TGF-β_2转染关节软骨细胞的实验研究

Inhibition of dedifferentiation of human articular chondrocytes by transfection with recombinant TGF-β_2 in vitro

目的观察人关节软骨细胞在体外单层培养过程中的去分化,以及转染TGF-β2在关节软骨细胞内的表达和对去分化的抑制作用。方法从手术中切除的软骨组织中分离培养成人关节软骨细胞,通过脂质体介导的方法将已构建的pcDNA3.1(+)/TGF-β2转染到体外单层培养的软骨细胞中。采用RT-PCR、ELISA、组织学染色、免疫组化和原位杂交的方法,分别对转染组和未转染组的第1、6、9代细胞进行检测,比较目的基因表达、软骨细胞形态以及胶原和多糖生物合成的差异。结果经多次传代后,未转染软骨细胞在体外单层培养过程中逐渐走向去分化,TGF-β2、Ⅱ型胶原和蛋白多糖聚糖体的表达逐渐减低,而Ⅰ型胶原的表达增高。目的基因在转染组各代软骨细胞内均得到表达,转染后细胞保持软骨细胞的形态,Ⅱ型胶原和蛋白多糖聚糖体表达虽有降低,但均高于未转染的同代细胞,而Ⅰ型胶原表达增高的程度低于未转染细胞。结论人关节软骨细胞在体外单层培养中有去分化趋势;pcDNA3.1(+)/TGF-β2真核表达载体转染人关节软骨细胞获得成功,在转染后关节软骨细胞内稳定表达,并对软骨细胞的去分化有抑制作用。

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-β 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-β 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-β 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-β 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-β 2 could produce endogenous TGF-β 2 .Transfection of pcD-NA3.1(+)/TGF-β 2 is able to provide transient and long lasting expression of TGF-β 2 in chondrocytes,and may be a feasible method to inhibit dedifferentiation of articular chondrocytes in vitro.