慢性腱病模型大鼠肌腱干细胞体外成脂和成肌腱的分化能力

背景：慢性腱病是一种常见的肌腱退行性病变，好发于运动员以及肌腱过度劳损的人群。由于慢性腱病的发病机制尚未阐明，临床上还缺乏有效的治疗手段。 目的：体外研究比较慢性腱病大鼠和正常大鼠来源肌腱干细胞成脂、成肌腱分化能力。 方法：从慢性腱病大鼠以及正常大鼠髌腱中分离培养原代肌腱干细胞，传代培养至第3代，体外观察细胞形态学变化。将两种来源肌腱干细胞（P3）单层培养至细胞融合，分为2组，成脂诱导组用成脂诱导培养基培养，对照组用基础培养基培养。成脂诱导分化21 d后，将两种来源肌腱干细胞的成脂诱导组和对照组分别行油红O染色定量分析。实时荧光定量PCR检测各组细胞成脂性基因C/EBPα和PPARγ2的mRNA的表达。将两种来源的肌腱干细胞体外单层培养至70%-80%融合时，行实时荧光定量PCR检测慢性腱病来源肌腱干细胞以及正常肌腱干细胞成肌腱相关基因Col1a1，Scx，Tnmd和Dcn的mRNA的表达。 结果与结论：肌腱干细胞体外培养至第3代时，正常肌腱来源的肌腱干细胞保持细长纺锤形的典型的干细胞形态，而慢性腱病来源的肌腱干细胞虽然形态发生改变，但仍保持纺锤形形态。肌腱干细胞（P3）体外成脂诱导分化21 d后，慢性腱病来源的肌腱干细胞胞体变大、变圆，可见大量油红O染色阳性的细胞，油红O染色阳性率显著高于正常大鼠（P=0.004）。实时荧光定量PCR结果显示，慢性腱病大鼠来源肌腱干细胞的成脂性基因（C/EBPα和PPARγ2）mRNA的表达均显著高于正常大鼠（P=0.004），肌腱特异性基因Col1a1，Scx， Tnmd以及Dcn mRNA表达量均明显低于正常大鼠（P =0.009）。说明与正常大鼠来源的肌腱干细胞相比，慢性腱病来源的肌腱干细胞体外成肌腱分化的能力减弱，而成脂分化的能力增强，此结果为进一步揭示慢性腱病发病机?

Adipogenic and tenogenic differentiation of tendon-derived stem cells isolated from an animal model of chronic tendinopathy in vitro

BACKGROUND：Chronic tendinopathy is a tendon disorder extremely common in athletes and in the general population with repetitive strain injuries of tendons. The pathogenesis of tendinopathy remains unclear and hence treatment of tendinopathy is usual y pal iative. 〈br〉 OBJECTIVE：To investigate the of adipogenic and tenogenic ability of patel ar tendon-derived stem cel s isolated 〈br〉 from chronic tendinopathy and healthy rats in vitro. 〈br〉 METHODS：Tendon-derived stem cel s were isolated from patel ar tendons of chronic tendinopathy and healthy rats respectively. The tendon-derived stem cel s were cultured to the 3rd passage in complete culture medium, and cel morphology was observed. The cel s were divided into adipogenic induction group and control group. Cel s in the adipogenic induction group were cultured in adipogenic induction medium, while those in the control group cultured in complete culture medium. The ability of adipogenic differentiation between tendon-derived stem cel s isolated from the tendon of chronic tendinopathy and healthy rats in vitro was examined by oil red O staining and quantification assay. The mRNA expressions of C/EBPαand PPARγ2 were detected by real-time quantitative PCR. When 70%-80%cel s were confluent, the mRNA expressions of Col1a1, Scx, Tnmd and Dcn were also detected by real-time quantitative PCR. 〈br〉 RESULTS AND CONCLUSION：At the third passage, slender spindle-shaped cel s were seen in both two groups, but there was a little change in the cel morphology in the chronic tendinopathy group. Lipid droplets were formed after the cel s were cultured in adipogenic induction medium for 21 days. This was not observed in the control group. We observed more oil red O-positive oil droplets in tendon-derived stem cel s from the tendons of chronic tendinopathy rats than healthy rats. The difference between them was statistical y significant （P=0.004）. The results of real-time quantitative PCR showed that the mRNA expressions of C/EBPαand PPARγ2 in the tendon