[1]张小丽,高建,贾茜,等.肝癌干细胞样细胞的分离及其耐药性受PI3K/Akt通路调节[J].第三军医大学学报,2013,35(02):99-104.
 Zhang Xiaoli,Gao Jian,Jia Qian,et al.Isolation of HCC cancer stem-like cells and chemo-resistance mediated by PI3K/Akt pathway[J].J Third Mil Med Univ,2013,35(02):99-104.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
35卷
期数:
2013年第02期
页码:
99-104
栏目:
论著
出版日期:
2013-01-30

文章信息/Info

Title:
Isolation of HCC cancer stem-like cells and chemo-resistance mediated by PI3K/Akt pathway
作者:
张小丽高建贾茜邓涛
重庆医科大学附属第二医院消化内科; Toronto General Research Institute,University of Toronto,M5G 2M1,Toronto,Ontario,Canada
Author(s):
Zhang Xiaoli Gao Jian Jia Qian Deng Tao
Department of Gastroenterology, Second Affiliated Hospital, Chongqing Medical University, Chongqing, 400010, China; Toronto General Research Institute, University of Toronto, M5G 2M1, Toronto, Ontario, Canada
关键词:
肝细胞癌肿瘤细胞培养的肿瘤干细胞表柔比星耐药性PI3K/Akt
Keywords:
hepatocellular carcinoma tumor cells cultured neoplastic stem cellsepirubicinchemoresistancePI3K/Akt
分类号:
R73-351;R730.23;R735.7
文献标志码:
A
摘要:
目的      分离肝癌干细胞样细胞,初步探讨PI3K/Akt通路调节其对化疗药物阿霉素的敏感性。      方法      将人肝癌细胞株PLC、HepG2、Hep3B置于无血清条件培养基中培养,形成细胞球,选用PLC细胞株进行后续实验。采用流式细胞仪、克隆形成实验、SCID小鼠体内成瘤实验鉴定PLC细胞球(肝癌干细胞样细胞)的肿瘤干细胞特性。MTT法、流式细胞仪测定PLC细胞球对化疗药物阿霉素的敏感性。流式细胞仪分析加入PI3K/Akt通路特异性抑制剂LY294002、阿霉素共同孵育细胞球后,其凋亡率的变化。 Western blot法比较PLC细胞球、PLC贴壁细胞中p-Akt1(Ser473)蛋白分子表达量及加入抑制剂LY294002作用于细胞球后,p-Akt1(Ser473)、Akt1蛋白分子表达量的变化。      结果      肝癌干细胞标志物CD90在细胞球中的表达较贴壁细胞显著升高(P<0.01)。细胞球的克隆形成数目(123.00±28.48)为贴壁细胞(56.33±7.37)的2.18倍(P<0.05)。同样细胞数接种于SCID小鼠皮下7周后,细胞球的致瘤率明显大于贴壁细胞。以5 μg/mL的阿霉素分别处理细胞球和贴壁细胞48 h后,细胞球的增殖率明显高于贴壁细胞[(71.83±12.30)% vs (45.68±5.95)%, P<0.05],凋亡率显著低于贴壁细胞[(11.73±3.77)% vs (41.22±6.73)%, P<0.01],而以阿霉素5 μg/mL和LY294002共同孵育细胞球后,其凋亡率[(35.44±6.65)%]显著增加(P<0.01)。Western blot检测到细胞球的p-Akt1(Ser473)蛋白分子表达量显著高于贴壁细胞(P<0.01),加入抑制剂LY294002处理细胞球后,p-Akt1(Ser473)蛋白表达量明显降低(P<0.05),Akt1的表达量无明显变化(P>0.05)。      结论      肝癌干细胞样细胞对化疗药物阿霉素具有耐药性,其耐药机制与Akt信号通路第473位点磷酸化Akt1分子有关。
Abstract:
Objective      To isolate hepatocellular carcinoma (HCC) cancer stem-like cells and investigate the role of PI3K/Akt pathway in the sensitivity of liver cancer stem-like cells to chemotherapeutic drug doxorubicin (DOX).        Methods      Human HCC cell lines PLC, HepG2, and Hep3B were cultured in serum-free condition medium to form tumor spheres, and PLC cell line was finally selected to proceed the subsequent experiments. Fluorescence-activated cell sorting (FACS) and colony formation assay and SCID mice tumorigenicity experiments in vivo were used to identify the traits of CSCs in PLC spheres(liver cancer stem-like cells). The sensitivity of the cancer stem-like cells to DOX was detected with MTT assay and FACS. The apoptotic rate of the cancer stem-like cells was analyzed with FACS after the treatment of DOX and LY294002, an inhibitor specific to PI3K/Akt signaling pathway. Western blotting was used to detect the expression of p-Akt1Ser473 and Akt1 protein in PLC spheres and in PLC monolayer cells in present or absent of the inhibitor LY294002.        Results      The expression of liver CSCs marker CD90 in the obtained spheres was obviously up-regulated as compared to monolayer cells (P<0.01). The cloning number of spheres (123.00±28.48)was 2.18 times higher than that of the monolayer cells(56.33±7.37, P<0.05). The tumorigenicity of spheres was evidently greater than the monolayer cells when same number of cells was subcutaneous injected into SCID mice after 7 weeks. The proliferation rate was greatly elevated after 48 hour-treatment of 5 μg/mL DOX in the spheres than in the monolayer cells [(71.83±12.30)% vs (45.68±5.95)%, P<0.05], while the apoptosis rate was sharply reduced [(11.73±3.77)% vs (41.22±6.73)%, P<0.01]. But the apoptotic rate of spheres was sharply increased after the treatment of 5 μg/mL DOX and LY294002. (35.44±6.65)%, P<0.01). The expression of p-Akt1Ser473 protein significantly exceeded in spheres than monolayer cells (P<0.01) and the p-Akt1Ser473 expression at protein levels was obviously decreased after addition of LY294002 (P<0.05). There was no change in the expression of Akt1 protein (P>0.05).        Conclusion      The liver cancer stem-like cells demonstrate drug resistance to chemotherapeutic drug DOX, and the mechanism is related to Akt signaling pathway molecule Akt1 phosphorylated at Ser473.

参考文献/References:

张小丽, 高建, 贾茜, 等. 肝癌干细胞样细胞的分离及其耐药性受PI3K/Akt通路调节[J].第三军医大学学报,2013,35(2):99-104.

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更新日期/Last Update: 2013-01-18