小鼠间充质干细胞三系分化中SOX9与Ⅱ型胶原mRNA的定量

Quantitative analysis of SOX9 and type Ⅱ collagen mRNA in the three-lineage differentiation of rat mesenchymal stem cells

背景:研究表明,软骨中的主要成分Ⅱ型胶原的基因-Col2a1在软骨细胞中的表达与SOX9的浓度呈剂量依赖正相关关系。目的:通过成骨、成软骨、成脂肪诱导干细胞分化,分析3种分化过程及不同时期的SOX9与Ⅱ型胶原mRNA含量的变化,探讨SOX9在不同时空分布的表达规律及与Ⅱ型胶原的相关关系。方法:取4周龄昆明小鼠骨髓间充质细胞,体外培养得到间充质干细胞并传达至第3代,对间充质干细胞进行流式细胞仪鉴定细胞表型,共分3组每组设3个时间段,通过成骨、成软骨、成脂肪3种诱导培养液对3组细胞进行诱导,另设不进行诱导的细胞作为对照组。分别在诱导3,7,14 d后收集提取细胞的总RNA,通过RT-PCR进行SOX9与Ⅱ型胶原的mRNA定量检测,同时对诱导后的细胞进行染色、免疫荧光染色,观察其分化状态及相关统计分析。结果与结论:第3代骨髓间充质干细胞生长良好,流式细胞仪鉴定细胞表型证实为干细胞,对诱导后细胞进行染色、免疫荧光染色结果证实细胞分化为骨、软骨、脂肪细胞。经RT-PCR检测,在3组诱导分化细胞中SOX9 mRNA含量由高到低分别是成软骨、成骨、成脂肪,Ⅱ型胶原mRNA含量由高到低分别是成软骨、成脂肪、成骨。在成软骨分化中SOX9在3,7 d表达不断升高,14 d呈下降趋势。Ⅱ型胶原在3,7,14 d均逐渐升高。在成骨分化中SOX9 mRNA含量随着时间推移而增加,而Ⅱ型胶原则随着时间推移而不断降低。在成脂肪分化中SOX9 mRNA表达与对照组比较差异无显著性意义(P>0.05);而Ⅱ型胶原的表达没有规律可循,时间点的延伸及检测未观察到。结果提示,SOX9在软骨分化中作用优于成骨、成脂肪组,且软骨分化中SOX9与Ⅱ型胶原存在相关性,可能在软骨分化的早期Ⅱ型胶原随着SOX9的变化而变化;且软骨分化和成骨分化过程中SOX9可能起到了一个互相协调促进平衡的关键作用。

BACKGROUND： The main component of cartilage, type Ⅱ collagen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner. OBJECTIVE： To observe the variation of SOX9 and typeⅡ collagen mRNA content at different periods in the differentiation process （osteogenic, chondrogenic, adipogenic induction） of mesenohymal stem cells, and to explore the correlation of SOX9 expression and type Ⅱ collagen.METHODS： Bone marrow mesenchymal stem cells were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cell phenotype was identified with flow cytometry. Cells were divided into three groups and subjected to three kinds of induction conditions favorable for adipogenic, chondrogenic and osteogenic differentiation, and each group was observed at three time points. In addition, the non-induced cells were used as a control group. The total RNA of cells was extracted at 3, 7, 14 days after induction, and SOX9 and type II collagen mRNA was quantifiedwith reverse transcription-polymerase chain reaction. The induced cells were stained by immunofluorescence to observe the differentiation and perform statistical analysis. RESULTS AND CONCLUSION： Passage 3 bone marrow mesenchymal stem cells grew well, and cell phenotype was confirmed as stem cells by flow cytometry. The staining results showed that, the cells differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cells were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type II collagen mRNA levels in the induced cells were the highest in chondrogenic differentiation group, then in adipogenic differentiation group, and the lowest in osteogenic differentiation group. SOX9 expression in chondrogenic differentiation group increased at 3 and 7 days, and then decreased at 14 days. While type Ⅱ collagen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ collagen expression gradually decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group （P 〉 0.05）, while type Ⅱ collagen expression was not regularly changed. Experimental findings suggest that, critical effect of SOX9 in chondrogenic differentiation is better than that in osteogenic and adipogenic differentiation. SOX9 is associated with type II collagen, which may alter along with the SOX9 in the early chondrogenic differentiation; SOX9 may play a fine-tuning role in the process of chondrogenic and osteogenic differentiation.