【作者】 孙雄华；

【导师】 王兆钺；

【作者基本信息】 苏州大学 ， 内科血液学， 2013， 博士

【摘要】 MYH9相关疾病（MYH9-related disease，MYH9-RD）是一种常染色体显性遗传性疾病，是由位于人染色体22q12.3的非肌性肌球蛋白重链9基因(MYH9)突变导致。MYH9基因编码非肌性肌球蛋白重链IIA (NMMHC-IIA)，分子量约220kDa，在细胞迁移、胞质分裂、细胞粘附、细胞形态、细胞极性形成和细胞趋向性等多项功能中均起着重要的作用。至今为止，对于MYH-RD的发病机制和NMMHC-IIA的正常功能尚未解释清楚。因此，本研究对17例MYH9-RD病人进行了临床特征和基因分析，同时进行了基因突变对于细胞形态的影响、NMMHC-IIA相互作用蛋白的鉴定、NMMHC-IIA突变后对细胞蛋白表达谱的影响等研究。这些研究对于深入阐述NMMHC-IIA的功能和MYH9-RD的分子致病机制具有重要的意义。目的1、分析17例MYH9-RD患者临床特征，进行MYH9基因诊断；2、构建5种不同NMMHC-IIA突变（K74E、D1424N、W33R、E1841K和R702H-IIA）的pEGFP-C3-IIA表达载体，筛选HEK293稳转细胞株，观察包涵体和细胞形态的变化；3、鉴定NMMHC-IIA相互作用蛋白；4、分析NMMHC-IIA突变后对细胞蛋白表达谱的影响。方法1、用全血细胞计数仪及手工法进行血小板计数；用瑞氏染色法和免疫荧光法观察血小板形态及中性粒细胞包涵体；抽提患者外周血基因组DNA，PCR扩增MYH9患者的40个外显子及两端侧翼序列，DNA测序并与基因序列比对以确定基因异常；用限制性核酸内切酶电泳法分析排除多态性。2、构建正常的pEGFP-C3-NMMHC-IIA表达载体，利用PCR介导的质粒定点突变技术，以K74E、D1424N、W33R、E1841K和R702H突变引物进行扩增，Dpn I消化后转入DH5α感受态细胞，挑选阳性克隆质粒，测序正确后扩大培养，无菌环境下提取高质量高浓度质粒；用脂质体转染法（Lipofectamine2000）转染HEK293细胞，加G418进行筛选，得到HEK293稳转细胞株；利用免疫荧光技术观察细胞形态、细胞内NMMHC-IIA包涵体形成情况。3、应用免疫共沉淀技术和高效液相色谱-质谱联用方法高通量鉴定非肌性肌球蛋白IIA相互作用蛋白组成；应用生物信息学软件Gominer分析非肌性肌球蛋白IIA相互作用蛋白的分子功能、亚细胞定位及生物过程等。4、应用稳定同位素标记氨基酸细胞培养（SILAC）定量蛋白质组学技术分析突变型非肌性肌球蛋白IIA对细胞蛋白表达谱的影响；应用生物信息学软件Gominer分析表达受到影响蛋白的分子功能分布情况；采用蛋白免疫印迹技术验证上述分析得到的表达发生变化的蛋白水平。结果1、17例患者除3例外，都有血小板计数下降、血小板体积增大、中性粒细胞内淡蓝色包涵体三联征；瑞氏染色没有观察到包涵体的3例病人，经过免疫荧光分析，都在胞浆中观察到了小的颗粒状的包涵体；基因分析共发现了9种突变， W33R、p.Q1443_K1445dup、K74E、D1447A、IVS25+1T→A、3个R702H、6个D1424N、2个R1933X、E1945X，其中前5种突变是首次发现的新突变。2、成功构建了K74E、D1424N、W33R、E1841K和R702H-IIA的pEGFP-C3-IIA突变质粒表达载体；得到了转染wt、K74E、D1424N、W33R、E1841K和R702H-IIA的HEK293稳转细胞株；除了R702H外，其它表达突变型非肌性肌球蛋白IIA的HEK293细胞中都有包涵体形成，且各种突变型细胞骨架形态变化都不同。3、在蛋白质组学水平分析了非肌性肌球蛋白IIA相互作用蛋白的组成，共计鉴定到4591个肽段，其对应151个蛋白，这些蛋白在分子功能、亚细胞定位以及生物过程等方面具有非常广泛的分布。4、大规模比较了表达突变型非肌性肌球蛋白IIA细胞中的蛋白表达谱与表达野生型非肌性肌球蛋白IIA细胞的蛋白表达谱，结果发现前者与后者相比有55个蛋白表达量明显下调，有37个蛋白表达量明显上调。这92个表达明显变化的蛋白具有广泛的功能分布；其中热休克蛋白70（HSP70）、非极性肌球蛋白IIB的蛋白免疫印迹表达情况与上述大规模定量蛋白质组学技术分析结果是一致的。结论1、确证17例MYH9-RD病人，发现了9种突变，其中W33R、p.Q1443_K1445dup、K74E、D1447A、IVS25+1T→A为国际上首次报道。2、转染wt、K74E、D1424N、W33R、E1841K和R702H-IIA的HEK293稳转细胞株已经建立，除了R702H外，都有包涵体，且各突变型细胞骨架形态变化都不同。3、建立了第一个非肌性肌球蛋白IIA相互作用蛋白数据库，包含151个蛋白。4、发现非肌性肌球蛋白IIA突变后引起了55个表达下调蛋白，以及37个表达上调蛋白，这些均为深入研究MYH9-RD的分子致病机制提供了目标。更多还原

【Abstract】 MYH9related disease (MYH9-related disease, MYH9-RD) is an autosomal dominantinherited disease, which is caused by mutation of non-muscle myosin heavy chain9gene(MYH9) located on human chromosome22q12.3. MYH9gene encodes non-musclemyosin heavy chain IIA (NMMHC-IIA) with molecular weight being about220kDa,which plays important roles in cell migration, cytokinesis, cell adhesion, cell morphology,cell polarity formation and cell tropism. However, MYH9-RD pathogenesis andNMMHC-IIA function are not yet explained clearly. Therefore, we investigated clinicaland genetic features of17patients with MYH9-RD; observed the effects of mutatedNMMHC-IIA on cell morphology; identified the NMMHC-IIA protein interactome;anlyzed the impact of NMMHC-IIA mutant on the proteome profiles. These studies mightbe in favor of clarifying NMMHC-IIA function and pathogenesis of MYH9-RD.Aim：1.Clinical features and genetic diagnosis of17patients with MYH9-RD;2.Preparation offive expression vectors of different NMMHC-IIA mutations (K74E, D1424N, W33R,E1841K and R702H-IIA), establishment of stable transfected HEK293cell lines, andobservation of inclusion bodies and morphology;3. Identification of NMMHC-IIAinteracting proteins;4. Analysis of proteome profiles by mutated NMMHC-IIA.Method：1. Hemocytometer and manual method for platelet count; Wright’s staining andimmunofluorescence staining for observation of platelet morphology and neutrophilinclusion bodies; extraction of DNA from peripheral blood of MYH9-related diseasepatients, amplification of40exons and flanking sequences at both ends using PCR, and DNA sequencing to determine genetic abnormalities; exclusion of polymorphism usingrestriction fragment length polymorphism analysis.2.Preparation of pEGFP-C3-NMMHC-IIA expression vector; using site-directedmutagenesis technique, PCR amplification of plasmid with K74E、D1424N、W33R、E1841K and R702H primers; Digested by DpnI and transformed into DH5α competentcells; Choosing and sequencing positive clones plasmid; Extraction of plasmid with highquality and high concentrations; transfection of these plasmids into HEK293cells byLipofectamine2000; Screening by G418; Observation of cell morphology andNMMHC-IIA inclusion bodies using immunofluorescence analysis.3. High-throughput identification of interacting protein of non-muscle myosin IIA usingco-immunoprecipitation technique and high performance liquid chromatography-tandemmass spectrometry method; Gominer analysis of non-muscle myosin IIA interactingproteins in molecular function, subcellular localization and biological processes.4. Proteome profiling of the impact of mutanted non-muscle myosin IIA using stableisotope-labeled amino acid cell culture (SILAC) quantitative proteomics technology;Gominer analysis of the distribution of molecular function of those changed proteins;Verifying the level of the above changed proteins using western blot technique.Results：1. With Wright’s staining the macrothrombocytopenia, light blue neutrophil inclusionbodies were observerd in14of17patients; small or faint inclusion bodies were observedin other3patients using immunofluorescence analysis, which were not found withWright’sstaining; Nine mutations of W33R, p.Q1443_K1445dup, K74E, D1447A, IVS25+1T→A,R702H, D1424N, R1933X, and E1945X were found, and the former five were firstlydiscovered;2. Five mutated pEGFP-C3-IIA vectors with mutation of K74E, D1424N, W33R, E1841Kand R702H-IIA were constructed; HEK293stable transfected cell lines were established;Inclusion bodies in HEK293cells except for overexpressed R702H were observed; thechanges of cytoskeleton morphological in those cell lines were different. 3. A total of4591peptides were identified, corresponding to151proteins interacting withNMMHC-IIA, and there is a very wide distribution of those proteins in molecular function,subcellular localization, and biological processes.4. Compared with the cells of overexpressed wild-type non-muscle myosin IIA, there weresignificant changes of the expression levels of92proteins in the cells of overexpressedmutated non-muscle myosin IIA. Among these, the levels of37proteins were remarkablyincreased, and the levels of55proteins were significantly decreased. These proteins had awide range of functional distribution; the expression levels of heat shock protein70(HSP70) and non-muscular myosin IIB were consistent with the results of large-scalequantitative proteomics analysis with Western Blotting.Conclusions：1.17cases of patients with MYH9-RD are confirmed, and nine mutations of which W33Rp.Q1443_K1445dup, K74E, D1447A, the IVS25+1T→A are firstly found.2. Construction the mutant plasmids of K74E, D1424N W33R, E1841K and R702H-IIA;After transfection and screening with G418, the HEK293stable cell lines overexpressingthese plasmids have been established; Except for R702H, inclusion bodies have beenobserved, and cells cytoskeletal have differently changed.3. The first database of NMMHC-IIA interacting proteins which contains151proteins wasestablished.4. The downregulation of55proteins, and the upregulation of37proteins caused byNMMHC-IIA mutant were identified, which are in favor of in-depth pathogenicmechanism studies for MYH9-related disease.更多还原