大鼠肺微血管内皮细胞衰老机制的研究
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摘要
肺功能不全是多种肺疾病发展至最后可能产生的共有的病理过程，并由此引发多器官功能衰竭，危及生命。人口老龄化是21世纪全球面临的重大课题。提示研究包括老年肺功能不全在内的老年性疾病发生机制的迫切性。肺微血管内皮细胞(pulmonary microvascular endothelial cells，PMVECs)在肺功能不全的发病学中起主导作用。本研究拟通过研究衰老PMVECs存活与凋亡信号通路的变化规律，阐明老化过程中肺功能不全的细胞分子机制。将对老年肺功能不全以及老年相关疾病的研究提供有益探索，具有极其重要的社会意义和经济意义。
目的
在成功分离培养大鼠PMVECs的基础上，建立稳定可靠的D-半乳糖(D-galactose，D-gal)诱导的大鼠PMVECs衰老模型；从细胞周期，细胞生长存活和细胞凋亡及其信号传导途径调控等方面探讨PMVECs衰老的细胞分子机制。
方法
1．大鼠PMVECs的分离培养与鉴定。联合应用胰蛋白酶和胶原酶消化肺边缘组织分离细胞，血清诱导内皮细胞聚集。根据内皮细胞典型的铺路石样的形态学特征，运用胰蛋白酶／EDTA局部消化内皮细胞克隆再进行扩大培养。用下述方法鉴定大鼠PMVECs：(1)免疫荧光标记法检测内皮细胞内vWF；(2)荧光显微镜观察PMVECs吞噬红色荧光染料DiI标记的乙酰化的低密度脂蛋白(DiI-Ac-LDL)的功能；(3)透射显微镜观察大鼠PMVECs的超微结构 (4)根据内皮细胞能吞噬DiI-Ac-LDL的功能，运用流式细胞仪计量有荧光标记的阳性细胞，以鉴定所分离大鼠PMVECs的纯度。
2．D-gal诱导大鼠PMVECs衰老模型的建立。运用四甲基偶氮唑盐微量酶反应比色法(MTT法)观察D-gal对大鼠PMVECs生存活力的影响；将原代分离的大鼠PMVECs传代，以10g／L D-gal的DMEM培养液培养细胞，传代3次继续培养7d后终止培养，即为用于本文实验的D-gal诱导的大鼠PMVECs衰老模型。以不含D-gal的上述培养液培养传代3次的细胞为对照组。此外，将PMVECs经D-gal诱
Aged pulmonary insufficiency is the possible common pathological process of many lung diseases , which can lead to multiple organ dysfunction syndrome(MODS),eventually death. It is urgent to study on molecular and cellular mechanisms of age-related diseases, including aged pulmonary insufficiency. Pulmonary microvascular endothelial cells( PMVECs )is the key point in pathogenesis of pulmonary insufficiency because of the special functions. We hope to clarify the celluar and molecular mechanisms of pulmonary insufficiency during ageing through research on the change rules by signal pathway in growth, survival and apoptosis . The subject not only will provide useful clues for the study of aged pulmonary insufficiency and age-related diseases but also has extremely important social and economic meaning..
Object
To establish a stable and relialble model of PMVECs ageing induced by D-gal based on isolating and culturing PMVECs from rats lung successfully; Explore cellular and molecular mechanisms of PMVECs senescence on cell cycle, growth and survival ,apoptosis ,signal pathways.
Methods
1. Isolation, culture, purification and identification of PMVECs from rats lung. The purified PMVECs were obtained after isolated from peripheral lung tissues , digested with trypsin and collagenase and aggregated by serum stimulation. According to typical cobble morphology of endothelial cells, PMVECs were selected and transferred by trypsin/EDTA local digestion to amplify the number of cells. The following methods were used to identify PMVECs from rats lung.(l) von
目的
在成功分离培养大鼠PMVECs的基础上，建立稳定可靠的D-半乳糖(D-galactose，D-gal)诱导的大鼠PMVECs衰老模型；从细胞周期，细胞生长存活和细胞凋亡及其信号传导途径调控等方面探讨PMVECs衰老的细胞分子机制。
方法
1．大鼠PMVECs的分离培养与鉴定。联合应用胰蛋白酶和胶原酶消化肺边缘组织分离细胞，血清诱导内皮细胞聚集。根据内皮细胞典型的铺路石样的形态学特征，运用胰蛋白酶／EDTA局部消化内皮细胞克隆再进行扩大培养。用下述方法鉴定大鼠PMVECs：(1)免疫荧光标记法检测内皮细胞内vWF；(2)荧光显微镜观察PMVECs吞噬红色荧光染料DiI标记的乙酰化的低密度脂蛋白(DiI-Ac-LDL)的功能；(3)透射显微镜观察大鼠PMVECs的超微结构 (4)根据内皮细胞能吞噬DiI-Ac-LDL的功能，运用流式细胞仪计量有荧光标记的阳性细胞，以鉴定所分离大鼠PMVECs的纯度。
2．D-gal诱导大鼠PMVECs衰老模型的建立。运用四甲基偶氮唑盐微量酶反应比色法(MTT法)观察D-gal对大鼠PMVECs生存活力的影响；将原代分离的大鼠PMVECs传代，以10g／L D-gal的DMEM培养液培养细胞，传代3次继续培养7d后终止培养，即为用于本文实验的D-gal诱导的大鼠PMVECs衰老模型。以不含D-gal的上述培养液培养传代3次的细胞为对照组。此外，将PMVECs经D-gal诱
Aged pulmonary insufficiency is the possible common pathological process of many lung diseases , which can lead to multiple organ dysfunction syndrome(MODS),eventually death. It is urgent to study on molecular and cellular mechanisms of age-related diseases, including aged pulmonary insufficiency. Pulmonary microvascular endothelial cells( PMVECs )is the key point in pathogenesis of pulmonary insufficiency because of the special functions. We hope to clarify the celluar and molecular mechanisms of pulmonary insufficiency during ageing through research on the change rules by signal pathway in growth, survival and apoptosis . The subject not only will provide useful clues for the study of aged pulmonary insufficiency and age-related diseases but also has extremely important social and economic meaning..
Object
To establish a stable and relialble model of PMVECs ageing induced by D-gal based on isolating and culturing PMVECs from rats lung successfully; Explore cellular and molecular mechanisms of PMVECs senescence on cell cycle, growth and survival ,apoptosis ,signal pathways.
Methods
1. Isolation, culture, purification and identification of PMVECs from rats lung. The purified PMVECs were obtained after isolated from peripheral lung tissues , digested with trypsin and collagenase and aggregated by serum stimulation. According to typical cobble morphology of endothelial cells, PMVECs were selected and transferred by trypsin/EDTA local digestion to amplify the number of cells. The following methods were used to identify PMVECs from rats lung.(l) von
引文
1. Lou J N, Mili N, Decrinch C, et al. An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung. In Vitro Cell Dev Biol Anim, 1998,4(7):529-536.
2. Kim N S, Kim S J. Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum. Yonsei Med, J, 1991, 32(4):303-314
3．杨红，斯琴，孙仁宇．肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000，16(9)：831-834
4. Oite T, Suzuki Y, Morioka T, et al. Efficient isolation of rat aortic endothelial cells by elimination of contaminating cells with a monoclonal antibody. Microvasc Res, 1995, 50(1): 113-118
5. Magee J C, Stone A E, Oldham K, et al. Isolation, culture,and characterization of rat lung microvascular endothelial cells.Am.J.Physiol,1994,267(4 pt 1):L433-441
6. Chen S F, Fei X, Li S H. A new simple method for isolation ofmicrovascular endothelial cells avoiding both chemical and mechanical injuries. Microvasc Res, 1995, 50 (1):119-128
7. Nishida M, Carley W W, Gerritsen M E, et al. Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol, 1993,264(2 pt 2): H639-652
8. Ryan U S, Linda A W, Lopez M, et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood, 1994,84(1):10-19
9．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001，46(19)：1636-1640
10．李姗姗，李澎，黄启福．脂多糖对体外人脐静脉内皮细胞分泌功能及活力的影响．中国病理生理杂志，2001，17(7)：658-661
11．崔旭，李文彬，张炳烈，等．D-gal对神经元和成纤维细胞拟老化作用研究的实验研究．中国应用生理学杂志，1997，13(2)：131-133
12. Shen J, Ham R G, Karmiol S. Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells. Microvasc Res, 1995, 50 (3):360-372
13．肖贞良，孙耕耘，钱桂生．肺循环灌注对大鼠肺微血管内皮细胞分离和培养的影响．中国病理生理杂志，1999，15(11)：1053-1055
14．龚国清，徐黻本．小鼠模型衰老模型的研究．中国药科大学学报，1991，12(2)：101-103
15．马宏，张宗玉，童坦君．衰老的生物学标志．生理科学进展，2002，33(1)：65-681. Xiao Z Q, Yu Y J, Khan A, et al. Induction of G_1 checkpoint in the gastric mucosa of aged rats.Am J Physiol Gastrointest Liver Physiol. 1999,277:G929-G934.
2. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence -associated apoptosis. Exp Gerontol. 2001,36:1327-47.
3. Stein GH, Dulic V, et al. Origins of G(1) arrest in senescent human fibroblasts. Bioessays,1995, 17:537-543.
4. Buchkovich, K, Duffy LA, Harlow E, et al. The retinoblastoma protein is phosphorlated during specific phases of the cell cycle. Cell. 1989,58:1097-1105.
5. Basheer R, Shiromani PJ. Effects of prolonged wakefulness on c-fos and AP-1 activity in young and old rats. Brain Res Mol Brain Res.2001, 89:153-7.
6. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000,278:G855-865
7. Warner HR. Recent progress in understanding the relationships among aging, replicative senescence, cell turnover and cancer. In vivo, 2002,16:393-396.
8. Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by p53. Eur J Biochem, 2001,268:2784-91.
9．刘晓秋，李卫东，唐惠琼等．D-半乳糖衰老大鼠自由基代谢状况分析．中国老年学杂志．2001，21：456-458．
10. Pagano M, Pepperkok R, Verde E Cyclin A is required at two points in the human cell cycle. EMBO J. 1992, 11:961-971.
11. Sherr CJ. G1 phase progression: Cvclin on cue, Ce11.1994.79:551-555.
12．彭黎明，王曾礼主编。细胞凋亡的基础与临床，北京：人民卫生出版社。2000，4-5
13. Sharpless NE, Depinho RA. p53:good cop/bad cop. Ce11,2002,110:9-12
14. Ide T, Tsuji Y, Ishibashi S, et al. Reinitiation of host DNA synthesis in senescent human dipoid cells by infection with simian virus. Exp Cell Res.1983, 143:343-349.
15. Ide T, Tsuji Y, Nakashima T, et al. Progress of aging in human diploid cells transformed with a tsA mutant of simian virus 40. Exp Cell Res.1984, 150:321-328.
16. Gorman SD& Cristofalo VJ. Reinitiation of cellular DNA synthesis in BrdU-selected nondividing senescent WI38 cells by simian virus 40 infection. J Cell Physiol. 1985,125:122-126.
17. Elmore LW, Rehder CW, Di X,Mcchesney PA, Jackson-Cook CK, Gewirtz DA. Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction. J Biol Chem 2002 Sep 20; 277(38):35509-15.
18. Donehower LA. Does p53 affect organismal ageing? J cell physiol.2002,Jul; 192(1):23-33
19. Lodygin D, Menssen A, Hermeking H. Induction of the Cdk inbibitor p21 by LY83853 inhibits tumor cell proliferation in a p53-independent manner. J Clin Invest.2002, 110:1717-27.
20. Chen X, Zhang W, Gao YF, et al. Senescence-like changes induced by expression of p21(waf/Cipl) in NIH3T3 cell line. Cell Res.2002, 12:229-233.
21. Sheerin A, Thompson KS, Goyns MH. Altered composition and DNA binding activity of AP-1 transcription factor during the ageing of human fibroblasts. Mech Ageing Dev.2001,122:1813-1824.
22. Medicherla R, Leers-Sucheta S, Luo Y, et al. Impaired activarion of AP-1 and altered expression of constituent proteins in rat adrenal during ageing. Mech Ageing Dev, 2001, 122: 1169-1186.1．刘汴生．衰老过程中的病理生理．实用老年医学．2002，16：64-65．
2. Zhang Y, Herman B. Ageing and apoptosis. Mech Ageing, 2002,123:245-60.
3. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence-associated apoptosis. Exp Gerontol, 2001, 36:1327-47.
4. Campici, J. The role of cellular senescence in skin aging. J Investig Dermatol. Symp.Proc, 1998,3:1-5.
5. Potestio M, Caruso C, Gervasi F, et al.Apoptosis and ageing.Mech Ageing Dev,102:221-37.
6. Kapasi AA, Singhal PC. Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53,bax,and caspase-3. Moi Cell Biol Res Commun, 1999,1:78-81.
7. Bennett MR. Apoptosis of vascular smooth muscle cells in vascular remodeling and atherosclerotic plaqur rupture. Cardiovasc Res, 1999,41:361-368.
8. Haake AR, Roublevskaia I, Cooklis M. Apoptosis: a role in skin aging? J Investing Dermatol Symp Proc, 1998, 3:28-35.
9．彭黎明，王曾礼，主编．细胞凋亡的基础与临床．北京：人民卫生出版社，2000．
10．黄英，张宗玉，童坦君．人衰老成纤维细胞凋亡的可诱导性．中华老年医学杂志，2000，19：170—173．
11．杨红，斯琴，孙仁宇．肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000,16：831-834．
12．王士雯．老年多器官功能不全肺启动机制若干问题初探．中华老年心脑血管病杂志，2000．2：149-151．
13. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res,2001,89:709-15.
14. Zhang J, Patel J M, Block E R, et al. Enhanced apoptosis in prolongfe cultures of senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev, 2002, 123: 613-25.
15. Muskhelishvili L, Hart RW, Turturro A, et al. Age-related changes in the intrinsic rate of apoptosis in livers of diet-restricted and ad libitum-fed B6C3F1 mice. Am J Pathol, 1995,147:20-24.
16. Higami Y, Shimokawa I, Tomita M, et al. Aging accelerates but life-long dietary restriction suppresses apoptosis-related Fas expression on hepatocytes. Am J Pathol, 1997,151:659-663
17. Taglialatela G, Gegg M, Perez-Polo JR, et al. Evidence for DNA fragments in the CNS of aged??Fischer-344 rats. Neuroreport, 1996,7:977-980.
18．刘小秋，李卫东，唐惠琼．D-半乳糖衰老大鼠自由基代谢状况分析，2001，21：456—458．
19. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing. Nature, 2000, 408: 239-247.
20. Leonard Haytick.The future of ageing. Nature, 2000, 408:267-269.
21. Ronald A.Depinho.The age of cancer. Nature, 2000,408: 248-254.
22. Remmen HV, Richardson A. Oxidative damage to mitochondria and aging. Exp Gerontol, 2001,36:957-968.
23．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001，46：1636-1640．
24. Zou H, Li Y, Liu X, et al. An APAF-lcytochrome c multimeric complex is a functional apoptosis that activates procaspase-9. J Biol Chem, 1999,274:11594-11556.
25. Rodriguez J, Lazebnik Y. Caspase-9 and APAF-1 from an active holoenzyme. Genes Dev, 1999,13:3179-3184.
26. Pollack M, Leeuwenburgh C. Apoptosis and aging: Role of the mitochondria. J Gerontol A Biol Sci, 2001, 56A: B475-B482.
27. Korsmeyer SJ, Wei MC, Sation M, et al. Pro-apoptotic cascade activites BID, which oligomerizes BAK or BAX into pores that result in the release of cytomchrome c. Cell Death Differ, 2000,7:1166-1173.1. Wang Q, Doerscuk CM. The p38 mitogen-activiated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation.J Immunol, 2001, 66:6877-6884.
2. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing.Nature 2000, 408: 239-247.
3. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res, 2001,89:709-715.
4. Awad M, Omori S, Hida M. MAP kinase in renal development. Nephrol Dial Transplant, 2002,17:5-7.
5. Zhen X, Uryu K, Cal G, et al. Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol A Biol Sci Med Sci, 1999,54:B539-48.
6. Kim HJ, Jung KJ, Yu BP, et al. Influence of ageing and calorie restriction on MAPKs activity in rat kidney. Exp Gerontol, 2000,37:1041-53.
7. Xiao Z Q, Yu Y J, Khan A, et al. Induction of G_1 checkpoint in the gastric mucosa of aged rats .Am J Physiol Gastrointest Liver Physiol.1999,277:G929-G934.
8. Kirk CJ, Freilich AM, Miller RA, Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes. Cell Immunol, 1999,197:75-82.
9. Chung JH, Kang S, Varani J, et al. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J Invest Dermatol, 2000,115:177-82.
10. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000,278:G855-65.
11. Liu Y, Guyton KZ, Gorospe M, et al. Age-related decline in mitogen-activated protein activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem, 1996,271:3604-7.
12. Suh Y. Age-specific changes in expression, activity, and activation of the c-jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by mythyl methanesulfonate in rats.MechAgeing,2001,122:1797-811
13．吴飞翔，章卫平．JNK信号转导与细胞凋亡．生命的化学．2002，22(4)：346-347．
14. Verbeke P, Fonager J, Clark BF, et al. Heat shock response and ageing: mechanism and applications. Cell Biol.Int, 2001,25:845-857.
15. Fargnoli J, Kunisada T, Fornace AJ Jr, Schneider EL, Holbrook NJ. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc Natl Acad Sci USA, 1990,87:846-50.
16. Chang E, Yang J, Nagavarapu U, et al. Aging and survival of cutaneous microvasculature. J Invest Dermatol, 2002,118(5): 752-758.
17. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci, 2001,114(pt16): 2903-2910.
18. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res.2000, 87:434-439.
19. Irani K. Oxidant signaling in vascular cell growth,death, and survival. Circ Res.2000, 87:179-183.
20．张兵，吴乔，陈睦传．蛋白激酶B的特性及其生物学功能．细胞生物学杂志．2002，24(4)：209-212．
21．邹伟，李兆育，李春雷．蛋白激酶B及其在磷脂酰肌醇3—激酶介导的信号转导中的作用．生理科学进展．2000，31(2)：120-124．1．杨红，斯琴，孙仁宇。肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000，16：831-834
2．王士雯。老年多器官功能不全肺启动机制若干问题初探．中华老年心脑血管病杂志，2000，2：149-151
3．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001．46：1636-1640
4. Lou J N, Mili N, Decrinch C, et al. An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung. In Vitro Cell Dev Biol Anim, 1998,4:529-536
5. Magee J C, Stone A E, Oldham K, et al. Isolation, culture, and characterization of rat lung microvascular endothelial cells. Am J Physiol, 1994,267:L433-441
6. Shen J, Ham R G, Karmiol S. Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells. Microvasc Res, 1995, 50:360-372
7. Kim N S, Kim S J. Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum. Yonsei Med, J, 1991, 32:303-314
8. Zhang Y, Herman B. Ageing and apoptosis. Mech Ageing, 2002,123:245-60.
9. Ostler EL, Wallis CV, Boalchamat B, et al. Telomerase and the cellular lifespan: implication of the aging process. J Pediatr Endocrinol Metab,2000,13:1467-1476
10．黄丹．细胞衰老的研究进展．细胞衰老的进展．暨南大学学报，2001，22：36-39
11. Cherif H, Tarry JL, Ozanne SE, et al. Aging and telomeres: a study into organ- and gender-specific telomere shortening. Nucleic Acids Res, 2003,31:1576-83
12. Kerr JER, Wyllie AH, Currie AR, et al. Apoptosis: a basic biological phenomenon with wide-rangeing implication in tissue kenetics. Br J Cancer,1972,26:239-257
13．彭黎明，王曾礼主编。细胞凋亡的基础与临床，北京：人民卫生出版社。2000，4-5
14. Pollack M, Leeuwenburgh C. Apoptosis and aging: Role of the mitochondria. J Gerontoi A Biol Sci, 2001, 56A: B475-B482
15. Hayflic L. Aging, longevity, and immortality in vitro. Exp.Gerontol. 1992, 27:363-368
16. Muskhelishvili L, Hart RW, Turturro A, et al. Age-related changes in the intrinsic rate of apoptosis in livers of diet-restricted and ad libitum-fed B6C3F1 mice. Am J Pathol, 1995,147:20-24
17. Higami Y, Shimokawa I, Tomita M, et al. Aging accelerates but life-long dietary restriction suppresses apoptosis-related Fas expression on hepatocytes. Am J Pathol, 1997,151:659-663
18. Potestio M, Caruso C, Gervasi F, et al.Apoptosis and ageing.Mech Ageing??Dev, 1998,102:221-237
19. Kapasi AA, Singhal PC. Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3. Mol Cell Biol Res Commun,1999,1:78-81
20. Fujita Y, Ozaki K, Yamamasu S, et al. DNA fragmentation of oocytes in aged mice. Hum Reprod, 1996,11:1480-483
21. Taglialatela G, Gegg M, Perez-Polo JR, et al. Evidence for DNA fragments in the CNS of aged Fischer-344 rats. Neuroreport, 1996,7:977-980
22. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence -associated apoptosis. Exp Gerontol,2001,36:1327-1347
23. Zhang J, Patel JM, Block ER. Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev, 2002, 123:613-25
24. Smale G, Nichols NR, Brady DP, et al. Evidence for apoptotic cell death in Alzheimer's disease. Exp Neurol, 1995,133:225-230
25. Lassman H, Bancher C, Breitschopf H, et al. Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ. Acta Neuropathol(Berl), 1995,89:35-41
26. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing. Nature, 2000, 408(9): 239-247
27. Leonard Hayrick.The future of ageing. Nature, 2000, 408,:267-269.
28. Ronald A.Depinho.The age of cancer. Nature 2000,408:248-254.
29. Remmen HV, Richardson A. Oxidative damage to mitochondria and aging. Exp G erontol.,2001,36:957-968
30. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998,281:1309-1312
31. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev, 2001,15:2922-2933.
32. Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. Biochem Soc Syrup, 1999,66:181-203.
33. Zou H, Li Y, Liu X, et al. An APAF-lcytochrome c multimeric complex is a functional apoptosis that activates procaspase-9. J Biol Chem, 1999,274:11594-11556
34. Rodriguez J, Lazebnik Y. Caspase-9 and APAF-1 from an active holoenzyme. Genes Dev,1999,13:3179-3184
35. Huang DCS, Strasser A. BH3-only proteins-essential initiators of apoptotic cell death. Cell,2000,103:839-842
36. Datta SR, Katsov A, Hu L, et al.14-3-3 Proteins and survival kinases coopertate to inactivate BAD by BH3 domain phosphorylation. Mol Cell, 1999,6:41-51
37. Korsmeyer S J, Wei MC, Sation M, et al. Pro-apoptotic cascade activites BID, which oligomerizes BAK or BAX into pores that result in the release of cytomchrome c. Cell Death Differ,2000,7:1166-117338. Chinnaiyan AM, O'Rourke K, Tewari M, ea al. FADD, a novel death domain containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 1995,81:505-512
39. Muzio M, Stockwell BR, Stennieke, HR, et al. An induced proximity model for caspase-8 activiation. J Biol Chem,273,2926-2930
40. Krammer PH.CD95's deadly mission in the immune system. Nature,2000,407:789-795
41. Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl2-interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Ce11,1998,94:481-490
42. Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase-8 mediates the mitochondrial damage in the apoptosis, Ceil, 1998,94:491-501
43．黄英，张宗玉，童坦君．人衰老成纤维细胞凋亡的可诱导性．中华老年医学杂志，2000，19：170-173
44. Wang E, Lee MJ, Mountz JD, et al. Control of fibroblasts senescence and activation of programmed cell death. J cell Biochem, 1994,54:432-439
45. Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2 function in an antioxidant pathway to prevent apoptosis. Cell, 1993,75:241
46. Gupta K, Kshirsagar S, Li W, et al. VEGF prevents apoptosis of human microvascular endothelial cells via oppsing effects on MAPK/ERK and SAPK/JNK signaling. Exp Cell Res, 1999,247:495-504
47. Liu Y, Yin G, Surapisitchat J, et al. Laminar flow inhibits TNF-induced ASKI activation by preventing dis-sociation of ASK1 from its inhibitor 14-3-3. J Chin Invest, 2001,107:917-923
48. Yue TL, Ni J, Rousseau S, et al. TL1, a novel tumor necrosis factor-like cytokine,induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases(stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3 like protease. J Biol Chem, 1999,274:1479-1486
49. Awad M, Omori S, Hida M. MAP kinase in renal development. Nephroi Dial Transplant,2002,17:5-7
50. Wang Q, Doerscuk CM. The p38 mitogen-activiated protein kinase mediates cytoskeletai remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation. J Immunol,2001,166:6877-84
51. Brouard S, Otterbein LE, Anrather J, et al. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med, 2000,192:1015-1026
52. Zhen X, Uryu K, Cal G, et al. Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol A Bioi Sci Med Sci, 1999,54:B539-48
53. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000, 78:G855-86554. Liu Y, Guyton KZ, Gorospe M, et al. Age-related decline in mitogen-activated protein activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem, 1996,271:3604-3607
55. Chung JH, Kang S, Varani J, et al. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J Invest Dermatol, 2000,115:177-82
56. Kirk CJ, Freilich AM, Miller RA, Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes. Cell Immunol,1999, 197:75-82
57．吴飞翔，章卫平．JNK信号转导与细胞凋亡．生命的化学，2002，22：346-347
58. Suh Y. Age-specific changes in expression, activity, and activation of the c-jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by mythyl methanesulfonate in rats. Mech Ageing, 2001,122:1797-1811
59. Verbeke P, Fonager J, Clark BF, et al. Heat shock response and ageing: mechanism and applications. Cell Biol.Int, 2001,25:845-857
60. Fargnoli J, Kunisada T, Fornace AJ Jr, Schneider EL, Holbrook NJ. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc Natl Acad Sci USA,1990,87:846-850
61．陈广生，许宋峰．热休克蛋白与凋亡．国外医学肿瘤学分册，1998，25：332-333
62. Chang E, Yang J, Nagavarapu U, et al. Aging and survival of cutaneous microvasculature. J Invest Dermatol, 2002,118:752-758
63. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci, 2001, 14:2903-2910
64. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res,2000, 87:434-439
65. Irani K. Oxidant signaling in vascular cell growth,death, and survival. Circ Res,2000,87:179-183
66．张兵，吴乔，陈睦传．蛋白激酶B的特性及其生物学功能．细胞生物学杂志．2002，24(4)：209—212
67．邹伟，李兆育，李春雷．蛋白激酶B及其在磷脂酰肌醇3—激酶介导的信号转导中的作用．生理科学进展，2000，3 1：120-124
68. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res, 2001,89:709-715
69. Jin HM, Liu QH, Cao X, et al. Dysfunction of microvascular endothelial cells induced by tumor necrosis(TNF-α): celluar, and molecular mechanism. Clincal Hemorheology and Microcirculation, 2000,23:109-112.
70．吴其夏，余应年，卢建．新编病理生理学。北京：中国协和医科大学出版社．1999，242-252．
2. Kim N S, Kim S J. Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum. Yonsei Med, J, 1991, 32(4):303-314
3．杨红，斯琴，孙仁宇．肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000，16(9)：831-834
4. Oite T, Suzuki Y, Morioka T, et al. Efficient isolation of rat aortic endothelial cells by elimination of contaminating cells with a monoclonal antibody. Microvasc Res, 1995, 50(1): 113-118
5. Magee J C, Stone A E, Oldham K, et al. Isolation, culture,and characterization of rat lung microvascular endothelial cells.Am.J.Physiol,1994,267(4 pt 1):L433-441
6. Chen S F, Fei X, Li S H. A new simple method for isolation ofmicrovascular endothelial cells avoiding both chemical and mechanical injuries. Microvasc Res, 1995, 50 (1):119-128
7. Nishida M, Carley W W, Gerritsen M E, et al. Isolation and characterization of human and rat cardiac microvascular endothelial cells. Am J Physiol, 1993,264(2 pt 2): H639-652
8. Ryan U S, Linda A W, Lopez M, et al. Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion. Blood, 1994,84(1):10-19
9．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001，46(19)：1636-1640
10．李姗姗，李澎，黄启福．脂多糖对体外人脐静脉内皮细胞分泌功能及活力的影响．中国病理生理杂志，2001，17(7)：658-661
11．崔旭，李文彬，张炳烈，等．D-gal对神经元和成纤维细胞拟老化作用研究的实验研究．中国应用生理学杂志，1997，13(2)：131-133
12. Shen J, Ham R G, Karmiol S. Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells. Microvasc Res, 1995, 50 (3):360-372
13．肖贞良，孙耕耘，钱桂生．肺循环灌注对大鼠肺微血管内皮细胞分离和培养的影响．中国病理生理杂志，1999，15(11)：1053-1055
14．龚国清，徐黻本．小鼠模型衰老模型的研究．中国药科大学学报，1991，12(2)：101-103
15．马宏，张宗玉，童坦君．衰老的生物学标志．生理科学进展，2002，33(1)：65-681. Xiao Z Q, Yu Y J, Khan A, et al. Induction of G_1 checkpoint in the gastric mucosa of aged rats.Am J Physiol Gastrointest Liver Physiol. 1999,277:G929-G934.
2. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence -associated apoptosis. Exp Gerontol. 2001,36:1327-47.
3. Stein GH, Dulic V, et al. Origins of G(1) arrest in senescent human fibroblasts. Bioessays,1995, 17:537-543.
4. Buchkovich, K, Duffy LA, Harlow E, et al. The retinoblastoma protein is phosphorlated during specific phases of the cell cycle. Cell. 1989,58:1097-1105.
5. Basheer R, Shiromani PJ. Effects of prolonged wakefulness on c-fos and AP-1 activity in young and old rats. Brain Res Mol Brain Res.2001, 89:153-7.
6. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000,278:G855-865
7. Warner HR. Recent progress in understanding the relationships among aging, replicative senescence, cell turnover and cancer. In vivo, 2002,16:393-396.
8. Itahana K, Dimri G, Campisi J. Regulation of cellular senescence by p53. Eur J Biochem, 2001,268:2784-91.
9．刘晓秋，李卫东，唐惠琼等．D-半乳糖衰老大鼠自由基代谢状况分析．中国老年学杂志．2001，21：456-458．
10. Pagano M, Pepperkok R, Verde E Cyclin A is required at two points in the human cell cycle. EMBO J. 1992, 11:961-971.
11. Sherr CJ. G1 phase progression: Cvclin on cue, Ce11.1994.79:551-555.
12．彭黎明，王曾礼主编。细胞凋亡的基础与临床，北京：人民卫生出版社。2000，4-5
13. Sharpless NE, Depinho RA. p53:good cop/bad cop. Ce11,2002,110:9-12
14. Ide T, Tsuji Y, Ishibashi S, et al. Reinitiation of host DNA synthesis in senescent human dipoid cells by infection with simian virus. Exp Cell Res.1983, 143:343-349.
15. Ide T, Tsuji Y, Nakashima T, et al. Progress of aging in human diploid cells transformed with a tsA mutant of simian virus 40. Exp Cell Res.1984, 150:321-328.
16. Gorman SD& Cristofalo VJ. Reinitiation of cellular DNA synthesis in BrdU-selected nondividing senescent WI38 cells by simian virus 40 infection. J Cell Physiol. 1985,125:122-126.
17. Elmore LW, Rehder CW, Di X,Mcchesney PA, Jackson-Cook CK, Gewirtz DA. Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction. J Biol Chem 2002 Sep 20; 277(38):35509-15.
18. Donehower LA. Does p53 affect organismal ageing? J cell physiol.2002,Jul; 192(1):23-33
19. Lodygin D, Menssen A, Hermeking H. Induction of the Cdk inbibitor p21 by LY83853 inhibits tumor cell proliferation in a p53-independent manner. J Clin Invest.2002, 110:1717-27.
20. Chen X, Zhang W, Gao YF, et al. Senescence-like changes induced by expression of p21(waf/Cipl) in NIH3T3 cell line. Cell Res.2002, 12:229-233.
21. Sheerin A, Thompson KS, Goyns MH. Altered composition and DNA binding activity of AP-1 transcription factor during the ageing of human fibroblasts. Mech Ageing Dev.2001,122:1813-1824.
22. Medicherla R, Leers-Sucheta S, Luo Y, et al. Impaired activarion of AP-1 and altered expression of constituent proteins in rat adrenal during ageing. Mech Ageing Dev, 2001, 122: 1169-1186.1．刘汴生．衰老过程中的病理生理．实用老年医学．2002，16：64-65．
2. Zhang Y, Herman B. Ageing and apoptosis. Mech Ageing, 2002,123:245-60.
3. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence-associated apoptosis. Exp Gerontol, 2001, 36:1327-47.
4. Campici, J. The role of cellular senescence in skin aging. J Investig Dermatol. Symp.Proc, 1998,3:1-5.
5. Potestio M, Caruso C, Gervasi F, et al.Apoptosis and ageing.Mech Ageing Dev,102:221-37.
6. Kapasi AA, Singhal PC. Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53,bax,and caspase-3. Moi Cell Biol Res Commun, 1999,1:78-81.
7. Bennett MR. Apoptosis of vascular smooth muscle cells in vascular remodeling and atherosclerotic plaqur rupture. Cardiovasc Res, 1999,41:361-368.
8. Haake AR, Roublevskaia I, Cooklis M. Apoptosis: a role in skin aging? J Investing Dermatol Symp Proc, 1998, 3:28-35.
9．彭黎明，王曾礼，主编．细胞凋亡的基础与临床．北京：人民卫生出版社，2000．
10．黄英，张宗玉，童坦君．人衰老成纤维细胞凋亡的可诱导性．中华老年医学杂志，2000，19：170—173．
11．杨红，斯琴，孙仁宇．肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000,16：831-834．
12．王士雯．老年多器官功能不全肺启动机制若干问题初探．中华老年心脑血管病杂志，2000．2：149-151．
13. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res,2001,89:709-15.
14. Zhang J, Patel J M, Block E R, et al. Enhanced apoptosis in prolongfe cultures of senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev, 2002, 123: 613-25.
15. Muskhelishvili L, Hart RW, Turturro A, et al. Age-related changes in the intrinsic rate of apoptosis in livers of diet-restricted and ad libitum-fed B6C3F1 mice. Am J Pathol, 1995,147:20-24.
16. Higami Y, Shimokawa I, Tomita M, et al. Aging accelerates but life-long dietary restriction suppresses apoptosis-related Fas expression on hepatocytes. Am J Pathol, 1997,151:659-663
17. Taglialatela G, Gegg M, Perez-Polo JR, et al. Evidence for DNA fragments in the CNS of aged??Fischer-344 rats. Neuroreport, 1996,7:977-980.
18．刘小秋，李卫东，唐惠琼．D-半乳糖衰老大鼠自由基代谢状况分析，2001，21：456—458．
19. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing. Nature, 2000, 408: 239-247.
20. Leonard Haytick.The future of ageing. Nature, 2000, 408:267-269.
21. Ronald A.Depinho.The age of cancer. Nature, 2000,408: 248-254.
22. Remmen HV, Richardson A. Oxidative damage to mitochondria and aging. Exp Gerontol, 2001,36:957-968.
23．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001，46：1636-1640．
24. Zou H, Li Y, Liu X, et al. An APAF-lcytochrome c multimeric complex is a functional apoptosis that activates procaspase-9. J Biol Chem, 1999,274:11594-11556.
25. Rodriguez J, Lazebnik Y. Caspase-9 and APAF-1 from an active holoenzyme. Genes Dev, 1999,13:3179-3184.
26. Pollack M, Leeuwenburgh C. Apoptosis and aging: Role of the mitochondria. J Gerontol A Biol Sci, 2001, 56A: B475-B482.
27. Korsmeyer SJ, Wei MC, Sation M, et al. Pro-apoptotic cascade activites BID, which oligomerizes BAK or BAX into pores that result in the release of cytomchrome c. Cell Death Differ, 2000,7:1166-1173.1. Wang Q, Doerscuk CM. The p38 mitogen-activiated protein kinase mediates cytoskeletal remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation.J Immunol, 2001, 66:6877-6884.
2. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing.Nature 2000, 408: 239-247.
3. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res, 2001,89:709-715.
4. Awad M, Omori S, Hida M. MAP kinase in renal development. Nephrol Dial Transplant, 2002,17:5-7.
5. Zhen X, Uryu K, Cal G, et al. Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol A Biol Sci Med Sci, 1999,54:B539-48.
6. Kim HJ, Jung KJ, Yu BP, et al. Influence of ageing and calorie restriction on MAPKs activity in rat kidney. Exp Gerontol, 2000,37:1041-53.
7. Xiao Z Q, Yu Y J, Khan A, et al. Induction of G_1 checkpoint in the gastric mucosa of aged rats .Am J Physiol Gastrointest Liver Physiol.1999,277:G929-G934.
8. Kirk CJ, Freilich AM, Miller RA, Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes. Cell Immunol, 1999,197:75-82.
9. Chung JH, Kang S, Varani J, et al. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J Invest Dermatol, 2000,115:177-82.
10. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000,278:G855-65.
11. Liu Y, Guyton KZ, Gorospe M, et al. Age-related decline in mitogen-activated protein activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem, 1996,271:3604-7.
12. Suh Y. Age-specific changes in expression, activity, and activation of the c-jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by mythyl methanesulfonate in rats.MechAgeing,2001,122:1797-811
13．吴飞翔，章卫平．JNK信号转导与细胞凋亡．生命的化学．2002，22(4)：346-347．
14. Verbeke P, Fonager J, Clark BF, et al. Heat shock response and ageing: mechanism and applications. Cell Biol.Int, 2001,25:845-857.
15. Fargnoli J, Kunisada T, Fornace AJ Jr, Schneider EL, Holbrook NJ. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc Natl Acad Sci USA, 1990,87:846-50.
16. Chang E, Yang J, Nagavarapu U, et al. Aging and survival of cutaneous microvasculature. J Invest Dermatol, 2002,118(5): 752-758.
17. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci, 2001,114(pt16): 2903-2910.
18. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res.2000, 87:434-439.
19. Irani K. Oxidant signaling in vascular cell growth,death, and survival. Circ Res.2000, 87:179-183.
20．张兵，吴乔，陈睦传．蛋白激酶B的特性及其生物学功能．细胞生物学杂志．2002，24(4)：209-212．
21．邹伟，李兆育，李春雷．蛋白激酶B及其在磷脂酰肌醇3—激酶介导的信号转导中的作用．生理科学进展．2000，31(2)：120-124．1．杨红，斯琴，孙仁宇。肺血管内皮细胞在大鼠急性损伤发生中的作用．中国病理生理杂志，2000，16：831-834
2．王士雯。老年多器官功能不全肺启动机制若干问题初探．中华老年心脑血管病杂志，2000，2：149-151
3．周建军，高云飞，王红芳，等．TNF-α诱导血管内皮细胞的衰老．科学通报，2001．46：1636-1640
4. Lou J N, Mili N, Decrinch C, et al. An improved method for isolation of microvascular endothelial cells from normal and inflamed human lung. In Vitro Cell Dev Biol Anim, 1998,4:529-536
5. Magee J C, Stone A E, Oldham K, et al. Isolation, culture, and characterization of rat lung microvascular endothelial cells. Am J Physiol, 1994,267:L433-441
6. Shen J, Ham R G, Karmiol S. Expression of adhesion molecules in cultured human pulmonary microvascular endothelial cells. Microvasc Res, 1995, 50:360-372
7. Kim N S, Kim S J. Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum. Yonsei Med, J, 1991, 32:303-314
8. Zhang Y, Herman B. Ageing and apoptosis. Mech Ageing, 2002,123:245-60.
9. Ostler EL, Wallis CV, Boalchamat B, et al. Telomerase and the cellular lifespan: implication of the aging process. J Pediatr Endocrinol Metab,2000,13:1467-1476
10．黄丹．细胞衰老的研究进展．细胞衰老的进展．暨南大学学报，2001，22：36-39
11. Cherif H, Tarry JL, Ozanne SE, et al. Aging and telomeres: a study into organ- and gender-specific telomere shortening. Nucleic Acids Res, 2003,31:1576-83
12. Kerr JER, Wyllie AH, Currie AR, et al. Apoptosis: a basic biological phenomenon with wide-rangeing implication in tissue kenetics. Br J Cancer,1972,26:239-257
13．彭黎明，王曾礼主编。细胞凋亡的基础与临床，北京：人民卫生出版社。2000，4-5
14. Pollack M, Leeuwenburgh C. Apoptosis and aging: Role of the mitochondria. J Gerontoi A Biol Sci, 2001, 56A: B475-B482
15. Hayflic L. Aging, longevity, and immortality in vitro. Exp.Gerontol. 1992, 27:363-368
16. Muskhelishvili L, Hart RW, Turturro A, et al. Age-related changes in the intrinsic rate of apoptosis in livers of diet-restricted and ad libitum-fed B6C3F1 mice. Am J Pathol, 1995,147:20-24
17. Higami Y, Shimokawa I, Tomita M, et al. Aging accelerates but life-long dietary restriction suppresses apoptosis-related Fas expression on hepatocytes. Am J Pathol, 1997,151:659-663
18. Potestio M, Caruso C, Gervasi F, et al.Apoptosis and ageing.Mech Ageing??Dev, 1998,102:221-237
19. Kapasi AA, Singhal PC. Aging splenocyte and thymocyte apoptosis is associated with enhanced expression of p53, bax, and caspase-3. Mol Cell Biol Res Commun,1999,1:78-81
20. Fujita Y, Ozaki K, Yamamasu S, et al. DNA fragmentation of oocytes in aged mice. Hum Reprod, 1996,11:1480-483
21. Taglialatela G, Gegg M, Perez-Polo JR, et al. Evidence for DNA fragments in the CNS of aged Fischer-344 rats. Neuroreport, 1996,7:977-980
22. Wanger M, Hampel B, Bernhard D, et al. Replicative senescence of human endothelial cells in vitro involves G1 arrest, polyploization and senescence -associated apoptosis. Exp Gerontol,2001,36:1327-1347
23. Zhang J, Patel JM, Block ER. Enhanced apoptosis in prolonged cultures of senescent porcine pulmonary artery endothelial cells. Mech Ageing Dev, 2002, 123:613-25
24. Smale G, Nichols NR, Brady DP, et al. Evidence for apoptotic cell death in Alzheimer's disease. Exp Neurol, 1995,133:225-230
25. Lassman H, Bancher C, Breitschopf H, et al. Cell death in Alzheimer's disease evaluated by DNA fragmentation in situ. Acta Neuropathol(Berl), 1995,89:35-41
26. Toren Finkel &Nikki J.Holbrook.Oxidants,oxidative stress and the biology of ageing. Nature, 2000, 408(9): 239-247
27. Leonard Hayrick.The future of ageing. Nature, 2000, 408,:267-269.
28. Ronald A.Depinho.The age of cancer. Nature 2000,408:248-254.
29. Remmen HV, Richardson A. Oxidative damage to mitochondria and aging. Exp G erontol.,2001,36:957-968
30. Green DR, Reed JC. Mitochondria and apoptosis. Science, 1998,281:1309-1312
31. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev, 2001,15:2922-2933.
32. Halestrap AP. The mitochondrial permeability transition: its molecular mechanism and role in reperfusion injury. Biochem Soc Syrup, 1999,66:181-203.
33. Zou H, Li Y, Liu X, et al. An APAF-lcytochrome c multimeric complex is a functional apoptosis that activates procaspase-9. J Biol Chem, 1999,274:11594-11556
34. Rodriguez J, Lazebnik Y. Caspase-9 and APAF-1 from an active holoenzyme. Genes Dev,1999,13:3179-3184
35. Huang DCS, Strasser A. BH3-only proteins-essential initiators of apoptotic cell death. Cell,2000,103:839-842
36. Datta SR, Katsov A, Hu L, et al.14-3-3 Proteins and survival kinases coopertate to inactivate BAD by BH3 domain phosphorylation. Mol Cell, 1999,6:41-51
37. Korsmeyer S J, Wei MC, Sation M, et al. Pro-apoptotic cascade activites BID, which oligomerizes BAK or BAX into pores that result in the release of cytomchrome c. Cell Death Differ,2000,7:1166-117338. Chinnaiyan AM, O'Rourke K, Tewari M, ea al. FADD, a novel death domain containing protein, interacts with the death domain of Fas and initiates apoptosis. Cell, 1995,81:505-512
39. Muzio M, Stockwell BR, Stennieke, HR, et al. An induced proximity model for caspase-8 activiation. J Biol Chem,273,2926-2930
40. Krammer PH.CD95's deadly mission in the immune system. Nature,2000,407:789-795
41. Luo X, Budihardjo I, Zou H, et al. Bid, a Bcl2-interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Ce11,1998,94:481-490
42. Li H, Zhu H, Xu C J, et al. Cleavage of BID by caspase-8 mediates the mitochondrial damage in the apoptosis, Ceil, 1998,94:491-501
43．黄英，张宗玉，童坦君．人衰老成纤维细胞凋亡的可诱导性．中华老年医学杂志，2000，19：170-173
44. Wang E, Lee MJ, Mountz JD, et al. Control of fibroblasts senescence and activation of programmed cell death. J cell Biochem, 1994,54:432-439
45. Hockenbery DM, Oltvai ZN, Yin XM, et al. Bcl-2 function in an antioxidant pathway to prevent apoptosis. Cell, 1993,75:241
46. Gupta K, Kshirsagar S, Li W, et al. VEGF prevents apoptosis of human microvascular endothelial cells via oppsing effects on MAPK/ERK and SAPK/JNK signaling. Exp Cell Res, 1999,247:495-504
47. Liu Y, Yin G, Surapisitchat J, et al. Laminar flow inhibits TNF-induced ASKI activation by preventing dis-sociation of ASK1 from its inhibitor 14-3-3. J Chin Invest, 2001,107:917-923
48. Yue TL, Ni J, Rousseau S, et al. TL1, a novel tumor necrosis factor-like cytokine,induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases(stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3 like protease. J Biol Chem, 1999,274:1479-1486
49. Awad M, Omori S, Hida M. MAP kinase in renal development. Nephroi Dial Transplant,2002,17:5-7
50. Wang Q, Doerscuk CM. The p38 mitogen-activiated protein kinase mediates cytoskeletai remodeling in pulmonary microvascular endothelial cells upon intracellular adhesion molecule-1 ligation. J Immunol,2001,166:6877-84
51. Brouard S, Otterbein LE, Anrather J, et al. Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med, 2000,192:1015-1026
52. Zhen X, Uryu K, Cal G, et al. Age-associated impairment in brain MAPK signal pathways and the effect of caloric restriction in Fischer 344 rats. J Gerontol A Bioi Sci Med Sci, 1999,54:B539-48
53. Xiao ZQ, Majumdar AP. Induction of transcriptional activity of AP-1 and NF-kappaB in the gastric mucosa during aging. Am J Physiol Gastrointest Liver Physiol, 2000, 78:G855-86554. Liu Y, Guyton KZ, Gorospe M, et al. Age-related decline in mitogen-activated protein activity in epidermal growth factor-stimulated rat hepatocytes. J Biol Chem, 1996,271:3604-3607
55. Chung JH, Kang S, Varani J, et al. Decreased extracellular-signal-regulated kinase and increased stress-activated MAP kinase activities in aged human skin in vivo. J Invest Dermatol, 2000,115:177-82
56. Kirk CJ, Freilich AM, Miller RA, Age-related decline in activation of JNK by TCR- and CD28-mediated signals in murine T-lymphocytes. Cell Immunol,1999, 197:75-82
57．吴飞翔，章卫平．JNK信号转导与细胞凋亡．生命的化学，2002，22：346-347
58. Suh Y. Age-specific changes in expression, activity, and activation of the c-jun NH(2)-terminal kinase and p38 mitogen-activated protein kinases by mythyl methanesulfonate in rats. Mech Ageing, 2001,122:1797-1811
59. Verbeke P, Fonager J, Clark BF, et al. Heat shock response and ageing: mechanism and applications. Cell Biol.Int, 2001,25:845-857
60. Fargnoli J, Kunisada T, Fornace AJ Jr, Schneider EL, Holbrook NJ. Decreased expression of heat shock protein 70 mRNA and protein after heat treatment in cells of aged rats. Proc Natl Acad Sci USA,1990,87:846-850
61．陈广生，许宋峰．热休克蛋白与凋亡．国外医学肿瘤学分册，1998，25：332-333
62. Chang E, Yang J, Nagavarapu U, et al. Aging and survival of cutaneous microvasculature. J Invest Dermatol, 2002,118:752-758
63. Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci, 2001, 14:2903-2910
64. Dimmeler S, Zeiher AM. Endothelial cell apoptosis in angiogenesis and vessel regression. Circ Res,2000, 87:434-439
65. Irani K. Oxidant signaling in vascular cell growth,death, and survival. Circ Res,2000,87:179-183
66．张兵，吴乔，陈睦传．蛋白激酶B的特性及其生物学功能．细胞生物学杂志．2002，24(4)：209—212
67．邹伟，李兆育，李春雷．蛋白激酶B及其在磷脂酰肌醇3—激酶介导的信号转导中的作用．生理科学进展，2000，3 1：120-124
68. Hoffmann J, Haendeler J, Aicher A, et al. Aging enhances the sensitivity of endothelial cells toward apoptotic stimuli: important role of nitric oxide. Circ Res, 2001,89:709-715
69. Jin HM, Liu QH, Cao X, et al. Dysfunction of microvascular endothelial cells induced by tumor necrosis(TNF-α): celluar, and molecular mechanism. Clincal Hemorheology and Microcirculation, 2000,23:109-112.
70．吴其夏，余应年，卢建．新编病理生理学。北京：中国协和医科大学出版社．1999，242-252．