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目的应用重组腺病毒介导的Wnt-3a激活急性T淋巴细胞白血病细胞株Jurkat中的经典Wnt/β-catenin信号途径,观察该途径激活后对Jurkat细胞增殖及细胞周期的影响,初步探讨其在急性T淋巴细胞白血病分子发病机制中的作用。方法将携带Wnt-3a基因的重组腺病毒(Ad5-Wnt-3a)感染Jurkat细胞,实验分为3组:实验组(Jurkat/Ad5-Wnt-3a)、空载体组(Jurkat/Ad5-GFP)和空白对照组(Jurkat)。转染后,采用RT-PCR方法检测Jurkat细胞内Wnt-3amRNA的表达;Westernblot检测细胞内β-catenin蛋白的表达;MTT法检测细胞增殖,FCM法检测细胞周期,RT-PCR检测Wnt信号通路下游靶基因c-myc和cyclinD1mRNA表达。结果重组腺病毒Ad5-Wnt-3a转染Jurkat细胞后48h,实验组有效表达Wnt-3a的基因。Westernblot结果显示,与对照组比较,实验组细胞内β-catenin蛋白表达明显增加(P<0.05)。MTT结果显示,Wnt-3a转染后48h和72h可明显促进Jurkat细胞的增殖,与对照组相比差异有统计学意义(P<0.05)。FCM检测发现,Wnt-3a转染后可引起细胞G0/G1期下降,S期升高(P<0.05)。RT-PCR检测结果显示细胞内c-myc和cyclinD1mRNA表达升高,与对照组相比差异有统计学意义(P<0.05)。结论腺病毒介导的Wnt-3a可以激活Jurkat细胞中的经典Wnt/β-catenin信号途径,使Jurkat细胞增殖,促进细胞周期进展,其可能的机制是通过调控其下游靶基因c-myc和cyclinD1的表达实现。
Objective To investigate the effect of Wnt-3a on Wnt / 3a activation of Jurkat cell line Jurkat induced by recombinant adenovirus and the effect of this pathway on the proliferation and cell cycle of Jurkat cells. Role of molecular pathogenesis in acute T lymphocytic leukemia. Methods Jurkat cells were infected with recombinant adenovirus carrying Wnt-3a gene (Ad5-Wnt-3a). The experiment was divided into three groups: experimental group (Jurkat / Ad5-Wnt-3a), empty vector group (Jurkat / Ad5- And blank control group (Jurkat). After transfection, the expression of Wnt-3 mRNA in Jurkat cells was detected by RT-PCR; the expression of β-catenin protein in Jurkat cells was detected by Western blot; the cell proliferation was detected by MTT assay; the cell cycle was detected by FCM; Target genes c-myc and cyclinD1 mRNA expression. Results 48h after transfection of Ad5-Wnt-3a with Jurkat cells, the experimental group effectively expressed Wnt-3a gene. Western blot results showed that compared with the control group, the intracellular β-catenin protein expression was significantly increased (P <0.05). The results of MTT showed that the proliferation of Jurkat cells was significantly enhanced at 48h and 72h after Wnt-3a transfection compared with the control group (P <0.05). The results of FCM showed that Wnt-3a transfected cells could decrease the G0 / G1 phase and increase the S phase (P <0.05). The results of RT-PCR showed that the expression of c-myc and cyclinD1 mRNA in the cells increased significantly compared with the control group (P <0.05). Conclusions Adenovirus-mediated Wnt-3a can activate the canonical Wnt / β-catenin signaling pathway in Jurkat cells and promote Jurkat cell proliferation and cell cycle progression. The possible mechanism is that Wnt-3a regulates the expression of c-myc and cyclinD1 The realization of the expression.