CM-DiI标记兔外周血平滑肌祖细胞可行性分析  

In vitro evaluation of fluorescent dye CM-DiI labeled smooth muscle progenitor cell cultured from rabbit peripheral blood

作　　者：周六化[1] 杨斌[2,3] 夏佳东[1] 王鹏基[1] 陈赟[1] 戴玉田[1] 

机构地区：[1]南京大学医学院附属鼓楼医院泌尿男科,南京210008 [2]上海市第十人民医院 [3]同济大学附属第十人民医院泌尿外科,上海200072

出　　处：《南京大学学报（自然科学版）》2014年第1期1-8,共8页Journal of Nanjing University（Natural Science）

基　　金：国家自然科学基金(31100702/C100307;81170563/H0415);高等学校博士学科点专项科研基金(20110072120054)

摘　　要：采用荧光染料CM-DiI标记兔外周血平滑肌祖细胞(SPC),评价其生物可行性.分离和培养SPC,采用CMDiI进行标记,通过荧光显微镜和流式细胞仪检测标记效果和传代后的细胞标记率.同时,进行细胞爬片培养后免疫荧光染色,观察标记后细胞表达平滑肌肌动蛋白(α-SMA)、钙调节蛋白(Calponin)和增殖细胞核抗原(PCNA)的情况.进行台盼蓝排斥实验、细胞粘附实验、细胞增殖实验和细胞迁移实验,观察标记后的SPC存活率以及粘附、增殖和迁移能力的变化.结果发现,培养4d左右可见SPC开始生长,一周后出现SPC克隆.通过免疫荧光和流式细胞术分析,CM-DiI标记率为96%左右,连续传代2周后标记率仍在40%以上.标记后的细胞可表达α-SMA、Calponin和PCNA.同时发现,标记后的细胞存活率无明显降低,细胞粘附能力、增殖能力和迁移能力无明显改变.本研究证明采用CM-DiI标记SPC操作简便,效果好,不改变细胞表型,不影响细胞的存活率和粘附、增殖及迁移能力,可作为SPC的荧光示踪剂.Smooth muscle progenitor cell（SPC）is a novel cell source with the capability of proliferating,migrating,and differentiating into smooth muscle cell.In our previous study,SPC had been successfully isolated from the peripheral blood of New Zealand Rabbit and demonstrated the potential of being used as a smooth muscle cell source for tissue engineering of urinary bladder.However,it is important and necessary to investigate whether SPCs can survive,pro-liferate,migrate,differentiate into smooth muscle cells and integrate into the host tissue after they are seeded into a scaffold and implanted for bladder augmentation.Cell tracer technique may help resolve this problem,which can be used to trace the cell fate after transplantation.It is important to select an appropriate cell tracer with a high labeling＆amp;nbsp;efficiency but without the side effects on cells.CM-DiI is such a fluorescent dye that has being used widely for cell la-beling in tissue engineering.Therefore,the obj ective of this study was to evaluate the feasibility of using CM-DiI as a tracer to label SPC cultured from rabbit peripheral blood.In this study,SPCs were isolated and cultured from the pe-ripheral blood of New Zealand Rabbit and labeled with CM-DiI according to the manufacturer’s instructions.Labeling rate was evaluated by fluorescence microscope and flow cytometry study.After cultured on coverslips,SPCs were detected by indirect immunofluorescent staining using monoclonal antibody against alpha-smooth muscle actin（α-SMA）,Calponin and Proliferating Cell Nuclear Antigen（PCNA）.The impact of CM-DiI on the growth of SPC was examined by trypan blue exclusion,cell adhesion,cell proliferation and cell migration assays.Our results demonstrated that SPCs emerged after four days of culture and could formed clones after one week in culture.The labeling rate using CM-DiI was about 9 6% as detected with fluorescence microscope and flow cytometry study.After continuous passage culture for two weeks,the labeling rate was still greater than 4

关 键 词：荧光示踪 平滑肌祖细胞 组织工程 

分 类 号：Q462[生物学—生理学]