CAJ | 학술논문

背景：目前临床对于慢性腱病缺乏有效的治疗手段，原因在于其发病机制至今尚未阐明。 　　目的：研究体外骨形态发生蛋白2对胶原酶诱导的大鼠慢性腱病模型髌腱来源肌腱干细胞的成骨、成软骨分化的作用。 　　方法：从大鼠慢性腱病模型的髌腱中分离培养出原代肌腱干细胞，传代培养至第3代细胞，行成骨、成脂、成软骨诱导分化鉴定其干细胞的特性。将肌腱干细胞(P3)单层培养至细胞融合，用重组人骨形态发生蛋白2干预。7 d后分别行茜素红染色，并行茜素红染色定量分析。将肌腱干细胞体外三维微球培养后分为2组，诱导组用重组人骨形态发生蛋白2干预，对照组不进行干预。21 d后三维微球行苏木精-伊红染色，阿利辛蓝染色以及Sox9和Ⅱ型胶原免疫组织化学染色。 　　结果与结论：慢性腱病大鼠来源原代肌腱干细胞体外培养呈克隆样集落生长，传代后细胞主要表现为多突的纺锤形和星形的扁平细胞，具有成纤维细胞样的特征。肌腱干细胞(P3)成脂诱导10 d，油红O染色阳性；成骨诱导7 d，茜素红染色阳性；成软骨诱导14 d，苏木精-伊红染色阳性可见软骨样细胞，Ⅱ型胶原免疫组化染色阳性。单层培养的肌腱干细胞用重组人骨形态发生蛋白2诱导7 d茜素红染色阳性，对照组为阴性，茜素红染色定量检测显示差异有显著性意义。重组人骨形态发生蛋白2诱导肌腱干细胞21 d，苏木精-伊红染色可见软骨样细胞形成、阿利辛蓝染色可见细胞内糖胺多糖沉积、Sox9和Ⅱ型胶原免疫组织化学染色均呈阳性。可见体外重组人骨形态发生蛋白2可以诱导慢性腱病来源的肌腱干细胞成骨、成软骨分化。这为进一步研究慢性腱病的发病机制提供了细胞生物学依据。
배경：목전림상대우만성건병결핍유효적치료수단，원인재우기발병궤제지금상미천명。 　　목적：연구체외골형태발생단백2대효원매유도적대서만성건병모형빈건래원기건간세포적성골、성연골분화적작용。 　　방법：종대서만성건병모형적빈건중분리배양출원대기건간세포，전대배양지제3대세포，행성골、성지、성연골유도분화감정기간세포적특성。장기건간세포(P3)단층배양지세포융합，용중조인골형태발생단백2간예。7 d후분별행천소홍염색，병행천소홍염색정량분석。장기건간세포체외삼유미구배양후분위2조，유도조용중조인골형태발생단백2간예，대조조불진행간예。21 d후삼유미구행소목정-이홍염색，아리신람염색이급Sox9화Ⅱ형효원면역조직화학염색。 　　결과여결론：만성건병대서래원원대기건간세포체외배양정극륭양집락생장，전대후세포주요표현위다돌적방추형화성형적편평세포，구유성섬유세포양적특정。기건간세포(P3)성지유도10 d，유홍O염색양성；성골유도7 d，천소홍염색양성；성연골유도14 d，소목정-이홍염색양성가견연골양세포，Ⅱ형효원면역조화염색양성。단층배양적기건간세포용중조인골형태발생단백2유도7 d천소홍염색양성，대조조위음성，천소홍염색정량검측현시차이유현저성의의。중조인골형태발생단백2유도기건간세포21 d，소목정-이홍염색가견연골양세포형성、아리신람염색가견세포내당알다당침적、Sox9화Ⅱ형효원면역조직화학염색균정양성。가견체외중조인골형태발생단백2가이유도만성건병래원적기건간세포성골、성연골분화。저위진일보연구만성건병적발병궤제제공료세포생물학의거。
BACKGROUND:The pathogenesis of tendinopathy remains unclear and hence treatment of tendinopathy is usualy paliative. OBJECTIVE:To investigate the effects of bone morphogenetic protein 2 on the osteogenic and chondrogenic differentiation of patelar tendon-derived stem cels isolated from colagenase-induced tendinopathy ratsin vitro. METHODS: Patelar tendon-derived stem cels were isolated from patelar tendons of colagenase-induced tendinopathy rats. The multi-differentiation potential of patelar tendon-derived stem cels at passage 3 was identified by osteogenic, adipogenic and chondrogenic differentiation assays. The patelar tendon-derived stem cels were cultured to the 3rd passage in complete culture medium, and then the cels were divided into two groups with (experimental group) or without recombinant human bone morphogenetic protein 2 (control group) until the cels reached confluence for 7 days. Their osteogenic response to bone morphogenetic protein 2in vitro was examined by alizarin red S staining of calciumnodule formation and quantification assay. The patelar tendon-derived stem cellpelets were cultured in complete culture medium with (experimental group) or without bone morphogenetic protein 2 (control grup) for 21 days. Chondrogenic differentiation of the cellpelets was evaluated by hematoxylin-eosin staining, alcian blue staining, immunohistochemical staining for Sox9 and colagen type II. RESULTS AND CONCLUSION:Primary patelar tendon-derived stem cels from the tendinopathy rats culturedin vitro showed clonal growth; after passage, spindle fibroblast-like and flat-like cels were detectable. The cels were positive for oil red O staining at 10 days after adipogenic induction, positive for alizarin red staining at 7 days after osteogenic induction, and positive for hematoxylin-eosin staining and immunohistochemical staining of colagen type II at 14 days after chondrogenic induction. After patelar tendon-derived stem cels were induced with recombinant human bone morphogenetic protein 2 for 7 days, the result of alizarin red staining was positive in the experimental group, but negative in the control group without recombinant human bone morphogenetic protein 2. The difference in the result of alizarin red staining between the two groups was statisticaly significant. After patelar tendon-derived stem cels were induced with recombinant human bone morphogenetic protein 2 for 21 days, the results of hematoxylin-eosin staining, alcian blue staining, immunohistochemical staining for Sox9 and colagen type II were al positive. In conclusion, bone morphogenetic protein 2 could stimulate the osteogenic and chondrogenic differentiation of patelar tendon-derived stem cels isolated from colagenase-induced tendinopathy rats in vitro, which can help to better understand the pathogenesis of tendinopathy.