CD44+/CD24+表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

doi:10.3969/j.issn.1007-3969.2015.10.005

中国癌症杂志 ›› 2015, Vol. 25 ›› Issue (10): 785-790.doi: 10.3969/j.issn.1007-3969.2015.10.005

CD44⁺/CD24⁺表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

刘　红¹，王玉净²，边　磊²，黎海莉²，房朝晖¹，吴小华³，程建新²

1. 河北医科大学第四医院妇瘤科，河北石家庄 050011 ；
2. 河北医科大学第四医院妇科，河北石家庄 050011 ；
3. 白求恩国际和平医院妇科，河北石家庄 050082

出版日期:2015-10-30 发布日期:2015-12-17
通信作者: 程建新　E-mail:jianxin_cheng@263.ne
基金资助:
河北省科技支撑计划(14277770D)；河北省杰出青年科学基金 (C2010002011)。

Study on the chemoresistance of CD44+/CD24+ Siha cells to cisplatin and its mechanisms

LIU Hong¹, WANG Yujing², BIAN Lei², LI Haili², FANG Zhaohui¹, WU Xiaohua³, CHENG Jianxin²

1.Department of Gynecological Tumor, Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China; 2.Department of Obstetrics and Gynecology, Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China; 3.Department of Obstetrics and Gynecology, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei, China

Published:2015-10-30 Online:2015-12-17
Contact: CHENG Jianxin E-mail: jianxin_cheng@263.net

摘要/Abstract

摘要： 背景与目的：肿瘤干细胞的存在是肿瘤细胞抗拒化疗的原因之一。该研究探讨CD44⁺/CD24⁺宫颈癌Siha细胞对顺铂的耐药性及相关机制。方法：体外培养Siha细胞，流式荧光激活细胞分选仪分选CD44⁺/CD24⁺Siha细胞，噻唑蓝(thiazolyl blue，MTT)法检测不同浓度顺铂对细胞的体外抑制情况，采用流式细胞仪检测10 μg/mL顺铂作用于CD44⁺/CD24⁺ Siha细胞24、48和72 h时的细胞凋亡率，实时定量聚合酶链式反应(quantitutive real-time polymerase chain reaction，qRT-PCR)和蛋白［质］印迹法(Western blot)检测CD44⁺/CD24⁺ Siha细胞中Oct-4、ABCG2、Bcl-2的表达，同时设立亲代Siha细胞作为对照。结果：不同浓度顺铂(0.1、1、5、10、15和20 μg/mL)对CD44⁺/CD24⁺ Siha细胞的增殖抑制作用较亲代Siha细胞小［(88.4±1.5)% vs (92.9±1.5)%，(79.9±1.0)% vs (84.7±1.1)%，(69. 8±0.8)% vs (75.1±2.9)%，(59.0±0.7)% vs (65.8±2.7)%，(49.6±0.9)% vs(52.1±0.5)%，(45.1±0.7)% vs (48.8±1.0)%，P<0.05］；与亲代细胞相比，当10 μg/mL顺铂作用于CD44⁺/CD24⁺Siha细胞时，发现在24、48和72 h时的细胞凋亡率均较小［(3.05±0.16)% vs (5.17±0.27)%，(17.94±2.02)%vs (32.60±4.28)%和(40.14±3.01)% vs (56.62±5.32)%，P<0.05］。qRT-PCR和Western blot实验均显示CD44⁺/CD24⁺ Siha细胞高表达Oct-4、ABCG2和Bcl-2，与亲代Siha细胞比较差异有统计学意义(P=0.015)。结论：CD44⁺/CD24⁺ Siha细胞可以抵抗顺铂诱导的细胞凋亡，具有化疗抵抗性，并且高表达肿瘤干细胞表面标志物如Oct-4和ABCG2，该结果在宫颈癌肿瘤干细胞的有效分选以及肿瘤靶向治疗方面有深远意义。

关键词: 宫颈癌, 肿瘤干细胞, CD44⁺, CD24⁺, 顺铂, 耐药, 凋亡

Abstract: Background and purpose: One of the reasons why cancer cells are resistant to chemotherapy is the existence of cancer stem cells. The purpose of this study was to investigate the chemoresistance of CD44⁺/CD24⁺ Siha cells to cisplatin and its mechanisms. Methods: Siha cells were cultivated in vitro. The CD44⁺/CD24⁺ Siha cells were sorted out by fluorescence activated cell sorter (FACS) and in vitro proliferation was detected by MTT assay after treatment with the different concentrations of cisplatin. The cell apoptosis rate was detected by flow cytometry after 10 μg/mL cisplatin acted on CD44⁺/CD24⁺ Siha cells for 24, 48 and 72 h. The relative mRNA and protein expressions of Bcl-2, Oct-4 and ABCG2 were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot, respectively. Results: The survival rates of CD44⁺/CD24⁺ Siha cells treated with different concentrations of cisplatin (0.1, 1, 5, 10, 15 and 20 μg/mL) were higher than those of their parental Siha cells [(88.42±1.51)% vs (92.87±1.5)%, (79.94±1.05)% vs (84.72±1.09)%, (69.78±0.81)% vs (75.13±2.86)%, (58.97±0.70)% vs (65.79±2.71)%, (49.60±0.88)% vs (52.10±0.52)%, (45.13±0.69)% vs (48.84±1.02)%, P<0.05]. Compared with their parental Siha cells, the apoptosis rates of CD44⁺/CD24⁺ Siha cells were lower after 10 μg/mL of cisplatin acting on them for 24, 48 and 72 h, respectively [(3.05±0.16)% vs (5.17±0.27)%, (17.94±2.02)% vs (32.60±4.28)% and (40.14±3.01)% vs (56.62±5.32)%, P<0.05]. The results from both qRT-PCR and Western blot indicated that Oct-4, ABCG2 and Bcl-2 were highly expressed on CD44⁺/CD24⁺ Siha cells. A significant difference was found in Oct-4, ABCG2 and Bcl-2 expression between CD44⁺/CD24⁺ Siha cells and their parental cells (P=0.015<0.05). Conclusion: CD44⁺/CD24⁺ Siha cells could be resistant to apoptosis induced by cisplatin and expressed high levels of cancer stem cell markers such as Oct-4 and ABCG2. This study lays the basis for useful isolation and further targeted therapy of cervical cancer stem cells.

Key words: Cervical cancer, Cancer stem cells, CD44⁺, CD24⁺, Cisplatin, Drug resistance, Apoptosis

刘　红,王玉净,边　磊,等. CD44⁺/CD24⁺表型的宫颈癌Siha细胞对顺铂的耐药及机制研究[J]. 中国癌症杂志, 2015, 25(10): 785-790.

LIU Hong, WANG Yujing, BIAN Lei, et al. Study on the chemoresistance of CD44+/CD24+ Siha cells to cisplatin and its mechanisms[J]. China Oncology, 2015, 25(10): 785-790.

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CD44⁺/CD24⁺表型的宫颈癌Siha细胞对顺铂的耐药及机制研究

Study on the chemoresistance of CD44+/CD24+ Siha cells to cisplatin and its mechanisms

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