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目的:通过检测经典Wnt信号通路在非霍奇金淋巴瘤(non-Hodgkin’s lymphoma,NHL)细胞株中是否存在异常激活,以探讨其与NHL发病的相关性。方法:选取人NHL细胞株SUDHL-4、Raji和Namalwa(以正常人淋巴细胞作为对照),采用蛋白质印迹法检测各细胞株中β-链蛋白(β-catenin)、糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)及其失活形式Ser9被磷酸化的GSK-3β[phospho-glycogen synthase kinase-3β(Ser9),p-GSK-3β(Ser9)]蛋白的表达情况;应用实时荧光定量-PCR技术检测β-catenin编码基因CTNNB1、经典Wnt通路上游相关调控因子低密度脂蛋白受体相关蛋白5(low density lipoprotein receptor related protein5,LRP5)基因及靶基因c-Myc的转录表达水平。结果:与正常对照组淋巴细胞相比较,3株NHL细胞中总蛋白及细胞核内β-catenin蛋白的表达水平明显上调;p-GSK-3β(Ser9)蛋白较正常对照也存在不同水平表达上调的现象,而GSK-3β蛋白的表达在各组间差异无统计学意义。NHL细胞中CTNNB1、LRP5及c-MycmRNA表达水平与正常对照相比,均有明显提高。结论:β-catenin作为经典Wnt信号通路重要的激活标志,其在NHL中的高表达及经典Wnt通路其他相关分子异常,说明经典Wnt信号通路可能是导致NHL的分子作用机制之一。
OBJECTIVE: To investigate whether classical Wnt signaling pathway is abnormally activated in non-Hodgkin’s lymphoma (NHL) cell lines and to explore its association with the pathogenesis of NHL. Methods: The human NHL cell lines SUDHL-4, Raji and Namalwa were selected. The normal human lymphocytes were used as control. The protein expression of β-catenin, glycogen synthase kinase-3β The phosphorylation of GSK-3β (Ser9), p-GSK-3β (Ser9)] of phosphorylated GSK-3β and its inactivated form Ser9 were detected by Western blotting. Real-time fluorescence quantitative PCR was used to detect the expression of β-catenin coding gene CTNNB1, the transcription factor of low density lipoprotein receptor related protein 5 (LRP5) and target gene c-Myc upstream of Wnt pathway The expression level. Results: Compared with normal control group, the expression of β-catenin in total protein and nucleus was significantly increased in three NHL cells. The expression of p-GSK-3β (Ser9) protein was also up-regulated compared with the normal control However, the expression of GSK-3β was not significantly different between groups. The expression levels of CTNNB1, LRP5 and c-MycmRNA in NHL cells were significantly increased compared with the normal control. CONCLUSION: β-catenin is an important activation sign of canonical Wnt signaling pathway. The high expression of β-catenin in NHL and the abnormality of other related molecules in canonical Wnt pathway indicate that the classical Wnt signaling pathway may be one of the molecular mechanisms that lead to NHL.