生长分化因子5诱导兔脂肪干细胞成软骨细胞分化的实验研究

EXPERIMENTAL STUDY ON CHONDROGENIC DIFFERENTIATION OF RABBIT ADIPOSE-DERIVED STEM CELLS TREATED WITH GROWTH DIFFERENTIATION FACTOR 5

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 feasibil ity and effect of inducing adi pose-derived stem cells （ADSCs） treated with growth differentiation factor 5 （GDF-5） to undergo chondrogenic differentiation in vitro. Methods Six healthy Japanese rabbits aged 3 months （2-3 kg） of clean grade were chosen, irrespective of sex. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by vimentin immunohistochemistry and CD44, CD49d, CD106 immunofluorescence staining. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 106/mL, then the ADSCs were treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium, respectively. The morphology changes of the induced ADSCs were observed by inverted contrast phase microscope and their growth state were detected by MTT. The mRNA quantities of Col II and proteoglycan expressed by the induced ADSCs were detected with RT-PCR. The Col II proteoglycan synthesized by the induced ADSCs were detected with alcian blue staining, toluidine blue staining, immunohistochemistry staining, and Western blot method. Results ADSCs mostly presented small sphere, fusiform and polygon shape with positive expression of CD44 and CD49d and negative expression of CD106 and vimentin. The ADSCs treated with 100 ng/mL GDF-5 presented sphere or sphere-l ike change and vigorous prol iferation. The mRNA quantities of Col II and proteoglycan synthesized by the induced ADSCs treated with 0, 10, 100 ng/mL GDF-5 and common cultural medium increased in a dose-dependent manner at 7 days. There were significant differences among all the groups （P 〈 0.05）, except that no significant difference was evident between the 0 ng/mL group and the 10 ng/mL group （P 〉 0.05）. When ADSCs were treated with 100 ng/mL GDF-5 for 14 days, the Col II and the mRNA and protein quantities of ptoteoglycan reached the peak, and the results of alcian blue, toluidine blue and Col II immunohistochemistry staining were positive. Conclusion ADSCs treated with certain concentration of GDF-5 have higher expression of Col II and proteoglycan and possess partial biological function of chondrocyte.