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.

abstractsObjective 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.