CMPN基因突变及其机制与生物学意义的探讨

Effects of the PI-3K/AKT signal transduction pathway on the chronic myeloproliferative diseases with JAK2 gene mutation

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 investigate the pathogenesis of myeloproliferative neoplaspsms（CMPN） with JAK2 gene mutation. METHODS： The patients of CMPN with JAK2/V617F gene mutation were selected by Taqman-MGB and RT-PCR, and the mutation rates were calculated. The JAK2/V617F gene mutation was detected by Sequence analysis software. The expression levels of G-CSFR, EPOR and TPOR-mRNA were measured by real-time fluorescent relative- quantification reverse transcriptional PCR （FQ-PCR）, the protein expression levels of PI3K, AKT and p-AKT were detec- ted by western blotting. RESULTS：The percentage of this gene mutation was 75.0% ,46.7% and 26.7% in PV,ET and IMF groups respectively. Compared with healthy volunteers group, the expression levels of G-CSFR mRNA were in- creased 263.16%,276.32% and 247.37%; the expression levels of EPOR mRNA were increased 213.95%,220. 93% and 218.61% ; the expression levels of TPOR mRNA were increased by 172.55% ,176.47% and 182.35% in PV,ET and IMF groups respectively. The expression levels of PI3K and p-AKT protein in PV, ET and IMF groups were increased markedly. The expression levels of PI3K protein were increased by 115.79 %, 92.11% and 100.00 %. The expression lev- els of p-AKT protein were increased by 226.09 %, 243.48 % and 200.00 % （P〈0.05）. The expression levels of AKT pro- tein had on significant difference （P〉0.05）. CONCLUSIONS： Many of CMPN patients have JAK2/V617F gene muta- tion. This mutation may participate in the mechanisms of CMPN by affecting the signal transduction of many cytokines and the pathway of PI-3K/AKT,which can inhibit the apoptosis and promote the proliferation of cells.