自噬对宫颈癌细胞干性及增殖能力的影响

发布时间：2018-04-15 19:03

  本文选题：自噬 + 宫颈癌干细胞　； 参考：《重庆医科大学学报》2017年11期

【摘要】：目的:研究自噬对宫颈癌细胞干性及增殖能力的影响。方法:采用悬浮培养法分离HeLa肿瘤干细胞,免疫荧光检测干细胞标志基因CD133和Nanog,吖啶橙染色观察自噬小体的形成;Western blot检测贴壁细胞和肿瘤干细胞中自噬相关基因Beclin1和LC3B的表达情况;贴壁细胞和肿瘤干细胞平衡盐溶液EBSS饥饿处理4 h和8 h后,吖啶橙染色观察自噬小体的形成,Western blot检测Beclin1和LC3B表达,实时细胞检测系统检测野生型和饥饿处理后HeLa细胞的增殖能力;自噬抑制剂3-methyladenine(3-MA)阻断宫颈癌贴壁细胞和干细胞的自噬后,Western blot检测Beclin1和LC3B表达,实时细胞检测系统检测野生型和自噬抑制剂处理后HeLa细胞的增殖能力;运用CRISPR/Cas9基因编辑技术降低自噬基因Beclin1的表达,通过实时细胞检测系统检测野生型和Beclin1突变HeLa细胞的增殖能力。结果:基础水平下,HeLa肿瘤干细胞比HeLa贴壁细胞自噬水平高。饥饿诱导处理后,HeLa肿瘤干细胞和HeLa贴壁细胞自噬水平均增高,均在4 h最强,8 h后有所减弱,且HeLa肿瘤干细胞在饥饿诱导各时间点均比HeLa贴壁细胞强;HeLa细胞饥饿处理4 h和8 h后增殖能力均有所下降,8 h下降更明显。应用3-MA后,HeLa肿瘤干细胞和HeLa贴壁细胞自噬均减弱,且HeLa贴壁细胞自噬减弱更为明显;细胞增殖能力也明显减弱。Beclin1突变后HeLa细胞较野生型HeLa细胞增殖能力增强。结论:自噬参与维持宫颈癌HeLa细胞的干性。同时,自噬也能影响宫颈癌HeLa细胞的增殖能力。通过基因打靶扰乱自噬关键基因(如Beclin1或LC3B等)而改变宫颈癌细胞自噬水平可为宫颈癌的治疗带来新的治疗策略。
[Abstract]:Objective: to study the effect of autophagy on the dry and proliferative ability of cervical cancer cells.Methods: HeLa tumor stem cells were isolated by suspension culture.The expression of autophagy related genes Beclin1 and LC3B in adherent cells and tumor stem cells were detected by immunofluorescence and acridine orange staining respectively.Acridine orange staining was used to observe the formation of autophagy bodies in adherent cells and tumor stem cells treated with equilibrium salt solution EBSS for 4 h and 8 h. The expression of Beclin1 and LC3B was detected by Western blot.Real-time cell detection system was used to detect the proliferative ability of HeLa cells treated with wild type and starvation, and autophagy inhibitor 3-methyladenine 3-MA) blocked the expression of Beclin1 and LC3B in cervical cancer adherent cells and stem cells after autophagy by Western blot.Real-time cell detection system was used to detect the proliferative ability of HeLa cells treated with wild type and autophagy inhibitor, and CRISPR/Cas9 gene editing technique was used to reduce the expression of autophagy gene Beclin1.The proliferative ability of wild-type and Beclin1 mutant HeLa cells was detected by real-time cell detection system.Results: the autophagy level of HeLa tumor stem cells was higher than that of HeLa adherent cells.The levels of autophagy of HeLa tumor stem cells and HeLa adherent cells were both increased after starvation induction, and both decreased after 8 hours after the strongest treatment at 4 h.The proliferation ability of HeLa tumor stem cells was significantly decreased at each time point than that of HeLa adherent cells after starvation for 4 h and 8 h, respectively.The autophagy of HeLa tumor stem cells and HeLa adherent cells was decreased after 3-MA application, and the autophagy of HeLa adherent cells was more obvious, and the proliferative ability of HeLa cells was significantly decreased than that of wild-type HeLa cells after the mutagenesis of .Beclin1.Conclusion: autophagy is involved in maintaining the dryness of cervical cancer HeLa cells.At the same time, autophagy can also affect the proliferation of cervical cancer HeLa cells.Changing the level of autophagy of cervical cancer cells by gene targeting and disturbing key genes (such as Beclin1 or LC3B) may lead to a new treatment strategy for cervical cancer.
【作者单位】： 武汉大学中南医院基因诊断中心;湖北医药学院生化教研室;
【基金】：国家自然科学基金资助项目(编号:81541147) 湖北医药学院博士研究生启动金资助项目(编号:2015QDJZR01)
【分类号】：R737.33
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本文编号：1755383