丙二醛与丙酮醛对人骨髓间充质干细胞的生长和凋亡的影响
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摘要
活性羰基类物质(reactive carbonyl species,RCS)是脂质过氧化和非酶糖基化的共同中间产物,即使在健康组织中,它们也普遍存在,特别是不饱和醛酮,如:丙二醛(malondialdehyde,MDA),丙酮醛(methylglyoxal,MGO),4-羟基壬稀醛,3-脱氧葡糖醛酮和乙二醛等,它们在机体内能造成酶功能抑制,免疫功能紊乱,细胞膜损伤等广泛的病理生理变化。正是如此,羰基应激衰老学说认为:羰-氨反应是脂类和糖类两大能源物质新陈代谢过程中不可避免的生化副反应,它在体内能产生广泛而又缓慢的交联,最终造成体内蛋白、脂质、核酸等生物大分子的不可修复性损伤,从而加速衰老过程。
人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hMSCs)是具有高度自我更新能力和多向分化潜能的一类成体干细胞,它参与了组织更新和损伤修复。随着年龄的增加,hMSCs在体内将逐渐出现数量减少和功能缺陷,从而与骨关节炎、骨质疏松症、动脉粥样硬化和糖尿病等多种衰老相关疾病的病理生理密切相关。MDA和MGO作为两种重要的RCS,它们分别是脂质过氧化和非酶糖基化的产物,MDA和MGO等多种RCS在循环中蓄积,与多种衰老相关疾病的形成相关。本文旨在研究MDA与MGO对hMSCs生长和凋亡的影响,揭示羰基应激对体外培养hMSCs的作用机理。
先通过细胞分化、表面抗原分析、形态学观察鉴定细胞为hMSCs,然后用不同浓度的MDA与MGO处理hMSCs,以细胞计数法测定细胞生长曲线,MTT检测细胞活性,流式细胞仪分析细胞的凋亡百分率。结果发现:低浓度的MDA与MGO对hMSCs生长和凋亡没有显著影响,较高浓度MDA与MGO能显著抑制细胞的生长,导致群体倍增时间显著延长,细胞的存活率下降,凋亡细胞的百分率增加,且呈典型的剂量依赖关系。这些结果表明:MDA与MGO都易造成hMSCs的损伤,影响hMSCs的生长、代谢、增殖和诸多其它细胞功能,羰基应激可能是机体内一系列衰老相关的病理生理改变的重要原因。
Reactive carbonyl species(RCS) are common intermediates of lipid peroxidation and non-enzymatic glycosylation(glycation).Even in healthy organism,there are amounts of RCS,particularly carbonyls with di- and multi-functional groups,including malondialdehyde(MDA), methylglyoxal(MGO),4-hydroxylnonenal,3-deoxyglucosone and oxalaldehyde.It has been reported that RCS are correlated with inhibition of enzymes,disturbance of immune functions and destruction of membranes.According to the carbonyl stress theory of aging,the carbonyl-amino reactions are inevitable biological side-reactions in the metabolism of the lipids and carbohydrates,which will cause a series of crosslinkings in vivo,damage biological macromolecules,such as protein, lipid and nucleic acid,and accelerate the aging process.
Human bone marrow mesenchymal stem cells(hMSCs) are adult stem cells that have huge potential capacity of regeneration and differentiation.Numerous evidences have demonstrated that the hMSCs are involved in the process of self-renewal and renovation of damaged tissues,while its amount and function will decrease with the advance of age.As a result,it is associated with many age-related diseases including osteoarthritis,osteoporosis,atherosclerosis and diabetes.Intriguingly, both MDA and MGO,representive RCS and respective products of lipid peroxidation and glycation,have also been reported to accumulate during various age-related diseases and play a critical role in the pathophysiological of the diseases.Aiming to reveal the mechanism of carbonyl stress to hMSCs,the effects of MDA and MGO on proliferation and apoptosis of hMSCs were investigated in this study.
The hMSCs were identified by cell differentiation,surface markers and morphologies.Then the hMSCs were cultured in DMEM treated with different concentrations of MDA and MGO,and the growth curves were estimated by cell counting,the cellular viability were detected by MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide) assay and the apoptosis were analyzed by flow cytometry(FCM).The results showed that there were not significant effects on the growth and the apoptosis of hMSCs under low concentration of MDA and MGO. However,the higher concentrations of MDA and MGO resulted in significant dose-dependent decrease of the cell growth and viability, prolongation of the population doubling time and increase of the apoptosis.These findings indicated that both MDA and MGO could damage hMSCS and result in alternations of the growth,proliferation, metabolism and other functions,and the carbonyl stress might be an important reason for a series of age-related pathophysiological changes.
人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hMSCs)是具有高度自我更新能力和多向分化潜能的一类成体干细胞,它参与了组织更新和损伤修复。随着年龄的增加,hMSCs在体内将逐渐出现数量减少和功能缺陷,从而与骨关节炎、骨质疏松症、动脉粥样硬化和糖尿病等多种衰老相关疾病的病理生理密切相关。MDA和MGO作为两种重要的RCS,它们分别是脂质过氧化和非酶糖基化的产物,MDA和MGO等多种RCS在循环中蓄积,与多种衰老相关疾病的形成相关。本文旨在研究MDA与MGO对hMSCs生长和凋亡的影响,揭示羰基应激对体外培养hMSCs的作用机理。
先通过细胞分化、表面抗原分析、形态学观察鉴定细胞为hMSCs,然后用不同浓度的MDA与MGO处理hMSCs,以细胞计数法测定细胞生长曲线,MTT检测细胞活性,流式细胞仪分析细胞的凋亡百分率。结果发现:低浓度的MDA与MGO对hMSCs生长和凋亡没有显著影响,较高浓度MDA与MGO能显著抑制细胞的生长,导致群体倍增时间显著延长,细胞的存活率下降,凋亡细胞的百分率增加,且呈典型的剂量依赖关系。这些结果表明:MDA与MGO都易造成hMSCs的损伤,影响hMSCs的生长、代谢、增殖和诸多其它细胞功能,羰基应激可能是机体内一系列衰老相关的病理生理改变的重要原因。
Reactive carbonyl species(RCS) are common intermediates of lipid peroxidation and non-enzymatic glycosylation(glycation).Even in healthy organism,there are amounts of RCS,particularly carbonyls with di- and multi-functional groups,including malondialdehyde(MDA), methylglyoxal(MGO),4-hydroxylnonenal,3-deoxyglucosone and oxalaldehyde.It has been reported that RCS are correlated with inhibition of enzymes,disturbance of immune functions and destruction of membranes.According to the carbonyl stress theory of aging,the carbonyl-amino reactions are inevitable biological side-reactions in the metabolism of the lipids and carbohydrates,which will cause a series of crosslinkings in vivo,damage biological macromolecules,such as protein, lipid and nucleic acid,and accelerate the aging process.
Human bone marrow mesenchymal stem cells(hMSCs) are adult stem cells that have huge potential capacity of regeneration and differentiation.Numerous evidences have demonstrated that the hMSCs are involved in the process of self-renewal and renovation of damaged tissues,while its amount and function will decrease with the advance of age.As a result,it is associated with many age-related diseases including osteoarthritis,osteoporosis,atherosclerosis and diabetes.Intriguingly, both MDA and MGO,representive RCS and respective products of lipid peroxidation and glycation,have also been reported to accumulate during various age-related diseases and play a critical role in the pathophysiological of the diseases.Aiming to reveal the mechanism of carbonyl stress to hMSCs,the effects of MDA and MGO on proliferation and apoptosis of hMSCs were investigated in this study.
The hMSCs were identified by cell differentiation,surface markers and morphologies.Then the hMSCs were cultured in DMEM treated with different concentrations of MDA and MGO,and the growth curves were estimated by cell counting,the cellular viability were detected by MTT (3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyl tetrazolium bromide) assay and the apoptosis were analyzed by flow cytometry(FCM).The results showed that there were not significant effects on the growth and the apoptosis of hMSCs under low concentration of MDA and MGO. However,the higher concentrations of MDA and MGO resulted in significant dose-dependent decrease of the cell growth and viability, prolongation of the population doubling time and increase of the apoptosis.These findings indicated that both MDA and MGO could damage hMSCS and result in alternations of the growth,proliferation, metabolism and other functions,and the carbonyl stress might be an important reason for a series of age-related pathophysiological changes.
引文
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[2]Finch CE,Tanzi RE.Genetics of aging[J].Science,1997,278(5337):407-411.
[3]Warner HR.Longevity genes:from primitive organisms to humans[J].Mech Ageing Dev,2005,126(2):235-242.
[4]Harman D.Aging:a theory based on free radical and radiation chemistry[J].J Gerontol,1956,11(3):298-300.
[5]Harman D.The free radical theory of aging[J].Antioxid Redox Signal,2003,5(5):557-561.
[6]印大中.衰老:生命与熵增之战[J].中国老年学杂志,2003,(9):555-559.
[7]Cerami A.Hypothesis:Glucose as a mediator of aging[J].1985,(33):626-634.
[8]Baynes JW.Role of oxidative stress in development of complication in diabetes[J].Diabetes,1991,40(4):405-412.
[9]Kristal BS,Yu BP.An emerging hypothesis:synergistic induction of aging by free radicals and Maillard reaction[J].J Gerontol,1992,47(4):B107-114.
[10]Yin D.Studies of age pigments evolving into a new theory of aging[J].Gerontology,1995,41(suppl 2):159-172.
[11]Glomb MA,Monnier VM.Mechanism of protein modification by glyoxal and glycolaldehyde,reactive intermediates of the Maillard reaction[J].J Biol Chem,1995,267(17):10017-10025.
[12]Thornalley PJ.Advanced glycation and development of diabetic complication:Unifying the involvement of glucose,methylglyoxal and oxidative stress[J].Endocrinol Metab,1996,(3):149-166.
[13]Wells-Knecht KJ,Zyzak DV,Litchfield JE,Thorpe SR,Baynes JW.Mechamism of autoxidative glycosylation:Identification of glyoxal and arabinose as intermediates in the autoxidative modification of protein by glucose[J].Biochemistry.Biochemistry,1995,34(11):3702-3709.
[14]Esterbauer H,Schaur RJ,Zollner H.Chemistry and biochemistry of 4-hydroxynonenal,malondialdehyde and related aldehydes[J].Free Radic Biol Med,1991,11(1):81-128.
[15]Uchida K,Kanematsu M,Sakai K,Matsuda T,Hattori N,Mizuno Y,Suzuki D,Miyata T,Noguchi N,Niki E,Osawa T.Protein-bound acrolein:Potential markers for osidative stress[J].Proc Natl Acad Sci USA,1998,95(9):4882-4887.
[16]Anderson MM,Hazen SL,Hsu FF,Heinecke JW.Human neutrophils employ the myeloperoxidase-hydrogen peroxide-chloride system to convert hydroxy-amino acids into glycolaldehyde,2-hydroxypropanol,and acrolein:A mechanism for the generation of highly reactive α-hydroxy and α,β-unsaturated aldehydes by phagocytes at sites of inflammation[J].J Clin Invest,1997,99(3):424-432.
[17]Stadtman ER.Protein oxidation and aging[J].Science,1992,257(5074):1220-1224.
[18]Stadtman ER,Levine RL.Free radical-mediated osidation of free amino acids and amino acid residues in proteins[J].Amino Acids,2003,25(3-4):207-218.
[19]Sayre LM,Smith MA,Perry G.Chemistry and biochemistry of oxidative stress in neurodegenerative disease[J].Curr Med Chem,2001,8(7):721-738.
[20]Horton ND,Biswal SS,Corrigan LL,Bratta J,Kehrer JP.Acrolein causes inhibitor kB-independent decreases in nuclear factor kB activation in human lung adenocarcinoma (A549) cells[J].J Biol Chem,1999,274(14):9200-9206.
[21]Morimoto RL Regulation of the heat shock transcriptional response:cross talk between a family of heat shock factors,molecular chaperones,and negative regulators[J].Genes Dev,1998,12(24):3788-3796.
[22]Zhang K,Kaufman RJ.Signaling the unfolded protein response from the endoplasmic reticulum[J].J Biol Chem,2004,(279): 25935-25938.
[23]Davis RJ.Signal transduction by the JNK group of MAP kinases[J].Cell,2000,(103):239-252.
[24]Chen C,Edelstein LC,Gelinas C.The Rel/NF-kB family directly activates expression of the apoptosis inhibitor Bclx(L) [J].Mol Cell Biol,2000,(20):2687-2695.
[25]Zong WX,Edelstein LC,Chen C,Bash J,Gelinas C.The prosurvival Bcl-2 homologue Bfl-l/Al is a direct transcriptional target of NF-kB that blocks TNFR-induced apoptosis[J].Genes Dev,1999,(13):382-387.
[26]Page S,Fischer C,Baumgartner B,Haas M,Kreusel U,Loidl G,Hayn M,Ziegler-Heitbrock HW,Neumeier D,Brand K.4-Hydroxynonenal prevents NF-kB activation and tumor necrosis factor expression by inhibiting IkB phosphorylation and subsequent proteolysis[J].J Biol Chem,1999,274(17):11611-11618.
[27]Stewart ZA,Pietenpol JA.p53 Signaling and cell cycle checkpoints[J].Chem Res Toxicol,2001,14(13):243-263.
[28]Martindale JL,Holbrook NJ.Cellular response to oxidative stress:Signaling for suicide and survival[J].J Cell Physiol,2002,192(1):1-15.
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