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。 Objective: To study the effect of autophagy on the proliferation of human cervical cancer cells. Methods: HeLa tumor stem cells were isolated by suspension culture. The stem cell marker genes CD133 and Nanog were detected by immunofluorescence staining and the formation of autophagy bodies by acridine orange staining. The expression of autophagy genes Beclin1 and LC3B in adherent cells and tumor stem cells were detected by Western blot The expression of autophagosomes was observed by acridine orange staining and the expression of Beclin1 and LC3B was detected by Western blot. Real-time cell detection system was used to detect the expression of wild-type And 3-methyladenine (3-MA) ​​blocked the autophagy of cervical cancer adherent cells and stem cells, the expression of Beclin1 and LC3B was detected by Western blot, and the real-time cell detection system was used to detect the expression of wild-type Type and inhibitor of autophagy. The expression of Beclin1, an autophagy gene, was reduced by using CRISPR / Cas9 gene editing technology. The proliferation of HeLa cells was detected by real-time cell detection system. Results: Under the basal level, HeLa tumor stem cells had higher autophagy than HeLa adherent cells. After starvation induction, the levels of autophagy in HeLa tumor stem cells and HeLa adherent cells were all the highest at 4 h and weakened after 8 h, and HeLa tumor stem cells were stronger than HeLa adherent cells at all time points of starvation induction After 4 h and 8 h treatment, the proliferation of HeLa cells decreased, and the decrease was more obvious at 8 h. After using 3-MA, the autophagy of HeLa tumor stem cells and HeLa adherent cells were weakened, and the autophagy of HeLa adherent cells was weakened more obviously; and the cell proliferation ability was obviously weakened. Beclin1 mutant HeLa cells proliferation than wild-type HeLa cells increased. 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 in cervical cancer cells through gene targeting disrupts the key genes of autophagy (such as Beclin1 or LC3B) may bring a new therapeutic strategy for the treatment of cervical cancer.