转染正常c-CBL基因后耐药GIST体外药物敏感实验研究

Study on the imatinib sensitivity of drug-resistant gastrointestinal stromal tumor cells by transfection of c-CBL gene in vitro

目的研究转染正常c-CBL基因后耐药胃肠道间质瘤(GIST)细胞在体外对甲磺酸伊马替尼作用的影响。方法选择的瘤源细胞来自1例29岁青年男性患者,诊断为小肠间质瘤复发伴腹腔转移。通过细胞爬片、Western blot、RT-PCR检测基因鉴定细胞。通过将携带正常c-CBL基因的反转录病毒载体转染耐药GIST细胞中,四唑盐比色法(MTT)测定转染前后耐药GIST细胞对甲磺酸伊马替尼药物敏感性。结果 Western Blot检测培养耐药GIST的C-kit稳定表达C-kit蛋白,基因测序显示耐药GIST细胞C-kit突变位点位于第9号外显子,通过检测显示培养耐药GIST保持了原有的生物学特性。甲磺酸伊马替尼对GIST细胞作用72h MTT实验结果显示:转染前耐药细胞在1.6μmol/L浓度时,抑制率未超过50%;然而转然后,在0.10μmol/L浓度抑制率达到76.5%,最佳浓度为0.10~0.20μmol/L,转染前后不同浓度的抑制率差异有统计学意义(P<0.01)。结论通过转染正常c-CBL至耐药GIST细胞,增强了对甲磺酸伊马替尼的敏感性。

Objective To study on the effect of drug-resistant gastrointestinal stromal tumor（GIST） cells on imatinib in vitro after transfection of normal c-CBL gene. Methods The tumor cells were derived from a 29-year-old young male patient diagnosed with GIST recurrence with abdominal metastasis.The cultured cells were detected by cell cover-slips,Western blot and reverse transcription-polymerase chain reaction （RT-PCR）.Retro-viral vector with a normal c-CBL gene was transformed into imatinib-resistant GIST,detected the imatinib sensitivity before and after transfection by methyl thiazolyl tetrazolium （MTT）. Results The C-kit protein of cultured imatinib-resistant GIST cells was detected by Western blot,gene sequencing showed that the imatinib-resistant GIST cells C-kit mutation site was exon 9,the cultured cells maintained the original biological characteristics.The MTT results of imatinib terms of GIST cells for 72 hours showed that the inhibition rate did not exceed 50% when the concentration was 1.6 μmol/L before transfection,however,the inhibition rate reached 76.5% when the concentration was 0.10 μmol/L after transfection,the best concentration was 0.10-0.20 μmol/L,there was statistically significant difference in the inhibition rates of different concentrations before and after transfection （ P 〈0.01）. Conclusion By transfection of normal c-CBL gene into imatinib-resistant GIST cells,it enhances the sensitivity to imatinib.