摘要
背景：内皮祖细胞在缺血性疾病治疗应用中存在数量低的问题，许多细胞因子影响其动员效果和功能。目的：观察粒细胞集落刺激因子动员对体外扩增骨髓源性内皮祖细胞数量及功能的影响。设计、时间及地点：细胞学体外观察，于2007-08／2008—03在北京大学人民医院肝病研究所完成。材料：SPF级健康成年SD雄性大鼠16只，随机分为动员组和对照组，8只／组。重组人粒细胞集落刺激因子为麒麟鲲鹏生物药业有限公司产品。方法：实验前动员组大鼠皮下注射经生理盐水稀释的重组人粒细胞集落刺激因子10ug／kg，2次，d，共5d。采用密度梯度离心法分别从两组大鼠骨髓获取单个核细胞，洗涤后按2×10^7／孔接种在包被大鼠纤连蛋白的6孔板上，采用贴壁法纯化得到内皮祖细胞。主要观察指标：骨髓单个核细胞集落数及细胞表型，骨髓源性内皮祖细胞贴壁情况及细胞表型，通过DiI-acLDL摄取、体外血管生成进行内皮祖细胞功能学鉴定，透射电镜观察细胞超微结构。结果：与对照组比较，动员组骨髓单个核细胞集落增多，CD45^＋／CD34^＋、CD133^＋和Flk-1^＋阳性率均明显升高（F=5．655～61．892，P〈0．05）。培养第7，9天与对照组比较，动员组骨髓源性内皮祖细胞贴壁数、CD45^＋／CD34^＋、CD133^＋和Flk-1^＋阳性率均明显升高（F=5．694～38．879，P〈0．05）。贴壁细胞F1TC—UEA-1／Dil—acLDL双荧光染色阳性率为90％，接种于Matrigel包被的培养板24h后形成毛细血管索样结构，胞质内有Weibel—Palade小体存在。结论：粒细胞集落刺激因子动员能增加骨髓源性内皮祖细胞的数量，并促进其分化、增殖功能。
BACKGROUND： The low amount of endothelial progenitor cells might limit its application in treatment of ischemic diseases. Several factors can affect mobilization and function of endothelial progenitor cells. OBJECTIVE： To investigate effects of granulocyte colony simulating factor mobilization on the amount and function of in vitro expanded endothelial progenitor cells from bone marrow. DESIGN, TIME AND SETTING： The cytology in vitro observation was performed at the Institute of Hepatopathy, People＇s Hospital, Peking University from August 2007 to March 2008. MATERIALS： Sixteen SPF healthy adult male Sprague Dawley rats were randomly assigned into a mobilization group and a control group, with 8 rats for each group. Recombinant human granulocyte colony simulating factor was obtained from Qilin Kunpeng, China. METHODS： Before the experiment, rats in the mobilization group were subcutaneously injected with 10 u g/kg recombinant human granulocyte colony simulating factor diluted in saline, twice a day, for 5 days. The mononuclear cells were harvested from rat bone marrow by Ficoll density gradient centrifugation, washed and cultured in rat fibronectin-coated 6-well culture plate at 2× 10^7 per well. Endothelial progenitor ceils were collected using the adherence method. MAIN OUTCOME MEASURES： Bone marrow mononuclear cell colony number and phenotype; Bone marrow endothelial progenitor cell adherence and phenotype; Biological functions of endothelial progenitor cells were examined by DiI-Ac-LDL internalization. Cell ultrastructure was observed by transmission electron microscope. RESULTS： Compared with the control group, bone marrow mononuclear cell colony, and CD45^＋/CD34^＋, CD 133^＋ and Flk-1^＋ positive rates were significantly increased in the mobilization group （F=5.655-61.892, P 〈 0.05）. At 7 and 9 days, compared with the control group, adhered bone marrow endothelial progenitor cells, CD45^＋/CD34^＋, CD 133^＋ and Flk-1 ^＋ positive rates were significantly increased in the mo
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2008年第51期10033-10036,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家重点基础研究发展计划(2007CB512900)
国家青年科学基金(30700350)~~
