人脂肪源性干细胞成骨诱导过程中Ⅰ型胶原的表达

Expression of collagen type Ⅰ in human adipose-derived stem cells during osteogenic differentiation and induction

目的:研究人脂肪源性干细胞(hADSCs)成骨诱导后其合成和分泌Ⅰ型胶原能力的变化,探讨hADSCs作为组织工程骨的种子细胞的效能。方法:从人脂肪抽吸物中分离基质细胞,体外培养、扩增及鉴定。采用成脂诱导剂诱导hADSCs向脂肪细胞分化,采用油红O染色鉴定。采用成骨诱导剂诱导hADSCs向成骨细胞分化,将成骨诱导组分为0、7、14、21和28d组,分别于0、7、14、21及28d行茜素红染色检测矿化结节、VanGieson胶原纤维特殊染色染胶原纤维、Ⅰ型胶原细胞免疫荧光染色检测细胞Ⅰ型胶原的表达。用FVViewer1.7软件对Ⅰ型胶原表达的强度进行定量检测。结果:hADSCs成脂诱导14d后油红O染色阳性;hADSCs成骨诱导21d茜素红染色阳性;成骨诱导21dVanGieson胶原纤维特殊染色阳性;hADSCs成骨诱导后Ⅰ型胶原染色21d所有的细胞均阳性表达,28d呈强阳性表达。统计结果显示:Ⅰ型胶原的表达从7d开始,到28d逐渐升高,7、14、21和28d组与0d组比较差异均有显著性(P<0.05)。结论:hADSCs成骨诱导后全部细胞呈现成骨表型,成骨能力强,可以作为骨种子细胞应用于临床骨再生治疗。

Objective To investigate the synthesis and secretion of collagen type Ⅰ（Col Ⅰ） in human adipose-derived stem cells（hADSCs） during osteogenic differentiation and induction,and approach the efficacy of hADSCs as seed cells for bone regeneration engineering.Methods hADSCs were separated,cultivated and amplified in vitro and were identified by cell phenotype detection.Multi-lineage differentiation potential was assessed by histological staining such as oil red O for lipid accumulation and alizarin red S for mineralization nodules during adipogenic and osteogenic induction.Osteogenic differentiation and induction group was divided into five subgroups：0,7,14,21 and 28 d groups.Alizarin red S staining,Van Gieson collagen fiber staining and Col Ⅰ immunofluorescence were used to detect the expression of Col Ⅰ.FV Viewer 1.7 software was used to analyze the data.Results The positive stainning of oil red O was found 14 d after adipogenic induction.For the osteogenic induction groups,the calcified nodules and the collagen fiber red staining were found in 21 d group.The expression of Col Ⅰ was found in 21 d group for all cells and strongly positive expression in 28 d group.Compared with 0 d group,the expression of Col Ⅰ in osteogenic induction group was significantly increased from 7 d to 28 d（P〈0.05）.Conclusion During osteogenic induction of hADSCs,all cells have osteogenic capability and the hADSCs have outstanding capability of osteoinductive and osteogenesis.hADSCs may be useful in clinical cell-based therapy for bone regeneration engineering.