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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 investigate the factors affecting the differentiation of dermal fibroblasts into chondrocytes induced by chondroitumor morphogenetic protein-1 (CDMP1) growth factor in vitro. Methods A total of 3 dermis tissues were removed after circumcision. The fibroblasts were isolated and cultured in vitro. F12 medium containing 10% fetal calf serum was added to CDMP1 (final concentration 100 ng / ml) for induction. CDMP1 as a control. The cell phenotype differentiation was observed after multiple passages (P2, P5, P10). The concentration of CDMP1 was adjusted to 10, 30, 100, and 300 ng / ml respectively. The chondrocyte phenotypes were observed after induction. The cultured fibroblasts were cultured in vitro for 7 and 14 days, respectively. The expression of BMPR, Ⅱ, Ⅳ and Ⅹcollagen were detected by reverse transcriptase polymerase chain reaction (RTPCR) Type Ⅱ collagen, SOX9 and proteoglycan. Flow cytometry was used to detect the expression of surface markers CD29, CD105, CD106, CD166 and type Ⅰ and type Ⅱ collagen. Results After cells were passaged for many times (P5, P10), the cartilage matrix type Ⅱ collagen and proteoglycan could still be expressed. There was no significant difference between the positive rate of type Ⅱ collagen and that after P2 induction (P> 0.05). No expression of type Ⅱ collagen and proteoglycan were found when the concentration of CDMP1 was 10 and 30 ng / ml for 7 and 14 days, respectively. There was no significant difference in the expression of type Ⅱ collagen and proteoglycan when the concentration of CDMP1 was 100 and 300 ng / ml. After cultured for 14 days in monolayer fibroblasts, the expression of cartilage matrix disappeared. After three-dimensional culture, the cartilage matrix was maintained for 14 days. The expression of ActRI / ALK2, BMPRIA / ALK3, BMPRIB / ALK6 gene was significantly increased after RTPCR detection. Conclusion Under the condition of CDMP1 growth factor, human dermal fibroblasts maintain the phenotypic differentiation into chondrocytes after being expanded in vitro. The cell differentiation is related to the concentration of CDMP1, the three-dimensional cell culture conditions and the mediation of BMPR.