喉癌细胞株Hep-2中侧群细胞分选条件优化

The optimizing conditions in sorting of side population in Hep-2

目的：探讨喉癌细胞株Hep-2中侧群细胞分选的条件优化。方法：取对数生长期的Hep-2细胞,予胰酶消化后,制成1×106个/ml单细胞悬液。①实验组中加入Hoechst33342至终浓度分别为5、9、10、11μg/ml,37℃水浴90min。②取单细胞悬液,分别于37°水浴中孵育Hoechst 50、70、90、110、130min。③实验组喉癌细胞悬液加入Hoechst后,分两管分别孵育于37°水浴及37°恒温箱中。两管保持Hoechst浓度及细胞密度一致。④取汇合度分别为100%、70%的2瓶细胞,同上制成单细胞悬液。染色结束后,重悬PBS中,上流式细胞仪进行检测鉴定。以上各组均设阴性对照组于Hoechst染色前加维拉帕米,37℃中孵育30min,其他各条件保持与相应实验组一致。结果：①当Hoechst33342浓度分别为5、9、10、11μg/ml时,SP比例分别为（39.96±0.24）%、（26.23±0.39）%、（18.79±0.02）%、（19.01±0.14）%;死细胞比例分别为（30.45±0.63）%、（49.90±0.42）%、（50.12±0.68）%、（64.16±0.39）%。②Hoechst在37°水浴中孵育90min时,流式细胞图形最典型,SP比例为（17.37±1.42）%;③孵育于37°水浴较孵育恒温箱中,实验组未被Hoechst33342标记的细胞分别为（18.67±0.45）%、（22.6±0.50）%;④汇合度90%以上较汇合度70%的细胞,SP比例由（19.08±0.46）%降到（16.073±0.52）%。以上结果均经SPSS 13.0分析,差异有统计学意义（P〈0.05）。结论：在流式细胞仪分选喉癌Hep-2侧群细胞中Hoechst33342的最佳浓度为10μg/ml,最佳孵育时间为90min,且水浴优于恒温箱中孵育,细胞密度最好选择80%~90%。

Objective：To investigate the optimizing conditions in isolation of the side population in laryngeal carcinoma cell line Hep-2.Method：Single-cell suspension cells were detached from the culture flask with trypsinEDTA,at a concentration of 1×106 cells/ml.①The trail Samples were incubated with Hoechst33342at a concentration of 5μg/ml,9μg/ml,10μg/ml,11μg/ml for 90minutes.②They were incubated with Hoechst for 50,70,90,110,130min in water bath individually.③The single-cell suspension were incubated Hoechst in water bath and in thermostat each.④The two different density of cells were harvested,which were 100%and 70%,and then digest into single-cell suspension.Once incubation finished,suspended in phosphate buffered saline（PBS）,then test SP% by flow cytometry.Among all groups,Verapamil hydrochloride was added to the control samples,incubated at 37℃for 30minutes,the other condition were keep the same with their trial groups.Result：①The percentage of Hoechst-negative cells in trial group was（39.96±0.24）%,（26.23±0.39）%,（18.79±0.02）%,（19.01±0.14）%at the concentration of 5μg/ml,9μg/ml,10μg/ml,11μg/ml respectively,when the PI-positive cells were（30.45±0.63）%,（49.9±0.42）%,（50.12±0.68）%,（64.16±0.39）%separately.②Varying the duration of staining incubation showed that there was a typical FACS pattern and SP% was constant when the incubation was at least 90min.③Compare to water bath,SP% was more than in thermostat,the SP% was（18.67±0.45）%,（22.6±0.50）%respectively;④Cell density is also responsible for SP%.The low density the cell is,the less in SP%.SPSS13.0was used in statistical analysis,the groups were compared using t-Test.P＆lt;0.05was considered statistically significant.Conclusion：The optimum concentration and duration of incubation of Hoechst33342in isolation of the side population cells in laryngeal carcinoma cell line Hep-2is 10μg/ml and 90min.Incubated in water bath is better than in thermostat.The best staining cell density is around 80%-90%.