人骨髓间充质干细胞生物学特性及腺病毒介导的GFP标记

目的建立人骨髓间充质干细胞(hMSCs)分离、培养以及鉴定的方法,同时应用腺病毒介导的绿色荧光蛋白(Ad-GFP)检测细胞转染的效率。方法应用人淋巴细胞分离液(Ficoll)对hMSCs进行分离,经贴壁纯化后用MTT法检测增殖能力;并对其成骨、成脂、成平滑肌诱导分别进行碱性磷酸酶(ALP)、油红O染色及α-SMA免疫细胞化学检测;采用流式细胞仪(FCM)检测细胞表面标志的表达。用Ad-GFP转染,并应用荧光显微镜与FCM检测转染效率。结果 hMSCs镜下为成纤维样形态,约在第4天进入对数增长期;分化诱导后ALP染色、Von kossa银染、油红O染色以及α-SMA均为阳性;FCM检测CD14、CD31、CD45表达阴性,CD44、CD73、CD106及PDGFRβ表达阳性;当感染复数MOI=200,细胞培养48h时感染效率较高,FCM检测显示为99.81%。结论经过对增殖、分化能力以及表面标志的分析,证明用Ficoll可以分离得到较纯的hMSCs,Ad-GFP可以很好地感染细胞,为组织工程种子细胞的体内示踪提供了途径。

Biological features of human mesenchymal stem cells and labelling with Ad-GFP transfection

Objective To establish a method for isolating,culturing and identifying human mesenchymal stem cells(hMSCs) so as to investigate the transient expression by using Ad-GFP transfection.Methods hMSCs were isolated and purified from human bone marrow with Ficoll density gradient centrifugation and by adhering to the culture plastic.The proliferation ability was evaluated by MTT assay.ALP Kit and red oil O staining were used to prove the differentiation ability of osteoblasts and adipoblasts,respectively.The potential to differentiate into smooth muscle cells(SMCs) was assayed with α-SMA monoclonal antibody.Surface markers in hMSCs were detected by flow cytometry(FCM).Then Ad-GFP was transfected into hMSCs and the transient expression was examined with fluorescence microscope and FCM.Results hMSCs were fibroblast-like in morphology with a high proliferation,and homogenously expressed CD44,CD73,CD106 and PDGFRβ were negative for CD14,CD31,and CD45.After induction,the cells were ALP and α-SMA positive and red oil O staining was positive,too.At 48-h culture,the transient expression was up to 99.81% when transfected at an MOI of 200.Conclusion Highly purified hMSCs can be obtained with Ficoll density gradient centrifugation.Ad-GFP is a preferable method to label cells and can be used as a source for tissue engineering in vivo.