磷酸特异化流式细胞术检测骨髓白血病细胞磷酸化信号蛋白的实验研究

Detecting Phospho-Signaling Protein of Bone Marrow Leukemia Cells by Phospho-Signaling Flow Cytometry

本研究通过建立磷酸特异化流式细胞术(phospho-specific flow cytometry,phospho-flow)检测白血病细胞信号通路磷酸化蛋白的实验方法,探讨Phospho-flow在白血病研究中的应用价值。取白血病患儿的骨髓,分别采用Phospho-flow单个核细胞法和Phospho-flow直接固定法进行处理。Phospho-flow单个核细胞法为:骨髓经提取单个核细胞并固定破膜处理后,加入磷酸化抗体(P-AKT,P-ERK1/2)孵育,然后用流式细胞仪分析;Phospho-flow骨髓直接固定法为:骨髓经固定/裂解液和90%甲醇破膜细胞处理,然后加入细胞表面CD抗体及P-AKT,P-ERK1/2孵育,然后上流式细胞仪分析处理。结果表明,26例白血病患儿骨髓经phospho-flow单个核细胞法处理,P-AKT和P-ERK1/2阳性率分别为46.2%和30.8%,经phospho-flow骨髓直接固定法处理,P-AKT和P-ERK1/2阳性率分别为50.0%和38.5%。经统计学分析,两种phospho-flow的骨髓处理方法在检测P-AKT和P-ERK1/2的阳性率方面无统计学差异(p>0.05)。结论:本实验室建立的phospho-flow骨髓直接固定法,能有效检测白血病患儿骨髓细胞的磷酸化蛋白,可用于白血病信号通路的实验研究。

The purpose of this study was to establish the phospho-specific flow cytometry（phospho-flow）to detect the phosphorylated signaling proteins of leukemia cells and to evaluate its useful value in leukemia study.The bone marrow of leukemia children was collected,and treated by phospho-flow of extracted mononulear cells（MNC） and phospho-flow of directly fixed bone marrow（BM） respectively.In phospho-flow of extracted MNC,the MNC extracted from BM were fixed and permeabilized,then were cultured with P-AKT and P-ERK1/2,finally were analyzed by flow cytometry.In phospho-flow of directly fixed BM,the BM was treated with fixation/lysis buffer and 90% methanol,then were incubated with the surface CD antibody,P-AKT and P-ERK1/2,finally the treated BM cells were analyzed by flow cytometry.The results showed that the positive rates of P-AKT and P-ERK1/2 in MNC treated by phospho-flow of extrated MNC of 26 leukemia children were 46.2% and 30.8% respectively,while the positive rates of P-AKT and P-ERK1/2 in BM treated by phospho-flow of directly fixed BM were 50.0% and 38.5% respectively.The comparison of positive rates of P-AKT and P-ERK1/2 between the 2 treatment protocol showed no difference（p0.05）.It is concluded that the phospho-flow of directly fixed BM established by our laboratory can be used to analyze the signaling proteins of leukemia cells.