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目的探讨应用生长分化因子5(growth differentiation factor5,GDF-5)诱导脂肪干细胞(adipose-derived stem cells,ADSCs)成软骨细胞分化的可能性及效果。方法3月龄清洁级健康日本大耳白兔6只,雌雄不限,体重2～3kg。取兔皮下脂肪4～6mL,采用胶原酶消化离心贴壁培养法培养ADSCs,取第3代细胞进行实验。波形蛋白免疫组织化学和CD44、CD49d、CD106免疫荧光染色鉴定ADSCs。调整细胞密度为1×106个/mL,分别用普通细胞培养液以及含0、10、100ng/mLGDF-5的软骨细胞诱导液诱导培养。倒置相差显微镜观察细胞形态变化;MTT法检测细胞增殖情况;RT-PCR检测诱导细胞ColⅡ和蛋白多糖mRNA的表达;免疫组织化学、阿利新蓝染色、甲苯胺蓝染色和Westernblot法检测诱导细胞ColⅡ和蛋白多糖表达。结果ADSCs呈小圆形、梭形、多角形分布,表面抗原标志CD44、CD49d呈阳性表达,CD106和波形蛋白呈阴性表达。100ng/mLGDF-5诱导的ADSCs呈圆形或类圆形,且细胞增殖旺盛。普通培养液和0、10、100ng/mLGDF-5成软骨细胞诱导培养后7d,ColⅡ、蛋白多糖mRNA表达均呈浓度依赖性增加,除0、10ng/mLGDF-5两组间差异无统计学意义(P>0.05)外,其余各组差异均有统计学意义(P<0.05);100ng/mLGDF-5诱导培养14d,ColⅡ、蛋白多糖mRNA以及蛋白表达达高峰,阿利新蓝、甲苯胺蓝染色以及ColⅡ免疫组织化学染色均呈阳性。结论经一定浓度的GDF-5诱导的ADSCs,ColⅡ和蛋白多糖表达明显增加,具有软骨细胞的部分生物学功能。
Objective To investigate the possibility and effect of inducing adipose-derived stem cells (ADSCs) into chondrocytes by using growth differentiation factor 5 (GDF-5). Methods 6-month-old Japanese white-ear rabbits with clean grade were males and females, weighing 2-3 kg. Rabbit subcutaneous fat 4 ~ 6mL, using collagenase digestion adherent culture cultured ADSCs, take the third generation of cells for experiments. Vimentin immunohistochemistry and CD44, CD49d, CD106 immunofluorescence staining identified ADSCs. The cell density was adjusted to 1 × 10 6 cells / mL, and induced by chondrocyte induction liquid with ordinary cell culture medium and 0, 10, 100 ng / mL GDF-5 respectively. The morphological changes of cells were observed by inverted phase contrast microscope. The proliferation of cells was detected by MTT assay. The mRNA expression of ColⅡ and proteoglycan were detected by RT-PCR. The expressions of ColⅡ and Proteoglycan mRNA were detected by immunohistochemical staining, Alcian blue staining, toluidine blue staining and Western blotting. Proteoglycan expression. Results ADSCs were small round, spindle-shaped, polygonal distribution, the surface antigen markers CD44, CD49d positive expression, CD106 and vimentin negative expression. ADSCs induced by 100ng / mLGDF-5 were round or round, and the cell proliferation was strong. After induced by 0,10,100ng / mL GDF-5 chondrocytes, the mRNA expression of ColⅡ and proteoglycan increased in a concentration-dependent manner, except for 0,10ng / mL GDF-5, there was no significant difference between the two groups (P <0.05). The expression of ColⅡ, proteoglycan mRNA and protein peaked at 100ng / mLGDF-5 for 14 days, And Col Ⅱ immunohistochemical staining were positive. Conclusion The expression of ColⅡ and proteoglycan in ADSCs induced by certain concentration of GDF-5 is obviously increased, which has some biological functions of chondrocytes.