荧光活性染料DiI标记人脂肪干细胞  

Fluorescent reactive dye DiI labeled human adipose derived stem cells

作　　者：张云松[1] 高建华[1] 鲁峰[1] 朱茗[1] 廖云君[1] 李华[1] 

机构地区：[1]南方医科大学附属南方医院整形外科

出　　处：《中国组织工程研究与临床康复》2007年第15期2897-2899,共3页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：目的:观察荧光活性染料DiI标记的人脂肪干细胞生长增殖情况,为脂肪干细胞研究寻找理想的细胞标记方法。方法:实验于2006-06/09在广州市创伤外科研究所完成。取人吸脂术中吸出的脂质部分,知情同意并签署知情同意书,用PBS缓冲液反复冲洗,剪刀剪碎。0.1%胶原酶37℃消化40min,使用等量体积含体积分数为0.1胎牛血清+DMEM+双抗完全培养基中和后,1300r/min离心5min,去上清液,沉淀混悬后150UM尼龙网过滤,按1×104～2×104接种于25cm2培养瓶中,置入37℃、体积分数为0.05的CO2孵箱中培养,2～3d换液。待细胞生长融合达80%即可传代,取第3～4代细胞供实验用。将收集细胞用PBS清洗离心2次,加入无血清DMEM制成细胞悬液,以1×109L-1密度加入5μLDiI应用液,37℃下孵育20min,1500r/min离心5min,PBS清洗离心2次,加入含体积分数为0.1胎牛血清DMEM培养基,应用荧光显微镜观察24,48,72h脂肪干细胞显色情况及形态变化。检测脂肪干细胞上清液乳酸脱氢酶含量及细胞XTT比色值,分析脂肪干细胞受损及增殖情况。结果:①DiI标记脂肪干细胞体外观察:台盼蓝染色见脂肪干细胞活力好,仅偶见蓝染细胞,荧光显微镜下观察,见全部细胞标记后均显红色荧光,标记的脂肪干细胞呈梭形,胞浆丰富,保持了良好的正常形态,DiI标记阳性率为100%。DiI标记后早期细胞形态呈荧光环状,48h后细胞中荧光颗粒增多,荧光增强,细胞核未染荧光。②培养脂肪干细胞上清液中乳酸脱氢酶含量及XTT法检测脂肪干细胞受损及增殖情况:未标记DiI的脂肪干细胞与标记DiI的脂肪干细胞形态、细胞上清液乳酸脱氢酶含量及XTT值差异不明显(P>0.05)。结论:①DiI能有效标记体外脂肪干细胞,并在细胞内稳定表达。②DiI标记的脂肪干细胞形态良好,对活体细胞无毒性。AIM： To observe the growth and proliferation of fluorescent reactive dye Dil labeled human adipose derived stem cells （ADSCs） and search for ideal labeling method for ADSCs. METHODS： The experiment was performed at the Guangzhou Trauma Institute from June to September 2006. Lipid was collected by liposuction. The subjects knew the fact and signed the informed consent. The lipid was washed repetitively with PBS buffer solution, smashed with shears, digested with 0.1% collagenase for 40 minutes at 37 %, neutralized with 0.1 volume fraction fetal bovine serum （FBS） ＋DMEM＋double antibody complete medium of the same volume, 1 300 r/min centrifugalized for 5 minutes, filtered with 150 UM nylon net after removing supernatant and precipitating suspension, incubated in 25 cm^2 culture flask at the density of 1×10^4-2×10^4, cultured in 0.05 volume fraction CO2 incubation box at 37 %, and 2-3 days later the liquid was changed. Cells were gone down to the future generation till the cells fused to 80%, and cells in 3-4 generations were collected for the trial, washed with PBS and centrifugalized twice. Serum-free DMEM was added to make into cell suspension before 5 μL Dil liquor at the density of 1×10^9 L^-1 was added, and then cells were incubated for 20 minutes at 37 %, 1 500 r/min centrifugalized for 5 minutes, washed with PBS and centrifugalized twice. DMEM medium containing 0.1 volume fraction FBS was added before coloration and morphologic change of ADSCs were observed at hours 24, 48 and 72 with fluorescent microscope. Content of lactic dehydrogenase in supernatant of ADSCs and XTT colorimetry value were detected to analyze injury and proliferation of ADSCs. RESULTS： ①in vitro observation of Dil labeled ADSCs： Trypan blue staining showed ADSCs had good activity, only few blue-stain cells. Fluorescent microscope showed all the cells was with red fluorescence after labeling and the ADSCs with plenty kytoplasm were fusiform shape, keeping good normal shape. Dil positive rate was 100%. In the

关 键 词：脂肪 干细胞 荧光标记 

分 类 号：R394.2[医药卫生—医学遗传学]