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目的:分析JAK2基因突变阳性的慢性骨髓增殖性疾病(myeloproliferative neoplaspsms,CMPN)患者多种细胞因子受体和关键蛋白的表达变化,探讨其可能的发病机制。方法:采用Taqman-MGB探针联合RT-PCR法从50例CMPN患者中筛选出JAK2V617F突变阳性患者,计算突变率,并对突变结果进行测序分析。实时荧光定量PCR检测真性红细胞增多症(polycythernia vera,PV)患者、原发性血小板增多症(essential thrombocytosis,ET)患者和原发性骨髓纤维化(idiopathic myelofibrosis,IMF)患者骨髓中G-CSFR、EPOR和TPOR mRNA的表达,蛋白质印迹法检测PV患者、ET患者和IMF患者骨髓中PI-3K、P-AKT和AKT总蛋白的表达。结果:PV、ET和IMF患者的突变率分别为75.0%(15/20)、46.7%(7/15)和26.7%(4/15)。与健康志愿者相比,PV、ET和IMF患者骨髓中G-CSFR mRNA相对表达水平分别增加了263.16%、276.32%和247.37%,EPOR mRNA表达量分别增加了213.95%、220.93%和218.61%,TPOR mRNA表达量分别增加了172.55%、176.47%和182.35%。PI-3K蛋白表达水平分别增加了115.79%、92.11%和100.00%,P-AKT蛋白表达水平分别增加了226.09%、243.48%和200.00%,差异均有统计学意义,P<0.05;总AKT蛋白水平各组之间比较差异无统计学意义,P>0.05。结论:JAK2V617F点突变存在于大多数CMPN中,此突变可能通过介导EPOR、TPOR和G-CSFR在内的多种细胞因子的信号转导,激活PI-3K/AKT信号转导通路,进而促进细胞增殖,抑制细胞凋亡参与CMPD的发病机制。
Objective: To analyze the expression changes of many cytokine receptors and key proteins in patients with JAK2 gene mutation-positive chronic myeloproliferative neoplaspsms (CMPN), and to explore the possible pathogenesis. Methods: The JAK2V617F mutation-positive patients were screened from 50 CMPN patients by Taqman-MGB probe combined with RT-PCR. The mutation rate was calculated and the mutation results were sequenced. Real-time fluorescence quantitative PCR was used to detect G-CSFR in bone marrow of patients with polycythemia vera (PV), patients with essential thrombocytosis (ET) and idiopathic myelofibrosis (IMF) EPOR and TPOR mRNA were detected by Western blotting. The protein expressions of PI-3K, P-AKT and AKT in the bone marrow of PV patients, ET patients and IMF patients were detected by Western blotting. Results: The mutation rates in patients with PV, ET and IMF were 75.0% (15/20), 46.7% (7/15) and 26.7% (4/15) respectively. Compared with healthy volunteers, the relative expression levels of G-CSFR mRNA in bone marrow of patients with PV, ET and IMF increased by 263.16%, 276.32% and 247.37%, respectively, and the expression of EPOR mRNA increased by 213.95%, 220.93% and 218.61%, respectively. TPOR mRNA expression increased 172.55%, 176.47% and 182.35% respectively. PI-3K protein expression increased by 115.79%, 92.11% and 100.00%, P-AKT protein expression increased by 226.09%, 243.48% and 200.00%, the difference was statistically significant, P <0.05; total AKT protein The level of the difference between the groups was not statistically significant, P> 0.05. CONCLUSION: JAK2V617F point mutation exists in most CMPNs. This mutation may activate the PI-3K / AKT signal transduction pathway through signal transduction of various cytokines including EPOR, TPOR and G-CSFR, thereby promoting Cell proliferation, inhibition of apoptosis participate in the pathogenesis of CMPD.