ET患者血浆MP水平测定及其与JAK2V617F突变关系的研究

Level of Microparticles in Patients with Essential Thrombocythemia and Its Relation with JAK2V617F Mutation

目的:检测原发性血小板增多症(ET)患者血浆中的微颗粒(MP)水平,并分析其与JAK2V617F突变的关系。方法:抽取56例ET患者及28例正常人空腹静脉血,枸橼酸钠抗凝(1∶9),以藻红蛋白标记的特异性荧光抗体CD235a-PE、CD61-PE、CD142-PE和CD62E-PE分别标记红细胞微颗粒(RMP)、血小板微颗粒(PMP)、组织因子微颗粒(TF+MP)及内皮细胞微颗粒(EMP),FCM检测4种MP绝对数。试剂盒抽提基因组DNA,PCR扩增目标DNA,琼脂糖凝胶电泳筛选26例JAK2V617F突变标本。结果:ET组的RMP(157.2±304.9/μl vs 21.3±18.4/μl)、PMP(1378.9±2454/μl vs 113.8±97.1/μl)、TF+MP(123±354.6/μl vs 21±15.7/μl)、EMP(1434.7±2601.9/μl vs 291.7±297.7/μl)高于正常对照组(P<0.05);8例血栓组的RMP(258.2±327.7/μl vs 55.6±63.8/μl)、PMP(1853±1874.7/μl vs 444.7±712/μl)、TF+MP(120.4±112.5/μl vs 60.3±128.3/μl)、EMP(1637.1±1563.5/μl vs 629.4±1000.2/μl)高于25例非血栓组(P<0.05);17例脾肿大组的RMP(306.8±491.1/μl vs 59.3±51/μl)、PMP(2944.8±3767.6/μl vs 334.8±420.2/μl)、TF+MP(162.9±166.8/μl vs 31.0±28.7/μl)、EMP(3031.8±4048.8/μl vs 701.5±729.2/μl)高于11例非脾肿大组(P<0.05);26例JAK2V617F突变组的TF+MP高于未突变组(154.7±516.3/μl vs 100.5±126.6/μl)(P<0.05),另外3种MP在这2组间无明显区别(P>0.05)。结论:ET组、血栓组、脾肿大组4种MP水平均高于对照组;JAK2V617F突变组患者TF+MP数高于未突变组,另3种MP水平在突变与未突变组之间无明显区别。

Objective:To detect the levels of microparticles(MP)in plasma of patients with esseutial thrombocythermia(ET)and analyze the relationship between the JAK2V617F mutant and MP in ET patients.Methods:The numerical values of MPs were analysed by using flow cytometry.Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate(1∶9).The RMP,PMP,TF+MP and EMP were detected by FCM using phycoerythrin(PE)-conjugated monoclonal antibodies to CD235a for red blood cells,CD61 for platelets,CD142 for tissue factor(TF)and CD62E for endothelial cells,respectively.Forward scatter was set in scale using fluorescent microspheres of 0.8μm.Standard fluorescent microbeads(0-0.8μm)in diameter were used to set the microparticles gate.Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction(PCR).According to the size of molecular weight,the amplified products were separated by electrophoresis on a 2%agarose gel to screen 26 JAK2V617F mutations.Results:The detection results showed that the MP levels in ET group were higher than those in normal control group:RMP(157.2±304.9/μl vs 21.3±18.4/μl),PMP(1378.9±2454/μl vs 113.8±97.1/μl),TF+MP(123±354.6/μl vs 21±15.7/μl)and EMP(1434.7±2601.9/μl vs 291.7±297.7/μl)(P<0.05).In 8 patients with thrombus,the levels of these four kinds of MP were higher than those of controls without thrombus:RMP(258.2±327.7/μl vs 55.6±63.8/μl),PMP(1853±1874.7/μl vs 444.7±712/μl),TF+MP(120.4±112.5/μl vs 60.3±128.3/μl)and EMP(1637.1±1563.5/μl vs 629.4±1000.2/μl)(P<0.05).The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly:RMP(306.8±491.1/μl vs 59.3±51/μl),PMP(2944.8±3767.6/μl vs 334.8±420.2/μl),TF+MP(162.9±166.8/μl vs 31.0±28.7/μl)and EMP(3031.8±4048.8/μl vs 701.5±729.2/μl)(P<0.05).The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation(P>0.05),except TF+MP(154.7±516.3/μl vs 100.5±126.6/μl)(P<0.05).Conclusion:The numerical values of MP detected are more in ET patients than those in healthy controls.The number of MP is higher in patients with thrombus than that without thrombus,so do in patients with splenomegaly and without splenomegaly.Patients with JAK2V617F mutation show higher number of TF+MP than that without JAK2V617F mutation.But the other three kinds of MP show no this difference.