诱导骨髓间充质干细胞向软骨细胞分化的体外研究

目的 探讨转化生长因子β1（transforming growth factor β1，TGF—β1）、胰岛素样生长因子1（insulinlike growth factor1，IGF-1）在诱导骨髓间充质干细胞（marrow mesenchymal stem ceils，MSCs）向软骨细胞分化过程中的相互作用，并研究细胞密度对MSCs向软骨细胞分化的影响。方法 取健康昆明种小白鼠骨髓，用全骨髓贴壁法筛选获得MSCs，体外培养传代。采用特定的诱导培养使MSCs向软骨细胞分化，按培养基内添加生长因子的不同分成3个实验组和对照组。实验组分别为：TGF—β1＋IGF-1联合应用组（TGF—β1 10ng／ml、IGF-1 50ng／m1）；TGF—β1单独应用组（TGF—β1 10ng／m1）；IGF-1单独应用组（IGF-1 50ng／m1）；对照组不添加任何生长因子。TGF—β1＋IGF-1联合应用组于诱导14d和21d，分别进行甲苯胺蓝染色及免疫荧光双染法鉴定；于诱导7、14和21d各组分别提取诱导细胞总RNA，进行RT—PCR扩增，检测TGF—β1、IGF-1对诱导细胞Ⅱ型胶原表达量的影响；比较MSCs在平板培养及细胞团培养时，Ⅱ型胶原表达量的差异。结果TGF—β1＋IGF-1联合应用组诱导培养14d，诱导软骨细胞甲苯胺蓝染色呈阳性，免疫荧光染色可见诱导软骨细胞的细胞外基质含有Ⅱ型胶原。各组基因扩增产物的凝胶电泳可见，TGF—β1＋IGF-1联合应用组和TGF—β1单独应用组Ⅱ型胶原扩增片段呈阳性；IGF-1单独应用组和对照组，未见Ⅱ型胶原扩增条带；凝胶成像系统灰度扫描示Ⅱ型胶原表达量TGF—β1＋IGF-1联合应用组各时间点均比TGF—β1单独应用组明显增加（P〈0．05）。细胞团培养模式下，诱导细胞表达Ⅱ型胶原比平板培养模式更加显著。结论 MSCs向软骨细胞诱导分化时，IGF-1对TGF—β1有明显的促进作用；细胞培养密度提高有利于MSCs成软骨细胞表型。

IN VITRO STUDY ON INDUCTION SYSTEMS FOR MARROW MESENCHYMAL STEM CELLS TO CHONDROCYTES

OBJECTIVE: To study the effect of transforming growth factor beta1 (TGF-beta1) and insulin-like growth factor 1 (IGF-1) during the induction course from marrow mesenchymal stem cells (MSCs) to chondrocytes and to observe the effect of cell density on cell induction. METHODS: Differential time adherent methods were used to purify MSCs obtained from the bone marrow of Kunming mice. MSCs were cultured under special conditions to induce them to differentiate into chondrocytes. Toluidine blue staining and immunofluoresence were used to identify those induced chondrocytes. TGF-beta1 and IGF-1 were used individually or in combination under two different culture patterns: pellet culture and monolayer culture. According to different growth factors, experiment included 3 experimental groups (TGF-beta1 + IGF-1 group, 10 ng/ml and 50 ng/ml respectively; TGF-beta1 group, 10 ng/ml; and IGF-1 group, 50 ng/ml) and control group (without growth factor). In TGF-beta1 + IGF-1 group, toluidine blue staining and immunofluoresence staining were carried out at 14 days and 21 days. The effect of TGF-beta1 and IGF-1 on the expression of collagen II gene was detected by RT-PCR at 7, 14 and 21 days of induction; the expressions of collagen II were compared between two culture patterns. RESULTS: In TGF-beta1 +-IGF-1 group, the histological examination and immunofluoresence showed that those inducted chondyocytes could express collagen II at 14 days. The gel electrophoresis results showed that the fragment of collagen II gene was seen in TGF-beta1 +IGF-1 group and TGF-beta1 group and that no fragment of collagen II gene was seen in IGF-1 group and control group. The expression of collagen II gene was stronger in TGF-beta1+ IGF-1 group than in TGF-beta1 group, showing significant difference (P<0. 05). Cells expressed more collagen II under pellet culture than under monolayer culture. CONCLUSION: IGF-1 could enhance the effect of TGF-beta1 during the induction course from MSCs to chondrocytes. A certain extent of high cell density is more effective for MSCs to differentiate into chondrocytes.