摘要
目的观察不同生长因子对成人关节软骨细胞(adulthumanarticularchondrocytes,AHAC)增殖的影响,探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞,条件培养基培养;传2代细胞分别用不同浓度成纤维细胞生长因子2(fibroblastgrowthfactor-2,FGF-2)、转化生长因子β1(transforminggrowthfactorβ1,TGF-β1)、血小板衍生因子bb(plateletderivedgrowthfactor-bb,PDGF-bb)、肝细胞生长因子(hepatocytegrowthfactor,HGF)或其不同组合作用。用MTT法比较细胞增殖情况,用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF-!1、PDGF-bb、HGF均有促AHAC增殖的作用,其最大效应剂量分别是50ng/ml、1ng/ml、1ng/ml、20ng/ml。5ng/mlFGF-2+1ng/mlTGF-β1有最强的促增殖作用,继续加用PDGF-bb和(或)HGF无进一步促进作用;用这一因子组合培养AHAC,可以传10代以上,细胞扩增2000倍以上,且传9代细胞仍弱表达Ⅱ型胶原和aggre-can。结论FGF-2、TGF-β1、PDGF-bb、HGF均对AHAC有一定的促增殖作用;5ng/mlFGF-2+1ng/mlTGF-β1有最大的促增殖效应,细胞短期内大量扩增,且在大量扩增的同时维持了一定的软骨细胞表型,因此是合适的AHAC体外大量扩增促进剂。
Objective To investigate the effects of transforming growth factor-β1（TGF-β1）, fibroblast growth factor-2 （FGF-2）, platelet derived growth factor-bb （PDGF-bb） and hepatocyte growth factor （HGF） on adult human articular chondrocytes （AHAC） proliferation and phenotype maintaining. Methods Isolated AHAC were cultured in DMEM/F-12 supplemented with 10% human AB serum, 50 μg,/ml ascorbic acid-2-phosphate, 0.4 mmol/L proline, 5 μg/ml insulin and 1 mmol/L non-essential amino acids （NEAA）. The cells of 2nd passage were used for proliferation kinetics studying： 2.0×10^3 cells/well were seeded on 96- well plate, 24 h later, the cells were stimulated with various growth factors or combinations of these growth factors respectively, and the proliferation kinetics were analyzed by MTT colorimetric method. The passaged chondrocytes＂ phenotype were assessed by safranine O staining and immunostaining for type Ⅰ, Ⅱ collagens and aggrecan. Results All four growth factors： FGF-2, TGF-β1, PDGF-bb and HGF, could promote the proliferation of AHAC, and the optimal concentrations,when used separately, were 50 ng/ml, 1 ng/ml, 1 ng/ml, 20 ng/ml respectively. While 5 ng/ml FGF-2 combined with 1 ng/ml TGF-β1 could achieve the best proliferation effect, additionally adding PDGF-bb, HGF or both could not enhance more. With the combination of FGF-2 and TGF-β1, the AHAC could expand over 2000-fold and passage over 10 times. Chondrocytes of 9th passage still excreted type Ⅱ collagen and glycosaminoglycan （GAG）. Conclusion 5 ng/ml FGF-2 combined with 1 ng/ml TGF-β1 is a very appropriated circumstance for in vitro expanding of AHAC.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
2006年第11期773-778,共6页
Chinese Journal of Orthopaedics
基金
国家863计划资助课题(2002AA205021)
关键词
软骨细胞
生长物质
软骨
关节
人类
Chondrocytes
Growth substances
Cartilage, articular
Humans
