转化生长因子β1联合软骨源性形态发生蛋白-2体外诱导成肌细胞向软骨细胞的分化  

Chondrogenesis of Canine Myoblast in vitro Induced by TGF-β1 and CDMP-2

作　　者：顾羊林[1] 朱国兴[1] 施克勤[1] 杨玉生[1] 陈鹏[1] 朱文辉[2] 王予彬[2] 

机构地区：[1]江苏省无锡市第二人民医院骨科,江苏无锡214002 [2]上海同济大学附属东方医院运动创伤关节外科

出　　处：《中国运动医学杂志》2013年第1期34-40,共7页Chinese Journal of Sports Medicine

基　　金：上海市自然科学基金项目(09ZR1425500)

摘　　要：目的:探讨转化生长因子β1(TGF-β1)联合软骨源性形态发生蛋白-2(CDMP-2)体外诱导犬成肌细胞向软骨细胞表型定向分化的可行性。方法:取成年比格犬大腿股直肌肌肉,以机械分解法与二步酶消化法分离获得成肌细胞,取第4代成肌细胞,以2.0×104细胞/cm2的密度接种于24孔培养板,使用含CDMP-2和(或)TGF-β1的完全培养液诱导分化成肌细胞,观察细胞生长情况。诱导14 d后获取细胞,进行HE染色、甲苯胺蓝染色,观察细胞形态及胞内高硫酸化的蛋白聚糖变化。免疫细胞化学染色检测II型胶原蛋白着色情况。RT-PCR检测I型、II型胶原蛋白、aggrecan及Sox9 mRNA表达。阿尔新蓝法定量检测细胞糖胺聚糖(GAG)含量。结果:3个加入细胞因子的实验组成肌细胞形态由长梭形向多角形、类圆形转变;HE染色、甲苯胺蓝染色和软骨特异性I型和Ⅱ型胶原免疫组化法检测及RT-PCR检测中,实验组均有不同程度的阳性表达;TGF-β1与CDMP-2联合应用组糖胺聚糖含量明显高于其他三组(P<0.05)。结论:CDMP-2和TGF-β1可以单独诱导高密度单层培养的犬成肌细胞向软骨细胞分化,表达软骨细胞特异性细胞表型,并且CDMP-2和TGF-β1有协同作用。Objective To explore the possibility of chondrogenesis of canine myoblast induced by CDMP-2 and TGF-β1 in vitro. Methods The muscle from the hindlimb of adult Beagle was obtained, and the skeletal myoblasts were isolated and harvested by mechanical decomposition and two-step enzyme digestion. Myoblasts of the fourth passage were digested and incubated in a medium containing CDMP-2 and（or） TGF-β1 in a 24-wetl plate at a density of 2.0×10^4/cm^2. After 14 days of inducement, the cells were harvested and stained by haematoxylin-eosin （HE）and toluidine blue, and changes in cell histology and proteoglycane were observed. Immunohistoehemistry was used to evaluate the monoclonal antibody specific to collagen I and collagen II. RT-PCR was used to detect the expression of collagen Ⅰ, collagen II,aggrecan,and Sox9 mRNA. Glycosaminoglyean （GAG） quantitative analysis was performed using Aleian Blue. Results The appearance of canine myoblasts changed from a spindle-like into apolygonal shape after induction. The chondrocytes could express GAG and collagen Ⅰ, collagen Ⅱ. The expression of collagen Ⅰ,collagen Ⅱ,aggrecan and Sox9 mRNA in group with combination of TGF-β1 and CDMP-2 were significantly higher than that in control group,and in group with only TGF-β1 or CDMP-2（P 〈 0.05 ）. Conclusion CDMP-2 induces chondrogenic differentiation of canine myoblasts,and the effect of combined use of CDMP-2 and TGF-β1 are more significant than single use of CDMP-2 in vitro.

关 键 词：成肌细胞 细胞因子 软骨分化 种子细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]