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目的探讨鼻咽癌细胞株侧群细胞所需荧光染料Hoechst33342最佳的孵育时间和浓度;研究细胞密度对侧群细胞比例的影响;测定其表面标记物CD133和ABCG2蛋白的表达。方法荧光染料Hoechst33342孵育细胞,选择不同的时间(30、60、90、120、150、180min)及不同的浓度(3、4、5、6、7、8mg/L),用流式细胞仪检测,倒置荧光显微镜观察选择合适的孵育时间和浓度。选取不同生长密度(70%、100%)的细胞,流式细胞仪检测侧群细胞比例的变化。荧光抗体CD133和ABCG2共孵育Hoechst33342,流式细胞仪测定细胞表面标记物CD133和ABCG2蛋白的表达。结果Hoechst33342孵育时间为70min,侧群细胞染色效果较佳。不同的鼻咽癌细胞株,合适孵育终浓度不同,如CNE2为6mg/L,而CNE1为2mg/L。另外,侧群细胞的比例呈生长密度依赖性。CD133蛋白总含量恒定在0.1%～0.2%,侧群细胞和主群细胞表达量没有显著差异,CD133并不富集于侧群细胞中。而侧群细胞ABCG2蛋白较主群细胞优势表达,比例达80%以上。结论鼻咽癌细胞株侧群细胞最佳的荧光染料染色时间为70min,不同细胞株孵育终浓度不同。侧群细胞的比例呈密度依赖,与ABCG2密切相关。
Objective To investigate the optimal incubation time and concentration of Hoechst 33342, a fluorescent dye required for nasopharyngeal carcinoma cell lines, to study the effect of cell density on the proportion of lateral colony cells and to determine the expression of CD133 and ABCG2. Methods Fluorescent dye Hoechst33342 was used to incubate cells. Different time (30,60,90,120,150,180min) and different concentration (3,4,5,6,7,8 mg / L) Inverted fluorescence microscopy to select the appropriate incubation time and concentration. The cells with different growth densities (70%, 100%) were selected and the proportion of side population cells was measured by flow cytometry. Hoechst33342 was co-incubated with fluorescent antibody CD133 and ABCG2, and the expression of cell surface marker CD133 and ABCG2 protein was measured by flow cytometry. Results The incubation time of Hoechst33342 was 70 min, and the staining effect of side population cells was better. Different nasopharyngeal carcinoma cell lines, the final incubation for different concentrations, such as CNE2 6mg / L, and CNE1 2mg / L. In addition, the proportion of side population cells in a density-dependent manner. The total content of CD133 protein was constant at 0.1% ~ 0.2%. There was no significant difference in the expression of side population cells and main population cells. CD133 was not enriched in the side population cells. While the side population cells ABCG2 protein dominant than the dominant group of cells, the proportion of more than 80%. Conclusion The optimal fluorescent dye staining time of side population cells of nasopharyngeal carcinoma cell line is 70min, and the final concentration of different cell lines is different. The proportion of side population cells was density-dependent, closely related to ABCG2.