CAJ | 학술논문

目的：研究CD59分子在LAT（ Linker for activated T cells ）介导的T系淋巴细胞活化中所起的作用。方法：构建LAT-GFP融合蛋白，以逆转录病毒为载体转染Jurkat细胞，建立稳定转染细胞株（ Jurkat-GFP）。分别使用CD59抗体刺激和pSUPER-siCD59干扰质粒（ GFP标记）沉默Jurkat-GFP细胞。免疫荧光观察CD59和LAT分子在细胞膜的表达和定位；MTT比色法检测细胞增殖率的变化；Western blot检测LAT活化信号转导通路中相关蛋白分子磷酸化水平。结果：荧光显微镜下可见Jurkat-GFP细胞绿色荧光表达于细胞膜，转染干扰质粒后细胞膜和细胞质均有绿色荧光表达。激光共聚焦显微镜下可见CD59抗体刺激前CD59和LAT分子均匀分布于细胞膜，且转染干扰质粒的Jurkat-GFP细胞CD59表达量下降。 CD59抗体刺激后CD59分子和LAT分子点状聚集共定位于细胞膜。 MTT结果表明相对于正常Jurkat-GFP细胞，抗体活化后细胞增殖速率明显升高（P＞0.05），而电转干扰质粒后细胞生长减慢。 Western blot 结果表明，CD59抗体刺激后细胞ZAP-70、LCK、PLC-r总蛋白表达无明显差异（ P＞0.05），而磷酸化蛋白表达显著增高（ P＜0.05）。结论：抗体活化后细胞膜上CD59和LAT分子聚集并共定位于细胞膜，且细胞信号转导通路下游分子磷酸化水平增高，进一步证实CD59分子经抗体活化后可促进LAT介导的T细胞信号转导。
목적：연구CD59분자재LAT（ Linker for activated T cells ）개도적T계림파세포활화중소기적작용。방법：구건LAT-GFP융합단백，이역전록병독위재체전염Jurkat세포，건립은정전염세포주（ Jurkat-GFP）。분별사용CD59항체자격화pSUPER-siCD59간우질립（ GFP표기）침묵Jurkat-GFP세포。면역형광관찰CD59화LAT분자재세포막적표체화정위；MTT비색법검측세포증식솔적변화；Western blot검측LAT활화신호전도통로중상관단백분자린산화수평。결과：형광현미경하가견Jurkat-GFP세포록색형광표체우세포막，전염간우질립후세포막화세포질균유록색형광표체。격광공취초현미경하가견CD59항체자격전CD59화LAT분자균균분포우세포막，차전염간우질립적Jurkat-GFP세포CD59표체량하강。 CD59항체자격후CD59분자화LAT분자점상취집공정위우세포막。 MTT결과표명상대우정상Jurkat-GFP세포，항체활화후세포증식속솔명현승고（P＞0.05），이전전간우질립후세포생장감만。 Western blot 결과표명，CD59항체자격후세포ZAP-70、LCK、PLC-r총단백표체무명현차이（ P＞0.05），이린산화단백표체현저증고（ P＜0.05）。결론：항체활화후세포막상CD59화LAT분자취집병공정위우세포막，차세포신호전도통로하유분자린산화수평증고，진일보증실CD59분자경항체활화후가촉진LAT개도적T세포신호전도。
Objective:To investigate the function of CD 59 in LAT induced T lymphocytes'proliferation and activation.Methods:Transfected LAT-GFP recombinant lentiviral vectors into Jurkat cells and established a fusion-protein stable express cell line ( Jurkat-GFP ).Junket-GFP cells were transfected with pSUPER-siCD59 plasmids by electroporetion or stimulated by anti-CD59 antibody.The cellular locations of CD 59 and LAT were observed under fluorescence microscope with the immunofluorescence cytochem -istry.The cells proliferation were measured by MTT assay.Furthermore,Western blot was used to detect the total and phosphorylation levels of several down-stream proteins after T cell activated .Results: Jurkat-GFP cells successfully transfected with pSUPER-siCD59 plasmids showed lower fluorescence staining.CD59 and LAT distributed uniformly on the cell surface before stimulated with anti-CD59 antibody and formed clusters once upon stimulation.Jurkat-GFP cells stimulated with anti-CD59 antibody showed a higher level of pro-liferation and protein phosphorylation ,compared with the others.Conclusion:CD59 contributed to LAT induced signaling transduction of T lymphocytes ,and stimulated CD59 molecule partly promoted T cell activation.