生长分化因子5诱导大鼠软骨前体细胞向成软骨方向分化

Growth differentiation factor 5 induces the chondrogenesis of precartilaginous stem cells in rats

背景:生长分化因子5和软骨前体细胞都在肢体纵向发育中发挥重要作用。目前尚无生长分化因子5对软骨前体细胞影响的文献报道。目的:实验创新性构思重组生长分化因子5干预软骨前体细胞,希望得到生长分化因子5诱导软骨前体细胞向软骨细胞分化的证据。设计、时间及地点:细胞形态学体外实验,于2007-11/2008-03在华中科技大学同济医学院附属同济医院矫形外科研究室完成。材料:纯系清洁级新生24h内的SD大鼠12只,用于制备软骨前体细胞。生长分化因子5为PeproTech公司产品。方法:免疫磁珠分离纯化软骨前体细胞。取第3代细胞,分别用含0,10,50和100μg/L的完全软骨形成培养基干预,诱导14d。对照组采用低糖DMEM培养基。主要观察指标:光镜观察细胞形态和生长增殖,四甲基偶氮唑盐检测细胞增殖活性,阿利新蓝染色蛋白聚糖,应用免疫组织化学和反转录-聚合酶链反应检测Ⅱ型胶原的表达。结果:分离纯化的软骨前体细胞贴壁牢固,生长旺盛,折光度好,光镜下呈多角形或梭形。软骨前体细胞的增殖在48h内呈剂量依赖性增高,100μg/L生长分化因子5组细胞的增殖率显著高于其他组(P<0.05)。诱导14d后阿利新蓝染色阳性,细胞外基质中有明显的蛋白聚糖形成。在生长分化因子5干预软骨前体细胞第7天,Ⅱ型胶原mRNA和Ⅱ型胶原蛋白出现表达,并且14d持续表达。结论:生长分化因子5能够促进软骨前体细胞的增殖分化。

BACKGROUND： Growth differentiation factor 5 （GDF-5） and precartilaginous stem cells （PSCs） both play an important role in the longitudinal growth of limbs. However, reports about GDF-5＇s effect on the differentiation of PSCs are still few. OBJECTIVE： To authenticate the inductive effect of GDF-5 on PSCs＇ differentiation into chondrocytes with an innovationally designed experiment of restructuring the GDF-5＇s intervention of PSCs. DESIGN, TIME AND SETTING： The cell morphology in vitro experiment was conducted in the Research Room of Orthopaedic Surgery, Tongji Hospital of Tongji Medical College of Huazhong University of Science and Technology from December 2007 to March 2008. MATERIALS： Twelve clean SD rats of less than 24 hours old were used for harvesting PSCs. GDF-5 were purchased from PeproTech Inc., USA. METHODS： PSCs were isolated and purified using immunomagnetic microbeads. At the third passage, PSCs were induced by complete chondrogenic medium containing 0,10, 50 and 100 μg/L GDF-5 for 14 days respectively. Cells cultured in the low-glucose DMEM served as controls. MAIN OUTCOME MEASURES： Light microscope was used to observe the morphous and proliferation of PSCs. Methyl thiazolyl tetrazolium （MTT） assay was used to detect the cell proliferation activity. Alcian bule was used to staine proteoglycan. Immunohistochemistry staining and reverse transcription-polymerase chain reaction was adopted to test the expression of type Ⅱ collagen. RESULTS： After being isolated and purified, PSCs attached the culture flask stably and exhibited polygon or fusiform shape under light microscope, with good growth condition and luminosity. The proliferation of PSCs was promoted by GDF-5 in a dose-dependent manner within 48 hours, and the close of 100 μg/L GDF-5 resulted in a higher proliferation activity of PSCs than the other doses （P 〈 0.05）. After 14 days of induction, alcian blue staining showed positive result, which indicated that there were proteoglycan formed obviously in the extracellular matrix of PSCs. Expression of type Ⅱ collagen mRNA and protein appeared since day 7 after induced by GDF-5, and continuously expressed till day 14. CONCLUSION： GDF-5 can promote the proliferation and differentiation of PSCs.