摘要
目的探讨雷公藤甲素(TPL)逆转人结直肠癌细胞对奥沙利铂(Oxa)耐药的作用及机制。方法采用浓度梯度递增法构建结直肠癌耐药细胞系LoVo/Oxa。将传代培养的LoVo/Oxa细胞分为LoVo/Oxa组和LoVo/Oxa+TPL组,另取LoVo细胞作为LoVo组;LoVo组、LoVo/Oxa组细胞常规培养,LoVo/Oxa+TPL组细胞加入无毒剂量的TPL。采用细胞计数试剂盒-8检测3组细胞的耐药性。将传代培养的LoVo/Oxa细胞分为LoVo/Oxa组和LoVo/Oxa+TPL组,LoVo/Oxa组细胞常规培养,LoVo/Oxa+TPL组细胞加入无毒剂量的TPL。采用克隆形成实验检测2组细胞的增殖能力,流式细胞术检测2组细胞的细胞凋亡率和细胞周期,Transwell迁移侵袭实验检测2组细胞的迁移和侵袭能力,免疫印迹法检测2组细胞中周期及耐药相关蛋白[细胞周期蛋白D1(Cyclin D1)、视网膜母细胞瘤蛋白(Rb)、P糖蛋白(P-gp)、多药耐药相关蛋白1(MDR1)]、凋亡相关蛋白[B细胞淋巴瘤/白血病-2(Bcl-2)、B淋巴细胞瘤-2基因相关的X蛋白(Bax)、天冬氨酸特异性半胱氨酸蛋白酶-3(caspase-3)、剪切型冬氨酸特异性半胱氨酸蛋白酶-3(Cl-caspase-3)]及迁移侵袭相关蛋白[波形蛋白(Vimentin)、上皮细胞钙黏附蛋白(E-cad)、基质金属蛋白酶(MMP)-2、MMP-9]的表达水平。结果Oxa对LoVo/Oxa组细胞的半抑制浓度(IC 50)显著高于LoVo组细胞(P<0.01),Oxa对LoVo/Oxa+TPL组细胞的IC 50显著低于LoVo/Oxa组细胞(P<0.01)。0、24 h时,LoVo/Oxa组和LoVo/Oxa+TPL组细胞的增殖能力比较差异无统计学意义(P>0.05);48、72、96 h时,LoVo/Oxa+TPL组细胞的增殖能力显著低于LoVo/Oxa组(P<0.05)。LoVo/Oxa+TPL组细胞克隆数显著多于LoVo/Oxa组(P<0.01)。LoVo/Oxa+TPL组细胞中G 1、G 2期细胞占比显著低于LoVo/Oxa组(P<0.01),LoVo/Oxa+TPL组细胞中S期细胞占比显著高于LoVo/Oxa组(P<0.01)。LoVo/Oxa+TPL组细胞的凋亡率显著高于LoVo/Oxa组(P<0.01)。LoVo/Oxa+TPL组迁移细胞数和侵袭细胞数显著低于LoVo/Oxa组(P<0.01)。LoVo/Oxa
Objective To investigate the reversal effect and mechanism of triptolide(TPL)on oxaliplatin(Oxa)-resistance human colorectal cancer cell line.Methods The Oxa-resistance human colorectal cancer cell line LoVo/Oxa was induced by continuously increasing the contact concentration.The subcultured LoVo/Oxa cells were divided into LoVo/Oxa group and LoVo/Oxa+TPL group,the LoVo cells were take as the LoVo group;the cells in the LoVo/Oxa group were cultured routinely,while the cells in the LoVo/Oxa+TPL group were added with non-toxic doses of TPL.The resistance of cells in the three groups was detected by cell counting kit-8.The subcultured LoVo/Oxa cells were divided into LoVo/Oxa group and LoVo/Oxa+TPL group,the cells in the LoVo/Oxa group were cultured routinely,while the cells in the LoVo/Oxa+TPL group were added with non-toxic doses of TPL.The cell proliferation activity in the two groups was detected by colony formation assay;the apoptosis rate and cell cycle in the two groups were detected by flow cytometry;the migration and invasion activity of cells in the two groups were detected by Transwell migration and invasion assay;the expressions of the cycle and drug resistance related protein[Cyclin D1,retinoblastoma protein(Rb),P-glycoprotein(P-gp),multidrug resistance 1(MDR1)],apoptosis related protein[B-celllymphoma/leukemia-2(Bcl-2),Bcl-2 associated X(Bax),cysteine-containing aspartate-specific protease-3(caspase-3),cleaved cysteine-containing aspartate-specific protease-3(Cl-caspase-3)]and migration-invasion related protein[Vimentin,epithelia-cadherin(E-cad),matrix metalloproteinase(MMP)-2,MMP-9]in cells in the two groups were detected by Western blot.Results The half maximal inhibitory concentration(IC 50)of Oxa on cells in the LoVo/Oxa group was significantly higher than that in the LoVo group(P<0.01);the IC 50 of Oxa on cells in the LoVo/Oxa+TPL group was significantly lower than that in the LoVo/Oxa group(P<0.01).At 0,24 h,there was no significant difference in the proliferation ability of cells between the LoV
作者
熊万成
平贯芳
贺德栋
陈发帅
赫鹏
XIONG Wancheng;PING Guanfang;HE Dedong;CHEN Fashuai;HE Peng(Department of General Surgery,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China;Department of Pharmacy,the First Affiliated Hospital of Xinxiang Medical University,Weihui 453100,Henan Province,China)
出处
《新乡医学院学报》
CAS
2023年第7期613-620,共8页
Journal of Xinxiang Medical University
基金
河南省医学科技攻关计划联合共建项目(编号:LHGJ20210529)。
