摘要
为了探讨壳寡糖(COS)对He La细胞和A549细胞自噬性死亡的影响,利用不同浓度COS处理细胞后MTT法检测细胞的增殖抑制情况,采用MDC染色法观察自噬泡的形成,免疫荧光法检测LC3在细胞内的表达,Western-blot分析COS处理He La和A549细胞不同时间后细胞内LC3-Ⅱ/LC3-Ⅰ变化。结果表明,5.0 mg·m L-1COS对两种细胞的增殖抑制率最大,分别为52.15%和57.63%(P<0.01)。与阴性对照相比,经COS处理后的细胞MDC染色细胞内荧光强度增加,且阳性信号聚集于核周区域;免疫荧光检测发现LC3阳性颗粒数明显增加,表明细胞内自噬体的数量增多。Western-blot检测分析随着COS处理细胞时间延长,LC3-Ⅱ/LC3-Ⅰ比值逐渐增大,显示细胞内自噬活性增强。由此推测COS能够通过诱导He La和A549细胞的自噬性死亡来抑制细胞增殖,为COS的抗肿瘤作用机制的研究提供理论依据。
To explore the effect of autophagic cell death on HeLa cell lines and A549 cell lines induced by Chitooligosaccharide（ COS）, the inhibited rate of cell proliferation were examined by MTT assay after HeLa cell and A549 cell were treated with different concentration of COS for 24 h. The change of the autophagicvacuoles in cells were detected by MDC staining and immunofluorescence. Western-blot was used to measure the ratio of LC3-Ⅱ/ LC3- I in HeLa cell and A549 cell which were treated with COS for 0 h, 4 h, 8 h and 12 h. The resuhs showed when the concentration of COS was 5.0 mg·mL^-1 , the inhibited rate of proliferation were 52. 15% and 57.63% （P 〈0. 01 ）, respectively. The fluorescence intensity were strengthen in ceils and the specific fluorescent particles were appearance in the cytoplasm with MDC staining. The accumulated LC3 protein which involved in the formation of autophagosome further detected by immunofluorescence in HeLa cell and A549 cell, LC3 remained virtually absent in the control groups. Western blotting illustrated the ratio of LC3-Ⅱ/ LC3- I were increased with the increase of treating time by COS, those demonstrated autophagic flux was addition. In conclusion, all of the results suggested that the mechanisms of COS inhibiting the proliferation of HeLa cell and A549 cell might relate with autophagic cell death. This study provides a reliable basis for the antitumor mechanism of COS.
出处
《科学技术与工程》
北大核心
2015年第7期24-29,共6页
Science Technology and Engineering
基金
国家自然科学基金(31171191)
河北省自然科学基金(C2012201106)
河北大学博士基金(2009-168)
河北大学人才引进基金(2011-215)资助
