CAJ | 학술논문

目的探讨淋巴瘤细胞系T2和非霍奇金淋巴瘤(NHL)组织中酪氨酸磷酸酶1基因启动子区甲基化状态,三氧化二砷(As2O3)对T2细胞中SHP-1的去甲基化作用及对T2细胞生长增殖的生物学影响.方法以不同浓度As2O3处理淋巴瘤细胞株T2,采用二苯基溴化四氮唑蓝(MTT)法检测T2细胞的生长变化,采用流式细胞术检测细胞凋亡率的变化,采用实时定量聚合酶链反应(FQ-PCR)和Western blot方法检测T2细胞SHP-1基因mRNA和蛋白表达及c-kit蛋白表达变化.采用甲基化特异性聚合酶链反应(MSP)检测T2细胞和32例NHL组织SHP-1基因启动子甲基化状态.结果 As2O3使T2细胞增殖受抑,凋亡增加,效应均具有时间和剂量依赖性.As2O3能逆转T2细胞SHP-1基因启动子甲基化,SHP-1基因恢复表达,同时c-kit蛋白磷酸化水平降低.SHP-1基因在对照组淋巴组织中呈完全性非甲基化状态,而在NHL组织中启动子甲基化出现率为87.5%(28/32).结论在淋巴瘤细胞和NHL组织中,SHP-1基因启动子区域存在高度甲基化.As2O3能逆转T2细胞DNA异常甲基化,诱导SHP-1基因表达,并可能通过抑制c-kit受体及其信号传导路径的活化,抑制肿瘤细胞增殖.
목적 탐토림파류세포계T2화비곽기금림파류(NHL)조직중락안산린산매1기인계동자구갑기화상태,삼양화이신(As2O3)대T2세포중SHP-1적거갑기화작용급대T2세포생장증식적생물학영향.방법 이불동농도As2O3처리림파류세포주T2,채용이분기추화사담서람(MTT)법검측T2세포적생장변화,채용류식세포술검측세포조망솔적변화,채용실시정량취합매련반응(FQ-PCR)화Western blot방법검측T2세포SHP-1기인mRNA화단백표체급c-kit단백표체변화.채용갑기화특이성취합매련반응(MSP)검측T2세포화32례NHL조직SHP-1기인계동자갑기화상태.결과 As2O3사T2세포증식수억,조망증가,효응균구유시간화제량의뢰성.As2O3능역전T2세포SHP-1기인계동자갑기화,SHP-1기인회복표체,동시c-kit단백린산화수평강저.SHP-1기인재대조조림파조직중정완전성비갑기화상태,이재NHL조직중계동자갑기화출현솔위87.5%(28/32).결론 재림파류세포화NHL조직중,SHP-1기인계동자구역존재고도갑기화.As2O3능역전T2세포DNA이상갑기화,유도SHP-1기인표체,병가능통과억제c-kit수체급기신호전도로경적활화,억제종류세포증식.
Objective To investigate the methylation of CpG island in the SHP-1 gene promoter and its significance in lymphoma. To evaluate the effects of As2O3 on demethylation of SHP-1 in human lymphoma cell line T2 and on proliferation of T2 cells. Methods T2 cells were treated with As2O3. Methylation specific PCR was used to detected the status of SHP-1 methylation in newly diagnosed lymphoma tissues and the T2 cells. The mRNA and protein expression of SHP-1 were determined by FQ-PCR and Western blot. The expression of phospha-c-kit was examined by Westren blot. MTT and flow cytometry were used to determine the growth and apoptosis in T2 cells. Results T2 cells contained completely methylated SHP-1. Furthermore, there was constitutive c-kit phosphorylation. The expression of SHP-1 was recoverd when the cells exposed to As2O3, and concomitant with increasing SHP-1, a parallel down-regulation of phosphorylated c-kit occurred, so that by day 3 pbesphorylated c-kit was barely detectable. As2O3 inhibited the cell growth, and the effects were dose- and time-dependent. As2O3 also increased apeptosis rate of T2 cells in a dose- and time-dependent manner, too, and on the 1,2, 3 of treatment with As2O3 (2.5 μmol/L), the apoptosis rates were 6. 12% ,26.53% ,50.90%, respectively. The frequency of methylation in SHP-1 gene promoter in lymphoma tissues was 87.5% (28/32). In the control group, however, 12 specimens of benign lymph node proliferation showed no methylation in CpG island of SHP-1 gene promoter. Conclusion Hypermethylation of SHP-1 gene promoter in lymphoma indicates the inactivation of SHP-1 gene and its possible role in the tumorigenesis of lymphoma. As2O3 can effctively cause demethylation and inhibit the growth of tumor by reactivating the SHP-1 gene transcription. SHP-1 methylation leading to epigenetic activation of c-kit may have a tentative role in the pathogenesis of lymphoma. Therefore, As2O3 is potentially useful in the treatment of lymphoma as a demethylating gent.