CAJ | 학술논문

干细胞%分化%肌腱干细胞%慢性腱病%髌腱%成脂分化%成肌腱分化%油红O染色%实时定量PCR%成脂性基因(C/EBPα%PPARγ2)%肌腱特异性基因(Col1a1%Scx%Tnmd以及Dcn)%国家自然科学基金榦細胞%分化%肌腱榦細胞%慢性腱病%髕腱%成脂分化%成肌腱分化%油紅O染色%實時定量PCR%成脂性基因(C/EBPα%PPARγ2)%肌腱特異性基因(Col1a1%Scx%Tnmd以及Dcn)%國傢自然科學基金
간세포%분화%기건간세포%만성건병%빈건%성지분화%성기건분화%유홍O염색%실시정량PCR%성지성기인(C/EBPα%PPARγ2)%기건특이성기인(Col1a1%Scx%Tnmd이급Dcn)%국가자연과학기금
tissue engineering%stem cel s%tendinopathy%rats

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制提供了细胞生物学依据。揹景：慢性腱病是一種常見的肌腱退行性病變，好髮于運動員以及肌腱過度勞損的人群。由于慢性腱病的髮病機製尚未闡明，臨床上還缺乏有效的治療手段。 　　目的：體外研究比較慢性腱病大鼠和正常大鼠來源肌腱榦細胞成脂、成肌腱分化能力。 　　方法：從慢性腱病大鼠以及正常大鼠髕腱中分離培養原代肌腱榦細胞，傳代培養至第3代，體外觀察細胞形態學變化。將兩種來源肌腱榦細胞(P3)單層培養至細胞融閤，分為2組，成脂誘導組用成脂誘導培養基培養，對照組用基礎培養基培養。成脂誘導分化21 d後，將兩種來源肌腱榦細胞的成脂誘導組和對照組分彆行油紅O染色定量分析。實時熒光定量PCR檢測各組細胞成脂性基因C/EBPα和PPARγ2的mRNA的錶達。將兩種來源的肌腱榦細胞體外單層培養至70%-80%融閤時，行實時熒光定量PCR檢測慢性腱病來源肌腱榦細胞以及正常肌腱榦細胞成肌腱相關基因Col1a1，Scx，Tnmd和Dcn的mRNA的錶達。 　　結果與結論：肌腱榦細胞體外培養至第3代時，正常肌腱來源的肌腱榦細胞保持細長紡錘形的典型的榦細胞形態，而慢性腱病來源的肌腱榦細胞雖然形態髮生改變，但仍保持紡錘形形態。肌腱榦細胞(P3)體外成脂誘導分化21 d後，慢性腱病來源的肌腱榦細胞胞體變大、變圓，可見大量油紅O染色暘性的細胞，油紅O染色暘性率顯著高于正常大鼠(P=0.004)。實時熒光定量PCR結果顯示，慢性腱病大鼠來源肌腱榦細胞的成脂性基因(C/EBPα和PPARγ2)mRNA的錶達均顯著高于正常大鼠(P=0.004)，肌腱特異性基因Col1a1，Scx， Tnmd以及Dcn mRNA錶達量均明顯低于正常大鼠(P =0.009)。說明與正常大鼠來源的肌腱榦細胞相比，慢性腱病來源的肌腱榦細胞體外成肌腱分化的能力減弱，而成脂分化的能力增彊，此結果為進一步揭示慢性腱病髮病機製提供瞭細胞生物學依據。
배경：만성건병시일충상견적기건퇴행성병변，호발우운동원이급기건과도로손적인군。유우만성건병적발병궤제상미천명，림상상환결핍유효적치료수단。 　　목적：체외연구비교만성건병대서화정상대서래원기건간세포성지、성기건분화능력。 　　방법：종만성건병대서이급정상대서빈건중분리배양원대기건간세포，전대배양지제3대，체외관찰세포형태학변화。장량충래원기건간세포(P3)단층배양지세포융합，분위2조，성지유도조용성지유도배양기배양，대조조용기출배양기배양。성지유도분화21 d후，장량충래원기건간세포적성지유도조화대조조분별행유홍O염색정량분석。실시형광정량PCR검측각조세포성지성기인C/EBPα화PPARγ2적mRNA적표체。장량충래원적기건간세포체외단층배양지70%-80%융합시，행실시형광정량PCR검측만성건병래원기건간세포이급정상기건간세포성기건상관기인Col1a1，Scx，Tnmd화Dcn적mRNA적표체。 　　결과여결론：기건간세포체외배양지제3대시，정상기건래원적기건간세포보지세장방추형적전형적간세포형태，이만성건병래원적기건간세포수연형태발생개변，단잉보지방추형형태。기건간세포(P3)체외성지유도분화21 d후，만성건병래원적기건간세포포체변대、변원，가견대량유홍O염색양성적세포，유홍O염색양성솔현저고우정상대서(P=0.004)。실시형광정량PCR결과현시，만성건병대서래원기건간세포적성지성기인(C/EBPα화PPARγ2)mRNA적표체균현저고우정상대서(P=0.004)，기건특이성기인Col1a1，Scx， Tnmd이급Dcn mRNA표체량균명현저우정상대서(P =0.009)。설명여정상대서래원적기건간세포상비，만성건병래원적기건간세포체외성기건분화적능력감약，이성지분화적능력증강，차결과위진일보게시만성건병발병궤제제공료세포생물학의거。
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. OBJECTIVE:To investigate the of adipogenic and tenogenic ability of patel ar tendon-derived stem cel s isolated from chronic tendinopathy and healthy rats in vitro. 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. 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-derived stem cel s from the tendons of chronic tendinopathy rats were significantly higher than those in tendon-derived stem cel s from the tendons of healthy rats (P=0.004);the mRNA expressions of Col1a1, Scx, Tnmd and Dcn in the tendon-derived stem cel s from the tendons of chronic tendinopathy rats were significantly lower than those in tendon-derived stem cel s from the tendons of healthy rats (P=0.009). In conclusion, tendon-derived stem cel s from chronic tendinopathy rats showed a higher ability of adipogenic differentiation, but a lower capacity of tenogenic differentiation compared to tendon-derived stem cel s from healthy rats, which might contribute to better understand the pathogenesis of tendinopathy.