衰老的大鼠模型。随着年龄的增长,SD大鼠的体重增长减慢,血清SOD活力、胸腺指数逐渐下降,血清MDA含量逐渐增加。第二部分増龄对大鼠肠黏膜上皮屏障的影响[背景]随着自然增龄,老年人机体在结构和功能上出现各种退行性变化,肠道作为机体抵御病原体的第一道防线,在多种急慢性疾病的发生发展中起着至关重要的作用。因此抗肠道衰老也逐渐成为老年医学领域研究的重点和热点。肠道的屏障分为上皮屏障(机械屏障)、免疫屏障、生物屏障和化学屏障四类。其中以上皮屏障和免疫屏障尤为重要。研究表明,肠道的上皮屏障受多种致病因素(如营养不良、氧化应激、感染、酒精、恶性肿瘤、非甾体类药物、创伤等)的影响。但增龄是否对肠黏膜上皮屏障功能产生影响,仍是老年医学研究领域中的空白,与衰老相关的肠黏膜屏障损害方面还缺乏科学证据。[目的]观察衰老大鼠肠黏膜上皮屏障结构随着增龄出现的变化,为肠道老化及延缓肠道衰老方面的研究提供理论依据。[材料和方法](1)选取3月龄SD大鼠10只(A组)、12月龄SD大鼠10只(B组)、24月龄衰老模型SD大鼠10只(C组),其中24月龄衰老模型大鼠由3月龄SD大鼠予D-gal按0.125g·kg-1·d1剂量皮下连续注射40d的方法建立。常规饲养40d后取各组大鼠末端回肠组织进行常规HE染色,观察形态学改变及绒毛高度、厚度等,运用免疫组化技术Envision两步法检测小肠黏膜组织标本中Occludin、ZO-1蛋白的水平；运用半定量RT-PCR技术分析小肠黏膜组织标本中Occludin mRNA、ZO-1 mRNA的表达水平；取各组大鼠肝脏、脾脏、肠系膜淋巴结组织进行细菌培养,计算肠道细菌移位率。(2)比较3月龄组(A组)、12月龄组(B组)和24月龄衰老模型组(C组)间小肠黏膜组织标本中Occludin和ZO-1蛋白水平、Occludin mRNA和ZO-1mRNA的表达水平、肠道细菌移位率的差异。[结果](1)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)小肠黏膜厚度及绒毛高度均较3月龄(A组)降低[小肠黏膜厚度：24月龄(87.6±6.32)μm,12月龄(131.8±5.22)μm,3月龄(162.9±7.28)μ m,P<0.05；绒毛高度：24月龄(56.4±5.38)μm,12月龄(76.7±5.40)μ m,3月龄(108.1±6.42)μ m,P<0.05],且24月龄衰老模型鼠小肠黏膜厚度及绒毛高度较12月龄亦降低(P<0.05)。(2)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)小肠黏膜组织中紧密连接蛋白(Occludin、ZO-1)表达均较3月龄(A组)减少[Occludin蛋白：24月龄(2.23±0.60)%,12月龄(4.21±0.61)%,3月龄(12.31±0.94)%,P<0.05；ZO-1蛋白：24月龄(2.03±0.54)%,12月龄(4.02±0.65)%,3月龄(12.21±0.81)%,P<0.05],且24月龄衰老模型鼠小肠黏膜组织中紧密连接蛋白(Occludin、ZO-1)表达较12月龄亦降低(P<O.05)。(3)24月龄衰老模型鼠(C组)、12月龄大鼠(B组)小肠黏膜组织中Occludin mRNA和ZO-1mRNA相对表达量较3月龄(A组)明显减少[Occludin mRNA:24月龄(0.20±0.03),12月龄(0.38±0.02),3月龄(0.66±0.03),P<0.05； ZO-1mRNA:24月龄(0.18±0.03),12月龄(0.37±0.02),3月龄(0.63±0.03),P<0.05],且24月龄衰老模型鼠小肠黏膜组织中Occludin mRNA和ZO-1 mRNA相对表达量较12月龄亦减少(P<0.05)。(4)3组大鼠间肠道细菌移位率无显著差异。[结论]随着年龄的增长,老年SD大鼠分别与壮年、青年SD大鼠相比,小肠黏膜厚度及绒毛高度降低,小肠黏膜组织中紧密连接蛋白(Occludin和ZO-1)减少,小肠黏膜组织中紧密连接蛋白mRNA (Occludin mRNA、ZO-1 mRNA)表达减少,但肠道细菌移位率无显著差异。更多还原

【Abstract】 Part One Establishment of Aging Model[Background] The senescence society is coming. Establishment of aging animal model is an effective way to study the aging process of human being. The researches on aging and anti-aging agents become a hot point which require aging model. But natural aging animals are very rare because of the long period of feeding time and the large spending. So experimental aging model being copied by chemical method is used to research and provide higher efficiency compared with natural aging model. So far, many methods have been reported to copy aging animal model successfully. For example, ozone induced aging model, thymus removed aging model, aging SAMP model and D-galactose induced aging model, et al. D-galactose induced aging model has been widely used because of its easy accessibility, less cost and stable performance. And the model rats had some similar physiological and biological changes compared with natural aging rats, which led to an accelerated aging.[Objectives] The aim of this study was to establish the D-galactose induced aging rat model, and evaluate its aging indexes such as the variation of weight, the levels of serum SOD and MDA, the index of the thymus weight and spleen weight by compared with young rat. Meanwhile, the D-galactose aging model provides aged test animals to the subsequent study.[Methods] (1) Ten 3-month-old and ten 12-month-old SD rats were set as group A and group B individually. Ten 24-month-old model rats (group C) were established by D-galactose injection subcultaneously for 40 days with the dose of 0.125 g · kg-1 · d-1. All groups were given regular feeding and water for 40 days. Rats body weight were measured once every 10 days. The levels of serum SOD and MDA were detected after 40 days feeding, and the indexes of thymus weight and spleen weight were calculated.(2) The difference of body weight growth, serum SOD, MDA levels and indexes of thymus weight and spleen weight in three groups were compared.[Results] (1) After 40 days normal diet feeding,24-month-old rats (group C) and 12-month-old rats (group B) had significantly lower body weight growth compared with 3-month-old SD rats (group A) [(41.3± 11. 1)g, (85.6±10.9)g vs. (125.3±15.2)g, both P<0.05]. And group C had lower body weight growth than group B (P<0.05).(2) 24-month-old rats (group C) and 12-month-old rats (group B) had significantly lower activity of serum SOD compared with 3-month-old SD rats (group A) [(50.48 ± 10.12)U/ml, (58.40 ± 5.57)U/ml vs. (66.38 ± 5.39)U/ml, both P<0.05]. And group C had lower activity of serum SOD than group B (P<0.05).(3) 24-month-old rats (group C) and 12-month-old rats (group B) had significantly higher level of serum MDA compared with 3-month-old SD rats (group A) [(11.83 ± 3.13)nmol/ml, (8.41 ± 2.04)nmol/ml vs. (4.81± 1.37) nmol/ml, both P<0.05]. And group C had higher level of serum MDA than group B (P<0.05).(4) 24-month-old rats (group C) and 12-month-old rats (group B) had significantly lower indexes of thymus and spleen weight [thymus:(0.089 ±0.006)g/100g, (0.107±0.005)g/100g vs. (0.230±0. 011) g/100g; spleen: (0.147±0.011) g/100g, (0.138±0.007) g/100g vs. (0.305±0.017) g/100g, both P<0.05]. And group C had lower index of thymus weight than group B (P<0.05).[Conclusion] The establishment of D-galactose induced aging model is successful. Along with aging, rat body weight increase was slow, the index of thymus weight and serum level of SOD became lower, and content of MDA became higher.Part Two the Effect of Aging onIntestinal Mucosa Epithelial Barrier in Rats[Background] With the increase of age, a variety of degenerative changes will appear in the body structure and function. As one of the very important organs, the intestine also changes with age. But the effect of aging on intestinal mucosal barrier is lack of evidence.[Objectives] To study the changes of intestinal epithelial barrier function in rats with aging.[Methods] (1) Ten 3-month-old and ten 12-month-old SD rats were set as group A and group B individually. Ten 24-month-old model rats (group C) were established by D-galactose injection subcultaneously for 40 days with the dose of 0.125 g ·kg-1 · d-1. After 40 days feeding, the terminal ileum was removed to make hematoxylin and eosin (HE) section, and the morphology of small intestine mucosa, intestinal villus altitude and thickness were observed under light microscope. The expressions of Occludin and Z0-1 protein in terminal ileum mucous membrane were detected by immunohistochemistry. The expressions of Occludin and Z0-1 mRNA were determined by semi-quantitative reveals transcription polymerase chain reaction (RT-PCR). The liver, spleen, mesenteric lymph node tissue of rats in each group were taken for bacterial culture to calculate bacterial translocation rate.(2) The expressions of Occludin and ZO-1 protein in the small intestinal tissue were compared. Bacterial translocation rate in three groups were also compared.[Results] (1) After 40 days normal diet feeding, the small intestinal mucosa thickness and villus height were lower in group C and B than group A [thickness:(87.6±6.32) μ m, (131.8±5.22) um vs. (162.9 ±7.28)μm; villus height:(56.4±5.38)μm, (76.7±5.40)μm vs (108.1 ±6.42) μm; both P<0.05], and the small intestinal mucosa thickness and villus height in group C were lower than that in group B (P<0.05).(2) Occludin and Z0-1 protein expressions in the small intestine tissue were reduced in group C and B as compared with group A [Occludin protein:(2.23±0.60)%, (4.21±0.61)% vs. (12.31±0.94)%; Z0-1 protein:(2.03±0.54)%, (4.02±0.65)% vs. (12.21±0.81)%; both P<0.05], and Occludin and Z0-1 protein expressions were less in group C than that in group B (P<0.05).(3) The levels of Occludin and ZO-1 mRNA in small intestine tissue were reduced in group C and B as compared with group A[Occludin:(0.20 ±0.03), (0.38±0.02) vs. (0.66±0.03); Z0-1:(0.18±0.03), (0.37±0.022) vs. (0.63±0.03); both P<0.05], and Occludin and ZO-1 mRNA expressions were less in group C than that in group B (P<0.05)(4) The bacterial translocation rate had no significant difference in 3 groups.[Conclusion] Our study indicated that age-related intestinal mucosa epithelial barrier changes may be associated with mucosal atrophy, less expressions of tight junction molecular (Occludin and ZO-1) mRNA and protein, but the difference of bacterial translocation rate is insignificant.更多还原