本文選題：白血病　切入點(diǎn)：實(shí)驗(yàn)性　出處：《腫瘤》2016年02期

【摘要】：目的 :探討甲氨蝶呤(methotrexate,MTX)是否可以逆轉(zhuǎn)磷酸核糖焦磷酸合成酶1(phosphoribosyl pyrophosphate synthetase 1,PRPS1)基因突變引起的急性淋巴細(xì)胞白血病細(xì)胞對(duì)6-巰基嘌呤(6-mercaptopurine,6-MP)的耐藥性,并初步探討其可能的作用機(jī)制。方法 :將攜帶野生型(wide type,WT)PRPS1基因以及A190T或S103T突變型PRPS1基因的重組慢病毒分別感染人急性淋巴細(xì)胞白血病Reh細(xì)胞以感染空載體GV303作為空白對(duì)照),采用細(xì)胞活力實(shí)驗(yàn)和蛋白質(zhì)印跡法驗(yàn)證上述細(xì)胞系構(gòu)建成功。分別用MTX和6-MP單藥以及MTX+6-MP聯(lián)合用藥處理各細(xì)胞系,然后錐蟲藍(lán)拒染法和AnnexinⅤ/7-AAD雙染法分別檢測(cè)細(xì)胞的增殖和凋亡情況;蛋白質(zhì)印跡法檢測(cè)DNA損傷相關(guān)蛋白和凋亡相關(guān)蛋白的表達(dá)水平;細(xì)胞活力實(shí)驗(yàn)和液相色譜-質(zhì)譜聯(lián)用法分別檢測(cè)MTX作用各細(xì)胞系后,6-MP對(duì)細(xì)胞的半數(shù)抑制濃度(half maximal inhibitory concentration,IC50)、6-MP代謝產(chǎn)物以及次黃嘌呤水平的改變。結(jié)果:外源性PRPS1蛋白在Reh細(xì)胞中成功過表達(dá),而且與對(duì)照組GV303和WT細(xì)胞系相比,感染PRPS1突變基因的A190T和S103T細(xì)胞系均對(duì)6-MP和MTX耐藥,差異均具有統(tǒng)計(jì)學(xué)意義(P值均0.001)。與MTX和6-MP單藥組相比,MTX+6-MP聯(lián)合用藥可以更有效地抑制A190T和S103T細(xì)胞的增殖(P值均0.001),促進(jìn)細(xì)胞凋亡(P0.01,P0.001),上調(diào)磷酸化組蛋白H2AX(phosphorylated H2AX,γH2AX)、磷酸化細(xì)胞周期檢驗(yàn)點(diǎn)激酶(2phosphorylated checkpoint kinase 2,p-Chk2)及聚(腺苷二磷酸核糖)聚合酶[poly(ADP-ribose)polymerase,PARP]剪切體的表達(dá)(P0.05,P0.01,P0.001)。此外,MTX處理各細(xì)胞后,6-MP對(duì)A190T和S103T細(xì)胞的IC50值遠(yuǎn)遠(yuǎn)低于MTX未處理組P值均0.001),且細(xì)胞中次黃嘌呤的水平明顯降低(P值均0.001),而6-MP的代謝產(chǎn)物硫代次黃嘌呤核苷酸(thioinosine monophosphate,TIMP)和硫代鳥嘌呤核苷酸(thioguanosine monophosphate,TGMP)的水平均明顯升高(P值均0.001)。結(jié)論 :MTX可以逆轉(zhuǎn)PRPS1基因突變引起的急性淋巴細(xì)胞白血病細(xì)胞對(duì)6-MP的耐藥性,其機(jī)制可能與其抑制次黃嘌呤的異常積累有關(guān)。
[Abstract]:Methods: human acute lymphoblastic leukemia (Reh) cells were infected with recombinant lentiviruses carrying wild type wide type WTPPS1 gene and A190T or S103T mutant PRPS1 gene respectively. The empty vector GV303 was used as a blank control. Cell viability test was used.And Western blot analysis showed that the above cell lines were successfully constructed.The cell lines were treated with MTX and 6-MP and MTX 6-MP respectively, and then the proliferation and apoptosis of the cells were detected by trypanosome blue exclusion and Annexin 鈪，

本文編號(hào)：1715229