TGF-β1对大鼠脂肪干细胞体外软骨形成能力影响的研究
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摘要
目的本文研究大鼠脂肪干细胞(adipose-derived stem cells ADSCs)在体外分离培养、诱导分化成软骨细胞的能力,初步观察不同浓度的TGF-β1对其增殖、分化成软骨细胞的影响,为软骨组织工程及临床应用提供科学依据。
方法从3周龄SD大鼠腹股沟脂肪垫分离出脂肪组织,通过Ⅰ型胶原酶消化法分离干细胞,接种于含有胎牛血清的DMEM培养液中分离传代。每日以倒置显微镜观察细胞形态及生长情况。取第三代脂肪干细胞分成5组,包含1个对照组(培养液为人脂肪干细胞完全培养基：高糖DMEM、10%胎牛血清、50nmol/L抗坏血酸、6.25mg/L胰岛素、100U/ml青霉素、100μg/ml链霉素、100nmol/L地塞米松,不含TGF-β1)和4个TGF-β1浓度组(浓度分别是1、10、40、80μg/L加人脂肪干细胞完全培养基)。采用MTT比色法进行分化和增殖活性比较,Ⅱ型胶原免疫细胞化学染色和细胞甲苯胺蓝染色检测成软骨细胞分化情况。
结果原代细胞接种4-6小时后即可见沉降贴壁,7-8天左右可见大量增殖,按一定方向性排列呈漩涡状或束状,传代后细胞增殖迅速,生长稳定。成软骨诱导后行细胞甲苯胺蓝染色示：10μg/LTGF-β1浓度组阳性,40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度染色较淡,染色细胞少,0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。Ⅱ型胶原免疫组织化学染色显示：10μg/L TGF-β1浓度组阳性；40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度组为弱阳性；0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。MTT实验显示10μg/L的TGF-β1可以促进细胞增殖(P<0.05)。
结论1.大鼠脂肪干细胞能向成软骨细胞诱导分化；2.10μg/L的TGF-β1是脂肪干细胞增殖、分化成软骨细胞的最佳浓度；3.0、1、40、80μg/L的TGF-β1均不利脂肪干细胞增殖及软骨细胞形成。
Purpose Our research is to study the ability of Chondrogenic Differentiation of Rat Adipose-derived Stem Cells by isolating Chondrogenic Differentiation of Rat Adipose-derived Stem Cells by isolating and culturing in vitro and to observe the influences of different concentrations of TGF-β1 on proliferation and differentiation, in order to get the foundation for
Methods The adipose tissue were isolated from the inguinal fat pads of the three-weeks old Sprague-Dawley rat, then digesed with collagenase-I and placed in Dulbeco Modified Eagle Medium(DMEM)with fetal bovine serum in primary cultivation.We observed the growth and appearance of the cells by a inversion microscope. Take the third generation of ADSCs into 5 groups, including a control group(ADSCs medium:high glucose DMEM,10% fetal bovine serum,50 nmol/L ascorbic acid,6.25 mg/L insulin, 100U/ml penicillin, 100μg/ml streptomycin 100nmol/L dexamethasone, without TGF-β-1) and 4 groups of TGF-β1 concentrations(respectively 1,10,40,80μg/L increase of ADSCs medium). Both of the proliferation and differentiation of ADSCs was examinated by MTT test,and chondrogenic differentiation of ADSCs was examinated by II collagen immunohistochemical staining and toluidine blue staining.
Results The primary cells began to adherence at the 4-6th hour after settlement.They proliferative quikely at the 7-8th day,arranging according to a certain direction,such as whirlpool or bundle.The cells proliferatived quick and steady after passaging. These cells stained with toluidine blue and type II collagen immunohistochemical staining showed:10μg/L group positive; 40μg/L group and 80μg/L group, weakly positive; 0μg/L group and 1μg/L group was negative. MTT test showed:10μg/L TGF-β1 can promote cell proliferation significantly (P <0.05).
Conclusions Rat Adipose-derived Stem Cells can be induced to differentiate into cartilage cells; 10μg/L is the optimal concentration of TGF-β1 for Adipose-derived Stem Cells proliferating and differentiating to cartilage,and 0,1,40,80μg/L were not suitable.
方法从3周龄SD大鼠腹股沟脂肪垫分离出脂肪组织,通过Ⅰ型胶原酶消化法分离干细胞,接种于含有胎牛血清的DMEM培养液中分离传代。每日以倒置显微镜观察细胞形态及生长情况。取第三代脂肪干细胞分成5组,包含1个对照组(培养液为人脂肪干细胞完全培养基：高糖DMEM、10%胎牛血清、50nmol/L抗坏血酸、6.25mg/L胰岛素、100U/ml青霉素、100μg/ml链霉素、100nmol/L地塞米松,不含TGF-β1)和4个TGF-β1浓度组(浓度分别是1、10、40、80μg/L加人脂肪干细胞完全培养基)。采用MTT比色法进行分化和增殖活性比较,Ⅱ型胶原免疫细胞化学染色和细胞甲苯胺蓝染色检测成软骨细胞分化情况。
结果原代细胞接种4-6小时后即可见沉降贴壁,7-8天左右可见大量增殖,按一定方向性排列呈漩涡状或束状,传代后细胞增殖迅速,生长稳定。成软骨诱导后行细胞甲苯胺蓝染色示：10μg/LTGF-β1浓度组阳性,40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度染色较淡,染色细胞少,0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。Ⅱ型胶原免疫组织化学染色显示：10μg/L TGF-β1浓度组阳性；40μg/LTGF-β1浓度组和80μg/LTGF-β1浓度组为弱阳性；0μg/LTGF-β1浓度组和1μg/LTGF-β1浓度组为阴性。MTT实验显示10μg/L的TGF-β1可以促进细胞增殖(P<0.05)。
结论1.大鼠脂肪干细胞能向成软骨细胞诱导分化；2.10μg/L的TGF-β1是脂肪干细胞增殖、分化成软骨细胞的最佳浓度；3.0、1、40、80μg/L的TGF-β1均不利脂肪干细胞增殖及软骨细胞形成。
Purpose Our research is to study the ability of Chondrogenic Differentiation of Rat Adipose-derived Stem Cells by isolating Chondrogenic Differentiation of Rat Adipose-derived Stem Cells by isolating and culturing in vitro and to observe the influences of different concentrations of TGF-β1 on proliferation and differentiation, in order to get the foundation for
Methods The adipose tissue were isolated from the inguinal fat pads of the three-weeks old Sprague-Dawley rat, then digesed with collagenase-I and placed in Dulbeco Modified Eagle Medium(DMEM)with fetal bovine serum in primary cultivation.We observed the growth and appearance of the cells by a inversion microscope. Take the third generation of ADSCs into 5 groups, including a control group(ADSCs medium:high glucose DMEM,10% fetal bovine serum,50 nmol/L ascorbic acid,6.25 mg/L insulin, 100U/ml penicillin, 100μg/ml streptomycin 100nmol/L dexamethasone, without TGF-β-1) and 4 groups of TGF-β1 concentrations(respectively 1,10,40,80μg/L increase of ADSCs medium). Both of the proliferation and differentiation of ADSCs was examinated by MTT test,and chondrogenic differentiation of ADSCs was examinated by II collagen immunohistochemical staining and toluidine blue staining.
Results The primary cells began to adherence at the 4-6th hour after settlement.They proliferative quikely at the 7-8th day,arranging according to a certain direction,such as whirlpool or bundle.The cells proliferatived quick and steady after passaging. These cells stained with toluidine blue and type II collagen immunohistochemical staining showed:10μg/L group positive; 40μg/L group and 80μg/L group, weakly positive; 0μg/L group and 1μg/L group was negative. MTT test showed:10μg/L TGF-β1 can promote cell proliferation significantly (P <0.05).
Conclusions Rat Adipose-derived Stem Cells can be induced to differentiate into cartilage cells; 10μg/L is the optimal concentration of TGF-β1 for Adipose-derived Stem Cells proliferating and differentiating to cartilage,and 0,1,40,80μg/L were not suitable.
引文
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[2]Conget DA, Minguell JJ. Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells. J Cell Physiol,1999, 181:67-73.
[3]Zuk PA, Zhu M, Mizuno H, et al.Multilineage cells from human adipose tissue:inplications for cell-based therapies[J]. Tissue Eng,2001,7 (2):211-228.
[4]Goessler UR, Bugert P, Bieback K, et al. In-vitro analysis of the expression of TGF-β-superfamily members during chondrogenic differentiation of mesenchymal stem cells and chondrocytes during dedifferentiation in cell culture. Cell Mol Biol Lett,2005, 10(2):345-362.
[5]细胞培养(修订版)主编：司徒镇强 吴军正 世界图书出版公司.
[6]Ogston N, Harrison AJ, Cheung HF, et al. Dexamethsaone and retinoic acid differentially regulate growth and differentiation in an immortalized human clonal bone marrow stromal cell line with osteoblastic characteristics. Steroids,2002,67(11):895-906.
[7]Rose FR, Oreffo R0. Bone tissue engineering:hope vs hype. Biochem biophys Res Commun,2002,292(1):1-7.
[8]Kolf CM, Cho E, Tuan RS. Mesenchymal stromal cells. Biology of Adult mesenchymal stem cells:Regulation of niche, self-renewal and differentiation. Arthritis Res Ther,2007,9(1):204.
[9]Wang CY, Li SLL. Advance on seed cells of tissue engineering [J]. Jichu Yu linchuang,2001,21(6):506-510.
[10]张胜利,邓展生,等.大鼠脂肪间充质干细胞的成骨分化[J].中国组织工程研究与临床康复,2007,11(6)：1022-1024.
[11]Zuk PA,Zhu M, Peter A, et al. Human Adipose Tissue Is a Source of Multipotent Stem Cells. Molecular Biology of the Cell,2002,13:4279-4295.
[12]Wosnitza M, Hemmrich K, Groger A, et al. Plasticity of human adipose stem cells to perform adipogenic and endothelial differentiation, 2007;75(1):12-23.
[13]Ning HX, Lin GT, Lue TF, et al. Neuron-like differentiation of adipose tissue-derived stromal cells and vascular smooth muscle cells, Differentiation.2006; 74 (9-10):510-518.
[14]董晓红,雷永红,付小兵,等.脂肪干细胞向表皮细胞表型转分化的研究[J].中华整形外科杂志,2007,23(2)：151-153.
[15]Mehlhorn AT, Schmal H, Kaiser S, et al. Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture. Tissue Eng,2006;12(6):1393-1403.
[16]Barry F, Boynton RE, Liu B, et al. Chondrogenic differentiation of mesenchymal stem cells from bone marrow:differentiation-dependent gene expression of matrix components. Exp Cell Res,2001;268 (2):189-200.
[17]Palmer GD, Steinert A, Pascher A, et al. Gene-induced chondrogenes is of primary mesenchymal stem cells in vitro. Mol Ther,2005;12 (2):219-228.
[18]Jin XB, Sun YS, Zhang K, et al. Ectopic neocartilage formation from predifferentiated human adipose-derived stem cells induced by adenoviral-mediated transfer of hTGF beta2. Biomaterials,2007; 28(19):2994-3003.
[19]Woodbury D, Schwarz EJ, Prockop DJ, et al. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res,2000,61:364-370.
[20]Johnstone B, Hering TM, Caplan AI, et al. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. J Exp Cell Res,1998,238(1):265-272.
[21]Xu Y, Balooch G, Chiou M, et al. Analysis of the material properties of early chondrogenic differentiated adipose-derived stromal cells(ASCs) using an in vitro three-dimensional micromass culture system. Biochemical and Biophysical Research Communications,2007; 359(2):311-316.
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