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目的探讨 p27~(kip1)及其出核相关分子激活蛋白1(AP-1)的辅助因子 Jab1在淋巴瘤细胞中的表达变化及相互关系。方法采用血清饥饿合并释放方法同步化处理淋巴瘤细胞系 Jurkat 和Raji 细胞,分别采用 Western blot 及 RT-PCR 技术检测 p27~(kip1)、Jab1在淋巴瘤细胞中的蛋白表达水平和mRNA 表达水平变化;采用来普霉素 B(LMB)刺激增殖过程中的淋巴瘤细胞,检测 p27~(kip1)、Jab1的表达变化;构建人 Jab1基因的 pcDNA3.1-myc-Jab1的真核表达质粒,脂质体转染 Jurkat 细胞,配合免疫荧光技术检测 p27~(kip1)的亚细胞定位情况;免疫沉淀检测 Jurkat 和 Raji 细胞中 p27~(kip1)与 Jab1的结合情况。结果血清饥饿导致 Jurkat 和 Raji 细胞生长周期停滞,p27~(kip1)蛋白总量增加,Jab1蛋白总量减少。血清释放后两者的蛋白水平呈现相反的表达变化,而 p27~(kip1)mRNA 水平无明显改变。LMB 可以抑制由血清释放引起的细胞增殖,在此过程中 p27~(kip1)表达上调,Jab1表达下调。转染 Jab1真核表达质粒的 Jar-kat 细胞 p27~(kip1)定位有明显的改变。免疫沉淀结果显示在 Jurkat 和 Raji 细胞中 p27~(kip1)与 Jab1相互结合。结论 Jab1可能通过与 p27~(kip1)结合来介导 p27~(kip1)的核内外分布并影响其表达,进而影响 p27~(kip1)的功能状态,从而参与调控淋巴瘤细胞的生长。
Objective To investigate the expression changes and correlation of p27 (kip1) and Jab1, a coreactor of its nuclear export-related molecular activator 1 (AP-1), in lymphoma cells. Methods The lymphocyte lines Jurkat and Raji were synchronized by serum starvation combined with release method. The expression of p27 kip1 and Jab1 in lymphoma cells and mRNA expression levels were detected by Western blot and RT-PCR respectively The expression of p27 kip1 and Jab1 was detected by LMB stimulation. The eukaryotic expression plasmid pcDNA3.1-myc-Jab1 of human Jab1 gene was constructed, The plastids were transfected into Jurkat cells, and the subcellular localization of p27 kip1 was detected by immunofluorescence. The binding of p27 kip1 to Jab1 was detected by immunoprecipitation in Jurkat and Raji cells. Results Serum starvation resulted in the arrest of the growth cycle of Jurkat and Raji cells. The total amount of p27 kip1 protein increased and the total amount of Jab1 protein decreased. Serum levels of the two proteins showed the opposite expression changes, while p27 (kip1) mRNA levels did not change significantly. LMB can inhibit cell proliferation caused by serum release, in the process p27 ~ (kip1) upregulation, Jab1 expression down. Jar-kat cells transfected with Jab1 eukaryotic expression plasmid p27 kip1 localization significantly changed. Immunoprecipitation results showed that p27 ~ (kip1) interacts with Jab1 in Jurkat and Raji cells. Conclusion Jab1 may mediate the intranuclear distribution of p27 kip1 and affect the expression of p27 kip1, which may affect the functional status of p27 kip1 and thus regulate the growth of lymphoma cells.