【作者】 范磊；

【导师】 阮长耿；

【作者基本信息】 苏州大学 ， 内科血液学， 2007， 博士

【摘要】 人类的Mer（c-Mer Nyk Eyk）基因是1994年由Graham首先从人类B淋巴母细胞λgt-11的cDNA文库中克隆得到,随后小鼠的Mer基因于1995年同样由Graham成功克隆,并发现和人类的Mer蛋白序列具有88%的同源性。Mer基因的命名主要是基于开始发现其在人类单核细胞（Monocytes）、上皮组织（Epithelial）和生殖系统（Reproductive tissue）表达较高。Mer属于Axl受体酪氨酸家族,此家族还包括Axl和Tyro3另外两个受体酪氨酸激酶,它们有着共同的配体-Gas6。Mer的基因结构和其家族其他两个成员类似,胞外区包括两个免疫球蛋白（Ig）样区,两个三型纤维连接蛋白（FNⅢ）样区,其中Ig样区为Mer和其配体Gas6结合区域,而纤维连接蛋白样区则在Mer和Gas6结合过程中起调节作用。胞内区为酪氨酸激酶样区,具有激酶的活性,并且具有该家族特有的KWIAIES序列。目前关于Axl受体酪氨酸激酶家族成员相关研究显示该家族成员和其配体Gas6相互作用后可以影响一系列包括细胞生存凋亡、增殖、移行和吞噬作等多种重要的生理过程。除此之外还可能参与炎症、自身免疫、血栓于止血、血管和肾脏疾病甚至恶性肿瘤的发生发展等病理过程。目前Axl家族成员的功能研究更多的是集中于Gas6/Axl途径展开,而自Mer自1994年被克隆被首次克隆以来,对于Gas6/Mer相关研究尚少,直到近年才有文章逐步涉及Mer的功能研究,其中包括Mer在细胞炎症、细胞凋亡、细胞吞噬作用、血栓与止血以及部分恶性疾病中的异常表达。一、受体酪氨酸激酶Mer的单克隆抗体制备及功能研究1.受体酪氨酸激酶Mer免疫球蛋白样片段的原核表达目前发现受体酪氨酸激酶Mer的胞外Ig样区是与其配体Gas6结合的重要结构域,只有Gas6特异性结合Mer的Ig样区后才能激活其胞内受体酪氨酸激酶的活性,因此制备针对Ig样区的单克隆抗体对于研究Gas6/Mer的功能有重要意义。利用Primer5软件设计Mer克隆引物,以K562细胞的cDNA为模板体外扩增,和PQE30表达载体连接后,成功构建Mer Ig-PQE30的表达载体,转化M15大肠杆菌后,利用IPTG诱导重组蛋白表达。12%的SDS电泳分析重组蛋白分子量约26kD,表达量约占总菌体总蛋白的15%,并主要以包涵体形式存在。Western blot显示该重组蛋白可以被anti-His抗体特异性识别。2.受体酪氨酸激酶Mer单克隆抗体的制备、鉴定和初步功能研究利用本科室已经建立的杂交瘤技术,将重组Mer IgG样区蛋白经次氮基三乙酸镍琼脂糖（Ni-NTA agarose）柱纯化浓缩后免疫Balb/c小鼠,尾静脉加强后取脾细胞融合,ELISA法筛选阳性克隆,制备腹水。运用Western blot对抗体进行鉴定观察抗体与原核重组蛋白、真核重组胞外区全长蛋白以及细胞裂解液中天然Mer反应情况,并进行免疫亚型鉴定和组织特异性鉴定。同时将所制各的抗体与PRP孵育以检测其对于血小板聚集功能的影响。结果可以看出:小鼠脾细胞与杂交瘤细胞融合后筛选获得一株持续分泌单克隆抗体的细胞株,命名为SZ-128。该株杂交瘤细胞单克隆后接种小鼠腹腔产生腹水,间接ELISA法测定腹水效价为1×10^-4。琼脂糖双扩散法显示该株抗体为IgG1亚类。腹水经蛋白G-Sephrose4B亲和层析柱纯化后浓度分别为0.8mg/ml。SZ-128单抗能够特异性的识别相对分子量为26kD的重组Mer IgC样区蛋白、Mer真核重组胞外区全长蛋白以及Jurkat和K562细胞裂解液中天然Mer蛋白。免疫组化提示:Mer在前列腺、肺脏和肾脏组织高表达,而在脾脏、小肠、大肠、肝脏和扁桃体表达较低,在食道的平滑肌几乎没有表达。血小板聚集实验中,当SZ-128和血小板PRP孵育以后可以抑制ADP（2μmol/L）和瑞斯托霉素（1.25mg/ml）诱导的血小板聚集,抑制率分别为30.2%±8.5%和25.6%±7.4%（SZ-128终浓度均为50μg/ml）,并且在20μg/ml、50μg/ml和100μg/ml不同剂量组之间抑制血小板聚集的效应没有明显的量效关系,这种现象可能因为Gas6/Mer信号途径本身并不直接参与血小板聚集而只是血小板聚集的中后期起到信号放大作用有关。二、受体酪氨酸激酶Mer对于内皮细胞的功能调节1.Mer对内皮细胞凋亡的影响凋亡是正常机体内细胞程序性死亡的过程,是体内细胞内环境稳定的重要因素。目前已经发现包括Mer家族成员Axl在内的多种受体酪氨酸激酶参与正常细胞的凋亡过程,其中大多数是生长因子受体。为了探讨Mer对于内皮细胞凋亡的影响,首先利用经典的生长因子撤离的方法诱导HMEC-1（人类微血管内皮细胞株）发生凋亡,并利用定量PCR和细胞ELISA的方法分别检测凋亡过程中Mer mRNA和蛋白水平的变化趋势,ELISA结果显示从凋亡开始后4h后Mer表达开始即到达较高水平,而后逐步下降,48小时基本恢复正常水平,mRNA水平的变化趋势与蛋白水平基本相符。同时我们利用脂质体法将人类Mer真核全长表达质粒和pcDNA3.1（阴性对照）分别转染到HMEC-1细胞中,并经过定量PCR和Western blot鉴定确认其可以稳定的高表达Mer蛋白。同样利用生长因子撤离的方法诱导转染细胞凋亡,发现在生长因子撤离48h后,转染pcDNA3.1的HMEC-1细胞发生凋亡的均值为20.8%±6.8%,而转染Mer的HMEC-1细胞发生凋亡的均值为5.8%±3.1%,两者存在显著差异,荧光定量PCR检测检测了转染细胞的bcl-2基因的表达,结果显示转染Mer的HMEC-1中bcl-2的表达为转染pcDNA3.1的HMEC-1细胞的178%。以上结果表明Mer在HMEC-1细胞发生凋亡后表达上调,并且高表达Mer的HMEC-1细胞对于生长因子撤离导致凋亡的抵抗能力增强,该效应是通过bcl-2抗凋亡基因发挥作用。2.Mer过表达对HMEC-1细胞血管形成的影响及其机制探讨Gas6/Axl在体外可以抑制脐静脉内皮细胞的血管形成能力,但是关于Mer在血管新生中的作用和具体机理目前尚没有相关研究报道。我们利用已经构建的高表达Mer的HMEC-1细胞株,并以pcDNA3.1-HMEC-1作为阴性对照。体外的细胞迁移实验结果可以看出,高表达Mer的HMEC-1在体外的迁移能力有所降低,transwell实验结果显示迁移到下槽的细胞数分别为21±6/视野和36±11/视野,P＜0.05,具有统计学差别。而体外基于Matrigel的血管形成实验结果可以看出两者形成封闭管腔的数目分别为6±4/视野和26±8/视野,平均抑制率达到76.92%。高表达Mer的HMEC-1细胞血管形成能力显著下降,两者具有显著差异（P＜0.01）。定量PCR检测了两种细胞中主要血管新生相关因子VEGF-A、VEGF-B、VEGF-C、VEGF-D、VEGFR-1和VEGFR-2的表达变化,可以看出Mer-HMEC-1中VEGF-C和VEGFR-2 mRNA水平的表达量显著下降,分别为对照组的44.7%（P＜0.01）和25.6%（P＜0.01）,而VEGF-A、VEGF-B、VEGF-D和VEGFR-1的表达没有显著差异（P＞0.05）。三、受体酪氨酸激酶Mer在急性白血病中的异常表达与机制研究1.受体酪氨酸激酶Mer在急性白血病中的异常表达受体酪氨酸激酶Mer在正常骨髓和外周血中的粒细胞,T、B细胞均没有表达,但是在部分恶性白血病细胞株中存在异常的高表达,由此想到是否Mer在某些白血病患者骨髓中存在异常表达?Mer的异常高表达是否参与白血病的发生发展过程呢?我们搜集了57例急性白血病患者,包括32例急性粒细胞白血病（AML）患者和25例急性淋巴细胞白血病（ALL）患者,利用流式细胞仪检测了正常人骨髓和急性白血病患者骨髓中Mer的表达情况,在其中所有32例AML患者中有23例患者存在Mer的阳性异常表达,其中3例患者高水平表达Mer、8例中等水平表达、12例低水平表达,其余9例极低水平表达或者不表达;在25例ALL患者中共有16例患者Mer阳性异常表达,其中高水平表达患者有1例,中等水平表达患者有6例,低水平表达有9例,其余9例患者未见表达或者极低水平表达,在所有阳性患者中T-ALL有5人,B-ALL有11人。并且结果在蛋白水平和mRNA水平是基本符合的。同时我们在AML患者中发现高中水平表达Mer的AML患者多为M1或者M2患者,而M3型患者多为低水平或不表达,T-ALL阳性患者也具有幼稚免疫表型的高表达。提示Mer可能和细胞的幼稚程度以及急性白血病发生发展过程有关。2.受体酪氨酸激酶Mer异常表达的机制研究既然发现受体酪氨酸激酶Mer在部分急性白血病患者中异位表达就有必要探讨其作用的具体原理和分子机制。我们检测了常见的白血病细胞株细胞膜上Mer的表达情况,选取表达较高的Jurkat作为模型。我们将经过验证、确实有效的anti-Mer siRNA转染到Jurkat细胞中,并以转染非相关序列的siRNA为阴性对照,特异性的阻断Jurkat细胞中Mer的表达。MTT增殖实验绘制敲除Mer前后Jurkat细胞生长增殖曲线,虽然敲除Mer的Jurkat细胞生长较阴性对照组缓慢,但是没有统计学差异（P＞0.05）。体外阻断Mer基因在Jurkat细胞中的表达后可以看出Jurkat抵抗凋亡能力明显下降,血清撤离48h诱导的凋亡率明显上升,达到（15.3±2.3）%,而转染非相关序列的对照组仅为（1.5±0.5）%,并且实时定量PCR检测主要凋亡相关因子bcl-2和Caspase-3,验证结果表明利用anti-Mer siRNA体外阻断Mer表达后,Jurkat细胞中Bcl-2分子的表达也明显下调,仅为对照组的（42.7±8.6）%,而Caspase3变化则不明显。提示Mer在恶性血液病中的异常表达可以增加肿瘤细胞抵抗凋亡的能力,并且这个效应可能通过上调bcl-2抗凋亡蛋白起作用。Mer作为一种新型的酪氨酸激酶在1994年被首次克隆后其功能被日益完善,本研究表明其参与内皮细胞的凋亡和血管新生的过程;并且Mer在部分急性白血病细胞株和患者中的异常表达也提示其可能在恶性血液病的发生发展中扮演重要的角色,因此针对Gas6/Mer信号转导途径的特异性靶向治疗,可能成为研制相关疾病新一代的药物的靶点。更多还原

【Abstract】 Human Mer tyrosine kinase（MerTK） was first cloned fromλgt-11 cDNA lymphoblastoid library by Graham,and mouse Mer was cloned successfully the next year by the same person.Comparison of the mouse and human c-mer amino acid sequences reveals an overall identity of 88%.Human Mer is so named after its original reported expression pattern—monocytes and epithelial and reproductive tissues.Mer belongs to Axl receptor tyrosine kinase（RTK） family,which is composed of Axl,Tyro3 and Mer,and they have the common ligand—Gas6.Axl RTK subfamily members each possess a combination of two immunoglobulin（Ig）-like domains and two fibroneetin typeⅢ（FNⅢ） repeats in their extracellular regions.Among these,IgG-like domains can bind their ligand-Gas6,and FNⅢrepeats play a regulation fuction in binding process.Intraeellular region of this family is tyrosine kinase domain,and also contains the special KWIAIES sequence.After binding their ligand-Gas6,each Axl RTK family member appears to signal for apoptosis,proliferation,migration and phagocytosis functional outcomes,besides these they also participate in many pathology processes,just like infammafion, autoimmunity,thrombosis and hemostasis,vascular and kidney disease and even the development of some malignant diseases.Now the focus of Axl RTK family is Axl,and many research were carried out through the Gas6/Axl axis.After cloned in 1994,there were few reports about Gas6/Mer until recnet years,more and more articles are involved in the Mer function,there includes inflammation,apoptosis,phagocytosis, thrombosis and hemostasis and abnormal expression of Mer in some malignant diseases.Preparation and function study of monoclonal antibody against Mer1.Prokaryotic expression of Mer receptor tyrosine kinase IgG-like domains The existed reports show that Mer Ig-like domains is most important during the process binding its ligand Gas6,and only binding Mer Ig-like domains,Gas6/Mer can activate its tyrosine kinase activity,so preparation of monoclonal antibody against Mer Ig-like domains has great significance to study the function of Gas6/Mer.We designed the cloning primers of Mer Ig-like domains according to the sequence in gene bank by using Primer 5 softwoare,interested fragment was amplified from K562 cDNA template,and after linked to PQE30 expression vector,we constructed Mer-Ig-PQE30 recombinated vector successfully.The recombinated vector was transformed to E.coli M15.The positive clones were chosen and recombinated Mer-Ig（rMer-Ig） prokaryotic protein was induced by IPTG.After analized by 12%SDS electrophoresis,the recombinated protein molecular weight is about 26kD,mainly existed in inclusion body and accounted for 15%of total bacterial protein.The purified rMer-Ig can be special recognized by anti-His antibody by Western blot.2.Preparation,characterization and function study of monoclonal antibody against MerBy using our established method,the recombined protein was purified by Ni-NTA agarose column and Balb/c mice were immunized.After the spleen cells of the mice and SP2/0 hybridoma cells were hybridized,positive clones was screened by ELISA assay and the selected hybridoma cells were used to prepare ascites.Western blot was used to detect whether the prepared McAb can bind prokaryotic rMer-Ig protein,eukaryotic Met extracellular region protein and natural Mer protein in cell lines.Characterization of immunize subclass and tissue specificity.And detected prepared McAb inhibition effect on platelet aggregation.The results show that:After the spleen cells and hybridoma cells SP2/0 were hybridized,we got one strain of cell that can last secreting McAb against Mer named SZ-128.the selected hybridoma cells was used to prepare ascites The titers of McAbs in ascites was 1×10^-5 detected by indirect ELISA.The heavy chain of the McAbs belonged to IgG1 subclass.The concentrations of purified anti-Mer McAb were 0.8mg/ml after purifying by proteinG-Sephrose4B affinity chromatography column.The antibodies could not only identify 26kD prokaryotic rMer-Ig but also eukaryotic rMer extracellular region and natural Mer protein in Jurkat and K562 cell specially.The results of immuno-labelling show that Mer protein is high-expression in human prostatae,lung,kidney;and relative low-expression in spleen,intestine,colon,liver and tonsilla,and almost no expression in esophagus smooth muscle.In platelet aggregometry,we found that SZ-128 could inhibit platelet aggregation induced by ADP（2μmol/L） and ristomycin（1.25mg/ml）,the inhibition ratio are 30.2%±8.5%and 25.6%±7.4%respectively（The final concentration of SZ-128 is 50μg/ml）,but there is no significant deviation between the inhibition ratio of different dose group（20μg/ml、50μg/ml and 100μg/ml）,so we couldn’t observe dose-dependent effect obviously.This phenomenon may due to Gas6/Mer signal pathway doesn’t participate in platelet aggregation diretly,they just plays a signal-amplification fuction role in midanaphase of platelet aggregation.The regulation effect of receptor tyrosine kinase Mer on endothelial cell1.The influence of receptor tyrosine kinase Mer on endothelial cell apoptosisApoptosis is a programmed death process in normal system,which is important to maintain the stability of internal environment.Many reports show that many receptor tyrosine kinase including Axl RTK patticipate in cell apoptosis,and most of them are growth factor receptors.To study the effect of Mer RTK on EC apoptosis process,we used the classic growth factor starvation method to induce apoptosis of HMEC-1,which is a acknowledged human microvascular endothelial cell line,realtime-PCR and cell-ELISA were used to detected Mer mRNA and protein expression alteration during apoptosis, according to the ELISA results,Mer expression increased gradually from the beginning, reached the climax at 4h,and then descended gradually,and return to the normal level at 48h,the Mer mRNA alteration trend is similar to the result of ELISA.Simultaneously,human Mer full length vector and pcDNA3.1（negative control） vector were transfected into HMEC-1 by using Lipofectamine following the protocol, after identified with realtime-PCR and Western blot,high expression Mer-HMEC-1 was established.And we also remove the growth factor to induced transfected HMEC-1 apoptosis,after evacuated 48h,the apoptosis of Mer-HMEC-1 and negative control were 5.8%±3.1%and 20.8%±6.8%respectively（P＜0.01）,there was significant difference between these two transfected cells.And bcl-2 mRNA expression was 178% in Mer-HMEC-1 compare to the negative control detected by realtime-PCR.We can observe from the above results that Mer expression was increased after apoptosis,and the anti-apoptosis ability of Mer-HMEC-1 is better than the negative control during growth factor starvation induced apoptosis through up-regulating the anti-apoptosis gene-bcl-2.2.Study of anti-angiogenesis effect induced by overexpressed Mer and its mechanismGas6/Axl can inhibit human umbilical vein endothelial cell angiogenesis in vitro, and there is few report about the similar function of Gas6/Mer.We used established Mer-HMEC-1 cell model,and used pcDNA3.1-HMEC-1 as a negative control.From transwell migration experiment,we can observe that the migration capacity of Mer-HMEC-1 decreased,the cell number migrated through the insert is 21±6 vs 36±11（negative control）（P＜0.05）,there is statistics difference.And the angiogenesis experiment based on Matrigel show that the angiogenesis capacity of Mer-HMEC-1 decreased significantly,and the number of tube-like form was 6±4 vs 26±8（negative control）,the average inhibition ratio was 76.92%,there was significant difference（P＜0.01） between these two cells.The alteration of main angiogenesis -associated growth factor VEGF-A,VEGF-B,VEGF-C,VEGF-D,VEGFR-1和VEGFR-2 were scanned by realtime-PCR,the results show that the mRNA expression of VEGF-C and VEGFR-2 decreased significantly in Mer-HMEC-1,it was 44.7%（P＜0.01）和25.6%（P＜0.01） compare to the negative control,the the expression of VEGF-A,VEGF-B,VEGF-D and VEGFR-1 were similar to negative control（P＞0.05）,Study on abnormal expression of Mer in acute leukemia and its mechanism1.Abnormal expression of Mer RTK in acute leukemiaThere is no expression on normal granulocyte,T and B lymphocyte,but there is abnormal high expression on some leukemia cell lines,so we can guess is there ectope epression of Mer RTK on leukemia patient? And does Mer RTK participant in the occurrence and development of leukemia?Bone marrows sample from 57 acute leukemia patient（32 AML,and 25 ALL） and 5 adults healthy donors were collected.Flow cytometry was used to analyse Mer expression on mononuclear cell of patient and normal control bone marrow.The flow cytometry results:there was no Mer expressed on granulocyte and lymphocyte of normal bone marrow;There were 23 Mer positive patients in total 32 AML patients; Mer expression level was high in 3 patients,middle in 8 patients,low in 12 patients and very low/negative in left 9 patients.Among 25 ALL patient,there were 16 Mer positive, Mer expression level was high in 1 patients,middle in 6 patients,low in 9 patients and very low/negative in left 9 patients,among all the positive patient,5 sample were from T-ALL patient,and 11 from B-ALL patient.The results of RT-PCR was similar to flow cytometry.And we found the most of the high and middle Mer expression AML patient were M1 or M2 according to FAB group,and M3 patient were almost low expression of Mer,and some of T-ALL Mer positive patient had the immature immunize type.This implied that Mer may take part in cell differentiation and development of leukemia.2.Study on the mechanism of Mer abnormal expression in acute leukemiaWe found abnormal expression of Mer in parts of acute leukemia patient,and it was necessary to explore its molecular mechanism.We screened Mer expression on common leukemia cell lines,and selected high Mer expression cell line-Jurkat as cell model to downstream experiment.We transfected verified and effective anti-Mer siRNA into Jurkat to block Mer expression specificially,and use uncorrelate siRNA sequence as negative control.After transfected with anti-Mer siRNA,the proliferation of Jurkat was a little slowly by MTT assay,but there was no statistics difference compare to the negative control（P＞0.05）.Blocking Mer expression in vitro Jurkat cell capacity of anti-apoptosis decreased significantly,after serum starvation for 48h,the apoptosis ratio of Jurkat transfected with anti-Mer siRNA was（15.3±2.3）%,while negative control was only（1.5±0.5）%,and we used realtime-PCR to analyze change of main apoptosis-associated factors-bcl-2 and Caspase-3,from the results we can observe that the bcl-2 mRNA expression of Jurkat transfected with anti-Mer siRNA was only （42.7±8.6）%compare to the negative control,and there was no obvious change of Caspase-3.All the above results suggested that Mer could increase Jurkat capacity of anti-apoptosis via bcl-2 signal pathway. From Human Mer gene was first cloned as a new member of receptor tyrosine kinase family in 1994,more and more research work were reported about its function. In the present study,we revealed that Mer participated in the apoptosis and angiogenesis process of endothelial cell;and Mer was ectopic expression in some acute leukemia patient,this indicated Mer play an important role in the occurrence and development of acute leukemia.Thus,targeting the Gas6/Mer signal patheway is hoped to become a new target of leukemia treatment in the future.更多还原