靶向二氢乳清酸脱氢酶改善吉西他滨在胰腺导管腺癌中的疗效  

Targeting dihydroorotate dehydrogenase improves gemcitabine efficacy in pancreatic ductal adenocarcinoma

作　　者：刘文博 李明豆 赵斌[1] 李志刚 焦作义[1] Liu Wenbo;Li Mingdou;Zhao Bin;Li Zhigang;Jiao Zuoyi(Department of General Surgery,Lanzhou University Second Hospital,Lanzhou University Second Hospital Clinical Medical School,Lanzhou 730030,China)

机构地区：[1]兰州大学第二医院、兰州大学第二临床医学院普通外科,兰州730030

出　　处：《中华实验外科杂志》2025年第1期33-36,共4页Chinese Journal of Experimental Surgery

基　　金：甘肃省科技重大专项(21ZD4FA015);国家自然科学基金面上项目(32170729)。

摘　　要：目的探究Kirsten大鼠肉瘤病毒癌基因同源物G12D突变(Kras G12D)介导胰腺导管腺癌(PDAC)吉西他滨耐药的机制以及来氟米特联合吉西他滨对PDAC的疗效。方法构建Kras G12D过表达PDAC细胞系,使用实时定量荧光聚合酶链反应(RT-qPCR)和蛋白质印迹法探究Kras G12D对DHODH表达的影响。使用Kras G12D/+(K)小鼠、转化相关蛋白R172H杂合突变(LSL-Trp53 R172H/+,P)小鼠、胰岛细胞同源框转录因子1-环化重组酶嵌合体(Pdx1-Cre,C)小鼠繁育KPC、KC、C基因工程小鼠,模拟PDAC发展过程,探究DHODH与PDAC发展的关系。细胞活力实验探究二氢乳清酸脱氢酶(DHODH)在Kras G12D介导的吉西他滨耐药中的作用。构建24只KPC同种异体移植模型,简单随机方法分为对照组、吉西他滨组、来氟米特组、吉西他滨和来氟米特联用组,每组各6只,研究来氟米特联合吉西他滨对PDAC的治疗效果。组间比较采用独立样本t检验。结果Kras G12D过表达HPNE细胞中,DHODH mRNA和蛋白相对水平高于对照组(1.88±0.22比1.01±0.16,t=5.556,P<0.01;1.63±0.13比1.00±0.00,t=8.610,P<0.01)。DHODH免疫组织化学(IHC)评分在KC小鼠中高于C小鼠[(103.33±47.26)分比(20.00±10.00)分,t=2.988,P<0.01],在KPC小鼠中高于KC小鼠[(265.00±22.91)分比(103.33±47.26)分,t=5.332,P<0.01]。Kras G12D过表达组BXPC3细胞72 h 450 nm波长吸光度(A 450)值高于对照组(1.61±0.01比0.92±0.01,t=160.469,P<0.01),Kras G12D过表达联合DHODH敲减组与DHODH敲减组A 450值比较,差异无统计学意义(0.16±0.01比0.17±0.01,t=1.546,P>0.05)并分别低于对照组(0.16±0.01比0.92±0.01,t=205.199,P<0.01;0.17±0.01比0.92±0.01,t=253.906,P<0.01)。吉西他滨单药组肿瘤体积与对照组比较,差异无统计学意义[(1132.43±164.96)mm^(3)比(1297.95±154.16)mm^(3),t=1.796,P>0.05],来氟米特和吉西他滨联合组肿瘤体积小于吉西他滨单药组[(320.80±42.45)mm^(3)比(1132.43±164.96)mm^(3),t=12.433,P<0.01]。吉西他滨单药组Ki-67和cleaved Objective To investigate how Kirsten rat sarcoma viral oncogene homolog p.Gly12Asp(Kras G12D)causes gemcitabine resistance in pancreatic ductal adenocarcinoma(PDAC),and study the therapeutic efficacy of leflunomide combined with gemcitabine.Methods Kras G12D over-expressed PDAC cell lines were established,and the effects of Kras G12D on DHODH expression were explored using quantitative real-time polymerase chain reaction(RT-qPCR)and Western blotting.KPC,KC and C mice were bred using Kirsten rat sarcoma viral oncogene homolog p.Gly12Asp(Kras G12D/+,K)mice,Pancreatic and Duodenal Homeobox 1-Cyclization Recombination Enzyme(Pdx1-Cre,C)mice and Transformation-Related Protein 53 p.Lys172His(Trp53 R172H/+,P)mice to simulate the progression of PDAC in order to investigate the relationship between DHODH and PDAC progression.The role of DHODH in Kras G12D-mediated gemcitabine resistance was explored using cell viability assays.A total of 24 KPC allografts were established and divided into 4 groups of control,gemcitabine,leflunomide,and gemcitabine combined with leflunomide,with 6 mice per group by a simple randomised method to study the therapeutic effect of leflunomide combined with gemcitabine.The comparisons between groups were made using independent samples t-test,with P<0.05 as statistically significant difference.Results The relative mRNA and protein level of DHODH in Kras G12D over-expressed HPNE cells were higher than those in the control group(1.88±0.22 vs.1.01±0.16,t=5.556,P<0.01;1.63±0.13 vs.1.00±0.00,t=8.610,P<0.01).The immunohistochemistry(IHC)score of DHODH in pancreatic tissues was greater in KC mice than in C mice(103.33±47.26 vs.20.00±10.00,t=2.988,P<0.01),and greater in KPC mice than in KC mice(265.00±22.91 vs.103.33±47.26,t=5.332,P<0.01),with statistically significance.After treatment with gemcitabine,the Kras G12D overexpressed BXPC3 cells showed higher absorbance at 450 nm(A 450)than the control group as measured after 72 h(1.61±0.01 vs.0.92±0.01,t=160.469,P<0.01),while the difference in A 4

关 键 词：胰腺导管腺癌 吉西他滨耐药 二氢乳清酸脱氢酶 

分 类 号：R735.9[医药卫生—肿瘤]