CM-Dil与DAPI联合标记人羊膜上皮细胞的可行性研究

Feasibility of CM-Dil combined with DAPI double-labeling human amniotic epithelial cells

目的建立人羊膜上皮细胞（human amniotic epithelial cell，HAEC）的体外培养方法，并探讨氯甲基苯甲酰胺（CM—Dil）与4’，6-二脒基-2-苯基吲哚（DAPI）对HAEC进行联合标记示踪的可行性。方法运用酶消化法获取HAEC，收集第2代细胞，流式细胞仪检测CD29、CD34、CD44、CIM5和CD105的表达率，SP免疫化学法鉴定HAEC。CM—Dil与DAPI对HAEC进行体外标记，荧光倒置显微镜下观察1d、7d和14d的标记情况，台盼蓝染色检测细胞活力，CCK-8法检测细胞增殖以明确联合标记对体外培养HAEC生长特性的影响。结果HAEC贴壁培养后呈扁平多角形，CD29、CD34、CD44、CD45和CD105的阳性率分别为99．64％、2．21％、32．41％、0．84％、36．70％，细胞角蛋白Keratin阳性表达。HAEC在cM—Dil和DAPI联合标记1d后，荧光显微镜下可观察到细胞膜和细胞核分别在不同波长下呈红色和蓝色荧光，标记率为100％；14d后，经传代培养的HAEC荧光强度与1d时相近，细胞形态无改变。台盼蓝染色显示标记细胞存活率为96．8％-98．9％，CCK-8检测标记细胞的增殖力较未标记组差异无统计学意义（P〉0．05）。结论CM—Dil和DAPI可有效标记HAEC，染色简单、无细胞毒性，荧光衰减较慢，可作为HAEC的标记及示踪方法。

Objective To establish a method to cultivate human amniotic epithe- lial cells and investigate the feasibility of CM-Dil combined with DAPI double-labeling and tracing human amulotic epithelial cell（HAEC）. Methods HAEC were obtained by enzyme digestion method, and the second generation of HAEC were collected. The posi- tive rates of CD29 ,CD34 ,CD44 ,CD45 and CD105 were detected by flow cytometry,and cytokeratin was identified by SP immunohistochemistry. CM-Dil and DAPI were used to double-label HAEC in vitro and the efficiency of labeling at 1 day, 7 days and 14 days were examined under fluorescent microscope. Moreover, cell viability was detected by trypan blue, cell proliferation was detected by CCK-8 method to determine the negative effect of CM-Dil and DAPI on the growth of HAEC cultured in vitro. Results HAEC after adherent culture were polygonal, the positive rate of CD29, CD34, CD44, CD45 and CD105 were 99.64% ,2.21% ,32.41% ,0. 84% ,36. 70% ,and keratin immunoeytx）chemistry staining was positive. After double-labeled by CM-Dil and DAPI 1 day later,the cell mem- brane and the nucleus of HAEC presented red and blue fluorescence respectively under different wavelengths with fluorescent microscopy,the labeling rate was 100% ;After 14 days,the fluorescence intensity of HAEC was similar with that at 1 day,and there was no morphological change. Trypan blue staining showed survival rate of labeled cells was from 96.8% to 98.9% , and CCK-8 method detected there was no statistical difference in prolif- eration ability between labeled group and unlabeled group（P 〉0.05）. Canclusion The procedure of CM-Dil and DAPI labeling is effective, simple, slow fluorescence decayed and non-toxic. This method is suitable for labeling and tracing HAEC.