土木香内酯对胶质瘤干细胞替莫唑胺耐药性的影响  

Effect of alantolactone on the resistance of glioma stem cells to temozolomide

作　　者：矫永庆[1] 吴文霄[1] 陈松 郭天林[1] 王迅[1] Jiao Yongqing;Wu Wenxiao;Chen Song;Guo Tianlin;Wang Xun(Neurosurgery Department,The Third People’s Hospital of Dalian,Dalian 116033,China)

机构地区：[1]大连市第三人民医院神经外科,大连116033

出　　处：《广西医科大学学报》2020年第11期1942-1947,共6页Journal of Guangxi Medical University

基　　金：辽宁省自然科学基金资助项目(No.2019-BS-056,No.20180550761)。

摘　　要：目的:探讨土木香内酯(ALT)对胶质瘤干细胞替莫唑胺(TMZ)耐药性的影响,并初步研究其作用机理。方法:体外培养人胶质瘤细胞(U251),诱导分化为人胶质瘤干细胞(U251-SC),随机分为对照组、ALT低剂量组、ALT中剂量组和ALT高剂量组;进一步采用腺苷酸活化蛋白激酶(AMPK)抑制剂处理U251-SC细胞,分为对照组、30μmol/L ALT组和30μmol/L ALT+AMPK抑制剂组。免疫荧光法检测U251-SC和U251的肿瘤干细胞标记物CD133和巢蛋白(Nestin)表达情况;CCK-8法检测各组U251-SC细胞存活情况;Western blotting检测各组U251-SC细胞AMPK、p-AMPK、哺乳动物雷帕霉素靶蛋白(mTOR)和p-mTOR表达情况。结果:U251细胞呈均匀分散生长状态,CD133和Nestin呈阴性;与U251细胞相比,U251-SC细胞呈“球体”生长状态,且表现为CD133和Nestin强阳性。与对照组相比,ALT低、中和高剂量组U251-SC细胞存活率、p-mTOR蛋白表达依次降低(P<0.05),凋亡率和p-AMPK蛋白表达依次升高(P<0.05)。与对照组相比,30μmol/L ALT组U251-SC细胞p-AMPK蛋白表达和凋亡率显著升高(P<0.05),p-mTOR蛋白表达和存活率显著降低(P<0.05);与30μmol/L ALT组相比,30μmol/L ALT+AMPK抑制剂组U251-SC细胞p-AMPK蛋白表达和凋亡率显著降低(P<0.05),p-mTOR蛋白表达和存活率显著升高(P<0.05)。结论:ALT可能通过激活AMPK/mTOR通路诱导胶质瘤干细胞凋亡,增加胶质瘤干细胞TMZ敏感性。Objective:To explore the effect and mechanism of alantolactone(ALT)on the drug resistance of temozolomide(TMZ)in glioma stem cells.Methods:Human glioma cells(U251)were cultured in vitro and differentiate into human glioma stem cells(U251-SC),which were randomly divided into control group,low-dose ALT group,medium-dose ALT group and high-dose ALT group.U251-SC cells were further treated with AMP-activated protein kinase(AMPK)inhibitor and divided into control group,30μmol/L ALT group and 30μmol/L ALT+AMPK inhibitor group.Immunofluorescence was used to detect the expression of tumor stem cell markers CD133 and Nestin in U251-SC and U251.CCK-8 method was used to detect the survival of U251-SC cells in each group.Western blotting was used to detect the expression of AMPK,p-AMPK,mammalian target protein of rapamycin(mTOR)and p-mTOR in U251-SC cells.Results:U251 cells were uniformly dispersed,CD133 and Nestin were negative.Compared with U251 cells,U251-SC cells showed“sphere”growth state,and showed strong positive for CD133 and Nestin.Compared with the control group,ALT reduced the survival rate of U251-SC cells and p-mTOR protein expression in a dose-dependent manner(P<0.05),while elevated the apoptosis rate of cells and p-AMPK protein expression(P<0.05).Compared with the control group,the expression of p-AMPK protein and apoptosis rate of U251-SC cells in 30μmol/L ALT group were significantly increased(P<0.05),while the expression of p-mTOR protein and survival rate were significantly decreased(P<0.05).Compared with 30μmol/L ALT group,the expression of p-AMPK protein and apoptosis rate of U251-SC cells in 30μmol/L ALT+AMPK inhibitor group decreased significantly(P<0.05),while the expression of pmTOR protein and survival rate increased significantly(P<0.05).Conclusion:ALT may induce apoptosis of glioma stem cells and increase TMZ sensitivity of glioma stem cells by activating AMPK/mTOR pathway.

关 键 词：土木香内酯 替莫唑胺耐药 腺苷酸活化蛋白激酶 哺乳动物雷帕霉素靶蛋白 胶质瘤干细胞 

分 类 号：R739.41[医药卫生—肿瘤]