Src蛋白在伊马替尼继发耐药的胃肠道间质瘤中的表达及意义

Expression of Src protein in gastrointestinal stromal tumors tissues with secondary resistance to Imatinib Mesylate

目的 分析Src蛋白在伊马替尼继发性耐药的胃肠道间质瘤组织中的表达,探讨GIST中除c-kit基因二次突变外伊马替尼耐药的可能机制.方法 收集2009年1月至2013年9月手术切除留存标本,术后伊马替尼辅助治疗过程中出现继发性耐药,但因肠梗阻及消化道出血接受二次手术的GIST患者19例,继发性耐药GIST二次手术切除的肿瘤组织行kit基因常见突变位点检测,Western blot和免疫组织化学检测组织中Src及p-Src蛋白的表达,以20例术前伊马替尼新辅助治疗的GIST及25例未治疗直接手术GIST分别设为阳性及阴性对照,并分析其基因与蛋白差异.结果 耐药组c-kit基因外显子11突变者17例,（其中4例同时合并外显子13突变,1例外显子17突变,2例外显子14突变,2例外显子18突变,此9例患者第一次手术标本均为单独的c-kit基因外显子11突变）,外显子9突变2例.阳性对照组中c-kit基因原发突变者20例,其中17例外显子11,3例外显子9突变.阴性对照组中c-kit基因原发突变者25例,其中18例外显子11突变,7例外显子9突变.Western blot发现Src蛋白相对表达水平在阴性对照组、阳性对照组及耐药组中分别为0.50±0.05、0.54±0.04及0.59±0.05,差异无统计学意义.p-Src蛋白的表达在阴性对照组、阳性对照组及耐药组中的表达分别为0.23±0.02,0.31±0.01及0.61±0.02,耐药组中p-Src蛋白表达较阳性及阴性对照组明显增加（P〈0.05）,与此同时阳性对照组p-Src的表达较阴性对照组升高（P〈0.05）.免疫组织化学检测Src蛋白表达的平均A值在阴性对照组、阳性对照组及耐药组分别为0.45±0.03、0.71±0.02及0.73±0.02,差异无统计学意义（P〉0.05）;p-Src蛋白表达的平均A值在阴性对照组、阳性对照组及耐药组分别为0.21±0.01、0.40＋0.02及0.72±0.01,耐药组中p-Src蛋白表达明显增加,差异具有统计学意义（P〈0.05）.两种检测方法亚组分析显示无基因二次突变的患者Src蛋白和p-Src表达与二次突变组的表达水平无明显区别.结论 Src蛋白磷酸化可能在c-kit基因二次突变以外的GIST耐药机制中发挥作用,p-Src蛋白检测能为GIST的继发性耐药提供新的研究靶点.

Objective To investigate the expression of Src in gastrointestinal stromal tumors （GIST） tissues with secondary resistance to Imatinib Mesylate and elucidate the mechanisms secondary resistance to Imatinib without c-kit/PIX}FRA gene mutation. Methods The mutation site of c-kit gene were respectively detected using PCR and sequencing in 25 GIST tissues without secondary resistance to Imatinib Mesylate and J9 GIST with secondary resistance to Imatinib Mesylate, meanwhile the ex- pression of Src and p-Src were detected by western blot and immunochemistry assay. 20 GIST patients treated with Imatinib before surgery and 25 GIST without any treatment were enrolled as positive and negative groups. Results Among 25 GIST tissues without secondary resistance to Imatinib Mesylate, mutations of c-kit exon were found in 20 patients,while mutations were found in 17 GIST with second- ary resistance to Imatinib Mesylate. Mutations in c-kit exon in positive and negative group were 20 and 25 respectively. We found secondary mutation of c-kit gene in 9 tissues, while mutations were not found in the other group. No differences of expression of Src protein were detected between tissues with or without secondary resistance to Imatinib Mesylate. The expression level of p-Src protein was significantly elevated in GIST tissues with secondary resistance to Imatinib Mesylate. Conclusion Our results suggest that p-Src may play an important role in the mechanisms of secondary resistance to Imatinib in GIST, and provide novel predicting targets for secondary resistance to Imatinib in GIST.