【作者】 陈娜；

【导师】 王欣；

【作者基本信息】 山东大学 ， 内科学， 2014， 博士

【摘要】 慢性淋巴细胞白血病(chronic lymphocytic leukemia,CLL,简称慢淋)是单克隆性小淋巴细胞恶性增殖性疾病,其特点为成熟样的B淋巴细胞在血液、骨髓、淋巴结、肝和脾大量蓄积,最后累及淋巴系统以外的组织。慢性淋巴细胞白血病病因尚未明了,感染、免疫与遗传等因素备受关注。CLL细胞形态上类似成熟淋巴细胞,实质上是一种免疫学上不成熟、功能不完善的细胞。尽管单克隆抗体应用及免疫治疗已经得到人们的广泛关注并取得一定疗效,但是CLL仍然是一种除造血干细胞移植以外不可治愈的疾病。研究证明CLL患者体内存在T、B细胞构成比例及功能失调,免疫耐受及免疫调节功能紊乱,因此开展对CLL中异常免疫和免疫调控的研究有望为CLL发生发展机理的深入探讨、肿瘤免疫治疗及新药开发等提供理论依据。近年来,白介素9(interleukin-9, IL-9)在肿瘤免疫中的作用已经得到了越来越多的关注。长期以来,人们认为IL-9是Th2类细胞因子,作用于多种炎症细胞和组织细胞,在对抗寄生虫感染和诱导变应性疾病,尤其是过敏性哮喘中发挥着重要的作用。最近的数据表明,T细胞分泌的IL-9介导了肥大细胞的募集,参与肿瘤免疫,促进细胞转化,促进增殖和抗凋亡活性,这些都表明IL-9在肿瘤进展中可能存在潜在作用。IL-9活化后也参与了IL-2,4,7,15参与的信号通路,这些信号通路是通过细胞因子的特异性受体链与STAT家族的γ链形成异二聚体所介导的。信号转导与转录激活因子-3(Signal transducer and activator of transcription,STAT3)是近年来研究异常活跃的转录因子,STAT3能够将细胞外的信号传递到细胞核,影响靶基因的转录,调控细胞功能,广泛参与细胞的增殖、分化、凋亡、免疫调节等过程,是细胞因子受体下游区重要的信号通路之一。多项研究结果显示,STAT3是在多种肿瘤组织与细胞株中异常表达,是促进炎症向肿瘤转化的关键因子,STAT3持续激活可导致细胞异常增殖和恶性转化,在肿瘤的生长和转移中发挥了举足轻重的作用,目前已被公认为癌基因。microRNAs(miRNAs)的表达水平在预测慢性淋巴细胞性白血病的临床预后方面是很有价值的。在结构上,niRNAs为一类短小的内源性单链非编码RNA片段,长度约为19-25个核苷酸。这些RNA是从初级转录本,也就是pri-miRNA,转变成为称为pre-miRNA的茎环结构,最后成为具有功能的niRNA. miRNA来自一些从DNA转录而来,但无法进一步转译成蛋白质的RNA(属于非编码RNA).miRNA通过与靶信使核糖核酸(mRNA)特异结合,从而抑制转录后基因表达。近年来的研究表明在CLL中存在几种染色体异常,如11Q-,13Q,17p-,和12号染色体三体的分子畸变,还包括miRNA15a和16-1的丧失或下调以及抗凋亡基因的过度表达。已有研究显示在恶性血液病肿瘤患者血液中存在miR-15a/16,miR-29及miR-155簇表达的缺失或下调,并且它们的表达缺失或下调与IgVH表达和ZAP-70状态呈正相关。最近的研究发现,miR-21在白血病患者中的过表达与各种染色体畸变有相关性,比如17P,提示miR-21的表达可能可以预测患者的生存率。IL-9,STAT3,以及microRNAs均与肿瘤的生长有关,但IL-9在CLL患者中的表达及它们之间的交互作用尚不清楚。在本研究中,我们证实了在CLL患者血清中IL-9的表达水平明显升高,IL-9表达的不同与CLL患者的临床特点及预后密切相关。而pSTAT3及microRNAs的表达也明显升高。对MEC-1细胞株的实验表明,在CLL细胞中可能存在一个“胞外L-9-STAT3-miR-155,miR-21-胞内IL-9”的正反馈系统。第一部分：CLL患者及正常对照组外周血中IL-9、STAT3及microRNA的检测目的：CLL是一组在组织形态学、分子生物学、细胞遗传学及临床预后方面均存在很大的异质性的侵袭性恶性肿瘤。CL1的发生发展是一个多因素参与的复杂过程,目前虽然做了大量研究,其病因及发病机制仍不清楚。本研究检测了IL-9、STAT3及microRNAs在CLL病人外周血中的表达,并探讨了IL-9的过表达与CLL患者预后因子之间的关系,为患者预后的评价提供理论依据。方法：1.标本收集2.分离外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)及CD19+B细胞3. ELISA检测分析4.提取RNA,进行实时定量RT-PCR5.蛋白提取及蛋白印迹分析6.统计学分析结果：1.通过47例CLL患者外周血血清样本的检测,发现CLL患者血清中IL-9的水平增高,血清IL-9的阳性检测率为52.4%。对20例IL-9血清水平增高的患者我们分别采用逆转录PCR及Western-blot检测IL-9的表达,最终确定,IL-9在患者的外周血单个核细胞的mRNA和蛋白水平均有表达。与正常人的CD19+细胞相比,CLL患者的外周血单个核细胞中拥有更高的IL-9mRNA水平(P<0.05)。此外,通过对western-blot蛋白条带的灰度分析,表明IL-9在CLL患者蛋白水平的表达也显著高于正常人(P<0.0001)。IL-9表达不同的CLL患者在年龄、性别水平上没有明显的统计学差异。但是患者血清IL-9的表达水平与临床分期(P<0.05),ZAP-70的表达水平(P=0.0272),B2微球蛋白的表达水平(P=0.0101),IgVH基因突变状态(P=0.0320)具有显著的统计学意义。CLL患者血清IL-9水平与预后之间存在相关性。2.通过对20例血清IL-9增高的CLL患者的外周血单个核细胞进行Western-blot检测发现STAT3均有磷酸化表达,通过对蛋白条带的灰度分析表明,pSTAT3蛋白水平明显高于正常对照,两组比较有统计学意义(P<0.0001)。3.我们继续对20例患者的外周血单个核细胞进行检测,发现microRNA155及nicroRNA21mRNA表达水平明显高于正常对照,差异有统计学意义(P<0.05)。结论：1.CLL患者IL-9水平明显增高,升高的IL-9水平与患者的临床分期,ZAP-70的表达水平,B2微球蛋白的表达水平,IgVH基因突变状态呈正相关。2.CLL’患者的pSTAT3水平明显增高。3.CLL患者的microRNA155及nicroRNA21mRNA表达水平明显高于正常对照。第二部分：CLL中IL-9与STAT3信号通路的相关调节作用目的：我们在第一部分的实验中已经证明,在CLL患者的血清中,IL-9的表达有上调,而在外周血单个核细胞的蛋白水平及mRNA水平上,也存在IL-9的表达,并且IL-9的表达与患者的不良预后有相关性。我们从IL-9过表达的患者中检测到pSTAT3水平明显增高。在接下来的实验中,我们将在CLL细胞株中,检测IL-9与STAT3的磷酸化是否具有相互调节的关系,并检测IL-9对CLL细胞增殖及凋亡是否有影响,并探索IL-9对细胞的影响是否是通过STAT3的磷酸化起作用的,而它们之间是否存在正反馈作用。从而对IL-9影响CLL细胞生物学行为的机制进行初步的探索,达到更深入了解IL-9介导的信号通路的目的。方法：1.细胞培养2.MEC-1细胞的预处理3.蛋白提取及蛋白印迹分析4.流式细胞术检测细胞凋亡5.CCK8法检测细胞增殖6.统计学分析结果：1.我们检测的CLL细胞株是MEC-1细胞株,在加入20ng/ml IL-9的MEC-1细胞株中,我们进行Western-blot检测发现随着时间的增加,STAT3的磷酸化水平逐渐增高,并有时间依赖性,在加入IL-9培养2h的时候,STAT3的磷酸化水平达峰值。加入抑制剂(STAT3抑制剂：Wp1066)处理后,两者的磷酸化水平明显减低。2.我们继续用Western-blot法检测预处理后的细胞株IL-9的表达水平,在加入20ng/ml IL-9的MEC-1细胞株中,IL-9的表达也有时间依赖性,在加入IL-9培养2h的时候,IL-9表达水平达峰值。加入Vp1066(10nM)处理后,IL-9的表达明显减低。3.我们检测预处理后细胞的增殖及凋亡情况。在加入或不加入抑制剂培养48h后,用IL-9终浓度为20ng/ml的IMDM完全培养基培养MEC-1细胞不同的时间点后,利用CCK8法检测细胞增殖,结果显示,随着IL-9的培养时间增加,细胞增殖作用逐渐增加,在培养2h后,细胞增殖增加约20%,而这种作用能被抑制剂消除,同样,在细胞加入IL-9培养2h后,流式细胞术检测细胞株的凋亡水平发现,能够减低细胞凋亡至基础水平的40%左右,而抑制剂也可以消除这种作用。结论：1.MEC-1细胞株经过IL-9预处理后,STAT3的磷酸化水平逐渐增高,抑制剂可消除这种作用。2.MEC-1细胞株经过IL-9预处理后,IL-9表达水平逐渐增高,抑制剂可消除这种作用。3.胞外IL-9可以促进细胞增殖,抑制细胞凋亡。4.胞外IL-9的刺激可引起STAT3的磷酸化,进而正反馈调节胞内IL-9的分泌。第三部分CLL中由microRNAs调控STAT3介导的IL-9过表达的免疫调节机制目的：最近的数据表明,IL-9参与肿瘤免疫是由T细胞和肥大细胞介导的。它在多个转化的细胞的促进增殖和抗凋亡活性也表明在肿瘤进展中可能存在潜在作用。本研究应用人慢性淋巴细胞性白血病细胞株MEC-1为研究对象,应用质粒转染技术诱导microRNAs高表达,进一步研究CLL中由microRNAs调控STAT3介导的IL-9过表达的免疫调节机制。材料与方法：1.细胞培养2.质粒载体介导的上调慢性淋巴细胞性白血病的microRNA基因3.上调microRNA基因细胞的预处理4.流式细胞术检测细胞转染率5.蛋白提取及蛋白印迹分析6.流式细胞术检测细胞凋亡7.CCK8法检测细胞增殖8.统计学分析结果：1.质粒转染诱导microRNA155和microRNA21高表达后,在加入20ng/ml IL-9的MEC-1细胞株中,我们进行Western-blot检测发现IL-9在CLL细胞中的蛋白表达水平升高(P<0.05),且这种作用可以被STAT3的抑制剂所抑制(Wp1066)。2.质粒转染诱导microRNA155和microRNA21高表达后,CLL细胞增殖增加(n=3,P<0.0001),细胞凋亡受抑(n=3,P<0.0001)。结论：1.microRNA155和microRNA21高表达引起的胞内IL-9的分泌可以促进CLL细胞增殖,并抑制其凋亡。2.经胞外IL-9的刺激后,上调microRNAl55和microRNA21基因表达后的细胞的胞内IL-9分泌明显增加.3.IL-9可能与CLL的发生发展密切相关,而且这种作用可能是通过STAT3介导的microRNA155和]microRNA21调节的。更多还原

【Abstract】 Chronic lymphocytic leukemia (CLL) remains an incurable disease with a monoclonal small lymphocytes malignant hyperplastic. It is characterized by the accumulation of neoplastic B lymphocytes in the blood sample, bone marrow, lymph nodes, liver and spleen, and the last involving organizations outside the lymphatic system. The mechanism of the disease development still remains unrevealed. However, infection, immune and genetic factors have received many concerns. CLL cells were morphology similar to mature lymphocytes, essentially were an immunologically immature, imperfect cells. Although the application of monoclonal antibodies and immune therapy has been widespread concern and achieve a certain effect, it is still is an incurable disease outside hematopoietic stem cell transplantation. Studies have shown that the presence of T, B cell composition ratio imbalance and dysfunction, immune tolerance and immune dysfunction in vivo in patients with CLL. Therefore carried out the research on abnormal immune and immune regulation is expected to explore the development of the mechanism of tumor immunotherapy in depth and provided a theoretical basis on new drug development.In recent years, the role of IL-9in tumor immunity has got more and more attentions. The cytokine IL-9has largely been regarded as a Th2cytokine that makes multifocal contributions to allergic disease. Recent data suggested that IL-9involved in tumor immunity mediated by Treg cells and mast cells. Its growth factor and antiapoptotic activities on multiple transformed cells also suggest a potential role in tumor progression. IL-9activities also involved in IL-2,4,7,15and21signaling which mediated by a specific receptor chain that forms a heterodimeric receptor with the common gamma chain members of the STATs family.STAT3is a latent cytoplasmic transcription factor, originally discovered as a transducer of signal from cell surface receptors to the nucleus. Compelling evidences suggested that STAT3was constitutively activated in many cancers and played a pivotal role in tumor growth and metastasis. It regulated cellular proliferation, invasion, migration, and angiogenesis which were critical for cancer metastasis.The expression levels of microRNAs were valuabe for predicting the clinical behavior of CLL. Structurally, miRNAs are short (19-to25-nucleotide) RNAs, processed from hairpin loop structures (pre-miRNAs;60-110nucleotides in length) that regulate the expression of protein-coding genes as a result of imperfect complementarity with targeting messenger RNAs. Several chromosomal abnormalities, such as11q-,13q-17p-, and trisomy12, and molecular aberrations, including loss or down-regulatipn of miRNA15a and16-1and overexpression of antiapoptotic genes, have been identified in CLL in recent years. An unique miRNA signature was found to be differentially expressed in patients with various IgVH and ZAP-70kinase statuses and composed of the most frequently deregulated miRNAs in the different hematologic malignancies (such as miR-15/16, the miR-29family, and miR-155). Recent studies have found that miR-21expression stratified survival of CLL patients with17p-as well as patients with various chromosomal aberrations, suggesting that miR-21expression could predict patient survival.Given the implication that IL-9, STATs, and microRNAs are involved in tumor growth, it was interesting to explore the cross-talking pathways among these important factors. In present study, we demonstrated that an elevated level of serum IL-9was detected in CLL and its level was correlated with the clinical characteristics of CLL patients. Expressions of pSTATs protein, miR-155and miR-21were found up-regulated concomitantly. Experiments on MEC-1cells showed that there was a novel "extracellular IL-9-STATs-miR-155, miR-21-intracellular IL-9" positive feedback system in CLL cells.Part1Expression of IL-9, STAT3and microRNAs in CLL patients and healthy controls Objective:Chronic lymphocytic leukemia (CLL) is an aggressive disease with variable histomorphological, biological and cytogenetic features, as well as clinical outcomes. The malignization of most lymphoma is a complex process with multifactor. In spite of extensive research, the pathogenesis of CLL has not been clearly understood. In this study, Expression of IL-9, phosphorylation of signal transducer and activator of transcription (pSTAT)-3and microRNAs (miR) were analyzed from CLL patient samples. And we investigate whether the level of IL-9was related to the progression and prognosis of CLL.Material and Methods:1. Specimen collection2. Peripheral blood mononuclear cells (PBMCs) and CD19+B cells isolation3. ELISA analysis4. RNA extraction and real-time quantitative RT-PCR5. Protein extraction and Western Blot6. Statistical analysis Results:1. Higher expression of serum IL-9was detected in20CLL patients from serum of47CLL patients. The positive detection rate was52.4%. The protein and mRNA levels of IL-9were also upregulated in20patients with higher serum levels of IL-9. In order to investigate whether the level of IL-9was related to the progression and prognosis of CLL, we detected the correlation between IL-9level and the clinical features of CLL. As shown in Table2, there was no correlation between IL-9expression and patient age (P=0.6580) and gender (P=0.4063). Nevertheless, the level of IL-9was strongly correlated with CLL clinical staging (P<0.05), ZAP-70expression (P=0.0272), B2M expression (P=0.0101) and IgVH status (P=0.0320). These results implied that IL-9could contribute to CLL progression. 2. Expression of pSTAT3protein can be detected in20samples with up-regulation of IL-9, but none was detected in10PBMC from normal controls. The analysis on the gray scale of the Western blot bands indicated that expression of pSTAT3was higher in CLL patients when compared with healthy controls.(P<0.0001).3. Expressions of miR-155and miR-21in20CLL patients and healthy controls were measured by RQ-PCR. We found that miR-155and miR-21were significantly overexpressed in CLL samples when compared with controls.Conclusions:1. IL-9levels increased obviously in patients with CLL. Elevated levels of IL-9were associated with CLL clinical staging, ZAP-70expression, B2M expression and IgVH status, in CLL patients.2. pSTAT3expression was significantly upregulated in CLL patients.3. microRNA155and microRNA21expression was obviously higher in CLL patients than normal controls.Part2Extracellular IL-9-pSTAT3intracellular IL-9positive feedback system in CLL cellsObjective:We had proved that IL-9was overexpression in the serum of patients with CLL in the first part of the experiment. The expressions of IL-9mRNA and protein were determined in20CLL patients. The overexpression of IL-9was associated with CLL clinical progression. High expression of pSTAT3was detected in patients. In the following experiment, we will elucidate whether there was a function link between IL-9and STAT3in CLL and detect the effect of IL-9on cell proliferation and apoptosis. We will explore whether there is a positive feedback effect between them. Thus Finally, the relationship between IL-9and STAT3pathway in the pathogenesis of CLL also needs to be determined.Material and Methods:1. Cells culture2. Cotreatment or coculture experiments 3. Real-time quantitative polymerase chain reaction (RT-PCR)4. Western blot analysis5. Assessment of cell apoptosis6. Assessment of cell proliferation7. Statistical analysisResults:1. Recombinant human IL-9(rIL-9) was added into the medium of MEC-1cells and the final concentration of rIL-9was20ng/ml. Expressions of pSTAT3in MEC-1cells were measured by Western-blot at indicated time. We found that expressions of pSTAT3increased at a time-dependent manner. Furthermore, this phenomenon could be inhibited by Wp1066, which is a STAT3inhibitor.2. Expressions of IL-9in MEC-1cells were measured by Western-blot at indicated time. We found that expressions of IL-9increased at a time-dependent manner. Furthermore, this phenomenon could be inhibited by Wp1066, which is a STAT3inhibitor. These results indicated that rIL-9(extracellular IL-9) could enhance MEC-1cells to produce IL-9through STAT3phosphorylation.3. To determine the role of extracellular IL-9in MEC-1cells growth, we first examined its effect on the proliferation of these cells. rIL-9could promote MEC-1cells proliferation when the co-culture time with rIL-9. However, pretreatment with WP1066could significantly abolish the proproliferative effects of rIL-9on MEC-1cells. In our following experiments, we examined the role of rIL-9in CLL cells apoptosis and necrosis. rIL-9could decrease cell apoptosis to approximately60%of the baseline level. Pretreatment with WP1066could significantly abolish the anti-apoptosis effects of rIL-9on MEC-1cells.Conclusions:1. Expressions of pSTAT3were increased in pretreated MEC-1cells. This phenomenon could be inhibited by Wp1066. 2. Expressions of IL-9were increased in pretreated MEC-1cells. This phenomenon could be inhibited by Wp1066.3. rIL-9could promote MEC-lcells proliferation and decrease cell apoptosis. This phenomenon could be inhibited by Wp1066.4. rIL-9(extracellular IL-9) could enhance MEC-1cells to produce IL-9through STAT3phosphorylation.Part3Overexpression of IL-9induced by STAT3phosphorylation was mediated by microRNAs in chronic lymphocytic leukemiaObjective:In recent years, the role of IL-9in tumor immunity has got more and more attentions. The cytokine IL-9has largely been regarded as a Th2cytokine that makes multifocal contributions to allergic disease. Recent data suggested that IL-9involved in tumor immunity mediated by Treg cells and mast cells. We investigated that the gained function of microRNAs through its upregulation induced by Lipofectamine2000(Invitrogen). Further explore a novel "extracellular IL-9-pSTAT3-miR-155, miR-21-intracellular IL-9" positive feedback system in CLL cells.Material and Methods:1. Cell culture2. microRNAs upregulation using Lipofectamine2000(Invitrogen) in CLL cells3. RNA extraction and real-time quantitative RT-PCR4. Protein extraction and Western Blot5. Assessment of cell apoptosis by FACScan flow cytometer6. Assessment of cell proliferation using CCK8incorporation method7. Statistical analysisResults:1. After transfected with miR-155and miR-21for48h, MEC-1cells were treated with rIL-9(20ng/ml) for120min and then collected to examine the expression of IL-9by Western blot. We found that IL-9expression increased in transfected cells when compared with untransfected cells. When miR-transfected MEC-1cells were pretreated with Wp1066for48h, Wp1066could abolish IL-9production in cells induced by rIL-9.2. the proproliferative effects of rIL-9on miR-155/miR-21transfected MEC-1cells were more apparent than MEC-1cells. Pretreatment with WP1066could also abolish the proproliferative effects of rIL-9on transfected MEC-1cells. When cultured without rIL-9, the apoptosis rate of miR-155/miR-21transfected MEC-1cells was lower than that in MEC-1cells, which documented that IL-9produced by CLL cells itself could also inhibit CLL cells apoptosis. Pretreatment with WP1066could abolish the anti-apoptosis effects of rIL-9on transfected MEC-1cellsConclusions:1. rIL-9could promote MEC-1cells proliferation and decrease cell apoptosis.2. The overexpression of miR-155and miR-21could promote the production of IL-9in CLL cells.3. It is reasonably plausible that the up-regulation of IL-9induced by STAT3phosphorylation was mediated by miR-155and miR-21.更多还原