本文選題：自噬 + 宮頸癌干細(xì)胞　； 參考：《重慶醫(yī)科大學(xué)學(xué)報(bào)》2017年11期

【摘要】：目的:研究自噬對(duì)宮頸癌細(xì)胞干性及增殖能力的影響。方法:采用懸浮培養(yǎng)法分離HeLa腫瘤干細(xì)胞,免疫熒光檢測(cè)干細(xì)胞標(biāo)志基因CD133和Nanog,吖啶橙染色觀察自噬小體的形成;Western blot檢測(cè)貼壁細(xì)胞和腫瘤干細(xì)胞中自噬相關(guān)基因Beclin1和LC3B的表達(dá)情況;貼壁細(xì)胞和腫瘤干細(xì)胞平衡鹽溶液EBSS饑餓處理4 h和8 h后,吖啶橙染色觀察自噬小體的形成,Western blot檢測(cè)Beclin1和LC3B表達(dá),實(shí)時(shí)細(xì)胞檢測(cè)系統(tǒng)檢測(cè)野生型和饑餓處理后HeLa細(xì)胞的增殖能力;自噬抑制劑3-methyladenine(3-MA)阻斷宮頸癌貼壁細(xì)胞和干細(xì)胞的自噬后,Western blot檢測(cè)Beclin1和LC3B表達(dá),實(shí)時(shí)細(xì)胞檢測(cè)系統(tǒng)檢測(cè)野生型和自噬抑制劑處理后HeLa細(xì)胞的增殖能力;運(yùn)用CRISPR/Cas9基因編輯技術(shù)降低自噬基因Beclin1的表達(dá),通過實(shí)時(shí)細(xì)胞檢測(cè)系統(tǒng)檢測(cè)野生型和Beclin1突變HeLa細(xì)胞的增殖能力。結(jié)果:基礎(chǔ)水平下,HeLa腫瘤干細(xì)胞比HeLa貼壁細(xì)胞自噬水平高。饑餓誘導(dǎo)處理后,HeLa腫瘤干細(xì)胞和HeLa貼壁細(xì)胞自噬水平均增高,均在4 h最強(qiáng),8 h后有所減弱,且HeLa腫瘤干細(xì)胞在饑餓誘導(dǎo)各時(shí)間點(diǎn)均比HeLa貼壁細(xì)胞強(qiáng);HeLa細(xì)胞饑餓處理4 h和8 h后增殖能力均有所下降,8 h下降更明顯。應(yīng)用3-MA后,HeLa腫瘤干細(xì)胞和HeLa貼壁細(xì)胞自噬均減弱,且HeLa貼壁細(xì)胞自噬減弱更為明顯;細(xì)胞增殖能力也明顯減弱。Beclin1突變后HeLa細(xì)胞較野生型HeLa細(xì)胞增殖能力增強(qiáng)。結(jié)論:自噬參與維持宮頸癌HeLa細(xì)胞的干性。同時(shí),自噬也能影響宮頸癌HeLa細(xì)胞的增殖能力。通過基因打靶擾亂自噬關(guān)鍵基因(如Beclin1或LC3B等)而改變宮頸癌細(xì)胞自噬水平可為宮頸癌的治療帶來新的治療策略。
[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.
【作者單位】： 武漢大學(xué)中南醫(yī)院基因診斷中心;湖北醫(yī)藥學(xué)院生化教研室;
【基金】：國(guó)家自然科學(xué)基金資助項(xiàng)目(編號(hào):81541147) 湖北醫(yī)藥學(xué)院博士研究生啟動(dòng)金資助項(xiàng)目(編號(hào):2015QDJZR01)
【分類號(hào)】：R737.33
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本文編號(hào)：1755383