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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。
Objective To establish a method for the isolation, culture and identification of human bone marrow-derived mesenchymal stem cells (hMSCs). At the same time, adenovirus-mediated green fluorescent protein (Ad-GFP) was used to detect the transfection efficiency. Methods Human hMSCs were isolated by using Ficoll, and the proliferation of hMSCs was detected by MTT assay. The osteogenic, adipogenic and smooth muscle-derived hMSCs were induced by alkaline phosphatase (ALP), oil Red O staining and α-SMA immunocytochemistry were used to detect the expression of cell surface markers by flow cytometry (FCM). Transfection with Ad-GFP, and the use of fluorescence microscopy and FCM transfection efficiency. Results The hMSCs were fibroblast-like morphologically and entered the logarithmic growth phase at about day 4. ALP staining, Von kossa silver staining, oil red O staining and α-SMA staining were all positive after differentiation induction. The expressions of CD14, CD31, CD45, CD44, CD73, CD106 and PDGFRβ were negative. When the MOI was 200, the infection rate was higher at 48h and the FCM showed 99.81%. Conclusions After analysis of proliferation, differentiation and surface markers, Ficoll can be used to isolate more pure hMSCs. Ad-GFP can infect cells well and provide a pathway for in vivo tracing of tissue-engineered seed cells.