摘要
目的通过比较酪氨酸激酶抑制药GW2974给药后母本细胞株BT474与耐药株r BT474之间代谢相关因子mRNA表达的差异,探讨r BT474可能的耐药机制。方法收集对数生长期r BT474和BT474细胞,接种于96孔板,加入含有GW2974的培养基,分别于孵育0,12,24和48 h后,通过噻唑蓝法检测BT474与r BT474细胞的增殖情况。用逆转录实时定量-聚合酶链反应检测12种相关代谢因子葡萄糖转运体4(GLUT4)、2,6-二磷酸果糖激酶(PFK-2)、丙酮酸激酶2(PKM2)、乳酸脱氢酶(LDHA)、丙酮酸羧化酶(PC)、6-磷酸果糖激酶(G-6-PD)、脂肪酸合成酶(FASN)、脂酰肉碱转移酶1 (CPT1A)、葡萄糖调节蛋白75(GRP75)、电压依赖性阴离子通道蛋白1 (VDAC1)、葡萄糖调节蛋白78(GRP78)和钙网蛋白(CALR)的mRNA在BT474细胞和r BT474细胞中给药前后的表达水平。结果给予GW2974后,母本细胞BT474中糖脂代谢相关因子GLUT4、PFK-2、PKM2、LDHA、PC、G-6-PD、FASN和CPT1A的mRNA表达水平均明显下调,线粒体应激因子GRP75、VDAC1与内质网应激因子CALR表达水平均明显下调,内质网应激因子GRP78表达明显上调。而在耐药株r BT474中给药后这12种代谢相关因子的mRNA表达水平均明显上调。结论耐药株r BT474可能通过调控糖脂代谢以及细胞应激等过程使细胞耐受性增强。
Objective To investigate the possible resistance mechanisms of rBT474 by comparing the mRNA expression of metabolism - related factors between parental cell line BT474 and resistant strain rBT474 cells after GW2974 administration. Methods The logarithmic phase rBT474 and BT474 cells were collected and inoculated into 96 hole cell culture plate. Then supplemented the medium with GW2974 and incubated 0, 12,24 and 48 h,respectively. The proliferation characteristics of BT474 and rBT474 cells were detected by methyl thiazolyl tetrazolium as- say. The expressions of mRNA of twelve metabolism - related factors in- cluding glucose transporter 4 ( GLUT4),6 - phosphofructo - 2 - kinase ( PFK -2),pyruvate kinase M2 isoform ( PKM2),lactate dehydrogen- ase A ( LDHA),phosphatidyl cholines ( PC),glucose 6 - phosphatede- hydrogenase ( G - 6 - PD),fatty acid synthase ( FASN),carnitine palmitoyltransferase - I ( CPT1A), glucose - regulated protein 75 ( GRP75),voltage - dependent anion channel ( VDAC1),glucose -regulated protein 78 ( GRP78) and calreticulin ( CALR) were detected by real - time quantitative polymerase chain reaction detecting system. Results After GW2974 administration,the mRNA of GLUT4,PFK - 2,PKM2,LDHA, PC,G -6 - PD,FASN and CPT1A,related with glucose and lipid metabolism in BT474 cells were down - regulated, the mitochondrial stress factors GRP75,VDAC1 and the endoplasmic reticulum stress factor CALR were down - regula- ted,the endoplasmic reticulum stress factor GRP78 was up - regulated. While the mRNA of twelve factors were up - regulated in resistant strains rBT474 after administration. Conclusion The glucose and lipid metabolism as well as cellu- lar stress tolerance and the up -regulation of other metabolic -related proteins make GW2974 - resistance in rBT474 cells.
作者
闫聪聪
李志寒
贺佳
马姝丽
吴光华
张淼
毛宇彬
YAN Cong-cong;LI Zhi-han;HE Jia;MA Shu-li;WU Guang-hua;ZHANG Miao;MAO Yu-bin(Department of Pharmacy,Children’s Hospital Affiliated to Zhengzhou University,Henan Children’s Hospital, Zhengzhou Children ’ s Hospital, Zhengzhou 450053,China;School of Medicine,Xiamen University, Xiamen 361102,Fujian Province,China)
出处
《中国临床药理学杂志》
CAS
CSCD
北大核心
2019年第15期1629-1632,共4页
The Chinese Journal of Clinical Pharmacology
基金
福建省自然科学基金面上项目(2012J01417)
