摘要
背景:脂肪间充质干细胞具有多向分化能力,并且相对更加容易获得,因此作为一种新的细胞工程的种子细胞日渐受到重视。目的:观察从大鼠皮下分离脂肪间充质干细胞的体外培养情况,并对其进行免疫细胞化学染色等鉴定。设计:动物实验。单位:解放军总医院心内科。材料:选用1只健康雄性Wistar大鼠,清洁级,鼠龄4个月,体质量200g;Ⅰ型胶原酶、DMEM培养基、特级胎牛血清(GIBCO公司);免抗大鼠CD13、CD34、CD44、105、Ⅷ因子、HLA-DR、VWF、Myosin等多克隆抗体及SABC试剂盒(武汉博士德公司);绿色荧光蛋白-重组腺病毒包装载体为北京本元正阳基因技术有限公司产品。方法:实验于2006-10在解放军总医院心内科完成。①细胞分离和培养:全麻下取Wistar大鼠腹股沟脂肪垫0.3g,剪碎,消化后培养,镜下观察有大量梭形贴壁细胞后二三天换DMEM培养液,观察细胞生长情况。调整细胞浓度为2×107L-1,接种于96孔细胞培养板,每孔100μL。从第2天起每日随机取3孔,胰蛋白酶消化细胞,分别用血球计数板在倒置显微镜下计数。②细胞存活率的计算:收集第3至第8代细胞,加入预配好的DMSO冻存液,2～4周后复苏,台盼蓝染色检测细胞存活率。③免疫细胞化学染色及鉴定:以2×107L-1的细胞数接种多孔培养板,24h后行CD13、CD34、CD44、CD45、CD105、Ⅷ、HLA-DR、VWF因子等细胞表面抗体的免疫细胞化学(SABC法)鉴定及油红染色。④多细胞系定向诱导分化及鉴定:用多孔板接种后加含特定诱导因子的培养基进行多系定向诱导分化,并进行油红O脂肪颗粒染色,对成骨诱导组进行碱性磷酸酶组化染色,对成心肌诱导组进行肌浆球蛋白免疫组化染色。⑤转染腺病毒携带的绿色荧光蛋白基:用96孔板接种第4代ADMSC,同时加入Ⅰ型腺病毒为载体的绿色荧光蛋白,以不同MOI值1∶50、1∶100、1∶150、1∶200转染,观察转染情况。主要观察指
BACKGROUND： Adipose tissue-derived mesenchymal stem cells （ADMSCs） have the multilineage differentiation potential, and are relatively easier to be obtained, thus they have attracted more and more attention as a new seed cell for cell engineering. OB3 EC＇I-IVE： To observe the in vitro culture conditions of ADMSCs isolated from rat＇s subcutaneous adipose tissue, and identify them using immunohistochemical staining. DESIGN： An animal experiment SETTING ： Department of Cardiology, the General Hospital of Chinese PLA MATERIALS： One healthy male Wistar rat of clean degree, 4 months old, weighing 200 g, was used. DMEM, fetal bovine serum were from GIBCO; Monoclone antibodies of rabbit-anti-rat CD13, CD34, CD44, CD45, CD105, D-related human leucocyte antigen （HLA-DR）, factor-VIII, vov Willebrand factor （VWF）, Myosin, SABC kits and DAB staining kit from Wuhan Booster Biological Engineering, Co.,Ltd; Adeno-associated virus encoding green fluorescent protein from Vector Gene Technology Company Limited （Beijing）. METHODS： The experiments were carried out in the Department of Internal Medicine, the General Hospital of Chinese PLA in October 2006. ①Cell isolation and culture： 0.3 g adipose tissue was cut from subcutaneous adipose tissue of Wistar rat＇s groin under aseptic condition, then minced and digested before culture, DMEM was changed at 2-3 days after plenty of fusiform-shaped attached cells were observed under microscope, and the cell growth was observed. The cell concentration was adjusted to 2x10^7 L^-1, then seeded into 96-well plate, and 100 p.L for each well. From the second day, 3 wells were randomly selected every day, the cells were released with tripsin, and counted with blood cell counting chamber under inverted microscope. ②Cell viability assay： ADMSCs of passages 3 to 8 were added to DMSO freeze medium, and thawed after 2-4 weeks, and the cell viability was assessed by trypan blue dye exclusion. ③Immunohistochemical staining and identification： 2 xl0^7 L^-1 cell
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第33期6701-6705,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金面上项目(30471924,30572445)~~
