摘要目的 探讨JAK2抑制剂Ruxolitinib对JAK2V617F突变阳性的骨髓增殖性肿瘤(MPN)细胞内基质金属蛋白酶(MMP)调控的影响.方法 ①收集2012年1月到2015年12月保定市第一医院收治的40例未经治疗的JAK2V617F阳性MPN患者,以15名健康志愿者为对照组.免疫组化法检测两组骨髓活检组织中磷酸化JAK2 (p-JAK2)、基质金属蛋白酶2(MMP-2)、MMP-9的表达水平.选取JAK2V617F阳性MPN患者骨髓细胞,体外应用Ruxolitinib干预,测定干预前后细胞迁移力和MMP-2、MMP-9表达.②不同浓度Ruxolitinib(0、50、100、250、500、1 000 nmol/L)作用于人红白血病细胞株HEL细胞不同时间后CCK-8检测细胞活力,Tanswell小室检测细胞迁移,荧光定量PCR检测JAK2、MMP-2、MMP-9 mRNA水平变化,Western blot检测p-JAK2、MMP-2、MMP-9蛋白表达.结果 ①MPN组p-JAK2、MMP-2、MMP-9蛋白表达均高于对照组[(78.56+24.55)％对(41.59±17.29)％、(48.25±18.74)％对(22.79±13.89)％、(53.29±19.28)％对(15.56±14.96)％,JP值均＜0.05].Spearman相关分析显示MMP-2、MMP-9蛋白水平与JAK2V617F突变量呈正相关(r=0.526,P=0.001;r=0.543,p=0.001).②Ruxolitinib能够呈时间和剂量依赖性抑制HEL细胞增殖.③迁移实验结果显示5 nmol/LRuxolitinib作用MPN原代细胞及HEL细胞24 h后迁移至下室细胞数均少于无Ruxolitinib组(154.7±27.5对320.3±67.3,t=13.47,P=0.001;70.7±10.5对135.3±16.7,t=13.89,P=0.001).④JAK2、MMP-2、MMP-9 mRNA及蛋白表达随Ruxolitinib剂量增加而降低.结论 Ruxolitinib通过调控JAK2信号通路抑制MMP-2、MMP-9表达而抑制MPN细胞迁移能力.

abstractsObjective To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase (MMP in JAK2V617F positive myeloproliferative neoplasms (MPN) cells.Methods ①Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Baoding No.1 Hospital between January 2012 and December 2015 were enrolled in this study.JAK2V617F/JAK2 ratio was detected by real-time-PCR;the expression levels of phosphorylation protein tyrosine kinase 2 (p-JAK2),MMP-2 and MMP-9 in pathological tissues of bone marrow were detected by immunohistochemistry.The bone marrow cells of JAK2V617F positive MPN patients were treated with ruxolitinib,then the migration ability and MMP-2,MMP-9 gene and protein expression levels were detected.② The human erythroleukemia cell line HEL cells were treated with different concentrations of ruxolitinib (0,50,100,250,500,1 000 nmol/L).The cell viability was detected by CCK-8 test;cell migration ability was tested by transwell chambers.The mRNA expression levels of JAK2,MMP-2 and MMP-9 were detected by real-time-PCR.The protein expression levels of p-JAK2,MMP-2 and MMP-9 were detected by Western blot.Results ①The expression levels of p-JAK2,MMP-2 and MMP-9 in the newly diagnosed group were significantly higher than control group respectively [(78.56±24.55)％ vs (41.59±17.29)％,P＜0.05;(48.25±18.74)％ vs (22.79±13.89)％,P＜0.05;(53.29±19.28)％ vs (15.56±14.96)％,P＜0.05].Spearman correlation analysis showed the positive correlation of MMP-2 and MMP-9 protein expression levels with JAK2V617F mutation (r=0.526,P=0.001;r=0.543,P=0.001).②The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner.③Cell migration test showed the number of cells leaked to the low chamber in MPN patients bone marrow cells and HEL cells treated with 5 nmol/L of ruxolitinib group were significantly lower than that without ruxolitinib treatment after 24 h [(154.7±27.5) vs (320.3+67.3),t=13.47,P＜0.05;(70.7±10.5) vs (135.3+16.7),t=13.89,P＜0.05].The mRNA and protein expression levels of JAK2,MMP-2 and MMP-9 decreased with the increased concentration of ruxolitinib.Conclusion Ruxolitinib inhibits MPN cell migration and expression of MMP-2 and MMP-9 via JAK2 signal pathway.