摘要目的 应用聚合酶链反应(PCR)芯片技术观察特异性抑制髓母细胞瘤细胞1型胰岛素生长因子受体(IGF-1R)信号通路的差异性基因表达,探讨IGF-1R在髓母细胞瘤细胞增殖中的作用.方法 体外培养髓母细胞瘤Doay细胞株,使用不同终质量浓度NVP-ADW742作用48 h和24 h后,检测细胞增殖和凋亡,分别使用IGF-1R酪氨酸蛋白激酶抑制剂NVP-ADW742终质量浓度为0、0.5、1.0、2.0、5.0、10.0、20.0、50.0μmol/L作用48 h后检测细胞存活率,以及终质量浓度为0.1、1.0、2.0、5.0、10.0μmol/L作用24 h后检测凋亡率,应用PCR芯片技术检测IGF-1R信号通路的差异性基因表达,并采用Western blot检测其蛋白表达.结果 在对应浓度的NVP-ADW742作用下Doay细胞的存活率分别为95.95%、93.95%、91.97%、86.60%、70.57%、15.01%、1.63%(P＜0.05),凋亡率分别为6.92%、8.83%、11.22%、17.27%、29.98%(P＜0.05),两者呈剂量依赖性,其浓度越高,抑制和促凋亡作用越强;在PCR芯片检测有14个差异表达基因;Western blot检测表明在2μmol/L的NVP-ADW742作用下,随时间延长PI3K、Akt、p38、GSK-3β和bcl-2蛋白表达逐渐下调.结论 髓母细胞瘤中IGF-1R信号通路异常可以促进肿瘤细胞的增殖,利用功能性PCR芯片可以有效地检测肿瘤细胞中相关基因在肿瘤增殖和凋亡中的作用.

abstractsObjective To investigate the insulin-like growth factors and their receptor (IGF-1R)pathway by human insulin signaling pathway reverse transcription-polymerase chain reaction (RT-PCR) array which profiles the expression of 84 genes related to insulin-responsive genes. Methods Doay cells were treated with different concentrations of specific IGF-IR kinase inhibitor, NVP-ADW742 (0, 0.5,1.0, 2.0, 5.0, 10.0, 20.0, 50.0 μmol/L) for48 h and were analyzed to confirm cell survival. Also Doay cells were incubated for 24 h in the presence of different concentrations of NVP-ADW742 (0.1, 1.0,2.0, 5.0, 10.0 μmol/L) and apoptotic rate was determined.. Eighty-four genes related to insulin-responsive genes were examined to determine the difference of gene expression. The result of PCR array was verified by Western blotting. Results Cell counting kit-8 assay indicated that the survival rate of Doay cells induced with corresponding different concentrations of NVP-ADW742 was 95.95%, 93.95%, 91.97%,86.60%, 70.57%, 15.01%, 1.63% (P ＜ 0.05). Flow cytometry using Annexin V-FITC test revealed that the apoptosis rate of Doay cells was 6.92%, 8.83%, 11.22%, 17.27%, 29.98% ( P ＜ 0.05 ).NVP-ADW742 resulted in Doay cell survival and apoptosis in a dose-dependent manner. Doay cells of PCR-Array revealed that 14 genes associated with PI3K, MAPK pathways and metabolism of carbohydrates were down-regulated in Doay cells following treatment of NVP-ADW742, including AKT1, ARAF, BRAF,CEBPB, GSK3, JUN, MAP2K1, NCK1, PIK3R2, PRKCG, PTPN1, RRAS, SORES1. Furthermore, we verified the differences in protein level by Western blotting which focused on PI3K, Akt, p38, GSK-3β,C/EBPβ. The phosphorylation of Akt, p38 and GSK-3β was down-regulated and the total levels of PI3K,Akt, p38, GSK-3β, C/EBPβ and bcl-2 were reduced by NVP-ADW742 treatment. Conclusion The expression of IGF-1R plays an important role in development of medulloblastoma by promoting abnormal tumor cellular proliferation. NVP-ADW742 inhibits IGF-1R pathway probably by inactivating PI3K/Akt/MAPK pathway.