荧光染料CM-DiI标记大鼠骨髓间充质干细胞体外实验.docVIP

荧光染料CM-DiI标记大鼠骨髓间充质干细胞体外实验 　　【摘要】 目的 探讨荧光染料CM-DiI标记大鼠骨髓间充质干细胞的效果及其对细胞活力、增殖能力、凋亡等生物学特性的影响。方法 用梯度离心加贴壁培养法获取、培养、扩增大鼠骨髓间充质干细胞。取第4代MSCs，分为实验组和对照组，用浓度为6 μmol/L的CM-DiI对实验组MSCs进行标记。用台盼蓝染色检测细胞的活力，CCK-8法检测细胞增殖能力，Hoechst 33258染色法检测细胞凋亡。细胞标记后用倒置相差荧光显微镜动态观察不同子代MSCs的阳性标记率、荧光衰减情况及细胞生长、形态变化。结果 CM-DiI标记后的MSCs发橙红色荧光，高倍镜下显示细胞上有密集排列明亮荧光颗粒。CM-DiI标记后1 d、4 d、8 d、16 d、24 d细胞标记阳性率分别为100%、（85.2±4.9）%、（42.3±5.1）%、（17.6±3.6）%、（4.4±1.3）%，细胞标记阳性率和荧光强度随传代次数和培养时间的增加而呈显著进行性下降。标记细胞的大小、形态、活力、增殖能力、细胞凋亡与未标记细胞相比无明显差异。结论 CM-DiI标记骨髓间充质干细胞简便、有效，对其生物学特性无明显影响，可用于MSCs的短期示踪。 　　【关键词】 骨髓间充质干细胞； CM-DiI； 标记 　　In vitro study of rat mesenchymal stem cells labeled with fluorescent dye CM-DiI CHEN Guo-dong,ZHU Dong-liang,TAN Li.The First Peoples Hospital of Guangzhou,Guangzhou 510180,China 　　【Abstract】 Objective To evaluate the efficiency of lipophilic fluorescent dye CM-DiI on labeling mesenchymal stem cells（MSCs） of rat，and the effect on cell viability,proliferation and apoptosis. Methods The MSCs cells were isolated from bone marrow of SD rat by density gradient centrifugation,and amplificated using adherent culture.The fourth generation MSCs were divided into control group and experimental group.Cells of experimental group were stainted with 6 μmol/L CM-DiI concentrations.Cell viability and proliferation was measured by the exclusion of trypan blue dye and Cell Counting Kit-8（CCK-8） assay.Hoechst 33258 staining were used to determine cell apoptosis.The percentage of positively labeled cells,dicrease of fluorescent intensity,morphologic change of MSCs was dynamic observed by inverted phased fluorescence microscope.Results Using fluorescent microscopy,CM-DiI-positive cells showed strong orange-red fluorescence,and intensive fluorescent particles was observed within the CM-DiI-labeled cells.The percentage of CM-DiI-positive cells was 100%、（85.2±4.9）%,（42.3±5.1）%,（17.6±3.6）% and （4.4±1.3）%,after day 1,4,8,16 and 24,respectively.The percentage of CM-DiI-positive cells and the fluorescence intensity decreased rough