胞内磷酸化蛋白酪氨酸激酶的流式细胞术检测  

Detection of intracellular phosphorylated protein tyrosine kinases by flow cytometry

作　　者：吕合作[1] 李柏青[1] 

机构地区：[1]蚌埠医学院免疫学教研室,安徽蚌埠233003

出　　处：《蚌埠医学院学报》2005年第2期95-97,共3页Journal of Bengbu Medical College

基　　金：国家自然科学基金资助 (No .3 0 0 70 72 1);安徽省自然科学基金资助 (No .990 44 5 42 )

摘　　要：目的 :建立流式细胞术检测细胞内磷酸化蛋白酪氨酸激酶 (PTKs)的方法。方法 :用佛波醇酯 (PMA)及植物血凝素(PHA)刺激Jurkat细胞 ,用两种不同的固定和透膜方法处理细胞 ,加磷酸化PTKs特异性单抗P Tyr 10 0及FITC标记的二抗 ,建立胞内磷酸化PTKs的流式细胞检测技术 ;同时 ,用免疫印迹方法检测磷酸化PTKs以资对照。用双色荧光技术 ,对结核杆菌抗原 (Mtb Ag)活化的外周血单个核细胞 (PBMC)中的γδT细胞进行胞内PTKs分析。 结果 :PHA及PMA刺激能够使Jurkat细胞中磷酸化PTKs明显增加 ,流式细胞术与免疫印迹方法检测的结果一致。通过双色荧光技术检测发现 ,Mtb Ag刺激PBMC ,可以优先诱导γδT细胞胞内PTKs的活化。结论Objective:To set up the method of detection of intracellular phosphorylated protein tyrosine kinases(PTKs) by flow cytometry.Methods:After stimulated with PMA and PHA,Jurkat cells were fixed and permeabilized by two different methods,and the phospho-tyrosine monoclonal antibody,P-Tyr-100,and a FITC labeled second antibody were used to detect intracellular phosphorylated PTKs by flow cytometry.Correspondingly the Western-blotting method was also used to compare with flow cytometry.Two-colour immunofluorescence labelling technique was further used to detect phosphorylated PTKs in Mtb-Ag activated γδT cells of peripheral blood mononuclear cells(PBMC).Results:Phosphorylated PTKs in Jurkat cells increased obviously after stimulated with PMA and PHA.The results of flow cytometry were consistent with those of Western-blotting.By using two-colour immunofluorescence labelling technique,the phophorylated PTKs was detected preferentially in γδT cells among PBMCs stimulated with Mtb-Ag.Conclusions:The intracellular phosphorylated PTKs could be detected in single cell by flow cytometry.

关 键 词：蛋白酪氨酸激酶 流式细胞术 ΓΔT细胞 

分 类 号：Q555.7[生物学—生物化学] R446.113[医药卫生—诊断学]