【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(IC₅₀)显著高于LoVo组细胞（P<0.01）,Oxa对LoVo/Oxa+TPL组细胞的IC₅₀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G₁、G₂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+TPL组细胞中Cyclin D1、Rb、MDR1、P-gp、Bcl-2、Vimentin、MMP-2和MMP-9蛋白的相对表达量显著低于LoVo/Oxa组，Bax、Cl-caspase-3、E-cad蛋白的相对表达量显著高于LoVo/Oxa组（P<0.01）;2组细胞中caspase-3蛋白的相对表达量比较差异无统计学意义（P>0.05）。结论 TPL通过抑制细胞增殖和侵袭，以及促进细胞凋亡来逆转结直肠癌细胞对Oxa的耐药性。更多还原

【Abstract】 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(IC₅₀) of Oxa on cells in the LoVo/Oxa group was significantly higher than that in the LoVo group（P<0.01）;the IC₅₀ 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 LoVo/Oxa group and the LoVo/Oxa+TPL group（P>0.05）;at 48,72,96 h, the proliferation ability of cells in the LoVo/Oxa+TPL group was significantly lower than that in the LoVo/Oxa group（P<0.05）.The number of cell clones in the LoVo/Oxa+TPL group was significantly higher than that in the LoVo/Oxa group（P<0.01）.The percentage of G₁ and G₂ phase cells in the LoVo/Oxa+TPL group was significantly lower than that in the LoVo/Oxa group（P<0.01）,and the percentage of S phase cells in LoVo/Oxa+TPL group was significantly higher than that in the LoVo/Oxa group（P<0.01）.The apoptosis rate of cells in the LoVo/Oxa+TPL group was significantly higher than that in the LoVo/Oxa group（P<0.01）.The numbers of migrating and invading cells in the LoVo/Oxa+TPL group were significantly lower than those in the LoVo/Oxa group（P<0.01）.The numbers of migration and invasion cells in the LoVo/Oxa+TPL group were significantly lower than those in the LoVo/Oxa group（P<0.01）.The relative expressions of Cyclin D1,Rb, MDR1,P-gp, Bcl-2,Vimentin, MMP-2,MMP-9 protein in cells in the LoVo/Oxa+TPL group were significantly lower than those in the LoVo/Oxa+TPL group（P<0.01）;the relative expressions of Bax, Cl caspase-3,E-cad protein in cells in the LoVo/Oxa+TPL group were significantly higher than those in the LoVo/Oxa+TPL group（P<0.01）;there was no significant difference in the relative expression of caspase-3 protein in cells between the two groups（P>0.05）.Conclusion TPL can reverses the resistance of colorectal cancer cells to Oxa by inhibiting cell proliferation, invasion and promoting cell apoptosis.更多还原