体外定向诱导鹿茸间充质干细胞向软骨细胞的分化

Differentiation of antler mesenchymal stem cells into chondrocytes in vitro

目的:间充质干细胞的诱导分化的研究多见于骨髓、脐血等组织,而在鹿茸组织中分离的间充质干细胞是否能诱导为软骨细胞目前尚不清楚。实验拟建立梅花鹿鹿茸生长中心间充质干细胞体外培养方法,观察转化生长因子β1体外诱导鹿茸间充质细胞分化为软骨细胞的可行性。方法:实验于2006-03/2007-06在吉林大学畜牧兽医学院基础兽医研究室完成。①实验材料:4岁龄健康雄性梅花鹿由中国农科院左家特产研究所提供,实验过程中对动物处置符合动物伦理学标准。②实验方法:于生长早期锯取梅花鹿鹿茸,在解剖显微镜下定位和切取间质层(突起部),即为鹿茸间充质干细胞所在的组织层。Ⅰ型胶原酶消化法原代分离培养鹿茸生长中心间充质干细胞,锥虫蓝染色显示细胞活性达90%以上,将活性确定后的细胞液氮冻存。取第3代细胞,用含10%胎牛血清以及10μg/L转化生长因子β1的条件培养基诱导培养。③实验评估:培养12d后于倒置显微镜下观察细胞形态,采用细胞化学及免疫细胞化学鉴定细胞。结果:①鹿茸生长中心间充质干细胞的分离与培养:Ⅰ型胶原酶消化法培养可以获得均一的间充质干细胞,贴壁的间充质干细胞形态均匀,呈长梭形,克隆样生长,增殖迅速。②转化生长因子β1体外定向诱导鹿茸间充质细胞分化为软骨细胞:转化生长因子β1诱导分化的鹿茸间充质细胞生长迅速,诱导后细胞形态明显改变,呈典型的软骨细胞形态,甲苯胺蓝染色阳性,Ⅱ型胶原表达阳性。结论:采用酶消化法可以从鹿茸生长中心间充质层中分离出间充质干细胞,在体外转化生长因子β1具有促进鹿茸生长中心间充质干细胞分化为软骨细胞的能力。

AIM： Studies on differentiation of mesenchymal stem cells into chondrocytes was common in vitro in the bone marrow, cord blood and so on, whereas if mesenchymal stem cells in the antler tissues could differentiated into chondrocyte is unknown. This article investigates the feasibility of antler organic center mesenchymal stem cells into chondrocyte lineage by transforming growth factor- β1 in vitro. METHODS： Experiments were carried out at Basal Veterinary Research Institute of College of Animal Science and Veterinary Medicine of Jilin University from March 2006 to June 2007. ①The healthy 4-years-old sika deer stag was provided by Institute of Speciality in Zuojia of Chinese Academy of Agriculture Science. Experiments were performed in accordance with the animal ethical standards. ②The calf was slaughtered when antler grow at earlier stage. Mesenchyme layer （evection part, the antler organic center cells layer） was taken under anatomecal microscope. The antler organic center cells were isolated from sika deer antler and cultured by digested with collagenase Ⅰ . Cell viability was always above 90%, measured using Trypan blue, and then the viable cells were frozen in liquid nitrogen. The third passage cells were incubated and differentiated into chondrocytes induced by cultivation of confluent cells in the presence of 10% fetal calf serum and 10 μ g/mL transforming growth factor- β 1. ③Twelve days later, cell appearance was observed under an invert microscope and cells were aidentified by the cytochemical and immunocytochemical methods. RESULTS：①Isolation and culture of antler organic center mesenchymal stem cells： The primary culture way purified by digested with collagenase Ⅰ could obtain homogeneous antler stem cells. Cultured antler mesenchymal stem cells were uniformly spindle-shaped and adhered to plastic surface, the cloning growth of the antler organic center mesenchymal stem cells proliferated rapidly. ②Differentiation of antler mesenchymal stem cells into chondrocytes by transforming growth factor- β1 in vitro： Induced antler mesenchymal stem cells proliferated rapidly, morphous of cells were fundamentally different and had typical appearance of chondrocytes. The toluidine blue heterochromia of the induced cells was very evident, the expression of protein of collagen type Ⅱ were positive. CONCLUSION： The antler mesenchymal stem cells can be separated from antler organic center by digested with collagenase Ⅰ. Transforming growth factor- β1 has the ability to promote antler organic center mesenchymal stem cells differentiating into chondrocyte lineage in vitro.