茶黄素和转化生长因子-β1对兔骨髓基质干细胞向软骨细胞增殖及诱导分化的影响
|
摘要
目的:茶黄素是红茶中的主要成分,具有调节血脂、抗氧化、抗肿瘤、抗心脑血管疾病等多种药理活性。以往研究茶黄素对骨髓基质干细胞转化较少,本实验探讨茶黄素和转化生长因子β1(TGF-β1)对体外培养兔骨髓基质干细胞(BMSCs)增殖及向软骨细胞诱导分化的影响。
方法:实验于2008-05/8在安徽医科大学附属省立医院中心实验室完成。①实验动物:清洁级8周龄家兔12只,由安徽医科大学动物试验中心提供,实验过程中对动物的处置符合动物伦理学标准。②实验方法:兔全身肝素化后麻醉状态下处死,取双侧股骨和肱骨,去除软组织,切除包括骺板在内的两侧骺端,采用全骨髓法分离培养骨髓基质干细胞,按5×10~4/L的细胞密度接种,当细胞生长至80%～90%融合时消化传代。取传至第三代的细胞按1×10~4/ml的密度接种,加入含重组人胰岛素10mg/L、地塞米松10~(-8)mmol/L、TGF-β_15ng/L的DMBM成软骨诱导液培养2周。取第3代BMSC分为三组:A对照组,B组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)。C组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)、茶黄素(30 mg/L)。③实验评估:倒置显微镜下观察细胞生长状况,甲苯胺蓝染色、阿利新蓝比色法测定各板每孔中GAG含量、以及Ⅱ型胶原免疫组化鉴别。
结果:①细胞生长曲线:骨髓基质干细胞具有旺盛的增殖能力,培养1d为细胞的适应期,3d为对数增长期,8d时进入平台期,之后细胞增殖迅速减慢,细胞数下降。②观察细胞生长:骨髓中分离获得的BMSCs在体外增殖旺盛,TGF-β1和茶黄素诱导后细胞生长明显减缓。与对照组相比,经过诱导2周后,诱导组BMSCs均与对照组有显著的差异性(P＜0.05),诱导组之间也存在差异性(P＜0.05)。③甲苯胺蓝染色和免疫组化鉴定结果:经TGF-β1和茶黄素诱导2周后,细胞甲苯胺蓝异染明显,Ⅱ型胶原化学染色阳性,表现为软骨细胞生物学特性。
结论:茶黄素(30 mg/L)在TGF-β1存在的条件下能有效促进BMSCs在体外向软骨细胞分化。
Objective:Theaflavins are the major constituent of black tea,and havea lot of pharmacological activity,for example,regulating blood fat,antioxygen, anti-tumor,resisting cerebrovascular disease,and so on.To go forward,the study of theaflavins' effect on mankind MSCs transforming
Were relatively scarce,but our expriment to abserve the effects of theaflavin and transforming growth factor beta 1 on the proliferation and chondrogenic differentiation of rabbit marrow-derived mesenchymal stem cells in vitro.
Methods:Our expriment completed in center laboratory of Anhui provincial hospitol affiliated Anhui medical university.①Exprimntal animal:12 rabbits of cleaning grade and 8 weeks old are provided by animal expriment center of Anhui medical university. The disposal of animal in process of expriment refer to ethical standard of animals.②exprical method:The rabbits are heparinized and put to death in drugged state,we obtain its sideway femur,tibia and humerus,remove its soft tissue,cut epiphysis of two sides including epiphyseal plate,isolate and culture the MSCs with whole bone marrow culture method technology,inoculate it according to the density of 5×10~4/L,and when the cells grew to the fusion of 80%-90%,digesting and going down to the future generation.We inoculate cells of the third generation according to the density of 1×10~4/ml,and add the DMEM chondrocytes-induced liquid contained rh-insulin 10mg/L,dexamethasone 10~(-8)mmol/L,and TGF-β_15ng/L to be cultured for 2 weeks.MSCs from the third passage were grown in complete medium(Group A),inductive medium with 5mg/ml FGF-β_1(Group B),and inductive medium with 30mg/l theaflavin and with 5mg/ml TGF-β_1(Group C) respectively.③Exprical evaluation:inverted phase contrast microscope,MTT assay toluidine blue staining,alcian blue staining,and cartilage specific collagenⅡwere used to investigate cell biological characteristics.
Resucts:①Cell gowth curve:MSCs have vigorous reproductive activity,1 days to be cultured is in the cell adaptive phase,after 3 days is in the increased logarithmic phase, 8 days in the platform pharse,and 8 days.cell proliferation quickly steps down.②Observing cell growth:MSCs isolated from rabbit bone marrow proliferated actively in vitro.MSCS in monolayer cultures treated with TGF-β_1 and theaflavin showed growth deceleration.In comparing with the control group,MTT assay demonstrated that the cell protiferation of goup A.B and C,showed a significant difference(p＜0.05) after two weeks.③Identification result of toluidine blue staining and collagen type immunohistocyto staining:after 2 weeks,BMS_(cs) treated with 5mg/ml TGF-β_1 and 30mg/l theaflavin were assessed by toluidine blue staining and collagen type immunohistocyto staining,indicating that cartilage spacific matrix was produced in vitro.
Conclusion:On the condition of TGF-β1,theaflavin combination of TGF-β1 and theaflavin in concentration(30mg/L) can not only facilitate the proliferation of BMSCs,but also induce the chondrogenic differentiation actively.
方法:实验于2008-05/8在安徽医科大学附属省立医院中心实验室完成。①实验动物:清洁级8周龄家兔12只,由安徽医科大学动物试验中心提供,实验过程中对动物的处置符合动物伦理学标准。②实验方法:兔全身肝素化后麻醉状态下处死,取双侧股骨和肱骨,去除软组织,切除包括骺板在内的两侧骺端,采用全骨髓法分离培养骨髓基质干细胞,按5×10~4/L的细胞密度接种,当细胞生长至80%～90%融合时消化传代。取传至第三代的细胞按1×10~4/ml的密度接种,加入含重组人胰岛素10mg/L、地塞米松10~(-8)mmol/L、TGF-β_15ng/L的DMBM成软骨诱导液培养2周。取第3代BMSC分为三组:A对照组,B组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)。C组:完全培养基加地塞米松(10~(-8)mmol/L)、TGF-β1(5ng/L)、维生素C(10mmol/L)、茶黄素(30 mg/L)。③实验评估:倒置显微镜下观察细胞生长状况,甲苯胺蓝染色、阿利新蓝比色法测定各板每孔中GAG含量、以及Ⅱ型胶原免疫组化鉴别。
结果:①细胞生长曲线:骨髓基质干细胞具有旺盛的增殖能力,培养1d为细胞的适应期,3d为对数增长期,8d时进入平台期,之后细胞增殖迅速减慢,细胞数下降。②观察细胞生长:骨髓中分离获得的BMSCs在体外增殖旺盛,TGF-β1和茶黄素诱导后细胞生长明显减缓。与对照组相比,经过诱导2周后,诱导组BMSCs均与对照组有显著的差异性(P＜0.05),诱导组之间也存在差异性(P＜0.05)。③甲苯胺蓝染色和免疫组化鉴定结果:经TGF-β1和茶黄素诱导2周后,细胞甲苯胺蓝异染明显,Ⅱ型胶原化学染色阳性,表现为软骨细胞生物学特性。
结论:茶黄素(30 mg/L)在TGF-β1存在的条件下能有效促进BMSCs在体外向软骨细胞分化。
Objective:Theaflavins are the major constituent of black tea,and havea lot of pharmacological activity,for example,regulating blood fat,antioxygen, anti-tumor,resisting cerebrovascular disease,and so on.To go forward,the study of theaflavins' effect on mankind MSCs transforming
Were relatively scarce,but our expriment to abserve the effects of theaflavin and transforming growth factor beta 1 on the proliferation and chondrogenic differentiation of rabbit marrow-derived mesenchymal stem cells in vitro.
Methods:Our expriment completed in center laboratory of Anhui provincial hospitol affiliated Anhui medical university.①Exprimntal animal:12 rabbits of cleaning grade and 8 weeks old are provided by animal expriment center of Anhui medical university. The disposal of animal in process of expriment refer to ethical standard of animals.②exprical method:The rabbits are heparinized and put to death in drugged state,we obtain its sideway femur,tibia and humerus,remove its soft tissue,cut epiphysis of two sides including epiphyseal plate,isolate and culture the MSCs with whole bone marrow culture method technology,inoculate it according to the density of 5×10~4/L,and when the cells grew to the fusion of 80%-90%,digesting and going down to the future generation.We inoculate cells of the third generation according to the density of 1×10~4/ml,and add the DMEM chondrocytes-induced liquid contained rh-insulin 10mg/L,dexamethasone 10~(-8)mmol/L,and TGF-β_15ng/L to be cultured for 2 weeks.MSCs from the third passage were grown in complete medium(Group A),inductive medium with 5mg/ml FGF-β_1(Group B),and inductive medium with 30mg/l theaflavin and with 5mg/ml TGF-β_1(Group C) respectively.③Exprical evaluation:inverted phase contrast microscope,MTT assay toluidine blue staining,alcian blue staining,and cartilage specific collagenⅡwere used to investigate cell biological characteristics.
Resucts:①Cell gowth curve:MSCs have vigorous reproductive activity,1 days to be cultured is in the cell adaptive phase,after 3 days is in the increased logarithmic phase, 8 days in the platform pharse,and 8 days.cell proliferation quickly steps down.②Observing cell growth:MSCs isolated from rabbit bone marrow proliferated actively in vitro.MSCS in monolayer cultures treated with TGF-β_1 and theaflavin showed growth deceleration.In comparing with the control group,MTT assay demonstrated that the cell protiferation of goup A.B and C,showed a significant difference(p＜0.05) after two weeks.③Identification result of toluidine blue staining and collagen type immunohistocyto staining:after 2 weeks,BMS_(cs) treated with 5mg/ml TGF-β_1 and 30mg/l theaflavin were assessed by toluidine blue staining and collagen type immunohistocyto staining,indicating that cartilage spacific matrix was produced in vitro.
Conclusion:On the condition of TGF-β1,theaflavin combination of TGF-β1 and theaflavin in concentration(30mg/L) can not only facilitate the proliferation of BMSCs,but also induce the chondrogenic differentiation actively.
引文
1.江和源,等.红茶中的茶黄素[J].中国茶叶,1998,(3):18-20.
2.安徽农学院.茶叶生物化学(第二版).北京:农业出版社,1984:215-227
3.安徽农学院主编.茶叶生物化学(第二版).P215-227
4.宛晓春,李大祥,夏涛.茶色素及其药理学功能.天然产物研究与开发,2001,13(4):65-69
5.Ying CJ,uu Jt.Ikeda K,et al.Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin Ⅱ induced hyperpermeability in endothelial cells[J].Hypertents Res.2003Oct,26(10):823-828
6.Martin DR,Cox NR,Hathcock TL.et al.Isolation and characterization of maltipotential rnesenchymal stem cells from feline bone marrow[J].Exp Hematol.2002,30(8):879-886
7.Barry.F,Boynton RE,Liu B,et al.Chonduogenic differentiation of bone mesenchymal stem cells from marrow:differentiatiorr-dependent gene expression of matrix components[J].Exp Cell Res,2001,268(2):189-200
8.Martin I.Padera RF,Vunjak-Novakovic G,et al.In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues[J].J Orthop Res.2006.16(2):181-189
9.Wakitani S,Goto T,Pineda S J,et al.Mesenchymal cell-based repair of large ful-thickness defects articular cartilage[J].J Bone Joint Surg Am,2004,76(4):579 -592
10.Johnstone B,Hering MT,Caplan AI,et al.In vitro chondrogenesis of bone marrow -derived mesenchymal progenitor cells[J].Exp Cell Res,2005,238(1):265-272
11.Azizi SA,Stroker D,Augelli B J,et al.Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats-similar to astrocyte grafts[J].Proc Nat Ia cad Sci USA ,2004,95(7): 3908-3913
12. BruderSP,Jaisw al N,Haynesworth SE.Growth kinetics.self-renew al,and the o steogenic potrential of purified human mesenchymal stem cells during extensive subculativation and following cryopreservation [ J] .J Cell Biochem,2004,64:278-294.
13. Colter DC,Class R,Digirolamo CM.el al.Rapid expansion of recycling stem cells in culture of plastic-adherent cells form human bone marrow.Proc Natl Acad Sci USA.2005;97(7):3213-3218.
14. Qiu Q.Ducheyne P.Gao H.et al.Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel.Tissue Eng,2006;4(1):19-34.
15. Glowacki J,Mizuno. S.Greenberger JS.Perfusion enchances functionss of bone marrow stromal cells in three-dimensionalculture.CellTransplant ,2005;7(3):319-326.
16. Phinney DG. J Cell Biochem Suppl,2002;38(5):7-12
17. Maraglia A, Canoodda R. Quario R. J Cell Sci,2000,113(part7): 1161-1166
18. Herberson A. Aubin JE. Bone,1997;21(6):491-500
19. Beresford JN. Bennett JH,Devlin C,,et al. J Cell Sci,1992;102(part2):341-351
20. Rjornson CR. Rictze RL. Beynolds BA, et al .Science, 1999;283(5401): 534-537
21. Lagasse E, Connors H, Al-Dhalimy M, et al. Nat Med, 2000:6(11): 1229-1234
22. WoodburyD,SchwarzEJ,Prokop DJ, et al. J Neurosci Res,2000;61(4):364-370
23. Chopp M,Li Y,Lancet Neurol.2002;1(2);92-100
24. Kadner A,Hoerstrup SRZund Get al. Eur J cardiothorac Surg, 2002; 21 (6): 1055-1060
25. Stanworth ST.Mewland AC.Stem cells:progress in research and edging towards the clinical setting.Clin Med.2007:l:378-382.
26. Caplan AI.Mesenchymal stem cells.J Orthop Res,1999;9:641-650.
27. Surani MA.Reprogramming of genome function through epigenetic inheritance.Nature,2001;414:122-128.
28. Reik W.Dean W.Walter J.Epigenetic reprogramming in mammalian development.Science,2004; 1089-1093.
29. Wakitania S.Satio TXaplan AL.M yogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine [J] .Science,2006,279:1528-1530.
30. Ferrari G,Cusella DE, Angelis G et al.Muscle regeneration by bone marrow -derivedmyogenicprogenitors[J] .Science.2005.279:1528-1530.
31. Wang JS.Shum-Tim,Galipeau J et al.Marrow stromal cell for cellular cardiom yoplasty feasibility and potential clinical advantages[J].Thorac Care iovasc Surg.2004,120(5):999-1005
32. Young RH,Bulter DL,Weber W et.al.Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair [J].J Orthop Res,2005,6:406-413
33. Award HA. Bulter DL, Hariss M T.et al.Autologous mesenchymal stem cell-mediated repair of tendon[J].TissueEng,2004,5(3):267-277
34. Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain:Cells bearing neuronal antigens generated in vivo from bone marrow.Science.2006.290:1779
35. Sanchez Ramos J, Song S, Cardozo Pelaez F,et al.Adult bone marr-Ow stromal cells differentiate into neural cells in vivo.Exp Neurol,2004,164:247
36. Woodlbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. Neurosci Res, 2007;61(4):364-370
37. Kopen GC, Prockop DJ, Phinney DC, et al. Marrow stromal cells imgrate throughout forebrain and cerebellum,and they differentiate into astrocytes after injection into neonatal mouse brains[J]. ProcNatl Acad Sci USA, 2006,96(19):10711-10716
38.Woodlbury D,Schwarz EJ,Prockop DJ,et al.Myogenic cells derived from rat bone marrow messenchymal stem cells exposed to 5-azacytidine[J].Muscle Nervd,2005,18(12):1417-1426
39.Pittenger F,Mackay A,Beck Set al.Multilineage potential of adult human mesenchymal stem cells[J].Science.1999.284143-284147
40.Martin I,Murgraglia A,Campanile G,et al.Endocrinology.2003;138(10):4456
41.Fromigue O,Marie PJ,Lomri A.J Cell Biochem,2002;68(4):411
42.Bruder SP,Kurth AA,Shea M,et al.J Ovthop Res.1998;16(2):155
43.Ribinson D,Bah I,Nevo Z,J Bone Miner Res,2004;10(5):690
44.Gao J,Dennis JE,Solchaga LA,et al.Tissue engineered fabricatiaon an Osteochondral composite waft using rat bone marrow-derived mesencymal stem cell[J].Tissue Eng,2005,7(2):363-371
45.单玉兴,刘一,徐苹香。兔骨髓间质干细胞用于构建软骨组织的初步报告[J].中国修复重建外科杂志,2001,15(1):49-52
46.Capla AI,Elyaderani M,Mbchizuki Y,Wakitani S.and ColdberyVM.Principles of cartilage repair and regeneration.Clinical Ortho-Paedics and Related Research 2005,10(2):248-252
47.Cassiede P,Dennis JE,Ma F,and Caplan AI.Ostcochondrogenic Potential of marrow mesenchymal progenitor cells exposed to TGF-β1 of PDGFBB as Assayed in vivo or in vitro.J Bone Min Res,2004;11(9):1264-1266
48.Mackay AM,Beck SC,Murphy JM,et al.Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[J].Tissue Eng,1998,4(4):415-428
49.Pereira RF,Ohara M,Laptev AV,Halford KW,Pollard MD,Class R,Simon D,Livezey K,and Prockop DJ Proc Natl.Acad Sci.USA,2004;95(3):1142-1146
50.Lemischka IR,Stem Cells,1997;15(suppl)1:63
51.Shah A J,Smogorzewska EM,Hannum C,et al.Blood,1996:87(9):3563-.3566
52.Wanda P,Fiorella S,Lucia G,et al.Blood,1997;89(8):2644-2647
53.Lisovsky M,Braun SE,Ge Y.et al.Leukermia,1996,10(6):1012-1015
54.Lorgeot V,Rougier F,Fixe P.et al.Cytokine,1997;9(10):754-757
55.Guerriero A,Worford L,Holland HK.et al.Blood,1997;90(9):3444-3447
56.Wanda P,Bab I,Nevo Z.J Bone Miner Res,1995;10(5):690-694
57.Horwitz EM,Prockop DJ,Fitzpatrick LA.et al.Transplant ability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.Nat Med,2004.5(3):309 -312
58.Wang JS,Shum Tim D,,Chedrawy E.et al.The coronary delivery Of marrow stromal cells for myocardal regeneration:pathophysiologic and therapeutic implications.J Thorac Cardio- Vasc Surg,2006,122(4):699-673
59.Toma C,Pittenger MF,Cahill ks,et al..Human mesenchymal stromal cell differentiate to a cardiomyocyte phentype in the adult Murin heart.Cituculation,2002,105(1):93-98
60.Chen J,,Li Y,,Wang L,et al.Therapectic benefit of intravenous Administration of bone marrow stromal cells after cerebral ischemia in rats.Stroke.2004,32(6):1005-1009
61.萧伟祥,钟瑾,肃慧等.茶红色素形成机理和制取.茶叶科学,1997,17(1):1-8
62.李大祥.茶儿茶素化学氧化的研究.安徽农业大学2000届硕士论文,P2
63.Jain AK,Shimaoi K,,Nakamura Y.et al.Crude tea extracts decrease the mutagenic activity of intragastric tract of rats.Murat Res.2002,210(1):1-6.
64.Wang ZY,Hong J,Huang MT.Inhibition of N-nitrosodiethy Lamine and 4-(methylnitrosamine)-1-(3-pyridyI)-1-butanone- Induced tumorigenesis in A/J mice by green tea and black tea.Cancer Res,2004,52(7):1943-1947.
65.Steele V E,Kelloff GJ.Balentine D et al.Comparative chemopreventive mechanism of green tea,black tea and selected polyphenol.Carinogenesis,2000.Japan:21(1):63-67
66.Matsumoto N,Kchri T.Okushiok et al.Inhibitory effects of tea catechins,black tea extract and oolong tea extract on hepato-car- cinogenesis in rat.Japan,J.Cancer Res.,2005,87(10):1034-1038
67.Wang Z Y.Huang M T,Wu Y R et al.Inhibitory effects of hack tea,green tea,decaffeinated black tea.decaffeinated green tea on ultraviolet B light induced skin carcinogenesis in 7.12-dimet-Hlberiz(a) anthracene2initiated SKH21 mice.Carc.Res,2006,54(13):3428-3435
68.杜琪珍,江和源,茶色素的药理及应用,中国茶叶,1997.19(5):36-37
69.曹进,赵燕.红茶及茶色素药理作用研究的动态.中国药学杂志,1999,34(6):361-363
70.李春美,谢笔均.儿茶素氧化聚合物体内外抗肿瘤作用及对正常小鼠免疫功能的影响.食品科学,2001,22(11):23-27
71.谢笔均,石煜,胡慰望.绿茶、乌龙茶、红茶中主要成分和酚类化合物抑制脂肪氧合酶活性和抗油脂自动氧化特性的研究.天然产物研究与开发,1999.6(4):20-23
72.韩驰.茶叶与防癌.中华茶人,1999,1(13):29-32
73.李春美,谢笔均.儿茶素氧化聚合产物药理作用研究概况,茶叶,2001.27(1):28-34
74.Xie Z,Kometiani P.Liu JI.Askari A,Intracellular reactive oxygen species mediate the linkage of Na/k-ATPase to bypertrophy and its marker genes in cardac myocytes.Bilo.Chem.2006.274:19323-19328
75.曹进.茶色素抗菌抗病毒作用的研究进展.中草药,1998,29(9):636-639
76.曹进,赵燕,刘箭卫.茶色素药理作用的国际动态农业考古·国茶文化专号,1997.14(4):299-311
77.#12
78.Toda M.et al.Antibacterial and Bactericidal Activities of Tea E- Xtracts and Catechins against Methicillin-Resistant Staphylcoc Cusaureus[J].Nippon Saikingaku Zasshi-Japanese Journal of Bacteriology,September 2006;46:839-845
79.KITANOY.RADUA,SHABBANA,etal.Selection enrich-ment,And Culture expansion of murine mesenchymal progenitorcells By retroviral transduction of cycling adherent bone marrow cell[J].Exp Hematol,2005,28(12):1460-1465
80.OREFFOROC,VIRDIAS,TRIFFITTJT.Retroviral marking of human bone marrow fibroblasts;in vitro expansion and localization in calvarial sites after subcutaneous transplantation in Vivo[J].Cell Physiol,2004,186(2):201-206
81.BalsamLB,WagersAJChristensenJL,et al.Haematopoietic stemcells adopt mature haematopoietic fates inischamic myocardium[J].Nature,2004,428(10):668-673.
82.Toda M,Okubo S.Ikigai H.et al.The protective activitity of tea against infection by vibrio cholerac[J].J Appl.Batter.1990,69(2):109-114
83.Massko S.O.Ikigai H,Suzuki T.et al.[J].JAppl.Bacter,2002,70(2):109-114
84.XiYZ,Tang PH.Major call in question sonplasticity or trans-differention of adult stem cells[J].Chin J Immunol.2003,19(5):457 -460
85.Nakayama M.et al.[J].Letters in AppI Microbio 2000(11):38-44
86.江和源等.红茶中的茶黄素[J].中国茶叶1998,6(3):18-20
87.Chark K.J.et al.An in vitro study of theaflavins extracted from black tea to neutralize bovvimene rotavirus and bovine coronavirus infectious[J].Veterinary-Microbiology 2004,63(2-4):147-157
88.张国营,何丽娜.红茶、青茶、黑茶抗人轮状病毒的实验研究[J].中国病毒学,1993.8(2):151-153
89.Tang PH.Controversy on adult stem cell plasticity and rules govering fetal tissue use in China.J Lab Clin Med,2004,(April).
90.Jamanish H.Sasaki YF,Ohta T et al.Tea tannin components modify the induction of sister chromosome exchange and chromosome aberration in mutagen treated cultured mammatian cells and Mice Mutation Res.,2002,25(9):79-87
91.饶光宇,陈秀芬,于燕妮等.茶色素对小鼠实验性肝损伤的保护作用,中国药学杂志,2000,35(12):813-815
92.谢笔钧,石煌,胡慰望等 绿茶、乌龙茶、红茶中的主要组分和酚类化合物抑制脂肪氧化合酶活性和抗油脂自动氧化特性的研究.天然产物研究与开发.1994.6(4):19-26
93.Honda M et al.Proceedings of international symposium on tea science[J].2003,5(3):26-31
94.阮宇成,茶叶多种利用的状况与发展.中国茶叶加工,2002,1(3):36-37
95.Huang W,Chung U I,Kronenberg HM,et al.The chondrogenic transcription factor Sox9 is a target of sigonaling by the parathyroid hormone-related peptide in the gowth plate of endochondral bones Proc N atl A cad sciSA,2004;98(1):160-165
1.Stokes D J,Liu GD,harmavarant R.et al,Regulation of type-Collagen gene expression during human chondrocyte differebtion and recovery of chondro-specific phenotype inculture involves sry-Type high-mohility-group box(SOX) transcription factors,Biochem[J].2001,3(6):461-470
2.Dragoo JL,Samimi B.Zhu M,et al.Tissue engineered caetilage and bone using cells from human infrapatellar fat pats[J].J Bone Joint Surg(Br),2003,85-B:740
3.Wakitan S,Goto T,Young RG,et al.Repair of large full-thickness articular Cartilage defects with albgraft articular chondrocytes em-bedded in a collagen gel[J].Tissue Eng,1998,4(4):429-444
4.Wong MW,Qin L,Tai JK,et al.Engineered albgenetic chondrocyte pellet for construction of fibrocartilage zone at bone-tendon Juntion a preliminary.histological observation[J].Biomed Mater Res,2004,15:70(2):362-367
5.杨志明.干细胞组织工程与再生医学[J].中国修复重建外科志,2006,,20(2):95-97
6.Gao J,Dennis JE,Solchaga LA.et al.Tissue engineered fabricatiaon an osteochondral composite graft using rat bone marrow-derived mesencymal stem cell J].Tissue Eng,2005,7(2):363-371
7.单玉兴,刘一,徐苹香.兔骨髓间质干细胞用于构建软骨组织的初步报告[J].中国修复重建外科杂志,2001.15(1):49-52
8.周广东,王晓云,刘天一,等.软骨细胞诱导骨髓基质细胞体内骨形成[J].中华实验外科杂志.2005,22(3):272-274
9.Pate DW,Southerland SS,Grande DA,et al.Isolation and differenatiation of mesenchymal stem cells from rabbit muscle[J].Surg Forum,2004,44(3):587-589
10.Erickson GR,Gimble M,Franklin DM.et al.Chondrogenic potenial of aspose tissue derived stromal cells in vitro and in vivo[J].B Iochom B Iophys Rescommun,2002,290(2):263-269
11.NAWATA M,WAKITANI S,NAKAYA H,et al.Use of bone morphogenetic protein 2 and diffusion chambers to engineer car- Tilage tissue for the repair of defects in articular cartilage[J].Arthritis Rheum,2005.52(1):155-163
12.FUCHS J R,HANNOUCHE D,TERADA S,et al.Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells[J].Stem Cells,2005,23(7):958-964
13.KRAMER J,HEGER C,GUAN K,et al.Embryonic stem cell- Derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4[J].Mech Dev,2000,9(2):193-205
14.KRAMER J,KLINGER M,KRUSE C.et al.Ultrastrunctural analysis of mouse embryonic stem cell-derived chondrocytes[J].Anat Embryoal (Berl),2005,21(3):175-185
15.HWANGN S,KMM S,SAMPA TTAVANICH S,et al.Effects of three-dimers-Ional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells[J].Stem Cells,2006,24(2):284-291
16.VATS A,BIELBY RC,TOLLEY N,et al.Chondrogenic differentia tion of human embryonic stem cells:the effect of the mi- Cro-environment[J].Tissue Eng,2006,12(6):1687-1697
2.安徽农学院.茶叶生物化学(第二版).北京:农业出版社,1984:215-227
3.安徽农学院主编.茶叶生物化学(第二版).P215-227
4.宛晓春,李大祥,夏涛.茶色素及其药理学功能.天然产物研究与开发,2001,13(4):65-69
5.Ying CJ,uu Jt.Ikeda K,et al.Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin Ⅱ induced hyperpermeability in endothelial cells[J].Hypertents Res.2003Oct,26(10):823-828
6.Martin DR,Cox NR,Hathcock TL.et al.Isolation and characterization of maltipotential rnesenchymal stem cells from feline bone marrow[J].Exp Hematol.2002,30(8):879-886
7.Barry.F,Boynton RE,Liu B,et al.Chonduogenic differentiation of bone mesenchymal stem cells from marrow:differentiatiorr-dependent gene expression of matrix components[J].Exp Cell Res,2001,268(2):189-200
8.Martin I.Padera RF,Vunjak-Novakovic G,et al.In vitro differentiation of chick embryo bone marrow stromal cells into cartilaginous and bone-like tissues[J].J Orthop Res.2006.16(2):181-189
9.Wakitani S,Goto T,Pineda S J,et al.Mesenchymal cell-based repair of large ful-thickness defects articular cartilage[J].J Bone Joint Surg Am,2004,76(4):579 -592
10.Johnstone B,Hering MT,Caplan AI,et al.In vitro chondrogenesis of bone marrow -derived mesenchymal progenitor cells[J].Exp Cell Res,2005,238(1):265-272
11.Azizi SA,Stroker D,Augelli B J,et al.Engraftment and migration of human bone marrow stromal cells implanted in the brains of albino rats-similar to astrocyte grafts[J].Proc Nat Ia cad Sci USA ,2004,95(7): 3908-3913
12. BruderSP,Jaisw al N,Haynesworth SE.Growth kinetics.self-renew al,and the o steogenic potrential of purified human mesenchymal stem cells during extensive subculativation and following cryopreservation [ J] .J Cell Biochem,2004,64:278-294.
13. Colter DC,Class R,Digirolamo CM.el al.Rapid expansion of recycling stem cells in culture of plastic-adherent cells form human bone marrow.Proc Natl Acad Sci USA.2005;97(7):3213-3218.
14. Qiu Q.Ducheyne P.Gao H.et al.Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel.Tissue Eng,2006;4(1):19-34.
15. Glowacki J,Mizuno. S.Greenberger JS.Perfusion enchances functionss of bone marrow stromal cells in three-dimensionalculture.CellTransplant ,2005;7(3):319-326.
16. Phinney DG. J Cell Biochem Suppl,2002;38(5):7-12
17. Maraglia A, Canoodda R. Quario R. J Cell Sci,2000,113(part7): 1161-1166
18. Herberson A. Aubin JE. Bone,1997;21(6):491-500
19. Beresford JN. Bennett JH,Devlin C,,et al. J Cell Sci,1992;102(part2):341-351
20. Rjornson CR. Rictze RL. Beynolds BA, et al .Science, 1999;283(5401): 534-537
21. Lagasse E, Connors H, Al-Dhalimy M, et al. Nat Med, 2000:6(11): 1229-1234
22. WoodburyD,SchwarzEJ,Prokop DJ, et al. J Neurosci Res,2000;61(4):364-370
23. Chopp M,Li Y,Lancet Neurol.2002;1(2);92-100
24. Kadner A,Hoerstrup SRZund Get al. Eur J cardiothorac Surg, 2002; 21 (6): 1055-1060
25. Stanworth ST.Mewland AC.Stem cells:progress in research and edging towards the clinical setting.Clin Med.2007:l:378-382.
26. Caplan AI.Mesenchymal stem cells.J Orthop Res,1999;9:641-650.
27. Surani MA.Reprogramming of genome function through epigenetic inheritance.Nature,2001;414:122-128.
28. Reik W.Dean W.Walter J.Epigenetic reprogramming in mammalian development.Science,2004; 1089-1093.
29. Wakitania S.Satio TXaplan AL.M yogenic cells derived from rat bone marrow mesenchymal stem cells exposed to 5-azacytidine [J] .Science,2006,279:1528-1530.
30. Ferrari G,Cusella DE, Angelis G et al.Muscle regeneration by bone marrow -derivedmyogenicprogenitors[J] .Science.2005.279:1528-1530.
31. Wang JS.Shum-Tim,Galipeau J et al.Marrow stromal cell for cellular cardiom yoplasty feasibility and potential clinical advantages[J].Thorac Care iovasc Surg.2004,120(5):999-1005
32. Young RH,Bulter DL,Weber W et.al.Use of mesenchymal stem cells in a collagen matrix for achilles tendon repair [J].J Orthop Res,2005,6:406-413
33. Award HA. Bulter DL, Hariss M T.et al.Autologous mesenchymal stem cell-mediated repair of tendon[J].TissueEng,2004,5(3):267-277
34. Mezey E, Chandross KJ, Harta G, et al. Turning blood into brain:Cells bearing neuronal antigens generated in vivo from bone marrow.Science.2006.290:1779
35. Sanchez Ramos J, Song S, Cardozo Pelaez F,et al.Adult bone marr-Ow stromal cells differentiate into neural cells in vivo.Exp Neurol,2004,164:247
36. Woodlbury D, Schwarz EJ, Prockop DJ, Black IB. Adult rat and human bone marrow stromal cells differentiate into neurons[J]. Neurosci Res, 2007;61(4):364-370
37. Kopen GC, Prockop DJ, Phinney DC, et al. Marrow stromal cells imgrate throughout forebrain and cerebellum,and they differentiate into astrocytes after injection into neonatal mouse brains[J]. ProcNatl Acad Sci USA, 2006,96(19):10711-10716
38.Woodlbury D,Schwarz EJ,Prockop DJ,et al.Myogenic cells derived from rat bone marrow messenchymal stem cells exposed to 5-azacytidine[J].Muscle Nervd,2005,18(12):1417-1426
39.Pittenger F,Mackay A,Beck Set al.Multilineage potential of adult human mesenchymal stem cells[J].Science.1999.284143-284147
40.Martin I,Murgraglia A,Campanile G,et al.Endocrinology.2003;138(10):4456
41.Fromigue O,Marie PJ,Lomri A.J Cell Biochem,2002;68(4):411
42.Bruder SP,Kurth AA,Shea M,et al.J Ovthop Res.1998;16(2):155
43.Ribinson D,Bah I,Nevo Z,J Bone Miner Res,2004;10(5):690
44.Gao J,Dennis JE,Solchaga LA,et al.Tissue engineered fabricatiaon an Osteochondral composite waft using rat bone marrow-derived mesencymal stem cell[J].Tissue Eng,2005,7(2):363-371
45.单玉兴,刘一,徐苹香。兔骨髓间质干细胞用于构建软骨组织的初步报告[J].中国修复重建外科杂志,2001,15(1):49-52
46.Capla AI,Elyaderani M,Mbchizuki Y,Wakitani S.and ColdberyVM.Principles of cartilage repair and regeneration.Clinical Ortho-Paedics and Related Research 2005,10(2):248-252
47.Cassiede P,Dennis JE,Ma F,and Caplan AI.Ostcochondrogenic Potential of marrow mesenchymal progenitor cells exposed to TGF-β1 of PDGFBB as Assayed in vivo or in vitro.J Bone Min Res,2004;11(9):1264-1266
48.Mackay AM,Beck SC,Murphy JM,et al.Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow[J].Tissue Eng,1998,4(4):415-428
49.Pereira RF,Ohara M,Laptev AV,Halford KW,Pollard MD,Class R,Simon D,Livezey K,and Prockop DJ Proc Natl.Acad Sci.USA,2004;95(3):1142-1146
50.Lemischka IR,Stem Cells,1997;15(suppl)1:63
51.Shah A J,Smogorzewska EM,Hannum C,et al.Blood,1996:87(9):3563-.3566
52.Wanda P,Fiorella S,Lucia G,et al.Blood,1997;89(8):2644-2647
53.Lisovsky M,Braun SE,Ge Y.et al.Leukermia,1996,10(6):1012-1015
54.Lorgeot V,Rougier F,Fixe P.et al.Cytokine,1997;9(10):754-757
55.Guerriero A,Worford L,Holland HK.et al.Blood,1997;90(9):3444-3447
56.Wanda P,Bab I,Nevo Z.J Bone Miner Res,1995;10(5):690-694
57.Horwitz EM,Prockop DJ,Fitzpatrick LA.et al.Transplant ability and therapeutic effects of bone marrow-derived mesenchymal cells in children with osteogenesis imperfecta.Nat Med,2004.5(3):309 -312
58.Wang JS,Shum Tim D,,Chedrawy E.et al.The coronary delivery Of marrow stromal cells for myocardal regeneration:pathophysiologic and therapeutic implications.J Thorac Cardio- Vasc Surg,2006,122(4):699-673
59.Toma C,Pittenger MF,Cahill ks,et al..Human mesenchymal stromal cell differentiate to a cardiomyocyte phentype in the adult Murin heart.Cituculation,2002,105(1):93-98
60.Chen J,,Li Y,,Wang L,et al.Therapectic benefit of intravenous Administration of bone marrow stromal cells after cerebral ischemia in rats.Stroke.2004,32(6):1005-1009
61.萧伟祥,钟瑾,肃慧等.茶红色素形成机理和制取.茶叶科学,1997,17(1):1-8
62.李大祥.茶儿茶素化学氧化的研究.安徽农业大学2000届硕士论文,P2
63.Jain AK,Shimaoi K,,Nakamura Y.et al.Crude tea extracts decrease the mutagenic activity of intragastric tract of rats.Murat Res.2002,210(1):1-6.
64.Wang ZY,Hong J,Huang MT.Inhibition of N-nitrosodiethy Lamine and 4-(methylnitrosamine)-1-(3-pyridyI)-1-butanone- Induced tumorigenesis in A/J mice by green tea and black tea.Cancer Res,2004,52(7):1943-1947.
65.Steele V E,Kelloff GJ.Balentine D et al.Comparative chemopreventive mechanism of green tea,black tea and selected polyphenol.Carinogenesis,2000.Japan:21(1):63-67
66.Matsumoto N,Kchri T.Okushiok et al.Inhibitory effects of tea catechins,black tea extract and oolong tea extract on hepato-car- cinogenesis in rat.Japan,J.Cancer Res.,2005,87(10):1034-1038
67.Wang Z Y.Huang M T,Wu Y R et al.Inhibitory effects of hack tea,green tea,decaffeinated black tea.decaffeinated green tea on ultraviolet B light induced skin carcinogenesis in 7.12-dimet-Hlberiz(a) anthracene2initiated SKH21 mice.Carc.Res,2006,54(13):3428-3435
68.杜琪珍,江和源,茶色素的药理及应用,中国茶叶,1997.19(5):36-37
69.曹进,赵燕.红茶及茶色素药理作用研究的动态.中国药学杂志,1999,34(6):361-363
70.李春美,谢笔均.儿茶素氧化聚合物体内外抗肿瘤作用及对正常小鼠免疫功能的影响.食品科学,2001,22(11):23-27
71.谢笔均,石煜,胡慰望.绿茶、乌龙茶、红茶中主要成分和酚类化合物抑制脂肪氧合酶活性和抗油脂自动氧化特性的研究.天然产物研究与开发,1999.6(4):20-23
72.韩驰.茶叶与防癌.中华茶人,1999,1(13):29-32
73.李春美,谢笔均.儿茶素氧化聚合产物药理作用研究概况,茶叶,2001.27(1):28-34
74.Xie Z,Kometiani P.Liu JI.Askari A,Intracellular reactive oxygen species mediate the linkage of Na/k-ATPase to bypertrophy and its marker genes in cardac myocytes.Bilo.Chem.2006.274:19323-19328
75.曹进.茶色素抗菌抗病毒作用的研究进展.中草药,1998,29(9):636-639
76.曹进,赵燕,刘箭卫.茶色素药理作用的国际动态农业考古·国茶文化专号,1997.14(4):299-311
77.#12
78.Toda M.et al.Antibacterial and Bactericidal Activities of Tea E- Xtracts and Catechins against Methicillin-Resistant Staphylcoc Cusaureus[J].Nippon Saikingaku Zasshi-Japanese Journal of Bacteriology,September 2006;46:839-845
79.KITANOY.RADUA,SHABBANA,etal.Selection enrich-ment,And Culture expansion of murine mesenchymal progenitorcells By retroviral transduction of cycling adherent bone marrow cell[J].Exp Hematol,2005,28(12):1460-1465
80.OREFFOROC,VIRDIAS,TRIFFITTJT.Retroviral marking of human bone marrow fibroblasts;in vitro expansion and localization in calvarial sites after subcutaneous transplantation in Vivo[J].Cell Physiol,2004,186(2):201-206
81.BalsamLB,WagersAJChristensenJL,et al.Haematopoietic stemcells adopt mature haematopoietic fates inischamic myocardium[J].Nature,2004,428(10):668-673.
82.Toda M,Okubo S.Ikigai H.et al.The protective activitity of tea against infection by vibrio cholerac[J].J Appl.Batter.1990,69(2):109-114
83.Massko S.O.Ikigai H,Suzuki T.et al.[J].JAppl.Bacter,2002,70(2):109-114
84.XiYZ,Tang PH.Major call in question sonplasticity or trans-differention of adult stem cells[J].Chin J Immunol.2003,19(5):457 -460
85.Nakayama M.et al.[J].Letters in AppI Microbio 2000(11):38-44
86.江和源等.红茶中的茶黄素[J].中国茶叶1998,6(3):18-20
87.Chark K.J.et al.An in vitro study of theaflavins extracted from black tea to neutralize bovvimene rotavirus and bovine coronavirus infectious[J].Veterinary-Microbiology 2004,63(2-4):147-157
88.张国营,何丽娜.红茶、青茶、黑茶抗人轮状病毒的实验研究[J].中国病毒学,1993.8(2):151-153
89.Tang PH.Controversy on adult stem cell plasticity and rules govering fetal tissue use in China.J Lab Clin Med,2004,(April).
90.Jamanish H.Sasaki YF,Ohta T et al.Tea tannin components modify the induction of sister chromosome exchange and chromosome aberration in mutagen treated cultured mammatian cells and Mice Mutation Res.,2002,25(9):79-87
91.饶光宇,陈秀芬,于燕妮等.茶色素对小鼠实验性肝损伤的保护作用,中国药学杂志,2000,35(12):813-815
92.谢笔钧,石煌,胡慰望等 绿茶、乌龙茶、红茶中的主要组分和酚类化合物抑制脂肪氧化合酶活性和抗油脂自动氧化特性的研究.天然产物研究与开发.1994.6(4):19-26
93.Honda M et al.Proceedings of international symposium on tea science[J].2003,5(3):26-31
94.阮宇成,茶叶多种利用的状况与发展.中国茶叶加工,2002,1(3):36-37
95.Huang W,Chung U I,Kronenberg HM,et al.The chondrogenic transcription factor Sox9 is a target of sigonaling by the parathyroid hormone-related peptide in the gowth plate of endochondral bones Proc N atl A cad sciSA,2004;98(1):160-165
1.Stokes D J,Liu GD,harmavarant R.et al,Regulation of type-Collagen gene expression during human chondrocyte differebtion and recovery of chondro-specific phenotype inculture involves sry-Type high-mohility-group box(SOX) transcription factors,Biochem[J].2001,3(6):461-470
2.Dragoo JL,Samimi B.Zhu M,et al.Tissue engineered caetilage and bone using cells from human infrapatellar fat pats[J].J Bone Joint Surg(Br),2003,85-B:740
3.Wakitan S,Goto T,Young RG,et al.Repair of large full-thickness articular Cartilage defects with albgraft articular chondrocytes em-bedded in a collagen gel[J].Tissue Eng,1998,4(4):429-444
4.Wong MW,Qin L,Tai JK,et al.Engineered albgenetic chondrocyte pellet for construction of fibrocartilage zone at bone-tendon Juntion a preliminary.histological observation[J].Biomed Mater Res,2004,15:70(2):362-367
5.杨志明.干细胞组织工程与再生医学[J].中国修复重建外科志,2006,,20(2):95-97
6.Gao J,Dennis JE,Solchaga LA.et al.Tissue engineered fabricatiaon an osteochondral composite graft using rat bone marrow-derived mesencymal stem cell J].Tissue Eng,2005,7(2):363-371
7.单玉兴,刘一,徐苹香.兔骨髓间质干细胞用于构建软骨组织的初步报告[J].中国修复重建外科杂志,2001.15(1):49-52
8.周广东,王晓云,刘天一,等.软骨细胞诱导骨髓基质细胞体内骨形成[J].中华实验外科杂志.2005,22(3):272-274
9.Pate DW,Southerland SS,Grande DA,et al.Isolation and differenatiation of mesenchymal stem cells from rabbit muscle[J].Surg Forum,2004,44(3):587-589
10.Erickson GR,Gimble M,Franklin DM.et al.Chondrogenic potenial of aspose tissue derived stromal cells in vitro and in vivo[J].B Iochom B Iophys Rescommun,2002,290(2):263-269
11.NAWATA M,WAKITANI S,NAKAYA H,et al.Use of bone morphogenetic protein 2 and diffusion chambers to engineer car- Tilage tissue for the repair of defects in articular cartilage[J].Arthritis Rheum,2005.52(1):155-163
12.FUCHS J R,HANNOUCHE D,TERADA S,et al.Cartilage engineering from ovine umbilical cord blood mesenchymal progenitor cells[J].Stem Cells,2005,23(7):958-964
13.KRAMER J,HEGER C,GUAN K,et al.Embryonic stem cell- Derived chondrogenic differentiation in vitro:activation by BMP-2 and BMP-4[J].Mech Dev,2000,9(2):193-205
14.KRAMER J,KLINGER M,KRUSE C.et al.Ultrastrunctural analysis of mouse embryonic stem cell-derived chondrocytes[J].Anat Embryoal (Berl),2005,21(3):175-185
15.HWANGN S,KMM S,SAMPA TTAVANICH S,et al.Effects of three-dimers-Ional culture and growth factors on the chondrogenic differentiation of murine embryonic stem cells[J].Stem Cells,2006,24(2):284-291
16.VATS A,BIELBY RC,TOLLEY N,et al.Chondrogenic differentia tion of human embryonic stem cells:the effect of the mi- Cro-environment[J].Tissue Eng,2006,12(6):1687-1697