【作者】 申徐良；

【导师】 陈方平；

【作者基本信息】 中南大学 ， 内科学， 2008， 博士

【摘要】 2005年以来数个独立的研究组报告了BCR/ABL阴性骨髓增殖性疾病(myeloproliferative disorders,MPD)中存在JAK2基因突变,JAK2基因突变位于JAK2基因第1849位,原来的鸟嘌呤(G)被胸腺嘧啶(T)所取代(G→T),导致原位于617位的缬氨酸错义编码为苯丙氨酸(JAK2V617F)。国外报告JAK2V617F突变见于65%～90%的真性红细胞增多症(PV)、23%～57%的特发性血小板增多症(ET)和35%～57%特发性骨髓纤维化(IMF),是MPD发生的重要分子机制。JAK2V617F突变引起人们广泛关注而称为“MPD的JAK2时代”。然而,JAK2V617F突变国内报道较少,其作为临床诊断依据的价值仍需大量的临床资料验证?JAK2V617F突变导致MPD的发病机制还不清楚?JAK2V617F突变相关的信号通路及其下游靶基因还有待进一步研究?我们在临床上对110例恶性血液病患者检测了JAK2V617F基因突变,并应用细胞模型探索JAK2V617F突变相关的信号通路及其靶基因,为相关靶向药物的研发提供新的靶点和理论依据,对阐明其作用的分子机制有重要意义。目的①在临床上探索JAK2V617F突变在BCR/ABL阴性MPD患者中的特异性、发生率及其临床意义,并确立一种敏感特异、简便快捷的实验诊断体系;②应用细胞模型,研究JAK2V617F突变相关的信号通路;③寻找JAK2V617F(JAK/STAT)信号通路调控的下游靶基因,探索与细胞增殖和凋亡异常的相关性,以期进一步阐明PV的发病机制。方法①临床上对110恶性血液病患者和10例正常对照的外周血或骨髓粒细胞提取基因组DNA,采用等位基因特异性PCR(AS-PCR)、限制性内切酶消化方法检测JAK2V617F突变,并对BCR/ABL阴性MPD 41例和正常对照2例进行基因测序鉴定;②言袢撕彀籽∠赴闔EL细胞为模型,用上述三种方法检测和验证HEL细胞模型中存在天然的JAK2V617F突变,并对HEL细胞的形态学、细胞化学染色和染色体核型进行观察;③应用Western blot方法研究HEL细胞中JAK2特异性抑制剂AG490干预对JAK2/STAT5信号通路中的JAK2、STAT5、磷酸化JAK2和磷酸化STAT5蛋白表达的影响;④Cell Titer-Glo(?)法和FITC-AnnexinV/PI双染后的流式细胞技术分别用于检测细胞增殖和凋亡情况;⑤应用RT-PCR技术筛选JAK2/STAT5信号通路可能的下游靶基因(GATA1、GATA2、PTTG1、bcl-x)。Western blot技术和流式细胞技术用于进一步检测HEL细胞中AG490对PTTG1的蛋白表达水平的影响;⑥双荧光素酶报告基因技术用于检测AG490对PTTG1启动子的影响。结果①AS-PCR和限制性内切酶的方法检测JAK2V617F阳性的结果为:PV 11/12例(91.7%)、ET 8/15例(53.3%)、IMF 4/7例(57.1%)。高嗜酸粒细胞增多症(HES)7例、慢性粒细胞白血病(CML)25例、急性白血病44例[包括急性髓细胞白血病(AML)24例、急性淋巴细胞白血病(ALL)18例、慢性粒细胞白血病急粒变1例、急性混合细胞白血病1例]均未检测到JAK2V617F基因突变。将所有BCR/ABL阴性MPD患者41例、正常对照2例一并进行基因测序鉴定,结果显示AS-PCR和限制性内切酶的方法检测JAK2V617F阳性的23例患者为JAK2V617F突变型,其他标本为野生型;②通过AS-PCR、限制性内切酶分析和DNA测序,我们证实了HEL细胞中存在JAK2V617F突变,并且细胞形态学和细胞化学观察也提示该细胞株具有红系细胞的形态特征;③Western blot结果表明在总蛋白上样量一致的情况下,随着AG490作用时间(0小时、12小时、24小时、48小时、72小时)的延长,JAK2蛋白总量表达变化不大,但是磷酸化JAK2的表达逐渐下降,最大下降95.3%;同样STAT5的总蛋白水平变化不大,磷酸化的STAT5表达水平明显降低,最大下降72.4%。在K562细胞中,在100μM的AG490作用72小时后蛋白JAK2、磷酸化JAK2、STAT5、磷酸化STAT5的表达水平均没有明显变化;④用50μM AG490干预后(0小时、24小时、48小时、72小时、96小时和120小时),HEL细胞增殖活力与对照组比较明显降低(P＜0.01);⑤通过FITC-AnnexinV/PI双染法和流式细胞技术,我们发现AG490作用之后HEL细胞的凋亡率明显升高,并呈现出明显的时间和剂量依赖性;⑥通过RT-PCR技术,发现AG490干预HEL细胞后癌基因PTTG1和凋亡相关基因Bcl-x(主要为bcl-x_L)mRNA的表达水平明显下调,并呈现出明显的时间和剂量依赖性;而转录因子GATA1和GATA2mRNA的表达水平无明显变化;⑦用100μM AG490(0小时、12小时、24小时、48小时、72小时)干预HEL细胞,Western blot技术观察到,随着AG490作用时间的延长,PTTG1的蛋白表达明显下降;流式细胞技术检测未加药时PTTG1表达阳性率为55.9%,随着AG490作用时间的延长,PTTG1阳性细胞的百分比下降,72小时后的细胞阳性率仅为9.0%;⑧通过双报告基因技术,我们发现AG490作用之后,PTTG1的启动子活性下降2.5倍。结论①90%以上的PV、50%以上的ET和IMF可检测到JAK2V617F基因突变;AS-PCR和限制性内切酶分析是JAK2V617F基因突变敏感特异、简便快捷的检测方法;JAK2V617F可以作为MPD诊断的分子标志,也可能是治疗的新靶点;②在HEL细胞中JAK2激酶特异性抑制剂AG490可以抑制突变的JAK2V617F激酶和JAK2V617F/STAT5信号通路,使HEL细胞增殖活力明显降低和凋亡率明显升高;而AG490导致Bcl-x(主要是Bcl-x_L)基因的mRNA表达明显降低,提示JAK2V617F可能通过上调Bcl-x_L抑制细胞凋亡;③用AG490抑制JAK2V617F/STAT5信号通路后,PTTG1基因的启动子活性、mRNA和蛋白水平的表达明显下调,提示PTTG1是JAK2V617F/STAT5信号通路的下游靶分子,JAK2V617F在转录水平上调PTTG1基因表达可能导致细胞的异常增殖。更多还原

【Abstract】 In early 2005,several independent groups published virtually simultaneously on the presence of a specific mutation in the JAK2 associated with BCR/ABL-negative MPD.The mutation of JAK2 was the same base change(G to T)at nucleotide 1849,resulting in a substitution of valine 617 for a phenylalanine in the JH2 domain of JAK2(JAK2V617F).The frequency of JAK2V617F mutation was 65%～97%among patients with PV,23%～57%among patients with ET and 35%～57%among patients with IMF,which indicates JAK2V617F mutation plays an important role in the pathogenesis of MPD. JAK2V617F mutation caused a widespread concern,and the period afterwards was named as "JAK2 era of myeloproliferative disorders" However,JAK2V617F mutation among Chinese people has been scarcely reported;The diagnostic value of JAK2V617F mutation should be assessed further by considerable clinical data;The pathogenesis role of JAK2V617F in myeloproliferative disorders is still unclear;The signal pathway and downstream target gene of JAK2V617F mutation remains to be studied further.In this investigation,JAK2V617F mutation in 110 patients with malignant hematologic disease was detected,and a cell model was used to characterize the signal pathway and identify the target gene of this mutation.The research provides new targets as well as a theoretical basis for research and development of JAK2V617F-mutation-targeted drug,and will be of great significances in elucidating the mechanisms underling the mutation.OBJECTIVE①To analyze the specificity,incidence of JAK2V617F mutation in patients with BCR/ABL-negative myeloproliferative disorders(MPD)and explore the clinical significances, and develop a sensitive and specific clinical diagnosis test system for patients with MPD.②A cell model was used to characterize the signal pathway associated with the JAK2V617F mutation;③To further elucidate the mechanism underling the polycythaemia vera(PV),the target genes of JAK2V617F(JAK/STAT)signal pathway was identified and the relevance between this mutation and apoptosis and proliferation was studied.METHODS①Genomic DNA was isolated from granulocytes originating from peripheral-blood or bone marrow.Allele-specific polymerase chain reactions(AS-PCR),restriction enzyme digestion were performed to detect the mutation in genomic DNA.110 patients and ten normal controls were detected.The PCR product originating from 41 patients with BCR/ABL-negative MPDs and two normal controls was subject to DNA sequencing.②HEL cell line,which has a genetic background of erythrocyte and carries a homozygous JAK2V617F mutation confirmed by the three methods mentioned above,was selected as a cell model.We also investigated the morphology,cytochemical staining and chromosome karyotype properties of this model.③Western blot was performed to determine the effect of AG490 on the JAK2/STAT5 signal pathway by the antibodies against the JAK2、STATS、phospho-JAK2 and phospho-STATS.④CeilTiter-Glo(?) Luminescent cell viability assay and FITC-Annexin V/PI double staining and then Flow cytometry(FCM)were performed to determine the proliferation ability and apoptosis rate of the HEL cell,respectively.⑤RT-PCR was performed to screen the candidate gene(GATA1、GATA2、PTTG1、and bcl-x)to identify the downstream target of the JAK2/STAT5 signal pathway.Western blot and Flow Cytometry(FCM) were performed to analyze the effect of AG490 on the expression of PTTG1 protein further.⑥Dual Luciferase Reporter Assay was performed to detect the effect of AG490 on the promoter activity of PTTG1.RESULTS①The positive rate of JAK2V617F detected by AS-PCR and restriction enzyme was shown as follows:11(91.7%)of 12 with PV,8(53.3%)of 15 with ET,4(57.1%)of 7 with IMF,while in other patients including 7 patients with HES,25 patients with CML,44 patients with AL(including 24 patients with AML,18 patients with ALL,1 patients with CML-blast phase,1 patients with mixed-lineage acute leukemia (MAL)),JAK2V617F could not be detected.The PCR product prepared from 41 patients with BCR/ABL negative MPD patients and 2 normal controls were further subjected to sequencing analysis,and the results demonstrated that the 23 patients carrying JAK2V617F mutation detected by AS-PCR and restriction enzyme digestion were JAK2V617F positive and the others were wild-type.②The presence of the JAk2V617F in the HEL cell was confirmed by AS-PCR,restriction enzyme digestion and DNA sequencing,and morphological experiments and cytochemical staining suggested that the HEL cell has a genetic background of erythrocyte.③Western blot demonstrated that under the condition that the total protein loaded in each well were much the same,as the time extended(0h,12h,24h,48h,72h),the JAK2 total protein changed little, while the pospho-JAK2 protein decreased gradually,with the greatest decreasing rate of 95.3%;Similarly the STAT5 total protein changed little, while the phospho-STAT5 protein decreased obviously,with the greatest decreasing rate of 72.4%;In K562 cells,the protein expression of JAK2, phospho-JAK2,STAT5,phospho-STAT5 has little change when treated by AG490 for 72h.④When treated by 50μM AG490(0h,24h,48h,72h, 96h),the proliferation rate of HEL cells decreased dramatically compared with the control(P<0.01).⑤FITC-AnnexinV/PI double staining and FCM demonstrated that the apoptosis rate of HEL cell increased obviously in a time and dose dependent manner after treated by AG490.⑥RT-PCR demonstrated that when treated by AG490,the mRNA of PTTG1 was down-regulated obviously,and the down-regulation was strongly time and dose dependent;but the expression of transcriptional factor GATA1 and GATA2 changed little.⑦Western blot demonstrated that when treated by 100μM AG490(0h,12h,24h,48h,72h),PTTG1 protein decreased obviously as time extended;Flow cytometry(FCM)showed that the PTTG1 positive rate of the HEL cells was 55.9%under normal condition,and as the AG490 treating time extended,the positive rate decreased,which was only 9.0%at 72h;⑧Dual-luciferase assay showed that the PTTG1 promoter activity decreased by 2.5 fold when treated by AG490.CONCLUSION①The frequency of JAK2V617F mutation was more than 90%among patients with PV,more than 50%among patients with ET and IMF.AS-PCR and restriction enzyme digestion assay were sensitive,specific,convenient and shortcut tests for the JAK2V617F mutation;This mutation might be a molecular marker as well as a treatment target of MPD in the future;②In HEL cells,the muted JAK2V617F kinase and JAK2V617F/STAT5 signal pathway can be inhibited by the specific inhibitor of JAK2 kinase,resulting in the significantly decreasing of the cell proliferation activity and increasing of the apoptosis rate;AG490 caused an obviously decrease of Bcl-x(Bcl-x_L mRNA was the main species of Bcl-x found),which suggests that JAK2V617F might inhibit cell apoptosis through upregulation of Bcl-x_L.③On the inhibition of the JAK2V617F/STAT5 signal pathway by AG490, PTTG1 mRNA and protein were decreased significantly,which suggested that PTTG1 might be a downstream target of JAK2V617F signal pathway. Upregulation of PTTG1 on the transcriptional level might promote cell proliferation abnormally.更多还原