CN114946765B - 一种先天性血小板减少综合征的斑马鱼模型及其应用 - Google Patents
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Abstract
本发明公开了一种先天性血小板减少综合征的斑马鱼模型及其应用。该斑马鱼模型为thposzy6突变体斑马鱼。该模型是利用CRISPR/Cas9基因编辑技术构建的血小板生成素(Thpo)蛋白功能丧失的thposzy6突变体斑马鱼模型。thposzy6突变体斑马鱼中thpo基因发生了突变，Thpo蛋白的表达显著降低，并出现先天性血小板减少症的表型。该突变体斑马鱼可以被用来评估THPO受体激动剂(THPO‑RAs)作为THPO缺陷血小板减少症治疗药物的作用，也可以进行药物的评价和新药的开发验证以及临床突变意义不明的人类THPO基因点突变的致病性的研究。
Description
技术领域
本发明属于生物技术领域，特别涉及血小板发育研究领域，具体涉及一种先天性血小板减少综合征的斑马鱼模型及其应用。
背景技术
血小板是血液系统中主要的组成部分。当血管破损时，血小板起到了止血和凝血的作用。当血小板数目减少或者功能减退，会导致机体止血功能障碍和血栓形成不良，并且引发相关的血小板疾病。先天性血小板减少综合征,遗传性血小板减少症是一组少见的遗传性血小板异常疾病，表现为血小板数量减少，并往往伴有血小板功能异常。患者在幼时有不同程度的出血倾向，并有血小板减少的家族史。血小板生成素(THPO)在调控造血干细胞分化及巨核细胞的发育成熟继而形成血小板的过程发挥着巨大的作用，之前的研究报导显示THPO基因外显子区域的突变会导致严重的先天性血小板减少综合征，但是同时也有众多的突变意义不明的THPO突变类型的致病性需要进一步的验证(Dasouki M J,Rafi S K,Olm-ShipmanAJ,et al.Exome sequencing reveals a thrombopoietin ligand mutationin a Micronesian family with autosomal recessive aplastic anemia.[J].Blood,2013,122(20):3440–3449.doi:10.1182/blood-2012-12-473538)。THPO突变导致的血小板减少症较为罕见但极其严重，目前除骨髓移植和血小板灌输外，对于先天性的血小板减少症的治疗仍缺乏足够的有效药物，新的药物开发和筛选是目前所急需的(Cornish N,Aungraheeta M R,Fitzgibbon L,et al.Monoallelicloss-of-function THPO variantscause heritable thrombocytopenia[J].BloodAdv(2020)4(5):920–924.doi:10.1182/bloodadvances.2019001293)。在之前的研究中，研究人员通过基因编辑技术分别制备了THPO整体敲除和肝脏THPO特异性敲除的小鼠THPO突变体模型，这两种突变体小鼠模型在外周血中的血小板的数量表现为一定量的减少并伴随着造血干细胞数目上一定程度的下降，但是该研究制备的模型只复制THPO突变导致的血液表型，并未有实际的运用研究。由于小鼠研究过程中成本的高昂以及利用小鼠开展给药实验和显微注射实验困难性，利用该模型用来进行新药的筛选开发验证或者临床突变意义不明的人类THPO基因突变的研究的可行性极小，同时利用细胞系开展相关实验的可靠性也远远不及体内实验(O'Neill A,Chin D,Tan D,et al.Thrombopoietin maintains cell numbers ofhematopoietic stem andprogenitor cells with megakaryopoietic potential[J].Haematologica,2019.241406.doi:10.3324/haematol.2019.241406.)。斑马鱼作为一种新的模式生物，其产卵量高、体外受精且发育早期胚胎透明、信号通路保守以及实验成本相对低廉等特点为快速验证和研究人类血液病候选基因提供强大的工具平台，利用斑马鱼进行显微注射构建特异性基因编辑动物模型相比于小鼠等传统模式生物更具技术上的优势，同时斑马鱼也已经被广泛运用到血液系统疾病的药物开发验证和筛选运用中并筛选出若干有效的药物进行了临床前或临床的研究(Konantz M,C S chürch,Hanns P,et al.Modelinghematopoietic disorders in zebrafish[J].Disease Models and Mechanisms,2019,12(9):dmm040360.doi:10.1242/dmm.040360.)。
为了更好地研究THPO在血小板中的作用以及该类先天性疾病的发病机制，为该种疾病筛选或开发专治药物提供便利，同时为众多的突变意义不明的THPO突变类型的致病性需要进一步的验证提供平台，可以用斑马鱼建立疾病模型来进行大规模的药物筛选及临床基因突变的致病性验证。
发明内容
为了克服现有技术存在的不足，本发明的目的是提供一种先天性血小板减少综合征的斑马鱼模型及其应用。该模型具体为一种血小板生成素(THPO)缺陷先天性血小板减少症的斑马鱼模型。本发明提供的先天性血小板减少综合征的斑马鱼模型是一种thpo基因突变致血小板减少的斑马鱼模型。
本发明为了更好地研究thpo在早期发育血小板中的作用以及相关的调控机制，并为后期的药物开发及筛选提供便利，构建斑马鱼thpo突变体并应用于制备相应的疾病模型(先天性血小板减少综合征)。由于斑马鱼产卵高，体外受精，早期胚胎发育透明的特性，有利于观察血小板的行为变化。同时，也有利于一些治疗先天性血小板减少综合征的大规模药物筛选。
本发明旨在于提供一种thpo突变导致的先天性血小板减少综合征的斑马鱼模型。本发明提供的thpo基因突变致血小板减少斑马鱼模型可以应用于先天性血小板减少综合征发病机制的研究。
本发明的目的至少通过如下技术方案之一实现。
本发明提供的先天性血小板减少综合征的斑马鱼模型，该斑马鱼模型为thposzy6突变体斑马鱼。
进一步地，thposzy6突变体斑马鱼的表型是由thpo基因异常表达引起，即由thposzy6突变引起。
进一步地，所述thposzy6突变体斑马鱼为斑马鱼thpo基因缺失2个并插入1个碱基，导致thpo基因提前终止表达，Thpo蛋白表达异常的突变体斑马鱼。所述thposzy6突变体斑马鱼的thpo基因翻译提前终止，为缺失重要的功能域的突变体。
进一步地，所述thposzy6突变体斑马鱼是通过CRISPR/Cas9靶向基因敲除thpo基因上的2个碱基并插入1个碱基得到的。
进一步地，所述thposzy6突变体斑马鱼是通过CRISPR/Cas9靶向基因敲除斑马鱼GP9基因的2号外显子序列ATG起始密码子之后的2个碱基并插入1个碱基得到的。
进一步地，所述thposzy6突变体斑马鱼的Thpo蛋白相对于野生型斑马鱼的Thpo蛋白缺失了EPO/THPO的功能域。
本发明提供了thpo基因突变致血小板减少综合征的斑马鱼模型在制备先天性血小板减少综合征的动物模型中的应用。
本发明提供了所述先天性血小板减少综合征的斑马鱼模型在药物筛选评价和点突变致病性预测中的应用。
进一步地，所述先天性血小板减少综合征的症状为胚胎期血小板减少，造血干/祖细胞的数目减少，该模型对于临床上用于治疗血小板减少症的药物响应良好，也可用相关点突变的验证。
本发明总体构思为：
(1)通过CRISPR/Cas9靶向基因敲除的技术，得到thpo基因序列缺失的突变体斑马鱼。
(2)利用标记不同阶段血小板的探针进行原位杂交试验来检测血小板分子标记的变化以及通过Q-PCR的方法检测与血小板发育成熟的相关基因的表达变化。
(3)在胚胎期利用临床药物处理thposzy6突变体验证其有效性。
(4)在胚胎期利用显微注射临床点突变位点来验证thposzy6突变体的可靠性。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明提供的突变体斑马鱼能应用在筛选对先天性血小板减少综合征有效的药物；该斑马鱼thposzy6突变体血小板的数目减少，造血干/祖细胞也存在缺陷的现象，具有与人类由THPO突变导致的先天性血小板减少综合征疾病的相似症状；由于斑马鱼产卵量多，养殖方便，饲养的费用较饲养小鼠低的特点，有利于高通量筛选治疗先天性血小板减少综合征的药物，花费较少；另外，thposzy6突变体胚胎期血小板的数目减少，可以在胚胎期对斑马鱼进行药物处理来筛选有效的药物，实验周期短。
(2)本发明中的先天性血小板减少综合征的斑马鱼模型，由于其稳定遗传性可以用于长期追踪疾病的发病机制，从胚胎期到成年期疾病的进展。并且thposzy6突变体具有稳定遗传的血小板减少的表型，thposzy6突变体还可以用来扩展研究血小板凝血功能障碍和血小板相关疾病的机制。
(3)本发明中thposzy6突变体具有以下表型：①thposzy6突变体胚胎期血小板的数目减少，②thposzy6突变体造血干/祖细胞的数目减少。③thposzy6突变体除血小板和造血干/祖细胞外的其他类型血液细胞数目基本维持不变。④thposzy6突变体可用于验证临床上THPO点突变是否具有致病性。⑤thposzy6突变体对于临床或实验中用于治疗血小板减少症的药物有较好对的响应。
附图说明
图1是CRISPR/Cas9靶向基因敲除技术获得斑马鱼突变体thposzy6示意图；其中图1中的A部分为CRISPR/Cas9靶向基因敲除设计方案，选择斑马鱼thpo基因的1号外显子序列作为靶点进行基因敲除。其中图1中的B部分为经测序得到突变体thposzy6在靶点位置缺失了2个碱基并插入了1个碱基，缺失后Thpo蛋白提前终止，缺乏重要的功能域。图1中的C部分为根据结构预测的thposzy6突变体蛋白相对于野生型缺乏EPO/TPO(Erythropoietin/thrombopoietin)结构域。
图2是4dpf的突变体鱼血小板数目及有关基因表达量的结果图；图2的A部分为抗体免疫荧光染色实验，图2的B部分为血小板数目统计图，显示突变体thposzy6的带有Tg(mpl:eGFP)标记的血小板数量较野生型显著减少；图2的C部分为qPCR检测基因表达量，显示突变体thposzy6血小板相关基因的表达减少(t-检验，P<0.05；均数±标准误，n＞25)。
图3是在不同时期检测到的其他各造血谱系的标记基因表达的整体原位杂交结果图。其中，图3的A部分为在36hpf野生型斑马鱼(thpoWT)和thposzy6突变体斑马鱼中造血干/祖细胞的标记基因cmyb表达量及统计图,显示与野生型相比，thposzy6突变体中造血干/祖细胞的标记物表达量的降低，提示造血干细胞的缺陷(采用连续性调整卡方统计量来确定统计学差异，*P<0.05)；图3的B部分为在4dpf野生型斑马鱼(thpoWT)和thposzy6突变体斑马鱼中红细胞的标记基因βe1表达量及统计图,显示与野生型相比，thposzy6突变体中红细胞的标记物表达量没有变化，提示突变体对红细胞无影响(采用连续性调整卡方统计量来确定统计学差异，P<0.05)；图3的C部分为在4dpf野生型斑马鱼(thpoWT)和thposzy6突变体斑马鱼体内粒细胞的标记基因mpo表达量及统计图，显示与野生型相比，thposzy6突变体中粒细胞的标记物表达量的不变(t-检验，P＞0.05；均数±标准误，n＞25)。；图3的D部分为5dpf野生型斑马鱼(thpoWT)和thposzy6突变体斑马鱼中淋巴细胞的标记基因rag1表达量及统计图，显示与野生型相比，thposzy6突变体中淋巴细胞的的标记物表达量的不变(采用连续性调整卡方统计量来确定统计学差异，P＞0.05)；
图4是利用thposzy6突变体模型验人源THPO基因点突变致病性的结果图；图4的A部分所选的点突变位点在人THPO蛋白上的位置，两个突变位点都位于THPO蛋白中重要的EPO/TPO(Erythropoietin/thrombopoietin)结构域前端，突变会导致该结构域的功能异常。图4的B和C部分是分别显微注射野生型和带有突变位点的人源THPO mRNA后thposzy6突变体斑马鱼体内血小板计数的变化图。数据显示注射野生型人源THPO mRNA可有效恢复突变体中血小板的数量，而注射了带有致病突变的人源THPO mRNA将对血小板没有恢复效果。
图5是利用临床药物验证thposzy6突变体模型的可靠性的结果图。图5的A部分是药物处理的示意图。图5的B和C部分是四种药物分别处理后thposzy6突变体中血小板数量的变化结果图；结果显示，这几种临床上用于治疗血小板减少症的药物均可有效的增加thposzy6突变体模型中血小板的数量。表明我们的模型对于临床药物的响应较好，具有可靠性。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
本发明中所使用的术语“野生型”或者“WT”都是指野生型斑马鱼。
本发明中所使用的术语“hpf”指受精后的小时数，“dpf”指受精后的天数。
实施例1
1、材料和方法：
(1)斑马鱼养殖
斑马鱼的养殖如文献(Westerfield M.The zebrafish book.Aguide for thelaboratory use of zebrafish(Danio rerio)[M].Eugene:University ofOregon Press,2000:385.)所述。
(2)本发明中用到如下的品系：AB野生型斑马鱼、thposzy6突变体斑马鱼、Tg(mpl:eGFP)转基因斑马鱼。其中，将各个转基因斑马鱼品系与thposzy6突变体斑马鱼杂交后再自交获得MPL转基因背景的thposzy5突变体斑马鱼，如Tg(mpl:eGFP)；thposzy6。
(3)CRISPR/Cas9-靶向基因敲除thpo基因(可参照图1所示)
选择thpo基因的1号外显子序列ATG起始密码子之后的序列作为靶向敲除的序列，根据http://www.crisprscan.org/网站预测的得分高，靶点效率高的，脱靶效率低的靶点序列GGGCAAAGTCACGCACCTGC，该序列之后AGG构成PAM区，最后合成引物的序列为T7(17bp)+target+gRNA FP(20bp)序列：5’-3’TAATACGACTCACTATAGGAGATTTCATGTTGTCGCTGTTTTAGAGCTAGAAATAGC，gRNARP序列:5’-3’AGCACCGACTCGGTGCCACT，以Z-cas9质粒为模板，用T7 RNA聚合酶(Thermo，EP0111)体外合成。
所得到的突变体为thpo基因的1号外显子缺失了2个碱基后插入了1个碱基，导致了移码突变，提前终止，产生了截短的Thpo蛋白；相对于野生型Thpo蛋白，缺失了与血小板生成有关的EPO/THPO(Erythropoietin/thrombopoietin)重要功能域。
实施例2
整体原位杂交(Whole-mount in situ hybridization)检测thposzy6突变体造血谱系相关基因的表达
按照以下标准实验操作流程完成(ThisseC,Thisse B.(2008).High-resolutionin situ hybridization to whole-mount zebrafish embryos.NATUREPROTOCOLS.VOL.3NO.1).
结果如图3的A-D部分所示，在各类造血细胞标记物的表达时间点,thposzy6突变体中除造血/祖干细胞的标记分子cmyb的信号相对于野生型较少外，其他造血谱系的标记基因表达没有变化，说明thposzy6突变体只存在血小板及造血干/祖细胞的变化，与小鼠模型相似。
实施例3Q-PCR检测thposzy6突变体血小板相关基因的表达
收集4dpf WT和3dpf thposzy6突变体的斑马鱼幼鱼，使用Tripure RNA IsolationReagent(Thermo,15596018)提取胚胎的总RNA，按照说明书的操作步骤实施。用M-MLV逆转录酶(Promega,M1701)逆转录成cDNA。用斑马鱼elf1a为内参基因进行Q-PCR检测，其中引物的序列信息如下表1所示。
表1
结果如图2的C部分所示，在4dpf,thposzy6突变体相对于野生型，血小板相关的基因cd41,mpl,nfe2，gp9的表达都下降，结合Tg(mpl:eGFP)；thposzy6突变体中荧光信号减少的结果，进一步说明了在胚胎期，thposzy6突变体中血小板的数目减少。
实施例4利用thposzy6突变体验证人类THPO基因突变位点致病性
通过PCR扩增人源THPO mRNA的编码序列，将其克隆到pCS2+载体中，然后用mMESSAGE mMACHINETM SP6体外转录试剂盒(Invitrogen,AM1340)体外合成THPO mRNA，利用天根快速点突变试剂盒(TIANGEN,KM101)得到带有点突变的突变型人源THPO mRNA。后续将转录得到的mRNA通过显微注射技术到单细胞时期的突变体斑马鱼胚胎中，结果如图4的B-C部分所示，在4dpf时期检测到带有点突变的人源THPO mRNA不能增加thposzy6突变体血小板数量，而野生型人源THPO mRNA可以拯救thposzy6突变体血小板缺陷的表型。
实施例5利用临床药物验证thposzy6突变体适用性
在1.5dpf的时期开始将胚胎分别浸泡在含有100U/mL rhTHPO(3SBIOINC)、50μMlusutrombopag(HY-19883,MedChem Express)、50μM avatrombopag(HY-13463,MedChemExpress,)和1μM eltrombopag(HY-15306,MedChem Express)的培养体系中，待到胚胎发育到4dpf时期，检测到四种药物对突变体中血小板数量的影响如图5的B-C部分所示，可以看到，临床上用于升高血小板计数的药物均可有效提升thposzy6突变体中血小板的数量，表明该模型可有效模拟血小板减少的表型并对治疗药物响应较好，模型较为可靠。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。
Claims (9)
1.一种先天性血小板减少综合征的斑马鱼模型，其特征在于，所述斑马鱼模型为thposzy6突变体斑马鱼，所述thposzy6突变体斑马鱼是通过CRISPR/Cas9靶向基因敲除斑马鱼thpo基因的2号外显子序列ATG起始密码子之后的2个碱基并插入1个碱基得到的。
2.根据权利要求1所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述thposzy6突变体斑马鱼的表型是由thpo基因异常表达引起。
3.根据权利要求1所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述thposzy6突变体斑马鱼为斑马鱼thpo基因缺失2个并插入1个碱基，导致thpo基因的异常表达造成Thpo蛋白结构缺失的突变体斑马鱼。
4.根据权利要求1所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述thposzy6突变体斑马鱼的thpo基因的突变导致氨基酸翻译成蛋白质过程的终止密码子在起始密码子后第十一个密码子处出现，翻译提前终止，为缺失重要的功能域的突变体。
5.根据权利要求1所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述thposzy6突变体斑马鱼是通过CRISPR/Cas9靶向基因敲除thpo基因上的2个碱基并插入1个碱基得到的。
6.根据权利要求1所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述thposzy6突变体斑马鱼的Thpo蛋白相对于野生型斑马鱼的Thpo蛋白缺失了EPO/THPO的功能域。
7.根据权利要求1-6任一项所述先天性血小板减少综合征的斑马鱼模型，其特征在于，所述先天性血小板减少综合征的症状为胚胎期血小板减少，造血干/祖细胞的数目减少。
8.权利要求1所述先天性血小板减少综合征的斑马鱼模型在药物筛选评价和点突变致病性预测中的应用。
9.根据权利要求8所述应用，其特征在于，所述先天性血小板减少综合征的斑马鱼模型对于临床上用于治疗血小板减少症的药物响应良好。
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