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目的构建T细胞活化接头蛋白(LAT)棕榈酰化位点突变的慢病毒,感染Jurkat细胞,建立稳定转染的细胞株,观察LAT棕榈酰化位点突变对CD59介导的T淋巴细胞活化信号转导的影响。方法构建阴性对照(neg-EGFP)、LAT-M-EGFP融合蛋白基因载体,包装成慢病毒,分别感染Jurkat细胞,建立稳定感染的细胞株。激光共聚焦显微镜观察病毒感染效率及融合蛋白在细胞上的定位;CCK-8法检测CD59单克隆抗体交联前后各组细胞的增殖活性,流式细胞术检测细胞凋亡情况,Western blot法检测磷脂酶Cγ1(PLC-γ1)、淋巴细胞特异性蛋白酪氨酸激酶(LCK)蛋白的表达。结果共聚焦显微镜观察发现LAT-M组细胞的LAT分子在细胞膜上散在分布,CD59抗体交联刺激后,未出现明显的点簇状聚集区。与阴性对照细胞相比,LAT-M细胞增殖活性明显降低,中晚期凋亡细胞明显增加;Western blot结果显示,LAT-M组的PLC-γ1、LCK蛋白表达水平和阴性对照组大致相同,经抗体活化后无明显变化,而阴性对照细胞的PLC-γ1、LCK蛋白表达量下降。结论 LAT棕榈酰化位点突变的Jurkat细胞脂筏定位功能缺失,细胞处于抑制状态,对CD59糖基磷脂酰肌醇(GPI)介导的T淋巴细胞活化信号转导有抑制作用。
Objective To construct a lentivirus with palmitoylation site of T cell activation adapter protein (LAT), infect Jurkat cells and establish a stable transfected cell line. To observe the effect of LAT palmitoylation site mutation on CD59-mediated T lymphocyte activation signal The impact of transduction. Methods The negative control (neg-EGFP) and LAT-M-EGFP fusion protein gene vectors were constructed and packaged into lentivirus. Jurkat cells were infected respectively and stable infected cell lines were established. Laser scanning confocal microscope was used to observe the virus infection efficiency and localization of the fusion protein on the cells. The proliferation activity of CD59 monoclonal antibody before and after cross-linking was detected by CCK-8 assay, apoptosis was detected by flow cytometry and Western blot Phospholipase Cγ1 (PLC-γ1), and lymphocyte specific protein tyrosine kinase (LCK) protein expression. Results Confocal microscopy revealed LAT-M cells scattered LAT molecules in the cell membrane, CD59 antibody cross-linked stimulation, there is no obvious cluster point clustering. Compared with negative control cells, the proliferation activity of LAT-M cells was significantly decreased and the number of apoptotic cells in middle and late stages was significantly increased. Western blot results showed that the expression levels of PLC-γ1 and LCK in LAT-M group were almost the same as those in negative control group. Antibody activation did not change significantly, while the negative control cells PLC-γ1, LCK protein expression decreased. Conclusions The Jurkat cells with LAT palmitoylation sites are deficient in lipid raft targeting function and inhibit the activation of CD59 glycosylphosphatidylinositol (GPI) -mediated T lymphocyte activation signal transduction.