BMP-2诱导人跟腱来源肌腱干细胞成软骨分化的实验研究

EXPERIMENT OF BONE MORPHOGENETIC PROTEIN 2 INDUCED CHONDROGENIC DIFFERENTIATION OF HUMAN Achilles TENDON-DERIVED STEM CELLS IN VITRO

目的探讨BMP-2体外诱导人跟腱来源肌腱干细胞(human Achilles tendon-derived stem cells,hATDSCs)成软骨分化的作用。方法无菌条件下取急性跟腱断裂患者自愿捐赠的跟腱组织,胶原酶消化获得有核细胞;以500个/cm2细胞密度接种,细胞贴壁呈克隆样生长,混合培养获得原代hATDSCs;体外传代培养至第3代,流式细胞仪检测hATDSCs免疫表型;油红O染色、茜素红染色及番红O/快绿染色分别鉴定hATDSCs成脂、成骨及成软骨分化能力。取第3代细胞体外三维培养成微球后分为两组,实验组用重组人BMP-2(recombinant human BMP-2,rhBMP-2)干预,对照组不进行干预。3周后行微球大体观察;组织学切片行HE染色、番红O/快绿染色、Ⅱ型胶原免疫组织化学染色观察;实时荧光定量PCR检测成软骨特异性基因SOX9、Ⅱ型胶原和软骨聚集蛋白多糖(Aggrecan)表达。结果原代hATDSCs体外培养呈克隆样集落生长;传代后以纺锤形、成纤维样细胞生长,形态均一。hATDSCs阳性表达MSCs特异性表面标志CD44、CD90、CD105,阴性表达CD34、CD45和CD146。多向分化潜能鉴定示hATDSCs成脂诱导3周油红O染色阳性,成骨诱导4周茜素红染色阳性,成软骨诱导3周番红O/快绿染色阳性。rhBMP-2诱导hATDSCs 3周,实验组微球形成,体积显著大于对照组;HE染色、番红O/快绿染色、Ⅱ型胶原免疫组织化学染色均呈阳性,实时荧光定量PCR检测示SOX9、Ⅱ型胶原和Aggrecan mRNA表达显著高于对照组(P<0.05)。结论体外BMP-2可促进hATDSCs成软骨分化和蛋白聚糖合成增加,为研究慢性腱病组织病理学变化提供细胞生物学依据。

Objective To investigate the effects of bone morphogenetic protein 2 （BMP-2） on the chondrogenic differentiation of human Achilles tendon-derived stem cells （hATDSCs） in vitro. Methods Achilles tendon was harvested from a voluntary donor with acute Achilles tendon rupture. And nucleated cells were obtained by digesting with collagenase and were cultured to the 3rd passage. The flow cytometry was used to measure the immunophenotyping; and Oil red O staining, alizarin red staining, and Safranin O/fast green staining were used to identify the adipogenic differentiation, osteogenic differentiation, and chondrogenic differentiation, respectively. The hATDSCs pellet was cultured in complete culture medium with （experimental group） or without recombinant human BMP-2 （rhBMP-2） （control grup） for 3 weeks. Chondrogenic differentiation of hATDSCs was evaluated by HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II; and the mRNA expressions of soxg, collagen type II, and Aggrecan were detected by real-time fluorescence quantitative PCR. Results Primary hATDSCs cultured in vitro showed clonal growth; after cell passage, homogeneous spindle fibroblast- like cells were seen. The cells were positive for CD44, CD90, and CD105, while negative for CD34, CD45, and CD146. The results were positive for Oil red O staining at 3 weeks after adipogenic differentiation, for alizarin red staining at 4 weeks after osteogenic differentiation, and for Safranin O/fast green staining at 3 weeks after chondrogenic differentiation. After hATDSCs were induced with rhBMP-2 for 3 weeks, pellets formed in the experimental group, and the size of pellets was significantly larger than that in the control group; the results of HE staining, Safranin O/fast green staining, and immunohistochemical staining for collagen type II were all positive. The results of real-time fluorescence quantitative PCR showed that the mRNA expressions of SOX9, collagen type II, and Aggrecan in the experimental group were significantly higher than those in the control group （P 〈 0.05）. Conclusion BMP-2 can promote proteoglycan deposition and induce chondrogenic differentiation of hATDSCs in vitro. The effect of BMP-2 on hATDSCs might provide a possible explanation for histopathological changes oftendinopathy.