红细胞裂解法分离及培养大鼠骨髓间充质干细胞(英文)  

Isolation and culture of bone marrow mesenchymal stem cells by red blood cell lysis method

作　　者：邓近平[1] 戴钟铨[2] 刘收[3] 聂捷琳[2] 李莹辉[2] 

机构地区：[1]湖南农业大学动物营养研究所 [2]中国航天员科研训练中心航天细胞分子生物学实验室,北京市100094 [3]华中科技大学生命科学与技术学院,湖北省武汉市430074

出　　处：《中国组织工程研究与临床康复》2007年第3期579-582,F0003,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然基金资助项目(30370365)~~

摘　　要：背景:骨髓间充质干细胞在骨髓中的含量非常少,而且与其他细胞特别是易与红细胞相混杂,因此在分离过程中需去除干扰,以获得尽可能多的高纯度的骨髓间充质干细胞。目的:采用红细胞裂解法分离培养大鼠骨髓间充质干细胞,同时进行生物学鉴定。设计:观察对比实验。单位:中国航天员科研训练中心航天细胞分子生物学实验室。材料:选用50只生后30d的雄性SD大鼠,体质量100g,SPF级,购自北京实验动物中心(合格证:SCXK(京)2002-0003)。DAB浓缩显色液(含过氧化氢,中杉公司),兔抗大鼠多克隆抗体(武汉博士德公司),FCS(PAA,Austria),LG-DMEM培养基(Sigma,USA)。方法:实验于2004-09/2005-09在中国航天员科研训练中心航天细胞分子生物学实验室完成。将大鼠脱臼处死,暴露骨髓腔,收集细胞悬液,采用全骨髓培养法对骨髓间充质干细胞分离与原代培养。①红细胞裂解实验:取A、B、C、D4管分别加入0.5mL过滤并充分吹打均匀后的骨髓冲洗液,分别对应加入红细胞裂解液、氯化氨2mL、磷酸盐缓冲生理盐水2mL和体积分数为0.04的乙酸溶液0.5mL,分别测量各组吸光度值及血红蛋白浓度,并观察细胞生长情况。②骨髓间充质干细胞生长曲线、倍增时间及表面标志分子表达情况的观察:根据公式:群体倍增时间(TD)=t[lg2/(lgNt-lgN0)](N0和Nt分别代表接种后和培养t小时的细胞数),计算出细胞的群体倍增时间并描绘并分析骨髓间充质干细胞第2、4、6代细胞的生长曲线;采用亚甲基兰染色测定骨髓间充质干细胞增殖情况;采用免疫细胞化学染色检测骨髓间充质干细胞表面标志分子表达情况。主要观察指标:①大鼠骨髓间充质干细胞分离和培养的观察结果。②不同处理方法对红细胞的裂解效果及其对骨髓间充质干细胞生长的影响。③第2、4、6代细胞的生长曲线及细胞倍增时间。④大鼠骨髓间充质干细胞表面标志BACKGROUND： Bone marrow mesenchymal stem cells （BMSCs） are few in bone marrow, and they are easily mingled with other cells, especially red blood cells. Therefore, intervention needs to be depleted in the process of isolation of red blood cells so as to obtain highly purified BMSCs as many as possible. OB3 ECTIVE： To isolate and culture BMSCs of rats with red blood cell lysis method, and perform biological identification DESIGN： Observation and controlled tnal. SEl-rING： Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts MATERIALS： Fifty 30-day-old male SD rats, weighing about 100 g, of SPF degree, were purchased from Beijing Expen- mental Animal Center （License No. SCXK （Jing） 2002-0003） and involved in this trial. DAB condensed chromogen （hydrogen dioxide included, Zhongshan Company）, rabbit anti-rat polyclonal antibody （Boster Co.,Ltd., Wuhan）, fetal calf serum （PAA, Austria） and LG-DMEM medium （Sigma Company, USA） were used in this trial. METHODS ： This trial was carried out in the Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts during September 2004 to September 2005. The rats were sacrificed by dislocation to ex- pose bone marrow cavity. Cell suspension was collected. BMSCs were isolated and cultured primarily by whole bone marrow culture method. （1） Red blood cells lysis test： A, B, C and D 4 tubes were chosen and filled with 0.5 mL bone marrow rinse solution which was filtered and fully beat upon. Then, red blood cell lysis buffer of 2 mL, ammonium chlo- ride of 2 mL, phosphate buffer normal saline of 2 mL and 0.04 volume fraction acetic acid of 0.5 mL were correspondingly added into the 4 tubes. In each tube, absorbance and hemoglobin concentration were measured and cell growth was observed.（2） Observation of growth curve, doubling time and surface marker molecule expression of BMSCs： Based on the formula, population doubling time （TD）

关 键 词：干细胞 骨髓 细胞分离 细胞培养 生物学鉴定法 

分 类 号：R394.2[医药卫生—医学遗传学]