蛇葡萄素对结肠癌细胞增殖抑制、自噬诱导及自噬对增殖的影响研究  

Effects of Ampelopsin on Proliferation Inhibition,Autophagy Induction and Autophagy on Proliferation of Colon Cancer Cells

作　　者：李晨红 宋长丰 林志康 赵宇侠 LI Chenhong;SONG Changfeng;LIN Zhikang;ZHAO Yuxia(Graduate School,Shanghai University of Traditional Chinese Medicine,Shanghai 200120;College of Medical Technology,Shanghai University of Medicine and Health Sciences,Shanghai 201318;College of Pharmacy,East China University of Science and Technology,Shanghai 200237)

机构地区：[1]上海中医药大学研究生院,上海200120 [2]上海健康医学院医学技术学院,上海201138 [3]华东理工大学药学院,上海200237

出　　处：《四川师范大学学报（自然科学版）》2021年第3期390-397,共8页Journal of Sichuan Normal University（Natural Science）

基　　金：国家自然科学基金(20130371);上海健康医学院种子基金(E1-0200-19-201132);上海健康医学院百人库项目(B1-0200-19-311133)对本文给予了资助。

摘　　要：明确蛇葡萄素(Ampelopsin,AMP)对人结肠癌HCT116细胞增殖的抑制,探讨自噬活化及自噬对AMP抑制细胞增殖作用的影响,为AMP临床研究抗肿瘤作用的自噬靶点提供依据.采用CCK-8法检测AMP(0~300μg/m L)作用HCT116细胞后细胞增殖变化,确定AMP的抗肿瘤作用;慢病毒感染法构建HCT116-RFP-GFP-LC3重组细胞株研究自噬标志蛋白LC3的荧光表达,结合蛋白质印迹法检测自噬标志蛋白LC3I/II和P62的表达确定细胞自噬活化状态;通过抑制剂Baf A1阻断HCT116细胞自噬晚期降解过程,比较AMP组与AMP+Baf A1组间AMP对HCT116细胞增殖的影响明确自噬对细胞增殖的影响.结果表明:AMP(150~300μg/m L)抑制细胞增殖,表明AMP具有抗肿瘤作用;对比空白对照组,AMP(50、75μg/m L)组重组细胞出现LC3荧光点状自噬体,自噬发生率分别为26.72%、19.47%;LC3II蛋白表达上调,P62下调,表明AMP诱导细胞自噬且自噬过程完整;与AMP组比较,加入Baf A1后,LC3II、P62蛋白及自噬点均显著增加,证明自噬晚期过程被阻断,但抑制自噬晚期降解过程对AMP组与AMP+Baf A1组间细胞增殖影响不显著(P>0.05),说明在实验浓度范围内,阻断自噬降解过程对细胞增殖无显著影响.结论表明:AMP(0~300μg/m L)能够抑制结肠癌细胞增殖,激活细胞自噬,且自噬晚期降解过程阻断对细胞增殖无显著影响,细胞的增殖抑制可能是通过其他途径实现.In this paper,we clarify the inhibitory effect of Ampelopsin(AMP)on the proliferation of human colon cancer HCT116 cells,explore the effect of autophagy activation and autophagy on inhibitory effect of AMP on cell proliferation,and provide a basis for clinical research of AMP on autophagy targets for anti-tumor effects.The CCK-8 method was used to detect the cell proliferation of HCT116 cells after AMP(0-300μg/m L)treated to determine the anti-tumor effect of AMP;the HCT116-RFP-GFP-LC3 recombinant cell line was construct by the lentiviral infection method to study the expression of autophagy marker protein LC3;the expression of autophagy markers LC3 I/II and P62 was detected by Western blot to determine the autophagy activation state;the inhibitor Baf A1 was used to block the late autophagy degradation process of HCT116 cells,and the effect of autophagy on HCT116 cell proliferation was compared between AMP group and AMP+Baf A1 group to clarify the effect of autophagy on cell proliferation.The results show that:AMP(150-300μg/m L)inhibited cell proliferation indicating that AMP has an anti-tumor effect;compared with the blank control group,recombinant cells in AMP(50,75μg/m L)group showed LC3 fluorescent punctate autophagosomes,and the incidence of autophagy was 26.72%and 19.47%,respectively;the expression of LC3 II protein was up-regulated,and P62 was down-regulated indicating that AMP induced autophagy and the autophagy process was integrity;compared with the AMP group,after adding Baf A1,the expression of LC3 II,P62 protein and autophagic point all increased significantly which proved that the late autophagy process was blocked,but the inhibition of the late autophagy degradation process is not significant(P>0.05)on the cell proliferation between the AMP group and the AMP+Baf A1 group indicating that within the experimental concentration range,blocking the autophagy degradation process has no significant effect on cell proliferation.The conclusion shows that:AMP(0-300μg/m L)can inhibit colon cancer cell prol

关 键 词：蛇葡萄素 结肠癌 细胞增殖 自噬 

分 类 号：R285.5[医药卫生—中药学]