CAJ | 학술논문

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。揹景：研究錶明，軟骨中的主要成分Ⅱ型膠原的基因-Col2a1在軟骨細胞中的錶達與SOX9的濃度呈劑量依賴正相關關繫。 　　目的：通過成骨、成軟骨、成脂肪誘導榦細胞分化，分析3種分化過程及不同時期的SOX9與Ⅱ型膠原 mRNA含量的變化，探討SOX9在不同時空分佈的錶達規律及與Ⅱ型膠原的相關關繫。 　　方法：取4週齡昆明小鼠骨髓間充質細胞，體外培養得到間充質榦細胞併傳達至第3代，對間充質榦細胞進行流式細胞儀鑒定細胞錶型，共分3組每組設3箇時間段，通過成骨、成軟骨、成脂肪3種誘導培養液對3組細胞進行誘導，另設不進行誘導的細胞作為對照組。分彆在誘導3，7，14 d後收集提取細胞的總RNA，通過RT-PCR進行SOX9與Ⅱ型膠原的mRNA定量檢測，同時對誘導後的細胞進行染色、免疫熒光染色，觀察其分化狀態及相關統計分析。 　　結果與結論：第3代骨髓間充質榦細胞生長良好，流式細胞儀鑒定細胞錶型證實為榦細胞，對誘導後細胞進行染色、免疫熒光染色結果證實細胞分化為骨、軟骨、脂肪細胞。經RT-PCR檢測，在3組誘導分化細胞中SOX9 mRNA 含量由高到低分彆是成軟骨、成骨、成脂肪，Ⅱ型膠原 mRNA 含量由高到低分彆是成軟骨、成脂肪、成骨。在成軟骨分化中SOX9在3，7 d錶達不斷升高，14 d呈下降趨勢。Ⅱ型膠原在3，7，14 d均逐漸升高。在成骨分化中SOX9 mRNA含量隨著時間推移而增加，而Ⅱ型膠原則隨著時間推移而不斷降低。在成脂肪分化中SOX9 mRNA錶達與對照組比較差異無顯著性意義(P >0.05)；而Ⅱ型膠原的錶達沒有規律可循，時間點的延伸及檢測未觀察到。結果提示，SOX9在軟骨分化中作用優于成骨、成脂肪組，且軟骨分化中SOX9與Ⅱ型膠原存在相關性，可能在軟骨分化的早期Ⅱ型膠原隨著SOX9的變化而變化；且軟骨分化和成骨分化過程中SOX9可能起到瞭一箇互相協調促進平衡的關鍵作用。
배경：연구표명，연골중적주요성분Ⅱ형효원적기인-Col2a1재연골세포중적표체여SOX9적농도정제량의뢰정상관관계。 　　목적：통과성골、성연골、성지방유도간세포분화，분석3충분화과정급불동시기적SOX9여Ⅱ형효원 mRNA함량적변화，탐토SOX9재불동시공분포적표체규률급여Ⅱ형효원적상관관계。 　　방법：취4주령곤명소서골수간충질세포，체외배양득도간충질간세포병전체지제3대，대간충질간세포진행류식세포의감정세포표형，공분3조매조설3개시간단，통과성골、성연골、성지방3충유도배양액대3조세포진행유도，령설불진행유도적세포작위대조조。분별재유도3，7，14 d후수집제취세포적총RNA，통과RT-PCR진행SOX9여Ⅱ형효원적mRNA정량검측，동시대유도후적세포진행염색、면역형광염색，관찰기분화상태급상관통계분석。 　　결과여결론：제3대골수간충질간세포생장량호，류식세포의감정세포표형증실위간세포，대유도후세포진행염색、면역형광염색결과증실세포분화위골、연골、지방세포。경RT-PCR검측，재3조유도분화세포중SOX9 mRNA 함량유고도저분별시성연골、성골、성지방，Ⅱ형효원 mRNA 함량유고도저분별시성연골、성지방、성골。재성연골분화중SOX9재3，7 d표체불단승고，14 d정하강추세。Ⅱ형효원재3，7，14 d균축점승고。재성골분화중SOX9 mRNA함량수착시간추이이증가，이Ⅱ형효원칙수착시간추이이불단강저。재성지방분화중SOX9 mRNA표체여대조조비교차이무현저성의의(P >0.05)；이Ⅱ형효원적표체몰유규률가순，시간점적연신급검측미관찰도。결과제시，SOX9재연골분화중작용우우성골、성지방조，차연골분화중SOX9여Ⅱ형효원존재상관성，가능재연골분화적조기Ⅱ형효원수착SOX9적변화이변화；차연골분화화성골분화과정중SOX9가능기도료일개호상협조촉진평형적관건작용。
BACKGROUND:The main component of cartilage, type Ⅱ col agen gene expression in chondrocyte is positively correlated with SOX9 concentration in a dose-dependent manner. OBJECTIVE:To observe the variation of SOX9 and type Ⅱ col agen mRNA content at different periods in the differentiation process (osteogenic, chondrogenic, adipogenic induction) of mesenchymal stem cel s, and to explore the correlation of SOX9 expression and type Ⅱ col agen. METHODS:Bone marrow mesenchymal stem cel s were isolated from 4-week-old Kunming mice, and cultured in vitro to passage 3. The cel phenotype was identified with flow cytometry. Cel s 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 cel s were used as a control group. The total RNA of cel s was extracted at 3, 7, 14 days after induction, and SOX9 and type Ⅱ col agen mRNA was quantified with reverse transcription-polymerase chain reaction. The induced cel s were stained by immunofluorescence to observe the differentiation and perform statistical analysis. RESULTS AND CONCLUSION:Passage 3 bone marrow mesenchymal stem cel s grew wel , and cel phenotype was confirmed as stem cel s by flow cytometry. The staining results showed that, the cel s differentiated into chondrocytes, adipocytes and osteoblasts. The SOX9 mRNA levels in the induced cel s were the highest in chondrogenic differentiation group, then in osteogenic differentiation group, and the lowest in adipogenic differentiation group. Type Ⅱ col agen mRNA levels in the induced cel s 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 col agen expression increased at 3, 7, 14 days. SOX9 mRNA levels increased as the osteogenic differentiation, while type Ⅱ col agen expression gradual y decreased. There was no significant difference in the SOX9 mRNA expression between adipogenic differentiation group and control group (P>0.05), while type Ⅱ col agen 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 Ⅱcol agen, 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.