摘要
干细胞(Stem cells)是一种具有自我更新和多向分化潜能的特殊细胞,依其分化能力的不同可分为全能干细胞(Totipotent stem cell, TSC)、多能干细胞(Pluripotential stem cell, PSC)和单能干细胞(Unipotent stem cell, USC)三类。骨髓来源的间充质干细胞(Bone marrow-derived mesenchymal stem cells, MSCs)具有多向分化潜能,在特定诱导条件下可分化为成骨细胞、软骨细胞、肌肉细胞、脂肪细胞等,是目前研究、应用最多的干细胞类型,已成为临床细胞移植治疗和组织工程的主要细胞来源。但MSCs在体外生长缓慢、增殖能力有限,因此如何促进MSCs的体外增殖并保持其多向分化潜能是广泛研究和临床应用的前提。
已有研究证明,细胞外基质(Extracellular matrix, ECM)、细胞因子(Cytokine)和机械刺激(Mechanical stimulation)等理化因素对细胞的生长和增殖起着重要的调节作用。I型胶原(Type I collagen, Col I)是ECM的主要成分之一,碱性成纤维细胞生长因子(Basic fibroblast growth factor, bFGF)对多种细胞的生长和增殖具有明显的促进作用,适当的机械刺激也能显著促进细胞的体外增殖。但Col I、bFGF和机械刺激及其联合作用对MSCs生长和增殖的影响尚未见详细报道。为此,本文以大鼠MSCs(Rat MSCs,rMSCs)为实验材料,采用MTT法考察Col I、bFGF和机械拉伸等理化因素及其联合作用对rMSCs体外生长和增殖的影响,寻找在体外促进rMSCs生长和增殖的适宜理化刺激条件。主要研究工作和结果如下:
1. rMSCs的原代分离培养
利用密度梯度离心结合差异贴壁法进行rMSCs的原代分离培养,结果表明:1.073 g/mL的percoll密度梯度离心结合差异贴壁分离得到的细胞为形态比较均一的单核细胞,用含10%胎牛血清(Fetal bovine serum, FBS)的DMEM培养基培养24小时后细胞开始贴壁,继而分裂增殖,呈集落式生长,约2周后接近融合,呈纺锤形或长梭形的成纤维细胞样形态。其间有漂浮不贴壁的造血细胞等杂细胞,在反复换液中被除去。传代后细胞贴壁、生长较快,约一周可达到融合,呈长梭形或纺锤形均匀分布。
2. rMSCs的鉴定和周期分析
姬姆沙染色显示培养细胞胞核呈圆形或椭圆形,为紫色,可见数个深紫色的核仁,胞浆为淡紫色。利用免疫染色方法考察培养细胞表面部分抗原的表达,以及流式细胞技术分析了细胞周期的分布。结果发现:细胞表面抗原CD34(造血干细胞标记物)呈阴性,CD44和CD29呈阳性表达。细胞周期分析显示G0/G1期细胞占(89.74±3.87)%,S期细胞占(2.49±2.20)%,G2/M期细胞占(7.70±3.70)%,表明大部分细胞处于非增殖状态。
3. Col I和bFGF及其联合作用对rMSCs增殖的影响
本文首先测定了rMSCs的生长曲线,然后考察了Col I和bFGF单独和联合作用对rMSCs增殖的影响。结果表明:第1~2天为rMSCs生长潜伏期,第3~7天为对数增长期,7天以后细胞的生长进入平台期。在0-40μg/mL的研究浓度范围内,Col I裱衬均能显著促进rMSCs的增殖,20μg/mL的Col I裱衬增殖效果相对更好。时间依赖实验发现20μg/mL Col I裱衬培养8天,最有利于rMSCs增殖。在0-20 ng/mL的研究浓度范围内,bFGF都能促进rMSCs增殖,7 ng/mL bFGF诱导的增殖效果相对更好。7 ng/mL bFGF诱导培养8天,为rMSCs增殖的最佳条件。Col I裱衬和bFGF联合作用显示,联合作用的促rMSCs增殖效果高于单因素作用的效果,表明二者对rMSCs增殖具有协同促进作用。
4.机械拉伸以及与Col I、bFGF联合作用对rMSCs增殖的影响
本文应用细胞拉伸加载装置,考察了1 Hz下不同应变大小(5%和10%应变)、不同作用时间(15分钟、30分钟和60分钟)的拉伸作用rMSCs,继续培养6小时后测定细胞的增殖情况。结果表明:与对照组比较,5%应变组15分钟和60分钟的拉伸加载能促进rMSCs的增殖,而且15分钟加载的增殖效果好于60分钟加载,30分钟拉伸对rMSCs增殖却表现出抑制作用。10%应变组的拉伸结果与5%应变组类似,但诱导的增殖效果优于5%应变组。结果证明1 Hz 10%应变加载15分钟是促进rMSCs体外增殖的适宜拉伸条件。进一步以此拉伸条件联合Col I、bFGF同时对rMSCs进行诱导刺激,发现三者联合作用促进rMSCs增殖的效果最好。
本文的研究结果对全面认识各种理化因素及其协同作用对MSCs生长和增殖的调节有重要意义,为寻求促进MSCs体外增殖的相关研究提供了实验依据和方法学参考。
Stem cells are the special cells that have self-renewal and multi-differentiation ability. According to the different differentiation potential, stem cells can be divided into three types: totipotent stem cell (TSC), pluripotential stem cell (PSC) and unipotent stem cell (USC). Under some special conditions, bone marrow-derived mesenchymal stem cells (MSCs) can be induced to differentiate into osteoblasts, chondrocytes, muscle cells, adipocytes and so on. At the present, MSCs are studied and used widely and have become the main source of cells for clinical cell transplantation and tissue engineering. For the slow growth ability and limited life span of MSCs in vitro, it is the prerequisite for the research and clinical application to expand MSCs in vitro and to maintain its multi-differentiation ability.
It has been proved that many physical and chemical factors play an important role on the proliferation of cells, such as extracellular matrix (ECM), cytokine and mechanical stimulation. Collagen I (Col I) is one of the main components of ECM. Basic fibroblast growth factor (bFGF) and appropriate mechanical stimulation can significantly promote the proliferation of kinds of cells. However, little is known about the effect of Col I, bFGF and mechanical stimulation on proliferation of MSCs. Thus, to find the appropriate physical and chemical stimulation conditions in promoting the proliferation of rat MSCs (rMSCs) in vitro, we focus on the effect of Col I, bFGF and mechanical stimulation on proliferation of rMSCs by using them alone and synergetically. The main research works and results are as follows:
1. Isolation and culture of rMSCs in vitro
The rMSCs were isolated by centrifugation with 1.073 g/mL percoll solution. The results indicated the isolated cells were homogeneous histoleucocyte. Cultured in DMEM with 10% FBS for 24 hours, the cells adhered, divided and grew into colony. After about 2 weeks they achieved confluence, and showed fibroblast-like morphology. The nonadhesive cells were removed by changing the medium. After about 1 week, the subcultured spindly cells achieved confluence.
2. Identification and cell cycle analysis of rMSCs
Nucleoli of cultured cells, stained by Gimsa, were round or ellipse. Several dark purple nucleoli were thus clear meanwhile cytoplasm was lilaceous. We detected that the surface antigen CD34 negative while CD44 and CD29 were positive, and G0/G1 phase cells were (89.74±3.87) %, S phase were (2.49±2.20) % and G2/M phase were (7.70±3.70) %.
3. Effect of Col I, bFGF and the combined on proliferation of rMSCs
This paper measured the growth curve of rMSCs, and then investigated the effect of Col I and bFGF on proliferation of rMSCs by using them alone and synergetically. The result showed that: rMSCs growth latency was for a period of 1 to 2 days, and the growth period was from 3 to 7 days, after seven days cells turned into the period of growth platform. The proliferation of rMSCs could be significantly promoted by the 0-40μg/ml Col I, especially 20μg/ml Col I. Cells cultured 8 days with 20μg /mL Col I was the best stimulative conditions for rMSCs proliferation. Also the proliferation of rMSCs could be significantly promoted by 0-20 ng/ml bFGF, especially by 7 ng/ml bFGF, and the best stimulative conditions for cell proliferation were 8 days with 7 ng /mL bFGF. The effect of Col I and bFGF were significantly higher by using them synergetically than by using them alone. Col I and bFGF can promote synergetically the proliferation of MSCs.
4. Effect of Mechanical strain and the co-effect of mechanical strain, Col I and bFGF on proliferation of rMSCs
This paper characters the effect of mechanical stretch on proliferation of rMSCs. The effect on proliferation of rMSCs by single axis mechanical stretch (5% and 10%, 1Hz, 15, 30 and 60minutes) was investigated. The result showed that: in contrast to control group, the proliferation of rMSCs could be promoted after exposure to 5% strain for 15 and 60 minutes, especially for 15 minutes. After strain duration time of 30 minutes, a lower proliferation rate was measured compared with control levels. The result of 10% strain on proliferation of rMSC was in similar to 5% strain and the effect was higher than 5% strain. So exposed to 1 Hz, 10% strain for 15 minutes was an appropriate strain condition for promoting proliferation of rMSC in vitro. Furthermore, we found that the effect of the co-effect of mechanical strain, Col I and bFGF on proliferation of rMSCs was the highest.
The result is helpful to understand the various physical and chemical factors and the co-effect on the proliferation of rMSCs. It provides experimental basis and reference methodology for seeking to promote proliferation of MSCs in vitro.
引文
[1] Abrahamsson SO. Similar effects of recombinant human insulin-like growth factor-I and II on cellular activities in flexor tendons of young rabbits: experimental studies in vitro [J]. J Orthop Res, 1997, 15(2): 256-262.
[2] Brighton CT, Strafford B, Gross SB, et al. The proliferative and synthetic response of isolated calvarial bone cells of rats to cyclic biaxial mechnical strain [J]. J Bone Joint Surg, 1991, 73A: 320.
[3] Torricelli P, Fini M, Giavaresi G. Biomimetic PMMA-based bone substitutes: A comparative in vitro evaluation of the effects of pulsed electromagnetic field exposure [J]. J Biomed Mater Res, 2003, 64(1): 182-188.
[4] Jaiswal N, Haynesworth SE, Caplan AL, et al. Osteogenic differetiation of purified, culture expanded human mesenchymal stem cells in vitro [J]. J Cell Bilchen, 1997, 64(2): 295-312.
[5] Bruder SP, Ricalton NS, Boynton RE, et al. Mesenchymal stein cell surface antigen SB-10 corresponds to activated leukocyte cell adhesion molecule and is involved in osteogenic differentiation [J]. J Bone Miner Res, 1998, 13(4): 655-663.
[6] Majumdar MK, Thiede MA, Mosca JD, et al. Phenotypic and functional comparision of cultures of marrow-derived mesenchymal stem cells (MSCs) and stromal cells [J]. J Cell Physoil, 1998, 176(1): 57-66.
[7] Dexter TM, Allen TD, Lajtha LG. Conditions for controlling the proliferation of hematopoietic stem cells in vitro [J]. J Cell Physiol, 1977, 91(3): 335-344.
[8] Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells [J]. Exp Cell Res, 1998, 238: 265-272.
[9]李洪鹏,许建中,周强,等.流体剪切应力对人骨髓间充质干细胞增值及细胞周期的影响[J].第三军医大学学报, 2005, 27: 1637- 1639.
[10] Lennon DP, Edmison JM, Caplan AI. Cultivation of rat marrow-derived mesenchymal stem cells in reduced oxygen tension: effects on in vitro and in vivo osteochondrogenesis [J]. J Cell Physiol, 2001, 187: 345 - 355.
[11] Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation [J]. J Cell Biochem, 1997, 64: 278-294.
[12] Martin I, Muraglia A, Caplam Al, et al. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow [J]. Endocrinology, 1997,138(10): 4456-4462.
[13] Han P, Gou XH, Story CJ. Enhanced expansion and maturation of megakaryocytic progenitors by fibronectin [J]. Cytotherapy, 2002, 4(3): 277-283.
[14]王伟铭,姚建,曹红娣.细胞外基质对肾小管上皮细胞增殖的影响[J].细胞与分子免疫学杂志, 1998, 14(3): 217- 218.
[15] Anselme K. Osteoblast adhesion on biomaterials [J]. Biomaterials, 2000, 21: 667-681.
[16]张桂茹,王晓明,郭晓峰.细胞外基质对培养咽癌细胞增殖及P53, PCNA基因表达的影响[J].细胞生物学杂志, 1998, 20(4): 181- 184.
[17] Mayr-Wohlfart U, Kessler S, Knochel W, et al. BMP-4 of Xenopus laevis stimulates differentiation of human primary osteoblast–like cells [J]. J Bone Joint Surg Br, 2001, 83(5): 777-784.
[18] Thomas T, Gori F, Spelsberg TC, et al. Response of bipotential human marrow stromal cells to insulin-like growth factors: effect on binding protein production, proliferation, and commitment to osteoblasts an adipocytes [J]. Endocrinology, 1999, 140: 5036-5044.
[19] Hosokawa R, Kikuzaki K, Kimoto T, et al. Controlled local application of basic fibroblast growth factor (FGF-2) accelerates the healing of GBR: An experimental study in beagle dogs [J]. Clin Oral Implants Res, 2000, 11(4): 345-353.
[20] Koter ES, Pitaru S, Prichen S, et al. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2 [J]. Connect Tissue Res, 2002, 43(4): 606-612.
[21] Simons M. Integratine in angiogenesis [J]. Mol Cell Biochem, 2004, 264(1-2): 99-102
[22] Relf M, Lejeune S, Scott PAE, et al. Expression of the angiogenetic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor beta-1, platelet-derived endothelial cell growth factor, placenta growth factor, and pleiotophin in human primary breast cancer and its relation to angiogenesis [J]. Cancer Res, 1997, 57 (5): 963 - 969.
[23] Fakhry A, Ratisoontorn C, Vedhachalam C, et al. Effect of FGF-2/-9 in calvarial bone cell cultures: differentiation stage-dependent mitogenic effect, inverse regulation of BMP-2 and noggin, and enhancement of osteogenic potential [J]. Bone, 2005, 36(2): 254-266.
[24] Matsusaki M, Ochi M, Uchio Y, et al. Effects of basic fibroblast growth factor on proliferation and phenotype expression of chondrocytes embedded in collagen gel [J]. Gen pharmac, 1998, 31: 759-764.
[25]侯锐,毛天球,陈书军,等. rhBMP-2及rhbFGF对骨髓间充质干细胞增殖的长期效应的实验研究[J].口腔医学研究, 2003, 19: 166- 169.
[26]钱奇春,刘彦普,杨维东,等.骨形成蛋白-2和碱性成纤维细胞生长因子对人骨髓基质细胞的生物学作用[J].口腔医学研究, 2003, 10: 350- 352.
[27] Hankemeier S, Keus M, Zeichen J, et al. Modulation of proliferation and differentiation of human Bone marrow stromal cells by fibroblast growth factor 2: Potential implications for tissue engineering of Tendons and Ligaments [J]. Tissue Eng, 2005, 11(1-2): 41-49.
[28] Kostenuik PJ, Halloran BP, Morey-Holton ER, et al. Skeletal unloading inhibits the in vitro proliferation and differentiation of rat osteoprogenitor [J]. Am J Physiol Endocrinol Metab, 1997, 273: 1133-1139.
[29] Daniela K, Seidl W, Neidlinger-wilke C, et al. Proliferation of human-derived osteoblast-like cells depends on the cycle number and frequency of uniaxial strain [J]. J Biomech, 2002, 35: 873-880.
[30]蒋淳,霍建忠,马振洲,等.微重力旋转培养系统对骨髓间充质干细胞分化增殖的影响[J].中国临床医学, 2005, 12: 269- 271.
[31] Basso N, Heersche JN.Characteristics of in vitro osteoblastic cell loading models [J]. Bone, 2002, 30: 347-351.
[32] Kapur S, Baylink DJ, Lau KH. Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways [J]. Bone, 2003, 32: 241-251.
[33] Riddle RC, Taylor AF, Genetos DC, et al. MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation [J]. Am J Physiol Cell Physiol, 2006, 290: C776-C784.
[34] Xu J, Liu MY, Tanswell AK, et al. Mesenchymal determination of mechanical strain-induced fetal lung cell proliferation [J]. Am J Physiol Lung Cell Mol Physiol, 1998, 275: 545-550.
[35] Barkhausen T, van Griensven M, Zeichen J, et al. Modulation of cell functions of human tendon fibroblasts by different repetitive cyclic mechanical stress patterns [J]. Exp Toxicol Pathol 2003, 55:153-158.
[36] Zeichen J, Briensven M, Bosch U, et al.The proliferative response of isolated human tendon fibroblasts to cyclic biaxial mechanical strain [J]. Am J Sports Med, 1999, 28(6): 888-892.
[37] Marx JC, Allay JA, Persons DA, et al. High-efficiency transduction and long-term gene expression with a murine stem cell retroviral vector encoding the green fluorescent protein in human marrow stromal cells [J]. Hum Gene Ther, 1999, 10(7): 1163-1170.
[38] Jaiswal N, Haynesworth SE, Caplan AI, et al. Osteogenic differentiation of purified, culture expanded human mesenchymal stem cells in vitro [J]. J Cell Biochem, 1997, 64: 295-312.
[39] Deryugina EI, Mullersieberg CE. Stromal cells in long-term cultures: key to the elucidation ofhematopoietic development [J]. Crit Rev Immunol, 1993, 13: 115-150.
[40]刘晓丹,郭子宽,李秀森,等.人骨髓间充质干细胞分离与培养方法的建立[J].军事医学院院刊, 2000, 24(4): 282-284.
[41]艾国平,粟永萍.骨髓间充质干细胞的分离与培养[J].第三军医大学学报, 2001, 23(5): 553-555.
[42]司徒镇强.细胞培养[M].世界图书出版公司. 1996.
[43] Zobar R, Sodek J, Mecalloch CA. Characterization of stromal progenitor cells enriched by flow cytometry [J]. Blood, 1997, 90 (9): 3471-3480.
[44] Conget PA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells [J]. J Cell Physiol, 1999, 181(1): 67-73.
[45] Takano N, Kawakami T, Kawa Y, et al. Fibronectin combined with stem cell factor plays an important role in melanocyte proliferation, differentiation and migration in cultured mouse neural crest cells [J]. Pigment Cell Res, 2002, 15 (3): 192-200.
[46] Lee CH, Singla A, Lee Y. Biomedical applications of Collagen [J]. Int J Pharm, 2001, 221: 1-22.
[47] PAN Hai-tao, ZHENG Qi-xin, GUO Xiao-dong, et al. Effect of type I collagen on adhesion, proliferation and osteogenic differentiation of rabbit bone marrow stromal cells on PLGA-[ASP-PEG] scaffolds [J]. Chin J Orthop Trauma, 2006, 8(10): 938-943.
[48] Lennon DP, Haynesworth SE, Young RG, et al. A chemically defined medium supports in vitro proliferation and maintains the osteochondral potiential of rat marrow-derived mesenchymal stem cells [J]. Exp Cell Res, 1995, 219: 211-222.
[49] Locklin RM, Oreffo RO, Trifftt JJ, et al. Effects of TGF and bFGF on the differentiation of bone marrow stromal fibroblasts [J]. Cell Biol Int, 1999, 23(3): 185-194.
[50] Tsutsumi S, Shimazu A, Miyazaki K, et al. Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF [J]. Biochem Biophys Res Commun, 2001, 288 (2): 413-419.
[51] Hu Yun-sheng, Fan Qing-yu, Ma Bao-an, et al. Induced differentiation of cultured rabbit mesenchymal stem cells by basic fibroblast growth factor and recombinant bone morphogenetic protein-2 [J]. Chinese Journal of Clinicial Rehabilitation, 2006, 1(1): 163-165.
[52] Bhindi R, Brieger D, Ishii H, et al. Fibroblast growth factor 2 and the transcription factor Egr-1 localise to endothelial cell microvascular channels in human coronary artery occlusion [J]. Thromb Haemost, 2005, 93(1): 172-174.
[53] Sotiropoulou PA, Perez SA, Salagianni M, et al. Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells [J]. Stem Cells,2006, 24(2): 462-471.
[54]王磊,黄远亮,潘可风,等.不同浓度重组人骨形成蛋白2和碱性成纤维生长因子对犬骨髓基质细胞的生物学作用[J].中国组织工程研究与临床康复, 2007, 11(46): 9276-9280.
[55] Tsutsumi S, Shimazu A, Miyazaki K, et al. Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF [J]. Biochem Biophys Res Commun, 2001, 228(2): 413-419.
[56] Reape TJ, Kanczler JM, Ward JP, et al. IGF-I increases bFGF induced mitogenesis and up regulates FGFR-1 in rabbit vascular smooth muscle cells [J]. Am J Physiol, 1996, 270: H1141-H1148.
[57] Locklin RM, Williamson MC, Beresford JN, et al. In vitro effects of growth factors and dexamethasone on rat marrow stromal cells [J]. Clin Orthop, 1995, 313 (1): 27-35.
[58] Simmons CA, Matlis S, Thornton AJ, et al. Cyclic s train enhances matrix mineralization by adult human mesenchymal stem cells via the extracellular signal-regulated kinase (ERK1/2) signaling pathway [J]. J Biomech, 2003: 36(8): 1087-1096.
[59] Kaspar D, Seidl W, Neidlinger-Wilke C, et al. Dynamic cell stretching increases human osteoblast proliferation and CICP synthesis but decreases osteocalcin synthesis and alkaline phosphatase activity [J]. J Biomech, 2000, 33(1): 45.
[60] Zhuang H, Wang W, Tahernia D, et al. Mechanical strain induced proliferation of osteoblastic cells parallels increased TGF-β1 mRNA [J]. Biochem Biophys Res Commun, 1996, 229(2): 449.
[61] van Griensven M, Diederichs S, Kasper C. Mechanical strain of bone marrow stromal cells induces proliferation and differentiation into osteoblast-like cells [J]. Tissue Eng, 2005, 2: 1-20.
[62] Hughes-Fulford M. Signal transduction and mechanical stress [J]. Sci STKE, 2004, 249: RE12.
[63] Riddle RC, Taylor AF, Genetos DC, et al. MAP kinase and calcium signaling mediate fluid flow-induced human mesenchymal stem cell proliferation [J]. Am J Physiol Cell Physiol, 2006, 290(3): C776-784.