论文部分内容阅读
mRNA差异显示技术克隆的喉癌相关基因LCRG1,对不表达该基因的喉癌细胞系(Hep-2)的生长具有明显抑制作用.软件分析推测,LCRG1可能在细胞信号传导中发挥作用.为进一步地研究LCRG1的功能,应用RT-PCR和平板克隆形成实验证实,经多次传代的Hep-2/LCRG1细胞,仍表达LCRG1,且LCRG1具有显著的抑制细胞增殖的能力.抽提Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系总蛋白质,应用固相pH梯度(IPG)双向凝胶电泳(2DGE),结合抗酪氨酸磷酸化抗体的免疫印迹和基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS),鉴定酪氨酸磷酸化的蛋白质.得到了分辨率较高、重复性较好的Hep-2/LCRG1和Hep-2/pcDNA3.1(+)细胞系的总蛋白质双向凝胶电泳图谱;结合免疫印迹反应、软件分析和质谱技术识别并鉴定了13个差异反应的酪氨酸磷酸化的蛋白质.这些蛋白质参与了细胞信号传导和细胞代谢等过程.推测LCRG1可能是通过调节这些蛋白质的酪氨酸磷酸化、去磷酸化状态,参与细胞增殖、代谢和凋亡等过程的调控,而发挥抑瘤作用.这为全面、真实地揭示LCRG1抑瘤作用的分子机理提供了新思路.
LCRG1 was cloned by mRNA differential display technique, which could significantly inhibit the growth of laryngeal carcinoma cell line (Hep-2) that does not express this gene.Analysis of software suggests that LCRG1 may play a role in cell signaling.For further To investigate the function of LCRG1, LCRG1 was still expressed in Hep-2 / LCRG1 cells after multiple passages by RT-PCR and plate clone formation assay, and LCRG1 had a significant ability of inhibiting cell proliferation.Extraction of Hep-2 / The total proteins of LCRG1 and Hep-2 / pcDNA3.1 (+) cell lines were analyzed by solid phase pH gradient (IPG) two-dimensional gel electrophoresis (2DGE) combined with immunoblotting with an anti-tyrosine phosphorylation antibody and matrix-assisted laser desorption ionization Time-of-flight mass spectrometry (MALDI-TOF-MS) to identify tyrosine-phosphorylated proteins. The higher resolution and reproducible Hep-2 / LCRG1 and Hep- 2 / pcDNA3.1 , And identified 13 differential tyrosine phosphorylated proteins by Western blotting, software analysis and mass spectrometry.These proteins are involved in the processes of cell signaling and cell metabolism, LCRG1 may be by adjusting these White matter tyrosine phosphorylation, dephosphorylation, involved in the regulation of cell proliferation, metabolism and apoptosis process, which play antitumor effect. This provides a new idea for comprehensively and truly revealing the molecular mechanism of LCRG1 antitumor effect .