【作者】 杨平；

【导师】 孟宪军；

【作者基本信息】 沈阳农业大学 ， 食品科学与工程， 2009， 博士

【摘要】 刺五加由于含有多种的天然活性成分,且无毒副作用,自古就倍受人们青睐。随着生物活性物质分离纯化技术的发展,人们目前已经开展了大量关于刺五加活性成分的分离提取和结构鉴定方面的研究,刺五加苷就是人们利用现代化手段提取从刺五加中提取的天然活性物质。本课题给成年SD大鼠连续腹腔注射D-半乳糖建立衰老动物模型后,利用不同浓度刺五加苷作用于该模型,以维生素E作为阳性对照,从机能和形态两个方面研究刺五加苷是否具有延缓衰老的作用。在得出刺五加苷具有延缓衰老作用的基础上,结合目前世界上比较公认的自由基、免疫、端粒长度、细胞凋亡衰老学说,从体内、外两个方面研究刺五加苷延缓衰老的机理。在体内,研究了刺五加苷对衰老大鼠抗氧化能力、免疫功能、p16蛋白和对衰老相关-β-半乳糖苷酶的影响;在体外,利用具有自发凋亡的胸腺细胞作为模型,观察了刺五加苷对小鼠胸腺细胞凋亡的作用。由于衰老过程涉及机体各个系统、不同结构层次,因此本研究在整体水平、组织器官水平、细胞水平以及分子水平,分别研究了刺五加苷延缓衰老的机制。本研究的研究结果有以下几方面:（1）D-半乳糖致衰老模型构建成功。模型大鼠组织中SOD、GSH-Px活性下降,iNOS活性升高,MDA、NO浓度升高,T淋巴细胞增殖功能下降,血清IL-2浓度下降,IL-6浓度和TNF-α浓度升高,组织SA-β-gal活性增强,p16蛋白表达增加。（2）刺五加苷可以改善衰老大鼠功能。刺五加苷能增加衰老大鼠的胸腺指数和脑指数,增强学习和记忆能力,升高血中HDL-C浓度,降低TG、TC、LDL-C浓度,降低血液粘滞性。高浓度刺五加苷降低TG浓度、增强学习和记忆能力、增加胸腺和脑指数的作用好于维生素E,中、低浓度刺五加苷降低LDL-C作用效果弱于维生素E,低浓度刺五加苷改善大鼠学习和记忆能力作用也弱于维生素E。（3）高浓度刺五加苷可以延缓卵巢组织形态衰老。刺五加苷作用后衰老大鼠卵泡细胞轮廓清,线粒体损伤减轻,核肿胀缓解,常染色质分布均匀,中、低浓度刺五加苷和维生素E作用后细胞器多种退行性改变仍很严重。刺五加苷可延缓神经细胞退行性改变。高浓度刺五加苷可使神经细胞轮廓清晰,核膜完整光滑,常染色质分布均匀,但其对神经细胞质水肿、线粒体嵴损伤改善仍不明显,中、低浓度刺五加苷和维生素E作用后细胞器退行性改变较严重。各浓度刺五加苷都可以抑制肝细胞退行性改变。刺五加苷可以使肝细胞轮廓清晰、线粒体形态基本完好,高浓度刺五加苷作用效果最好,胞质内可见大量粗面内质网呈板层状排列,线粒体丰富,糖原数量多。（4）刺五加苷具有抗氧化作用。各浓度刺五加苷都可以升高衰老大鼠组织SOD、GSH-Px活性,抑制iNOS活性,降低MDA、NO浓度。高浓度刺五加苷提高GSH-Px、SOD活性、降低MDA和NO浓度、抑制iNOS活性的作用强于维生素E,中浓度提高GSH-Px活性、降低MDA浓度作用也强于维生素E,低浓度刺五加苷降低MDA和NO浓度作用弱于维生素E。（5）刺五加苷具有延缓免疫衰老作用。中、高浓度刺五加苷都可以提高衰老大鼠T淋巴细胞增殖功能和IL-2浓度,降低IL-6浓度,高浓度还可以降低TNF-α浓度。高浓度刺五加苷提高T淋巴细胞增殖功能和IL-2浓度作用,降低IL-6和TNF-α浓度作用都强于维生素E;中浓度提高T淋巴细胞增殖功能和IL-2浓度作用也强于维生素E,低浓度刺五加苷调节机体细胞免疫功能的作用与维生素E比较差异不明显。（6）刺五加苷具有抑制衰老相关-β-半乳糖苷酶活性和降低p16蛋白表达的作用。中、高浓度刺五加苷具有抑制大鼠衰老相关-β-半乳糖苷酶活性,降低组织p16蛋白表达的作用,低浓度只能抑制衰老相关-β-半乳糖苷酶活性。中、高浓度刺五加苷抑制组织衰老相关-β-半乳糖苷酶活性,降低组织p16蛋白表达作用强于维生素E;低浓度抑制组织SA-β-gal活性作用弱于维生素E。（7）小鼠胸腺细胞随着培养时间延长,会发生时间依赖性凋亡,表现为S期和G₂/M期细胞比例减少,增值能力下降。50、100μmol/L刺五加苷可以增加胸腺小鼠细胞增值指数、抑制细胞凋亡,作用好于维生素E;20μmol/L刺五加苷能抑制细胞凋亡,作用效果与维生素E没有显著差异。通过以上研究,本课题得出刺五加苷具有延缓机体衰老的作用,其作用机理与刺五加苷提高机体抗氧化能力、增强细胞免疫功能、降低p16蛋白表达、促进胸腺细胞增殖、抑制细胞凋亡相关。更多还原

【Abstract】 Ciwujia containing a wide range of natural active ingredients,and non-toxic side effects, with the advantages of both temporary and permanent cures,people on the popular favor since ancient times.With the isolation and purification of bioactive substances in the development of technology,people have carried out a large number of active ingredients on the ciwujianoside and structural identification studies.Ciwujianoside is the use of modern means of extracting ciwujia from natural active substances.In this study,by continuous subcutaneous injecting D-galactose to adult SD rats establish an animal model of aging,using of different concentrations ciwujianoside in rats,studying anti-aging from both functional and morphological.The result is ciwujianoside with anti-aging.Therefore,we research the current aging theory,such as the free radical theory,the immune theory of aging,telomere length theory,theory of apoptosis,study the mechanism of anti-aging ciwujianoside both in vitro and in vivo.In vivo,the studied is carried out by ciwujianoside on antioxidant capacity,immune function,p16 protein and senescence-associated-β-galactosidase impact in aging rats.In vitro, the studied is carried out by the ciwujianoside on the role ofapoptosis in thymocytes,with the spontaneous apoptosis of thymocytes as a model.As the aging process involves various body systems,the study relate to the level of different structures,the mechanisms of ciwujianoside anti-aging of in the overall level,the level of tissues and organs,cells and molecular level.The results of this study are the following:（1） The D-gal-induced aging model do not change rat body weight,electrocardiogram, heart rate,WBC,RBC,HGB,PLT,MCV,ALT,AST,TP,ALB,GLO,not change in liver, heart,brain,spleen,kidney tissue vitality tNOS.However,it decrease SOD,GSH-Px activity, increase iNOS activity,MDA,NO concentration increase;T lymphocyte proliferation function,serum IL-2,IL-6 concentrations decrease,TNF-αconcentrations increase;tissue SA-β-gal activity and p16 protein expression enhance.（2） Ciwujianoside can increase the rat thymus and brain aging index,and enhance learning and memory capacity,higher blood HDL-C concentration and lower TG,TC,LDL-C concentrations and lower blood viscosity,and high concentrati0ns of ciwujianoside obvious effect better than the low and middle.In addition,the high concentration of ciwujianoside reduct TG concentration,enhance learning and memory capacity,increase thymic index,and the role of the brain better than vitamin E.But effect of enhancing learning and memory capacity in the low concentration lower than that of the vitamin E.（3） High concentrations of ciwujianoside follicle cells in aging rats could outline a clear and reduce the extent of mitochondrial injury,to alleviate the status of nuclear swelling,so that distribution euchromatin with delay ovarian aging patterns.However,in other concentrations of ciwujianoside and vitamin E,follicles occupy the cytoplasm is obvious edema in follicles,mitochondrial degeneration seen,a variety of cell degenerative changes are still very serious.In addition,high concentrations of ciwujianoside can make the outline of nerve cells in aging rats clear,smooth nuclear membrane integrity,euchromatin distribution, delay nerve cell degeneration,but the cytoplasm of nerve edema,mitochondrial cfistae damage does not improve significantly.（4） Ciwujianoside can increase the SOD,GSH-Px activity,inhibiting iNOS activity and lower MDA,NO concentration.The role of high concentrations of ciwujianoside results significantly better than middle and low concentration groups,which is the role of a dose-dependent.High concentrations of ciwujianoside on increase T lymphocyte proliferation, IL-2 concentration,IL-6 concentrations,lower TNF-αconcentrations is better than the effect of vitamin E.Middle concentration ciwujianoside in aging rats only increase T lymphocyte proliferation and enhance IL-2 concentration better than vitamin E,The effect of low concentration ciwujianoside reducting MDA and NO concentration is lower than the vitamin E.（5） Ciwujianoside to postpone the role of immunosenescence in a dose-dependent way.High concentrations of ciwujianoside can delaying immunosenescence by increasing T lymphocyte proliferation,IL-2 concentration,lowing IL-6 and TNF-αconcentrations.Middle concentrations of ciwujianoside increase T lymphocyte proliferation and IL-2 concentration better than vitamin E;Low concentration of ciwujianoside in the regulation of aging cellular immune function in rats is not obvious.High concentrations of ciwujianoside in aging rats increase T lymphocyte proliferation,IL-2 concentration,lower concentrations of IL-6 concentrations,TNF-αconcentrations effects are better than the vitamin E.Low concentration of ciwujianoside in the regulation cellular immune function compare with the vitamin E the difference is not obvious.（6） Middle and high concentrations of ciwujianoside can inhibit SA-β-gal activity and lower p16 protein expression in a dose-dependent manner.Low concentrations of ciwujianoside only can inhibit SA-β-gal activity.The high and middle concentrations of ciwujianoside can inhibit SA-β-gal activity and reduce p16 protein expression better than vitamin E.Low concentration of ciwujianoside inhibition in SA-β-gal activity less the effect of vitamin E.（7） Cultured mouse thymocytes can be naturally occurring time-dependent apoptosis with the culture time period of the extension of S and G₂/M cells,a significant reduction in the proportion,decline in value-added.In the 20-100μmol/L ciwujianoside have cell intoxicity to cultured mouse thymocytes.Ciwujianoside can anti-aging by increasing index of thymus cells in mice,inhibition of apoptosis,which effect better than the vitamin E.20μmol/L ciwujianoside only inhibit apoptosis,no significant impact on value-added index,which effects no significant difference with vitamin E.This study conclude that ciwujianoside have anti-aging effect,which is to achieve by enhancing antioxidant capacity and cellular immune function,reducing the expression of p16 protein,promoting thymocyte proliferation,inhibiting apoptosis of thymocytes.更多还原