CAJ | 학술논문

目的:探讨胃肠道间质瘤相关基因突变体KIT Ins IPYD579和PDGFRA L839P对格列卫治疗的药物敏感性.方法:采用脂质体法将胃肠道间质瘤相关基因突变体KIT De1559-560、KIT Ins IPYD579、PDGFRA D842V及PDGFRA L839P重组质粒分别瞬时转染中国仓鼠卵巢上皮细胞系(CHO),表达24h后与格列卫共孵育90min,用Western-blot法检测的相关蛋白表达及其磷酸化状态.另外,用MTT法检测各组稳转PDGFRA基因突变体的CHO细胞与不同浓度格列卫共孵育72h后的增殖变化.结果:当格列卫浓度为0.1μM时,转染KIT De1559-560重组质粒的CHO细胞磷酸化KIT蛋白表达量明显降低.当格列卫浓度为1μM时,转染KIT Ins IPYD579和PDGFRA L839P重组质粒的CHO细胞的相应蛋白磷酸化形式不能检出.而转染PDGFRA D842V重组质粒的CHO细胞直至格列卫浓度提高至5μM时,其磷酸化PDGFRA蛋白表达水平仍无明显降低.MTT法显示:当格列卫浓度提高至1μM时,转染PDGFRA L839P重组质粒的CHO细胞增殖百分比明显降低,而转染PDGFRA D842V重组质粒的CHO细胞增殖百分比直至格列卫浓度为5μM时仍无明显变化.结论:格列卫在短时间内可使PDGFRA L839P或KIT Ins IPYD579突变体磷酸化蛋白表达减少,长期作用时对表达PDGFRA L839P突变体的细胞增殖具有明显的抑制作用.
목적:탐토위장도간질류상관기인돌변체KIT Ins IPYD579화PDGFRA L839P대격렬위치료적약물민감성.방법:채용지질체법장위장도간질류상관기인돌변체KIT De1559-560、KIT Ins IPYD579、PDGFRA D842V급PDGFRA L839P중조질립분별순시전염중국창서란소상피세포계(CHO),표체24h후여격렬위공부육90min,용Western-blot법검측적상관단백표체급기린산화상태.령외,용MTT법검측각조은전PDGFRA기인돌변체적CHO세포여불동농도격렬위공부육72h후적증식변화.결과:당격렬위농도위0.1μM시,전염KIT De1559-560중조질립적CHO세포린산화KIT단백표체량명현강저.당격렬위농도위1μM시,전염KIT Ins IPYD579화PDGFRA L839P중조질립적CHO세포적상응단백린산화형식불능검출.이전염PDGFRA D842V중조질립적CHO세포직지격렬위농도제고지5μM시,기린산화PDGFRA단백표체수평잉무명현강저.MTT법현시:당격렬위농도제고지1μM시,전염PDGFRA L839P중조질립적CHO세포증식백분비명현강저,이전염PDGFRA D842V중조질립적CHO세포증식백분비직지격렬위농도위5μM시잉무명현변화.결론:격렬위재단시간내가사PDGFRA L839P혹KIT Ins IPYD579돌변체린산화단백표체감소,장기작용시대표체PDGFRA L839P돌변체적세포증식구유명현적억제작용.
Objective: To explore the sensitivity of Kit or PDGFRA mutants related to gastrointestinal stromal tumor (GIST) to Gleevec.Methods: The recombinant plasmids of KIT Del559-560, KIT Ins IPYD579, PDGFRA D842V and PDG-FRA L839P gene mutants were transiently transformed into the CHO cells by liposome methods.Western blot was used to detect the expression of the related protein and their phosphorylated forms after the cells were incubated with Gleevec for 90 min.At 72 hours after incubation with Gleevec, MTT was used to detect cell proliferation.Results: Western blot results showed that Gleevec at 0.1 μM can notably reduce phosphorylation of KIT Del559-560.Gleevec at 1μM completely blocked phosphorylation of KIT Ins IPYD579 and PDGFRA L839P, but did not affect PDGFRA D842V phosphorylation.MTT analy-sis indicated that growth of CHOPDGFRA L839P was inhibited by Gleevec at 1μM, however, CHOPDGFRA D842V was re-sistant to Gleevec at 5 μM.Conclusion: Gleevec can decrease the expression of phosphorylated protein CHOPDGFRA L839P and CHOKIT Ins IPYD579, and can remarkably inhibit the proliferation of cells containing PDGFRA L839P mutant.