CAJ | 학술논문

背景:氯甲基-1,1十八烷基-3,3,3’,3’-四甲基-吲哚-羧花青-高氯酸盐(chlormethylbenzamido-1,1-dioctadecyl-3,3,3’,3’-tetramethylin-docarbocyamine,CM-DiI)和4’,6-联脒-2-苯基吲哚二盐酸盐(4’,6-diamidino-2-phenylindole,DAPI)是常用的活细胞示踪剂,分别用来标记细胞膜和细胞核.目的:观察应用细胞膜及细胞核标记物CM-DiI及DAPI联合示踪人脐血单核细胞的可行性及双标后人脐血单核细胞体外培养过程中细胞形态及活性的变化.方法:将新鲜分离的人脐血单核细胞用示踪剂CM-DiI及DAPI进行双标记,体外培养双标后的人脐血单核细胞,倒置相差显微镜下观察细胞形态变化,锥虫蓝染色检测不同时间细胞活性,同时倒置荧光显微镜观察不同时间经CM-DiI和DAPI双标的人脐血单核细胞荧光染色阳性数.结果与结论:人脐血单核细胞在CM-DiI和DAPI联合标记15 min后,荧光显微镜下可见标记细胞的细胞膜、细胞核分别在不同波长下分别呈现红色和蓝色的荧光.CM-DiI/DAPI双标后的人脐血单核细胞体外培养1,3,7,14,21 d, CM-DiI和DAPI双染的阳性细胞数各时间点比较差异无显著性意义.锥虫蓝染色观察人脐血单核细胞存活率为95.6%-98.8%.双标后的人脐血单核细胞体外培养过程中细胞形态变化与未经示踪标记的脐血单核细胞相比差异不明显,仍保持了良好的生长状态、贴壁能力和细胞增殖能力.由此证实,CM-DiI和DAPI可有效标记人脐血单核细胞,两种示踪剂对活细胞无毒不良反应,荧光衰减较慢,适用于干细胞标记及示踪.
배경:록갑기-1,1십팔완기-3,3,3’,3’-사갑기-신타-최화청-고록산염(chlormethylbenzamido-1,1-dioctadecyl-3,3,3’,3’-tetramethylin-docarbocyamine,CM-DiI)화4’,6-련미-2-분기신타이염산염(4’,6-diamidino-2-phenylindole,DAPI)시상용적활세포시종제,분별용래표기세포막화세포핵.목적:관찰응용세포막급세포핵표기물CM-DiI급DAPI연합시종인제혈단핵세포적가행성급쌍표후인제혈단핵세포체외배양과정중세포형태급활성적변화.방법:장신선분리적인제혈단핵세포용시종제CM-DiI급DAPI진행쌍표기,체외배양쌍표후적인제혈단핵세포,도치상차현미경하관찰세포형태변화,추충람염색검측불동시간세포활성,동시도치형광현미경관찰불동시간경CM-DiI화DAPI쌍표적인제혈단핵세포형광염색양성수.결과여결론:인제혈단핵세포재CM-DiI화DAPI연합표기15 min후,형광현미경하가견표기세포적세포막、세포핵분별재불동파장하분별정현홍색화람색적형광.CM-DiI/DAPI쌍표후적인제혈단핵세포체외배양1,3,7,14,21 d, CM-DiI화DAPI쌍염적양성세포수각시간점비교차이무현저성의의.추충람염색관찰인제혈단핵세포존활솔위95.6%-98.8%.쌍표후적인제혈단핵세포체외배양과정중세포형태변화여미경시종표기적제혈단핵세포상비차이불명현,잉보지료량호적생장상태、첩벽능력화세포증식능력.유차증실,CM-DiI화DAPI가유효표기인제혈단핵세포,량충시종제대활세포무독불량반응,형광쇠감교만,괄용우간세포표기급시종.
BACKGROUND:Chlormethylbenzamido-1, 1-dioctadecyl-3, 3, 3’, 3’-tetramethylin-docarbocyamine (CM-DiI) and 4',6-diamidino-2-phenylindole (DAPI) are two kinds of tracers which are commonly used for labeling cel ular membrane and nucleus respectively. OBJECTIVE:To investigate the feasibility of CM-DiI combined with DAPI labeling human umbilical cord blood mononuclear cel s and to observe the changes in morphology and activities of the cel s cultured in vitro after double labeling. METHODS:The freshly isolated mononuclear cel s derived from human umbilical cord blood cel s were double labeled with CM-DiI and DAPI, and the double labeled human umbilical cord blood cel s were cultured in vitro, the changes of the morphology was observed under inverted phase contrast microscope, the activity of the cel s at different times was detected though Trypan blue staining, at the same time, human umbilical cord blood cel s double labeled by CM-DiI and DAPI were counted under the inverted fluorescence microscope at different times. RESULTS AND CONCLUSION:The membrane and nucleus of human umbilical cord blood cel s present red and blue fluorescence respectively under different wavelengths with fluorescence microscope after double labeling by CM-DiI and DAPI for 15 minutes. The CM-DiI/DAPI double labeled human umbilical cord blood cel s were in vitro cultured for 1, 3, 7, 14 and 21 days, and there was no significant difference in the number of positive cel s double labeled by CM-DiI and DAPI at different time points under fluorescence microscope. The survival rates were 95.6%-98.8%. There was no significant difference in cel morphology between the in vitro cultured double labeled cel s and the cel s without labeling, and the growth state, adherent ability and cel proliferation ability of the double labeled cel s were not changed. Human umbilical cord blood mononuclear cel s can be effectively labeled by CM-DiI and DAPI at the same time. Both of the tracers have slower fluorescence decay and non-toxic adverse reactions on the living cel s and are suitable for labeling the stem cel s.