Ruxolitinib对JAK2V617F阳性骨髓增殖性肿瘤细胞基质金属蛋白酶调控的研究

Regulation of Ruxolitinib on matrix metalloproteinase in JAK2V617F positive myeloroliferativeneoplasms cells

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-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、1000nmol／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-9mRNA水平变化，Westernblot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）％，P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5nmol／LRuxolitinib作用MPN原代细胞及HEL细胞24h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-9mRNA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。

Objective To investigate the regulation of JAK2 tyrosine kinase inhibitor ruxolitinib on extracellular matrix metalloproteinase （MMP in JAK2V617F positive myeloproliferative neoplasms （MPN） ceils. Methods （1）Forty cases of newly diagnosed JAK2V617F positive MPN patients and 15 healthy volunteers as control in Banding 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.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 The expression levels ofp-JAK2, MMP-2and 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 ）. @The proliferation of HEL cells was inhibited by different concentrations of ruxolitinib in time and dose dependent manner. @Cell 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.