摘要
目的探讨使成熟软骨细胞转化成代谢活跃、增殖迅速的再生软骨祖细胞,而后利用这种细胞构建自体源性工程组织,修复成熟关节软骨的缺损。方法成熟兔关节软骨细胞进行普通单层培养和转化生长因子(TGF)β1、碱性成纤维细胞生长因子(bFGF)联合诱导培养。培养细胞进行细胞计数、Ⅱ型胶原免疫组织化学染色和逆转录聚合酶链反应(RTPCR)检测。将诱导培养的再生软骨祖细胞与聚乳酸载体(PDLLA)一道构建自体源性工程软骨,修复成熟关节软骨缺损。修复组织进行组织形态学和免疫组织化学研究。结果研究发现成熟关节软骨细胞在体外单层培养中增殖缓慢,而联合TGFβ1、bFGF诱导培养可促进成熟软骨细胞的增殖,10d内细胞增殖189倍。标准单层培养4～5代细胞经密集培养不表达Ⅱ型胶原,而联合细胞因子诱导培养6代的细胞在体外密集培养下,可很快恢复Ⅱ型胶原的表达。利用再生软骨祖细胞构建的自体源性工程软骨可修复软骨缺损,修复组织表达Ⅱ型胶原。结论成熟关节软骨细胞经TGFβ和bFGF联合诱导培养可形成再生软骨祖细胞,此细胞可用于构建自体源性工程组织,修复成熟关节软骨的缺损。
Objective To investigate if the mature chondrocytes could be transformed to the secondary chondroprogenitors which could metabolize and proliferate actively by definitely inducing culture methods,so as to expand enough cells to construct the autogenous engineered cartilage and repair the defects of mature articular cartilage.Methods Chondrocytes were isolated from the articular cartilage of mature New Zealand White rabbits.The cells were cultured by common monolayer technique or exposed to a combination of TGF-β1 and bFGF to induce the secondary chondroprogenitors.Cell counting was made and collagen type-Ⅱ expression was detected by immunohistochemical staining and RT-PCR.The secondary chondroprogenitors were expanded,then seeded onto three-dimentional PDLLA scaffold to construct the autogenous engineered cartilage,which was used to repair the defects of mature articular cartilage.The repair tissue was harvested for histological and immunohistochemical exminations.Results The mature chondrocytes proliferated extremely slowly with common monolayer culture in vitro,but they could be stimulated for accelerated multiplication by combined used of TGF-β1 and bFGF.Cells underwent 189 folds in number in 10 days.Cells subcultured to 4-5 passages with common monolayer technique ceased the production of collagen type-Ⅱ,and didn't express chondrocyte phenotype in high density culture,but if with the inducing culture,they could restore the expression of the collagen type-Ⅱ quickly.The autogenous engineered cartilage which was constructed with the secondary chondroprogenitors could resurface the cartilage defects well,and the repairing tissue express the collagen type-Ⅱ.Conclusion The mature chondrocytes could be transformed to the secondary chondroprogenitors by combined use of TGF-β1 and bFGF,so as to expand enough cells to construct the autogenous engineered cartilage and repair the defects of mature articular cartilage.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2005年第3期293-296,i005,共5页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目(39970745)
