机构地区:[1]上海第二医科大学附属第九人民医院整形外科,200011 [2]上海组织工程研究与开发中心
出 处:《中华医学杂志》2005年第33期2331-2337,共7页National Medical Journal of China
基 金:国家高技术研究发展计划基金资助项目(2001AA216021;2002AA205011);上海市青年科技启明星计划基金资助项目(00QB14025);上海市重点学科基金资助项目(沪教委科200033);上海市重点实验室基金资助项目(04DZ11604)
摘 要:目的探讨软骨形态发生蛋白1(CDMP1)生长因子体外诱导真皮成纤维细胞向成软骨细胞表型分化的影响因素。方法取包皮环切术后丢弃的真皮组织共3例,分离成纤维细胞体外培养扩增,以含10%胎牛血清的F12培养液加入CDMP1(终浓度为100ng/ml)进行诱导,未加CDMP1的作为对照。观察细胞多次传代后(P2、P5、P10)细胞表型分化情况;调整CDMP1浓度分别为10、30、100、300ng/ml,观察诱导后软骨细胞表型的变化;单层培养与微团块培养成纤维细胞分别体外诱导7、14d,逆转录聚合酶链反应(RTPCR)检测细胞诱导前后骨形态发生蛋白受体(BMPR)、Ⅱ、Ⅳ、Ⅹ型胶原表达;Western印迹检测细胞诱导前后Ⅱ型胶原、SOX9、蛋白聚糖的表达;流式细胞术检测表面标志CD29、CD105、CD106、CD166及细胞诱导后Ⅰ、Ⅱ型胶原表达。结果细胞多次传代后(P5、P10)仍可表达特异软骨基质Ⅱ型胶原与蛋白聚糖,Ⅱ型胶原阳性细胞率与P2诱导后差异无统计学意义(P>0.05)。CDMP1浓度为10、30ng/ml诱导7、14d,均未见Ⅱ型胶原与蛋白聚糖表达;CDMP1浓度为100、300ng/ml时,Ⅱ型胶原与蛋白聚糖表达强度差异无统计学意义。单层培养成纤维细胞诱导14d后,软骨基质表达消失;微团块三维培养诱导14d,软骨基质仍保持表达。RTPCR检测诱导后ActRI/ALK2,BMPRIA/ALK3,BMPRIB/ALK6基因表达明显增强。结论在CDMP1生长因子诱导下,人真皮成纤维细胞体外扩增后可保持向成软骨细胞表型分化能力,细胞分化与CDMP1浓度、细胞三维培养条件及BMPR介导有关。Objective To explore the factors influencing the differentiation of fibroblasts into chondrocyte phenotype induced by a growth factor, cartilage-derived morphogenetic protein 1 (CDMP1). Methods Fibroblasts isolated from foreskin obtained during circumcision were cultured in the forms of micromass and monolayer culture. The culture fluid of the fibroblasts at the passage 2, 7, and 10 was added with CDMP1 of the concentrations at the concentrations of 10, 30, 100, and 300 ng/ml respectively and cocultured for 7 days. RT-PCR was used to detect the expression of bone morphogenetic protein receptor (BMPR), activin receptor-like kinase (ALK) receptor, collagen types Ⅱ , Ⅳ, and Ⅹ before and after CDMP1 induction. Western blotting was used to detect the protein expression of collagen types Ⅱ , a transcriptional factor Soxg, and aggrean before and after the induction. Flow cytometry was used to detect the superficial markers CD29, CD105, CD106, and CD166, and the expression of collagen types Ⅰ and Ⅱ. Results Western blotting showed that the collagen type Ⅱ positive cell rate in the passage 5 cells was 74. 3% ±0. 4% , not significantly different from that of the passage 2 cells (73.4% +0. 5% ). When the concentrations of CDMP1 were 10 and 30 ng/ml no expression of aggrecan and collagen type Ⅱ was detected, When the concentrations of CDMP1 was 100 and 300 ng/ml, the expression of aggrecan and collagen type Ⅱ could be detected and without significant differences between these 2 concentrations. The expression of aggrcan and collagen type Ⅱ mRNA disappeared in the monolayer cultured P2 and P5 fibroblasts induced by CDMP1 for 14 days, However, RT-PCR and Western blotting showed expression of collagen type Ⅱ, aggrcan and SOX9 in the micromass cultured fibroblasts. RT-PCR showed that all fibroblasts cultured in vitro expressed ActR-I/ALK-2, BMPR-IA/ALK-3, and BMPR-IB/ALK-6 genes, and the expression of these genes significantly increased after CDMP1 for 7 days. Conclusion CDMP1 stimulates t
关 键 词:软骨细胞 成纤维细胞 骨形态发生蛋白质类 分化 骨形态发生蛋白受体 真皮成纤维细胞 成软骨细胞 表型分化 流式细胞术检测 逆转录-聚合酶链反应
分 类 号:R329.2[医药卫生—人体解剖和组织胚胎学] R619.6[医药卫生—基础医学]
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