摘要
【目的】1.研究云南白药对富血小板血浆促兔骨髓基质干细胞增殖和分化的影响;2.研究经口服云南白药干预后的富血小板血浆对兔骨髓基质干细胞增殖和分化的影响。
【方法】取4月龄新西兰大耳白兔骨髓,采用贴壁筛选法并结合传代对其进行纯化,并采用流式细胞术对其进行鉴定;观察第1代、第2代、第3代、第4代、第5代、第6代、第7代、第8代细胞的增殖情况,绘制生长曲线;以100mg/kg,1次/d,给兔灌药,连续7d,分别于给药第0、3、5、7d采静脉血;采用Landesberg法分别制备未经云南白药干预的PRP和经全身用药后的PRP;分别用不同浓度懂得云南白药配合未干预的PRP及用口服云南白药干预后的PRP培养rBMSCs;用四唑盐比色法(MTT)和酶动力学方法检测云南白药对PRP促rBMSCs增殖和分化的影响。
【结果】云南白药体外直接干预组与对照组相比,60μg/ml(0.53±0.03)、65μg/ml(0.56±0.05)组对rBMSCs的增殖作用有显著性差异(P<0.05);65μg/ml(1.21±0.08)、70μg/ml(1.28±0.05)、75μg/ml(1.24±0.06)组云南白药可显著增强rBMSCs的ALP活性(P<0.05)。口服云南白药干预组与对照组相比,服药5d(0.30±0.02)和7d(0.34±0.08)组对rBMSCs增殖作用有显著性差异(P<0.05);服药7d(0.88±0.09)组可显著增强rBMSCs的ALP活性(P<0.05)。
【结论】云南白药能够促进PRP对rBMSCs的增殖和分化作用,其中,60μg/m、65μg/m的云南白药能够促进PRP对rBMSCs的增殖作用;65μg/ml~75μg/m的云南白药能够促进PRP对rBMSCs的分化:服用云南白药5d和服用云南白药7d能够促进PRP对rBMSCs的增殖作用;服用云南白药7d能够促进PRP对rBMSCs的分化。
[Objective]To study the direct impact of YunnanBaiyao on platelet-rich plasma (PRP) for proliferation and differentiation of the bone marrow stromal cells of the rabbits;to study the impact of YunnanBaiyao which were taken though oral on PRP for proliferation and differentiation of the bone marrow stromal cells of the rabbits.
[Methods]The bone marrow stromal cells were isolated from the 4-month-old New Zealand rabbits as samples by using the adherent method.The Microscopy was used to study the cells's shape,and the flow cytometer was used to identify the cells.
Observed the proliferation status of the 1~(st),2~(nd),3~(rd),4~(th),5~(th),6~(th),7~(th),and 8~(th) generation cells,and drew the growth curve of the cells.Fed the rabbit with 100mg/Kg YunnanBaiyao once per day for 7 days.Obtain the blood samples of the rabbit on the 3~(rd),5~(th) and 7~(th) dav respectively.Obtained one pure PRP sample from the rabbit that has not been fed with YunnanBaiyao and one PRP sample from the rabbit that has been fed with YunnanBaiyao by using the Landesberg method.Then,check the jinfluence of YunnanBaiyao on the PRP for the proliferation and differentiation of rBMSCs by the MTT and enzyme kinetics methods.
[Results]Comparison between the YunnanBaiyao in vitro direct intervention groups and the control group:The difference between 60μg/m(0.53±0.03) and 65ug/m(0.56±0.051 sample goups on proliferation of rBMSCs(P<0.05) is distinct.
Sample goup 65μg/ml(1.21±0.08).70μg/ml(1.28±0.05),and 75μg / ml(1.24±0.06) proves that YunnanBaiyao can enchance the ALP activity(P<0.05) of the rBMSCs.
Comparison between the YunnanBaiyao oral intervention group and the control group: For the sample groups that have taken YunnanBaiyao for 5 days(0.30±0.02) and 7days(0.34±0.08),the one that has taken YunnanBaiyao for 7 days(0.88±0.06)proves that YunnanBaiyao can better enhance the ALP activity(P<0.05) of the rBMSCs.
[Conclusion]PRP,activated by YunnanBaiyao,can promote the proliferation and differentiation of rBMSCs.The 60μg / ml and 65μg / ml sample group and the sample group that has taken YunnanBaiyao for 5 davs and 7 days show the best effect on proliferation of rBMSCs;and the sample group 65μg/ml,70μg / ml,and 75μg / ml that have taken YunnanBaiyao for 7days show the best effect on differentiation of rBMSCs.
引文
[1]Miyanishi K,Trindade MC,Lindsey DP,et al.Effects of hydrostatic mesenchymal stem cell chondrogenesis in vitro.Tissue Eng,2006,12:1419-1428
[2]Nakayama N,Lee J,Chin L.Vascular endothelial growth factor synergistically enhances bone morphogenetic protein-4-dependent lymphohematopoietic cell generation from embryonic stem cells in vitro.Blood,2000,95(7):2275-2283
[3]吴在德.外科学.第5版,北京:人民卫生出版社,2000:804-806
[4]刘兴文.PRP在骨组织修复再生中的应用.国外医学生物医学工程分册.2003,26(6):251-254
[5]Huang LH,Neiva RE,Soehren SE,et al.The effect of platelet-rich plasma on the coronally advanced flap root coverage procedure:a pilot human trial.J Periodontol 2005,76(10):1768-1777
[6]Belli E,Longo B,Balestra FM.Autogenous platelet-rich plasma in combination,with bovine-derived hydroxyapatite xenograft for treatment of a cystic lesion of the jaw.J Craniofac Surg 2005,16(6):978-980
[7]张晔,曾炳芳,张长青等.富血小板血浆对体外培养骨髓间充质干细胞增殖及成骨活性的作用.中国修复重建外科杂志,2005,19(2):109-113
[8]刘兴文,刘宏伟,张秀白等.富血小板血浆对骨髓基质细胞BMSCs碱性磷酸酶活性和总蛋白含良的影响.临床口腔医学杂志,2004,20(5):259-261
[9]张卫兵,洪光祥,康皓.富血小板血浆对兔骨髓间充质干细胞增殖的作用.组织工程研究与临床康复.2008,12(34):6630-6642
[10]李威,阎福华,卢友光等.富血小板血浆对牙髓成纤维细胞附着、增殖的影响.口腔医学研究,2004,20(2):126-128
[11]阮敏.PRP技术及其在口腔种植医学中的应用.中国口腔种植学杂志,2004,9(3):141-144
[12]张宇,林野,邱立新等.富血小板血浆促进口腔种植骨再生的临床应用研究.中华口腔医学杂志,2004.39(4):269-272
[13]林敏魁,闫福华.富血小板血浆用于牙周组织再生的研究Ⅰ、PRP的提取物对PDLFs 增殖的影响.临床口腔医学杂志,2003,19(11):656-658
[14]张小梅,莫治强,李俊明等.云南白药对围手术期血小板表面糖蛋白及超微结构的影响研究.昆明医学院学报.2004,25(2):56-59
[15]Landesberg R,Roy M,Glickman RS.Quantification of growth of platelet-rich plasma gel preparation.J Oral Maxillofac Surg,2000,58(3):297-300
[16]Enrico Lucarelli,Amira Beccheroni,Davide Donati,et al.Platelet-derived growth factors enhance proliferation of human stromal stem cells.Biomaterials,2003,24:3095-3100
[17]范海蛟.补肾中药对大鼠骨髓基质干细胞增殖分化的影响[博士论文].广州.广州中医药大学,2008
[18]杨光,范东艳,陈强等.贴壁法培养不同龄小鼠骨髓间充质干细胞的生物学特点中国实验诊断学,2007,11(1):13-15
[19]魏凌云.兔骨髓间充质干细胞体外扩增培养影响因素的实验研究[硕士学位论文].广西,广西医科大学,2008
[20]Friendenstein A J,Gorskaja J F,Kulagina N N.Fibroblast Precursors in Normal and Irradiated Mouse Hematopoietic Organs.Exp Hematol,1976,4(5):267-274
[21]Hung SC,Chen N J,Hs IEH SL,et al.Isolation and characterization of size-sieved stem cells from human bone marrow.Stem cell 2002,20(3):262-272
[22]迟作华,张洹,陆琰等.人骨髓间充质干细胞分离方法的比较.中国临床康复,2006,10(1):20-24
[23]Kitano Y,Radu A,Shabban A,et al.Selection,enrichment and culture expansion of murine mesenchymal progenitor cells by retroviral transduction of cyciine adherent bone marrow cells.Exp Hematol,2000,28(12):1460-1469
[24]Deryugina EI,Mullersieburg CE.Stromal cells in longtern cultures:key to the clucidation of hematopoietic development.Crit Rev Immunol,1993,13:115-150
[25]Rao MS,Mattson MP.Stem cellsand aging:expanding the possibilities.Mech Ageing,2001,122:713-721
[26]阮绪芝,陈霞萍,严世荣等.骨髓基质干细胞的分离纯化及培养.生物学杂志,2004,21(2):25-27
[27]Coelho MJ,Cabral AT,Femande MH.Human bone cell cultures in biocomapatibility testing.Part Ⅰ:osteoblastic differentiation of serially passaged human bone marrow cells cultured in alpha-MEM and in DMEM.Biomatarials,2000,21(11):1087-1094
[28]Tennant GB,Truran LN,Bailey Wood R,et al.Centrel of PH in human longterm bone marrow eultues wit low-glucose med ium containing zwittionbuffer lengthens the period of haemopo ietic acivity.Br J Haematol,2000,109(4):785-787
[29]Pittenger MF,Mackay AM,Beck BC,et al.Multilineage potential of adult human mesenchymal stem cells.Science,1999,284-143
[30]Barry F P.Mesenchynal stem cells therapy in joint disease.Novartis Found Symp,2003,249:86-96
[31]袁霆,张长青.骨组织及软组织修复作用中富血小板血浆的制作及其原理.中国临床康复,2004,8(35):7939-7941
[32]Dugrillon A,Eichler H,Kern S,et al.Autologous concentrated platelet-rich plasma(cPRP)for local application in bone regeneration.Int J Oral Maxillofac Surg,2002,31(6):615-619
[33]刘彩霞,周健等.临床口腔医学杂志,2007,23(1):6-8
[34]杨晓辉,白彦华编译.机械公式应用手册.科学出版社,2005:35-38
[35]张良,徐立,袁冬萍等.中药血清药理学方法的研究进展.南京中医药大学学报(自然科学版),2002,18(4):254-256
[36]李仪奎,吴健宇.血清药理实验中采血时间的通法方案.中国药理学通报,1999,15(6):569-570
[37]赵婷秀,陈振发.中药血清药理学方法的研究现状[J]。湖北中医学院学报,2003,5(3)45-46
[38]贺石林,葛金文等.质疑血清药理学,加强多层次半体内实验研究.中国药理学通报.2005,21(3):277-279
[39]贺石林,李元健.医学科研方法学[M].北京:人民军医出版社,2003,411-413
[40]刘成海.重要复方体外药理研究思考.中药新药与临床药理,2000,7(1):53-56
[41]Maniatopoulos C,Soderek J,Melcher A H.Bone Formation in Vitro by Stromal Cell Obtained from Bone Marrow of Young Adults Rats.Cell Tissue Res,1995,254(3):207
[42]Otsuka E,Hirose S,et al.Characterization of Osteoblastic Differentiation of Stromal Cell;Line ST2 that is Induced by Aseorbic Acid.Am J Physioi,1999.277(12):132-138.
[43]Turksen K.Aubin JE.Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells.J Cell Biol.I991,114:373-377
[44]Malaval L,Modrowski D,Gupta AK,et al.Cellular expression of bone related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures.J Cell Physiolo,1994,158(3):555-558
[45]Ollignon H,Davecco MJ,Barlet JP.Isolation of cells from ovine fetal long bone and characterization of their osteoblastic activities during in vitro mineralization.Arch Physiol Biochem,1997,105(2):158-162
[46]Fromigue O.Marie PJ.Lomri A.Bone morphogenetic protein-2 and transforming growth factor-beta2 interact to modulate human bone marrow stromal cell proliferation and differentiation.J Cell Biochem,1998,68(4):411-426
[47]李才良.不同生长因子对骨髓基质细胞体外培养生物学特性的影响[硕士学位论文].广州.第一军医大,2002
[48]Ducy P,Schinke T,Karsenty G.The osteoblast:a sophisticated fibroblast under contral surveillance.Science,2000,289(5484):1501-1504
[48]Marx RE,Carlson ER,Eichstaedt RM,et al.Platelet2rich p lasma:Growth factor enhancement for bone grafts.Oral Surg Oral Med Oral Pathol Oral Radiol Endod,1998,85(6):638-646
[1]Weibrich G,Kleis W K,Harrier G.Growth factor levels in the platelet-rich plasma produced by 2 different methods:Curasan-type PRP kit versus PCCS PRP system[J],Int H Oral Maxillofac Implants,2002,17(2):184-190
[2]刘彩霞,周健,王银龙,等.不同方法制备的富血小板血浆对欠张成骨的影响[J].临床口腔医学杂志,2007,23(1):6-8
[3]宋扬,宫萍,侯睿,等.传统二次离心法和改良法制备富血小板血浆的比较研究[J].实用口腔医学杂志,2005,21(5):629-631
[4]Bouletreau P J,Warren S M,Spector J A,et al.Factors in the fracture microenvironment induce primary osteoblast angiogenic cytokine production[J].Plast Teconstr Surg,2002,110(1):139-148
[5]马忠雄,闫福华,钟声.富血小板血浆对牙周韧带成纤维细胞在牙骨质表面附着的增殖的影响[J].组织工程与重建外科杂志,2006,2(2):66-69
[6 De-Obarrio,Arauz D,Chamaberlain TM,et al.The.use of autologous growth factors in periodontal surgical therapy:plateletel biotechnology-case reports[J],Int J Perodontics Restorative Dent,2000,20(5):486-497
[7]Kawase T,Okuda K,Wolft L E,et al.Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro[J].J Perodontal,2003,74(6):858-864
[8]Camargo P M,Lekovic V,Weinlender M,et al.Plateler-rich plasma and bovine porous bone mineral combined with guided tissue regeneration in the treatment of intrabony defects in human [J].J Periodontal Res,2003,37(4):300-306
[9]Petrungaro P S.Using platelet-rich plasma to accelerate soft tissue maturation in esthetic periodontal surgery[J].Compen Contin Educ Eent.2001.11(9):729-732:734-736
[10]Fontana S,Olmedo D G,Linares J A,et al.Effect of platelet-rich plasma on the peri-implant bone response[J].Implant Dent,2004,13(1):73-78
[11]徐淑兰,周磊,黄建生,等.富血小板血浆促进种植体周围炎骨缺损修复的效果[J].广东医学,2008,29(5):801-804
[12]Fennis J P M,Stoelinga P J W,Janse J A.Mandibular reconstruction:Z clinical and radiographic animal study on the use of autogenous scaffolds and platelet-rich plasma[J].Int J Oral Maxillofac Surg,2002,31:281-296
[13]Thor A.Reconstruction of the anterior maxilla with platelet gel,autogenous bone,and titianium mesh:a case report[J].Clin implant Dent Relat Res,2002,4(3):150-155
[14]Anitua E.Plasma rich in growth factors:preliminary results of use in the preparation of future sites for implants[J].Int J Oral Maxillofac Implants,1999,14(4):529-535