Inhibition of dedifferentiation of human articular chondrocytes by transfection with recombinant TGF-?_2 in vitro / 中华骨科杂志

ABSTRACT

Objective In cartilage tissue engineering,it was a difficult problem to keep chongdro-cytes phenotype for long time.The aim was made to investigate the dedifferentiation of human articular chon-drocytes,and to verify the inhibition to dedifferentiation by transfection with recombinant pcDNA3.1(+)/TGF-? 2 in vitro.Methods Free chondrocytes were isolated from healthy articular cartilage harvested from6adult patients underwent operation because of other condition.The recombinant pcDNA3.1(+)/TGF-? 2 con-structed in advance was transferred by liposome mediated transfection to monolayer cultured articular chon-drocytes.The first,sixth and ninth generation chondrocytes being transfected and non-transfected were as-sayed by histological examination of HE and Safranine-O staining to observe their morphological changes.RT-PCR,ELISA,immunohistochemistry analysis and prime hybridization were performed to these cells to i-dentify the expression and biosynthesis of TGF-? 2 and cartilage-associated genes and proteins.Results The non-transfected articular chondrocytes in monolayer culture demonstrated the tendency of dedifferentia-tion,and lost the characteristics of their phenotype progressively in subculture.The expression of TGF-? 2 ,collagen typeⅡand proteoglycan in non-transfected cells were decreased,but expression of collagen typeⅠincreased gradually with time going on.On the contrary,as for the transfected chondrocytes,the trans-ferred gene was expressed in cells of all examined generations,and the expression levels were higher than that of non-transfected cell of same generations.The transfected chondrocytes maintained their phenotype and characteristics of articular cartilage.They expressed and produced collagen typeⅡand proteoglycan in a higher level,and collagen typeⅠin a lower level than that of non-transfected cells.Conclusion Human articular chondrocytes in monolayer culture have the tendency of dedifferentiation.The results suggested that chondrocytes tranfected by pcDNA3.1(+)/TGF-? 2 could produce endogenous TGF-? 2 .Transfection of pcD-NA3.1(+)/TGF-? 2 is able to provide transient and long lasting expression of TGF-? 2 in chondrocytes,and may be a feasible method to inhibit dedifferentiation of articular chondrocytes in vitro.