摘要
目的 探讨宫颈癌HeLa细胞中Bcrp1^＋细胞的生物学特性.方法 采用细胞免疫荧光标记及流式细胞仪分选HeLa细胞中Bcrp1^＋及Bcrp1^-细胞,透射电镜观察两种表型细胞的形态,倒置显微镜观察两种表型细胞培养不同时间（24、48、72 h）后的生长情况;采用流式细胞仪检测两种表型细胞的细胞周期及细胞凋亡情况;采用蛋白印迹法检测两种表型细胞中增殖周期相关蛋白--增殖细胞核抗原（PCNA）及半胱氨酸天冬氨酸蛋白酶3（caspase-3）的表达情况（结果均以＂表达丰度＂表示）.结果 （1）分选结果显示,宫颈癌HeLa细胞中,Bcrp1^＋细胞占7.1%,Bcrp1^-细胞占92.9%.透射电镜观察,与Bcrp1^-细胞相比较,Bcrp1^＋细胞核大、核仁明显、胞质内线粒体丰富、粗面内质网增多.倒置显微镜观察,分选后的Bcrp1^＋细胞迅速进入分裂状态,培养24 h即贴壁生长,而Bcrp1^-细胞培养48 h方有少量贴壁生长;培养24、48、72 h时,Bcrp1^＋贴壁细胞分别占72.8%、81.1%、80.4%,Bcrp1^-贴壁细胞分别占3.3%、18.7%、12.6%,各个时间点两种表型细胞的贴壁细胞所占百分比分别比较,差异均有统计学意义（P〈0.05）.（2）流式细胞仪检测显示,Bcrp1^＋细胞中S期细胞比例（54.1%）和增殖指数（55.6%）均高于Bcrp1^-细胞中S期细胞比例（21.1%）和增殖指数（47.0%）,差异均有统计学意义（P〈0.05）;而Bcrp1^-细胞中G0/G1及G2/M期细胞比例（分别为53.0%、25.9%）均高于Bcrp1^＋细胞（分别为44.4%、1.5%）,差异均有统计学意义（P〈0.05）.Bcrp1^＋细胞的凋亡率为0.2%,Bcrp1^-细胞为5.3%,两者比较,差异有统计学意义（P〈0.05）.（3）蛋白印迹法检测显示,在Bcrp1^＋细胞中PCNA蛋白的表达水平（表达丰度为3140）高于Bcrp1^-细胞（表达丰度为2255）,而caspase-3蛋白的表达水平（表达丰度为1970）低于Bcrp1^-细胞（表达丰度为3551）,分别比较,差异均有统计学意义（P〈0.05）.结论 宫颈癌HeLa细胞中Bcrp1^＋细胞具有较强的抗凋亡能力及旺盛的生命力,高表达PCNA蛋白、低表达caspase-3蛋白,是一群具有肿瘤干细胞特征的细胞.Bcrp1^＋的HeLa细胞中可能含有宫颈癌干细胞.
Objective To make sure whether Bcrp1 is the marker of cervical cancer stem-like cells or not by studying the characterization of Bcrp1^＋ HeLa cells.Methods Immunofluorescence stained flow cytometry and electron microacope were used to sort and observe uhrastructures of Bctp1^＋ and Bcrp1^- HeLa cells.Flow cytometry wag used to identify the cycle and the rate of apoptosis with annexin V in two group cells.The expression of proliferating cell nuclear antigen （PCNA）and caspase-3 were tested using western blot methed.Results （1）There were 7.1% Bcrp1^＋ cells and 92.9% Bcrol- cells in HeLa cells.Bcrp1^＋ HeLa cells were large in size of nuclear and nucleoli are clear.and there were rich of cytomicrosome and rough endoplasmic reticulum.After sorted and cultured for 24,48,72 hours,the adhesion in Bcrp1^＋ cells were 72.8%,81.1%,80.4%,respectively.While,they were 3.3%,18.7%,12.6%at each time for Bcrpl- cells（all P〈0. 05 ）. （2） There are more S phase cells in Bcrp1^＋ cells than that in Bcrp1^- cells （54. 1% vs 21.1%, P 〈0. 05） ,while the percentage of G0/G1 and G2/M in Bcrp1^- cells were highter than those in Bcrp1^＋ cells （53.0% vs 44. 4% ,25.9% vs 1.5% ; all P 〈0. 05 ）. The rate of apoptosis in Bcrp1^＋ cells was lower than that in Bcrp1^- cells （0. 2% vs 5.3%, P 〈 0. 05 ）. （ 3 ） The expression of PCNA in Bcrp1^＋ cells was higher than that in Bcrp1^- cells （3140 vs 2255, P〈 0. 05 ）, while the expression of caspase-3 of Bcrpl ＋ cells was lower than that in Bcrp1^- cells （ 1970 vs 3551, P 〈 0. 05 ）. Conclusion There are more vigor and ability of proliferation and lower rate of apeptosis in Bcrp1^＋ HeLa cells than those in Bcrp1^- cells ,which may be some characters of cervical cancer stem cells.
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2010年第7期525-529,共5页
Chinese Journal of Obstetrics and Gynecology
基金
国家自然科学基金（30901589）
吉林省科技发展计划（200705155）
