CN113473849A - 具有人il-34的非人动物及其用途 - Google Patents
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Abstract
本发明涉及在体内具有人白细胞介素34(interleukin 34:IL‑34)的非人动物。另外,还涉及具有人小胶质细胞的非人动物的制备方法,包括向所述非人动物中移植人CD34阳性造血干细胞。另外,还涉及人小胶质细胞的制造方法,包括自所述具有人小胶质细胞的非人动物中得到人小胶质细胞。
Description
技术领域
本发明涉及具有人IL-34的非人动物及其用途。特别涉及具有人IL-34基因的非人动物、使用了所述非人动物的制造人小胶质细胞(microglia)的方法,以及可自所述非人动物得到的人的小胶质细胞。另外,还涉及具有人小胶质细胞的非人动物的制备方法,以及HIV感染非人动物的制备方法。
本申请基于2019年1月28日于美国提交的62/797,369号主张优先权,将其内容援引至本申请中。
背景技术
小胶质细胞是中枢神经系统(central nervous system:CNS)的常驻巨噬细胞,对脑的发育、免疫防御作出了贡献。然而,人的小胶质细胞的正确起源、发生和特异性标志物逐渐成为争论的对象。尚不清楚成年人的脑中,在正常及患病状态下,小胶质细胞是仅来源于自胚胎发育阶段就存在于脑内的细胞、或是自造血干细胞(hematopoietic stem cell:HSC)或单核细胞向CNS中流入。单核细胞可以进入CNS的实质中,转变为与小胶质细胞在形态学上相似的细胞。然而,这样的转变仅发生于特定的条件下,而这些细胞是否确为小胶质细胞尚有争议。小鼠至小鼠的移植实验显示,在介由放射线、化学物质、或表达小胶质细胞特异性自杀基因而使内源小胶质细胞耗尽的小鼠中,来自供体的HSC产生小胶质细胞样细胞(例如,非专利文献1)。
另一方面,迄今为止,在免疫缺陷小鼠的脑中高效地使人小胶质细胞生成的尝试均未成功。将人CD34+细胞移植到免疫缺陷小鼠的实验中,仅确认到微量的人小胶质细胞(例如,非专利文献2)。
现有的人源化小鼠为通过在NOD/ScidIL2Rg-/-(NSG)小鼠等中移入HSC(例如,非专利文献3、非专利文献4)或人胚胎的肝脏和胸腺(例如,非专利文献5)使人免疫细胞稳定地重建而成的。然而,小鼠的脑中,脑膜和血管周围的巨噬细胞的数量和分布少,几乎没有发现小胶质细胞(非专利文献4、6)。小胶质细胞为HIV-1的主要靶标,是CNS中的HIV-1的主要储库(resevoir),因此,目前可用于脑内小胶质细胞分析的人源化小鼠模型对于重现人中HIV-1对CNS的感染而言是不充分的(非专利文献4)。
现有技术文献
非专利文献
非专利文献1:Capotondo,A.等,Brain conditioning is instrumental forsuccessful microglia reconstitution following hematopoietic stem celltransplantation.Proc Natl Acad Sci U S A 109,15018-15023(2012).
非专利文献2:Asheuer,M.等,Human CD34+cells differentiate intomicroglia and express recombinant therapeutic protein.Proc Natl Acad Sci U SA 101,3557-3562(2004).
非专利文献3:Arainga,M.,Su,H.,Poluektova,L.Y.,Gorantla,S.&Gendelman,H.E.HIV-1cellular and tissue replication patterns in infected humanizedmice.Sci Rep 6,23513(2016).
非专利文献4:Gorantla,S.等,Links between progressive HIV-1infection ofhumanized mice and viral neuropathogenesis.Am J Pathol177,2938-2949(2010).
非专利文献5:Denton,P.W.&Garcia,J.V.Novel humanized murine models forHIV research.Curr HIV/AIDS Rep 6,13-19(2009).
非专利文献6:Dash,P.K.等,Loss of neuronal integrity during progressiveHIV-1infection of humanized mice.The Journal of neuroscience:the officialjournal of the Society for Neuroscience 31,3148-3157(2011)
发明内容
发明所要解决的课题
如前文所述,以往的人源化小鼠中不能保持人小胶质细胞,或仅能保持极少的量,不适于重现人中的HIV感染等。
因此,本发明以提供人小胶质细胞的保持量多的非人动物及其制备方法为课题。此外,还以提供所述非人动物的利用方法作为课题。
用于解决课题的手段
本发明包括以下方式。
[1]非人动物,其在体内具有人白细胞介素34(interleukin 34:IL-34)。
[2]如[1]所述的非人动物,其被移植有人CD34阳性造血干细胞。
[3]如[1]或[2]所述的非人动物,其脑内存在人小胶质细胞。
[4]如[3]所述的非人动物,其中,所述人小胶质细胞表达选自由CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE组成的组中的至少1种基因。
[5]如[3]或[4]所述的非人动物,其中,所述人小胶质细胞分泌选自由CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a和CXCL10组成的组中的至少1种细胞因子。
[6]如[1]~[5]中任一项所述的非人动物,其感染有人免疫缺陷病毒(Humanimmunodeficiency Virus:HIV)。
[7]人小胶质细胞的制造方法,其包括自[3]~[6]中任一项所述的非人动物中得到人小胶质细胞。
[8]具有人小胶质细胞的非人动物的制备方法,其包括向在体内具有人IL-34的非人动物移植人CD34阳性造血干细胞。
[9]如[8]所述的具有人小胶质细胞的非人动物的制备方法,其中,所述在体内具有人IL-34的非人动物为免疫缺陷非人动物。
[10]如[8]或[9]所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞存在于脑内。
[11]如[8]~[10]中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞表达选自由CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE组成的组中的至少1种基因。
[12]如[8]~[11]中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞分泌选自由CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a和CXCL10组成的组中的至少1种细胞因子。
[13]HIV感染非人动物的制备方法,其包括通过[8]~[12]中任一项所述的具有人小胶质细胞的非人动物的制备方法制备具有人小胶质细胞的非人动物、然后使HIV感染所述具有人小胶质细胞的非人动物。
此外,本发明还包括以下方式。
[14]如[1]~[6]中任一项所述的非人动物,其分泌人IL-34。
[15]如[14]所述的非人动物,其具有人IL-34基因。
[16]如[14]或[15]所述的非人动物,其具有人小胶质细胞。
[17]如[1]~[6]和[14]~[16]中任一项所述的非人动物,其为啮齿类。
[18]如[17]所述的非人动物,其为小鼠。
[19]如[7]~[12]中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述在体内具有人IL-34的非人动物具有人IL-34基因。
[20]如[7]~[12]和[19]中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述在体内具有人IL-34的非人动物分泌人IL-34。
[21]如[7]~[12]和[19]~[20]中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述非人动物为啮齿类。
[22]如[21]所述的具有人小胶质细胞的非人动物的制备方法,其中,所述啮齿类为小鼠。
发明效果
通过本发明,可提供人小胶质细胞的保持量多的非人动物及其制备方法。另外,可提供所述非人动物的利用方法。
附图说明
图1A:图1A~D为示出了NOD.Cg-PrkdcscidIl2rgtm1Sug Tg(CMV-IL-34)1/Jic(NOG-hIL-34)小鼠的制备和表征的概况的图。图1A示出了为了制备NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL-34)1/Jic小鼠而向NOG-hIL-34转基因小鼠中导入的载体。将包含导入基因(Tg)、hIL-34的载体插入至CMV启动子的下游。
图1B:图1B示出了使用提取自纯合子小鼠的耳的DNA的PCR分析结果。NOG-hIL-34转基因小鼠(IL-34+/+)中扩增出hIL-34(358bp)的条带,对照的非转基因NOG小鼠中未检测出该条带。
图1C:图1C示出了使用提取自NOG-hIL-34小鼠的脑、脾脏、肺、肾脏、肝脏和皮肤的RNA的实时PCR结果。与NOG小鼠(对照)相比,NOG-hIL-34(IL-34)中确认到hIL-34在所有组织中的表达。
图1D:图1D示出了确认到血浆中的hIL-34表达的ELISA的结果。
图2:示出了表示小鼠脑的不同区域中的人IL-34的表达的RNAScope测定结果。使用以人IL-34的38~1774bp作为靶标的反义探针、Hs-IL-34-NoXMm进行RNAScope测定,在5μm厚的脑切片中以单个的棕色点或成簇的点的形式检测到人IL-34。NOG小鼠中未确认到显示人IL-34存在的信号,倍率为40倍。
图3A:图3A~B为示出了CD34-NOG-hIL-34小鼠中的人末梢血淋巴细胞的分化、增殖的图。图3A示出了末梢血的FACS分析的结果。对CD45淋巴细胞群设门,可以确认到分化45(CD45)+、以及人CD3、CD19和CD14细胞的人类集群(human cluster)的代表性的图。
图3B:图3B为示出了重建小鼠的末梢血中的人细胞亚群的比例的图。每个符号表示一只小鼠。
图4A:图4A~F示出了CD45-NOG-hIL-34小鼠脑中的人小胶质细胞的存在。图4A为在CD34-NOG-hIL-34和CD34-NSG小鼠之间、对组织巨噬细胞重建进行比较而得的图。对脑切片而言,针对HLA-DR进行染色,对肝脏和脾脏而言,针对CD68进行染色。
图4B:图4B为脑中的用人P2RY127、CD14、CD68、CD163抗体进行染色的结果,可确认人细胞。
图4C:图4C为从针对HLA-DR进行染色的CD34-NOG-hIL-34小鼠的脑将嗅球(OB,20倍)、皮质(CTX,20倍)和海马(HC,10倍)扩大而得的图。
图4D:图4D为将小胶质细胞的形态以更高倍率示出的图。
图4E:图4E为针对HLA-DR和Iba-1进行染色的脑的共聚焦图像。
图4F:图4F示出了全部Iba-1+细胞中的HLA-DR/Iba-1双阳性人小胶质细胞的比例。脑干(BS)、中脑(MB)、小脑(CB)、大脑皮质(CC)、海马体(HC)、嗅球(OB)。
图5A:图5A~B为示出了小鼠脑中的人小胶质细胞的重建的图。将5μm厚石蜡包埋脑切片针对HLA-DR进行染色。图5A以2倍的倍率示出了代表性的CD34-NOG-hIL-34小鼠脑。可确认整个小鼠脑区域的人小胶质细胞分布的整体图像。嗅球(OB)、大脑皮质(CTX)、海马体(HC)、中脑(MB)、小脑(CB)、纹状体(STR)、海马体(HC)、黑质(SN)、丘脑(TH)、脑干(BS)。利用Ventana iScan HT以200倍的原始倍率进行拍摄。脑区域(图4C的OB、CTX和HC)的放大图(20倍的物镜)示出了具有小胶质细胞形态的HLA-DR+细胞。
图5B:图5B示出了显示缺少人小胶质细胞的典型的CD34-NSG小鼠脑切片(2x)。在放大图(20倍)中示出了脑膜和血管周围区域(四边形包围的区域)中发现的少量HLA-DR+细胞。
图6:图6为示出小鼠脑中的人和小鼠胶质细胞的分布的图。为针对人MHC II型(HLA-DR)和小胶质细胞(Iba-1)进行染色而得的石蜡包埋脑组织的矢状切面。原始的共聚焦图像为630倍的倍率下通过Zeiss710 System收集而得的。
图7:图7为示出小鼠神经细胞与人小胶质细胞的相互作用的图。为针对人MHC II型(HLA-DR)、小鼠神经元标志物(MAP-2)、神经丝(NF-H)或突触素(SYN)进行染色而得的石蜡包埋脑组织的矢状切面。原始倍率为400倍。
图8A:图8A~D为示出CD34-NOG-hIL-34小鼠中的全身性HIV感染的成立的图。图8A为示出利用COBAS Amplicor System求得的末梢血的病毒量的图。每个符号表示各只感染小鼠。
图8B:图8B示出了针对人CD4和CD8阳性T淋巴细胞的脾细胞的流式细胞术分析的结果。示出了CD4和CD8阳性细胞的比例。
图8C:图8C示出了针对人CD4和CD8阳性T淋巴细胞的脾细胞的流式细胞术分析的结果。示出了脾脏中的HIV+群(n=12)和对照群(n=7)中的CD4/CD8比。
图8D:图8D为示出了脾脏切片的免疫组织学分析结果的图,所述结果显示了HLA-DR+细胞、和针对HIV-1p24进行染色的HIV-1感染细胞的存在。使用以854~8291bp的HIV-1作为靶标的反义探针、V-HIV1-Clade-B(ACD cat#416111)进行RNAScope测定,在5μm厚的脾脏切片中以单个棕色点或点簇的形式检测到HIV-1RNA。未感染小鼠中未检测到与病毒RNA的存在对应的信号。图像是利用Nuance multiplex系统以200倍的原始倍率拍摄得到的。
图9A:图9A~D为示出人源化小鼠脑中的HIV感染的图。图9A示出了显示HIV-1p24+感染细胞的存在的、脑区域的免疫组织学分析的结果。使用反义探针、V-HIV1-Clade-B进行RNAScope测定,以单个棕色点或点簇的形式检测到HIV-1RNA。原始倍率为200倍。
图9B:图9B示出了人小胶质细胞(HLA-DR+)附近的HIV-1感染小鼠脑中的小鼠星状胶质细胞(GFAP)和HIV-1p24阳性人小胶质细胞的免疫荧光染色的结果。原始倍率为400倍。
图9C:图9C示出了通过半巢式RT-PCR求得的、CD34-NSG小鼠和CD34-NOG-hIL-34小鼠的脑中的病毒RNA水平的比较结果。
图9D:图9D示出了感染小鼠脑中的各HIV-1基因的表达水平。RNAseq读段(read)与HIV-1ADA序列进行了比对。
图10A:图10A~图10C为示出了HIV感染的人源化NOG hIL-34小鼠中的人免疫细胞的分布的图。图10A示出了将脑的石蜡包埋5μm厚矢状切片针对人特异性免疫细胞标志物(CD4和CD8)进行染色的结果。图像是利用Nuance multiplex系统以200倍的原始倍率拍摄得到的。
图10B:图10B示出了人CD4和CD8T细胞的定量结果。针对HIV感染小鼠(n=4)和对照小鼠(n=3),每只小鼠以至少2个切片进行计数。黑色柱表示对照小鼠,灰色柱表示HIV感染小鼠。
图10C:图10C示出了CD4/CD8之比。针对HIV感染小鼠(n=4)和对照小鼠(n=3),每只小鼠以至少2个切片进行计数。
图11:图11示出了典型的CD34-NOG-hIL-34小鼠的脑,其示出了针对HIV-1p24进行染色的整个小鼠脑区域范围内的HIV-1感染的整体图像。以2倍的倍率拍摄。
图12A:图12A~E示出了CD34-NOG-hIL-34的脑组织中的转录产物的变化。示出了以Transcripts Per Kilobase Million(TPM)值计具有p<0.05的差异表达基因(Differentially expressed genes:DEG)。通过比较未感染CD34-NOG-hIL-34小鼠和感染CD34-NOG-hIL-34小鼠而得的、针对人基因组(h19)的基因比对,发现了687个DEG人基因。其中,261个基因经过HIV感染而被上调,426个基因经过HIV感染而被下调。图12A示出了在CD34-NOG-IL-34小鼠脑中表达的典型小胶质细胞标志物的排名列表。
图12B:图12B~C示出了人小胶质细胞和脑中,在HIV未感染CD34-NOG-IL-34和HIV感染CD34-NOG-IL-34中差异性表达的HIV感染相关基因的对数差异倍数的排名。图12B示出了HIV感染CD34-NOG-IL-34中被上调的DEG。
图12C:图12C示出了HIV感染CD34-NOG-IL-34中被下调的DEG。
图12D:图12D示出了HIV感染中被上调的人基因(261个)与干扰素信号传导、PRP和TLR信号传导相关。
图12E:图12E示出了HIV感染中被下调的人基因(426个)与EIF2信号传导和氧化磷酸化通路密切相关。
图13A:图13A为示出了下述687个DEG人基因的分类的饼图,所述基因是通过将HIV未感染CD34-NOG-hIL-34小鼠和HIV感染CD34-NOG-hIL-34小鼠进行了比较的、相对于人基因组(h19)的基因比对而发现的。261个基因经过HIV感染而被上调,426个基因经过HIV感染而被下调。
图13B:图13B为针对HIV未感染CD34-NOG-hIL-34小鼠vs HIV感染CD34-NOG-hIL-34小鼠而制成的、全部DEG(人)的火山图。X和Y轴分别示出了log2差异倍数和log10(p值)。蓝色点示出了被上调的基因,绿色点示出了被下调的基因,黑色点示出了被中性调控的基因。
具体实施方式
定义
本说明书中,只要没有明确记载,则“IL-34”意指IL-34蛋白质,“IL-34基因”意指编码IL-34的氨基酸序列的基因。“基因”是指包含至少一个编码特定蛋白质的开放阅读框的多核苷酸,可以包含外显子和内含子这两者。
本说明书中,“IL-34活性”是指导入至免疫缺陷小鼠的情况下,在该小鼠的体内诱导人单核细胞、小胶质细胞分化的活性。人IL-34活性包括在免疫缺陷小鼠的体内从人HSC诱导人小胶质细胞的活性。
本说明书中,氨基酸序列或碱基序列彼此之间的序列同一性(或同源性)可以作为以下形式求得:将两条氨基酸序列或碱基序列以对应的氨基酸或碱基一致情况最多的方式向相当于插入和缺失的部分填入空位而进行并列,求出相对于将所得到的比对中不包括空位的氨基酸序列全长或碱基序列全长而言的、一致的氨基酸或碱基的比例。氨基酸序列之间或碱基序列之间的序列同一性可以使用本技术领域已知的各种同源性检索软件求得。例如,氨基酸序列的序列同一性的值可以通过以经由已知的同源性检索软件BLASTP得到的比对为基础的计算来得到,碱基序列的序列同一性的值可以通过以经由已知的同源性检索软件BLASTN得到的比对为基础的计算来得到。
本说明书中,“严格的条件”可举出例如Molecular Cloning-ALABORATORY MANUALTHIRD EDITION(Sambrook等,Cold Spring Harbor Laboratory Press)中记载的方法。作为严格的条件,可举出例如下述条件:通过在由6×SSC(20×SSC的组成:3M氯化钠、0.3M柠檬酸溶液、pH7.0)、5×Denhardt溶液(100×Denhardt溶液的组成:2质量%牛血清白蛋白、2质量%Ficoll、2质量%聚乙烯吡咯烷酮)、0.5质量%的SDS、0.1mg/mL鲑鱼精子DNA、和50%甲酰胺组成的杂交缓冲液中于42~70℃孵育数小时至过夜,使其杂交。作为孵育后的洗涤之时使用的洗涤缓冲液,可举出优选含有0.1质量%SDS的1×SSC,更优选含有0.1质量%SDS的0.1×SSC溶液。
“免疫缺陷非人动物”是指具有T细胞和B细胞等功能性免疫细胞的缺少、DNA修复的缺陷、编码淋巴细胞上的抗原特异性受体的基因的重建中的缺陷、以及IgM、IgG1、IgG2a、IgG2b、IgG3和IgA等免疫功能分子的缺少中的一种以上的非人动物。免疫缺陷非人动物可以具有导致非人动物的异常免疫功能的上述缺陷或其他缺陷中的任一者。
本说明书中,涉及多核苷酸而使用的术语“有效地连接”意指充分靠近第二碱基序列地配置第一碱基序列,第一碱基序列能影响第二碱基序列或第二碱基序列的控制下的区域。例如,多核苷酸“有效地连接至启动子”是指该多核苷酸以在该启动子的控制下进行表达的方式被连接。
本说明书中,“启动子能够发挥作用”是指在作为对象的非人动物的细胞内,能够使有效地连接至该启动子的多核苷酸表达。
本说明书中,“能够表达的状态”是指在导入了多核苷酸的细胞内,该多核苷酸处于能够被转录的状态。
本说明书中,“表达载体”是指包含对象多核苷酸的载体,其具备在导入了该载体的细胞内使对象多核苷酸成为能够表达的状态的体系。
本说明书中,“标志物”是指特定种类的细胞中特异性表达的蛋白质。“标志物”优选为存在于细胞表面的蛋白质。
[具有IL-34的非人动物]
本发明的第一方式为在体内具有人白细胞介素34(interleukin 34:IL-34)的非人动物。
IL-34为介由集落刺激因子1受体(colony-stimulating factor-1receptor:CSF1R)促进单核细胞和巨噬细胞的分化和存活的细胞因子之一。
人IL-34的基因序列和氨基酸序列是已知的,其序列信息可自GenBank等已知的数据库获得。例如,作为人IL-34的基因序列和氨基酸序列,可举出以登录号No.NM_152456.2登录于GenBank中的序列(序列号1、2)等。人IL-34不限于具有上述序列,还包括上述序列的同源序列(直系同源、旁系同源)及它们的突变体。
人IL-34包含例如如下对象。
(1)包含序列号2中记载的氨基酸序列的多肽。
(2)由在序列号2中记载的氨基酸序列中缺失、取代、添加或插入有1个或多个氨基酸的氨基酸序列组成、且具有人IL-34活性的多肽。
(3)由与序列号2中记载的氨基酸序列具有80%以上序列同一性的氨基酸序列组成、且具有人IL-34活性的多肽。
人IL-34基因包含例如如下对象。
(4)编码包含序列号2中记载的氨基酸序列的多肽的多核苷酸。
(5)编码由在序列号2中记载的氨基酸序列中缺失、取代、添加或插入有1个或多个氨基酸的氨基酸序列组成、且具有人IL-34活性的多肽的多核苷酸。
(6)编码由与序列号2中记载的氨基酸序列具有80%以上的序列同一性的氨基酸序列组成、且具有人IL-34活性的多肽的多核苷酸。
(7)包含序列号1中记载的碱基序列的多核苷酸。
(8)由在序列号1中记载的碱基序列中缺失、取代、添加或插入有1个或多个碱基的碱基序列组成、且编码具有人IL-34活性的多肽的多核苷酸。
(9)由与序列号1中记载的碱基序列具有80%以上的序列同一性的碱基序列组成、且编码具有人IL-34活性的多肽的多核苷酸。
(10)可与由序列号1中记载的碱基序列组成的多核苷酸在严格的条件下进行杂交、且编码具有人IL-34活性的多肽的多核苷酸。
上述(2)和(5)中,缺失、取代、添加或插入的氨基酸的数量没有特别的限定,只要作为结果所生成的多肽具有人IL-34活性即可。上述(8)中,缺失、取代、添加或插入的碱基的数量没有特别的限定,只要作为结果所生成的多核苷酸编码具有人IL-34活性的多肽即可。缺失、取代、添加或插入的氨基酸或碱基的数量可以是例如1~80个,优选1~60个,更优选1~50个,可示例1~30个、1~20个、1~10个、1~5个、1~3个、1个或2个等。
上述(3)、(6)或(9)中,序列同一性没有特别的限定,只要是80%以上即可。序列同一性优选为85%以上,更优选为90%以上,进一步优选为95%以上,特别优选为97%以上。
另外,对序列号2中记载的氨基酸序列而言,1~20位为信号肽。由于信号肽于被分泌至细胞外之际被切割,因此,成熟的IL-34由序列号2中记载的氨基酸序列中的21~242位所记载的氨基酸序列构成。
因此,人IL-34还包含如下对象。
(11)包含序列号2中记载的氨基酸序列的21~242位的氨基酸序列的多肽。
(12)由在序列号2中记载的氨基酸序列21~242位的氨基酸序列中缺失、取代、添加或插入有1个或多个氨基酸的氨基酸序列组成、且具有人IL-34活性的多肽。
(13)由与序列号2中记载的氨基酸序列的21~242位的氨基酸序列具有80%以上的序列同一性的氨基酸序列组成、且具有人IL-34活性的多肽。
上述(12)中,缺失、取代、添加或插入的氨基酸的数量可举出与上述(2)的情况相同的例子。上述(13)中的序列同一性的数值可举出与上述(3)的情况相同的例子。
另外,人IL-34基因包含编码在上述(11)~(13)的任一项的多肽的N末端侧连接有用于胞外分泌的信号肽的多肽的多核苷酸。所述信号肽不限于人IL-34的信号肽,也可以是其他蛋白质的信号肽。作为这样的信号肽,可举出例如IL-34以外的细胞因子的信号肽。
非人动物只要是人以外的动物即可,没有特别的限定,优选为哺乳类。作为非人动物,例如,非人灵长类(猿、黑猩猩、大猩猩等)、啮齿类(小鼠、大鼠、豚鼠等)、狗、猫、兔、牛、猪、马、山羊、绵羊等,但不限于这些。其中,由于易于获得、实验,优选啮齿类,更优选小鼠。
从人细胞可以植入的方面考虑,非人动物优选为免疫缺陷非人动物。非人动物为小鼠的情况下,免疫缺陷小鼠的特征可在于Rag1和Rag2等与免疫功能相关的基因中的1个以上缺损(参见例如Oettinger,M.A等,Science,248:1517-1523,1990;Schatz,D.G.等,Cell,59:1035-1048,1989)。免疫缺陷小鼠可以具有导致小鼠的异常免疫功能的上述缺陷或其他缺陷中的任一者。
作为免疫缺陷小鼠,可举出例如NOG小鼠(NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic)、NSG小鼠(NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ)、NOD/ShiJcl小鼠等,但不限于这些。免疫缺陷小鼠已有市售,可以不受特别限定地使用这些市售的免疫缺陷小鼠。
本方式的非人动物的特征在于在体内具有人IL-34。作为在体内具有人IL-34的非人动物的制备方法,可举出例如将人IL-34施予至非人动物的方法、将人IL-34基因导入至非人动物的方法、将分泌人IL-34的细胞移入至非人动物的方法等。
将人IL-34施予至非人动物的情况下,只要人IL-34被保持在非人动物的体内即可,施予方法并无特别的限定,但非口服施予是优选的。作为非口服施予的途径,可举出例如肌肉注射、皮下注射、血管内注射等。只要人IL-34被保持在非人动物的体内,人IL-34的施予可以是单次施予,也可以是多次施予。
非人动物在体内是否具有人IL-34可以通过采集非人动物的血浆、测定该血浆中的人IL-34浓度来进行确认。对于血浆中的人IL-34浓度的测定方法而言,没有特别的限定,可举出使用抗人IL-34抗体的免疫化学方法。作为这样的方法,可举出例如ELISA法、EIA法、RIA法、Western印迹法等。血浆中的人IL-34的测定中可使用市售的人IL-34测定用ELISA试剂盒等。
作为血浆中的人IL-34的浓度,可举出例如20pg/mL以上,优选30pg/mL以上,更优选50pg/mL以上,进一步优选80pg/mL以上,特别优选100pg/mL以上。血浆中的人IL-34的浓度的上限没有特别的限定,可举出例如1000pg/mL以下、800pg/mL以下、或700pg/mL以下等。
将人IL-34基因导入至非人动物的情况下,优选将人IL-34有效地连接至在作为导入对象的非人动物中能够发挥作用的启动子的下游。作为在哺乳动物中能够发挥作用的启动子,可举出例如CMV(巨细胞病毒)启动子、SRα启动子、SV40早期启动子、逆转录病毒的LTR、RSV(劳氏肉瘤病毒)启动子、HSV-TK(单纯疱疹病毒胸苷激酶)启动子、EF1α启动子、金属硫蛋白启动子、热激蛋白启动子等,但并不限于这些。
人IL-34基因以可表达的状态被导入至非人动物,例如,以表达载体的形态被导入至非人动物。除人IL-34基因和启动子以外,表达载体还可含有增强子、多聚A附加信号、终止子等控制序列、耐药基因等标记基因。
载体的种类没有特别的限定,可以不受特别限定地使用通常使用的表达载体。载体可以是链状,也可以是环状,可以是质粒等非病毒载体,也可以是病毒载体(例如,慢病毒等逆转录病毒载体),还可以是基于转座子的载体。
向非人动物导入人IL-34基因的方法没有特别的限定,可以适用转基因动物的制备中通常使用的方法。作为向非人动物导入人IL-34基因的方法,可举出例如将含有人IL-34基因的表达载体通过显微注射等导入至作为导入对象的非人动物的受精卵的方法等。非人动物为小鼠的情况下,作为受精卵可示例如使NOG小鼠(NOD.Cg-PrkdcscidIl2rgtm1Sug/ShiJic)与NOD/ShiJcl小鼠交配而得的受精卵等,但不限于此。
对于导入了人IL-34基因的受精卵而言,于37℃培养18~24小时左右后,使其移植·着床于孕母的子宫,产下幼崽,由此可以得到具有人IL-34基因的非人动物。
如上所述得到的非人动物是否具有人IL-34基因可以通过从采集自该非人动物的试样提取基因组DNA实施PCR法等来确认。
另外,该非人动物是否表达人IL-34基因可以通过从采集自该非人动物的试样提取RNA实施RT-PCR法等、或通过使用采集自该非人动物的组织试样进行原位(in situ)杂交等来确认。或者,通过使用抗人IL-34抗体,对采集自该非人动物的试样中的人IL-34进行检测(例如,ELISA法、EIA法、RIA法、Western印迹法、EIA法、RIA法、免疫组织染色等)来确认。
导入有人IL-34基因的非人动物优选分泌人IL-34。在本文中,非人动物分泌人IL-34主要是指人IL-34从该非人动物的细胞释放至体液(血液、组织液、淋巴等)中。该非人动物是否分泌人IL-34可以通过在自该非人动物采集的血浆中测定该血浆中的人IL-34浓度来确认。血浆中的人IL-34浓度的测定方法可示例与上述相同的方法。
将分泌人IL-34的细胞(人IL-34分泌细胞)移入至非人动物的情况下,作为人IL-34分泌细胞,可举出例如来自人器官、血液的细胞、癌细胞等。作为所述来自人器官的细胞,可举出例如来自脾脏、胸腺、肝脏、小肠、大肠、前列腺、肺、心脏、脑、肾脏、精巢、子宫等的细胞等。作为所述来自人血液的细胞,可举出例如包含于末梢血单核细胞组分中的血细胞等。例如,可以自由上述人细胞建立的细胞株中挑选分泌人IL-34的细胞,作为用于移入非人动物的人IL-34分泌细胞使用。或者,也可以将导入有人IL-34基因的非人动物的细胞作为人IL-34分泌细胞使用。所述非人动物的细胞优选为与作为细胞的移入对象的非人动物属于同物种的非人动物的细胞。例如,例如,作为细胞的移入对象的非人动物为小鼠的情况下,导入人IL-34基因的细胞优选为小鼠的细胞。导入人IL-34基因的非人动物的细胞没有特别的限定,可举出例如来自器官、血液的细胞或其细胞株、造血干细胞(CD34阳性造血干细胞等)、癌细胞等。作为所述来自非人动物的器官、血液的细胞,可举出来自与上述人IL-34分泌细胞中所示例的相同的器官、血液组分的非人动物的细胞。与上述相同地,人IL-34基因以可表达的状态被导入至非人动物细胞,例如,以表达载体的形态被导入至非人动物细胞。向非人动物细胞导入人IL-34基因的方法没有特别的限定,可适用作为基因导入方法通常使用的方法。作为这样的方法,可举出例如病毒感染法、脂质体转染、显微注射法、磷酸钙法、DEAE-葡聚糖法、电穿孔法、使用转座子的方法、粒子枪法等,但不限于这些。
所述人细胞株或所述人IL-34基因导入细胞是否分泌人IL-34可以通过测定所述细胞或细胞株、或者所述人IL-34基因导入细胞的培养液中的IL-34浓度来确认,培养液中的人IL-34浓度的测定方法可示例与上述相同的方法。
向非人动物移入人IL-34分泌细胞的方法没有特别的限定,可适用通常用于向非人动物移入细胞的方法。作为向非人动物移入人IL-34分泌细胞的方法,可举出例如根据所使用的细胞的种类,将人IL-34分泌细胞施予至脾脏内、肝脏内、皮下或静脉内等的方法等。
移入有人IL-34分泌细胞的非人动物优选分泌人IL-34。在本文中,非人动物分泌人IL-34主要是指人IL-34从移入至该非人动物的人IL-34分泌细胞释放至体液(血液、组织液、淋巴等)中。该非人动物是否分泌人IL-34可以通过在自该非人动物采集的血浆中测定该血浆中的人IL-34浓度来确认。血浆中的人IL-34浓度的测定方法可示例与上述相同的方法。
另外,作为本方式的非人动物的制备方法,还可举出向非人动物直接接种组入了人IL-34基因的慢病毒、腺病毒等病毒,使其感染从而分泌人IL-34的方法。该方法包含在上述的将人IL-34基因导入至非人动物的方法中。
本方式的非人动物优选为具有人IL-34基因的动物,更优选为分泌人IL-34的动物。非人动物为具有人IL-34基因、且分泌人IL-34的动物的情况下,作为血浆中的人IL-34的浓度,可举出与上述相同的浓度。
<具有人小胶质细胞的非人动物>
通过将人CD34阳性造血干细胞移植至具有人IL-34的非人动物,从而在该非人动物的体内从该CD34阳性造血干细胞诱导人小胶质细胞。即,通过向具有人IL-34的非人动物移植人CD34阳性造血干细胞,从而可以制备具有人小胶质细胞的非人动物。
因此,本方式的非人动物可以是向具有人IL-34的非人动物移植人CD34阳性造血干细胞而得的动物。另外,本方式的非人动物可以是具有人IL-34和人小胶质细胞的非人动物。
另外,在一个实施方式中,本发明提供了具有人小胶质细胞的非人动物的制备方法,其包括将人CD34阳性造血干细胞移植至在体内具有人IL-34的非人动物。
人CD34阳性造血干细胞可以从人的脐带血、骨髓、血液等得到。从上述试样中得到人CD34阳性造血干细胞的方法没有特别的限定,可举出例如在实施这些试样的密度梯度离心法后、通过使用了抗人CD34抗体的磁珠法分离CD34阳性细胞的方法等。得到的人CD34阳性造血干细胞可通过流式细胞术等来确认纯度。
人CD34阳性造血干细胞的、向非人动物的移植方法没有特别的限定,可适用通常用于造血干细胞移植的方法。作为人CD34阳性造血干细胞的、向非人动物的移植方法,可举出例如,将出生后的非人动物(例如,出生后0~1天)用放射线进行全身处理后,将人CD34阳性造血干细胞施予至肝脏内、静脉内的方法等。
移植的人CD34阳性造血干细胞的数量没有特别的限定,例如,优选103个以上,更优选104个以上。移植的人CD34阳性造血干细胞的数量的上限没有特别的限定,可示例如,1010个以下、109个以下、或108个以下等。
对于非人动物是否被移植有人CD34阳性造血干细胞而言,可通过基于使用了针对人免疫细胞标志物(CD45、CD3、CD19、CD8、CD14等)的抗体的流式细胞术等,对非人动物的血液试样、脾脏组织试样进行分析,来进行确认。
将人CD34阳性造血干细胞移植至具有人IL-34的非人动物后,在该非人动物的体内,从CD34阳性造血干细胞诱导·分化人小胶质细胞,因此,能够得到具有人小胶质细胞的非人动物。对于非人动物是否具有人小胶质细胞而言,可通过使用了针对人小胶质细胞特异性的标志物(例如,HLA-DR和Iba1的双阳性)的抗体的免疫组织染色等,来进行确认。
具有人IL-34的非人动物中,人小胶质细胞存在的部位没有特别的限定,但小胶质细胞通常存在于中枢神经系统,因此优选在该非人动物中也存在于中枢神经系统,更优选存在于脑内。
本方式的非人动物所具有的人小胶质细胞优选为表达选自由CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE组成的组中的至少1种基因的细胞。这些基因是作为经典的巨噬细胞/小胶质细胞的标志物而为人所知的基因。另外,本方式的非人动物所具有的人小胶质细胞优选表达后述的表2A~2H中记载的基因的一部分或全部。
另外,本方式的非人动物所具有的人小胶质细胞优选为分泌选自由CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、和CXCL10组成的组中的至少1种细胞因子的细胞。
<感染有人免疫缺陷病毒的非人动物>
如下述实施例所示,具有人IL-34的非人动物可以较之不具有人IL-34的非人动物而言具有较多的人小胶质细胞。因此,通过使人免疫缺陷病毒(Human ImmunodeficiencyVirus:HIV)感染至具有人IL-34和人小胶质细胞的非人动物,能够适当地重现人脑中的HIV感染。
因此,本方式的非人动物可以是使HIV感染具有人IL-34的非人动物而得的动物。所述非人动物优选为具有人IL-34和人小胶质细胞的动物。
另外,在一个实施方案中,本发明提供了HIV感染非人动物的制备方法,其包括通过上述的方法制备具有人小胶质细胞的非人动物(具有人IL-34和人小胶质细胞)后、使HIV感染所述具有人小胶质细胞的非人动物。
用于感染的HIV可以是HIV-1和HIV-2中的任何。HIV向非人动物感染的方法没有特别的限定,可适用通常使用的感染方法。作为HIV向非人动物感染的方法,可举出例如将HIV施予至腹腔内的方法等。用于感染的病毒量只要是HIV感染能够成立的量即可,没有特别的限定,可举出例如500~5000TCID50,优选700~3000TCID50,作为具体例,可示例1000TCID50。
在非人动物中HIV感染是否成立可以通过测定采集自该非人动物的末梢血中的HIV量来确认。例如,在末梢血中检测出HIV量为106RNA拷贝/mL左右的情况下,可以判断HIV感染成立。另外,HIV向脑的感染可以通过在该非人动物的脑组织切片中实施以HIV特异性蛋白质、RNA作为靶标的免疫染色、原位(in situ)RNA杂交等来确认。本方式的非人动物优选HIV对脑进行了感染。
本方式的非人动物通过在体内具有人IL-34,可以适当地重建人的血液淋巴系统。尤其使在以往的免疫缺陷小鼠等非人动物中困难的、脑内的人小胶质细胞的重建成为可能。因此,可用于制备以小胶质细胞作为主要储库的HIV等的病毒感染模型。
本方式的非人动物可用于以小胶质细胞作为靶标的病毒感染机制的阐明、病毒感染治疗药的筛选·评价等。另外,本方式的非人动物可用于由小胶质细胞介导的中枢神经系统疾病的机制的阐明、治疗药的筛选·评价等。
[人小胶质细胞的制造方法]
本发明的第二方式为人小胶质细胞的制造方法,其包括从所述第一方式的非人动物中得到人小胶质细胞。
如上所述,第一方式的非人动物由于能够保持较多的人小胶质细胞,因而可用于人小胶质细胞的制造。因此,在一个方式中,本发明提供使用了第一方式的非人动物的、人小胶质细胞的制造方法,和通过该制造方法得到的人小胶质细胞。
本方式的人小胶质细胞的制造方法可包括以下步骤:
步骤(a)向非人动物施予人IL-34或导入人IL-34基因,得到在体内具有人IL-34的非人动物;
步骤(b)向所述步骤(a)中得到的非人动物移植人CD34阳性造血干细胞,得到具有人小胶质细胞的非人动物;以及
步骤(c)从所述步骤(b)中得到的非人动物中得到人小胶质细胞。
所述步骤(a)和步骤(b)可以如上述“[具有IL-34的非人动物]”的项目中所记载的方式实施。
所述步骤(c)可以通过从步骤(b)中得到的非人源化动物的血液试样、脑组织中分离人小胶质细胞来实施。例如,可以通过实施密度梯度离心法、使用了针对人小胶质细胞特异性标志物的抗体的磁珠法等,从这些试样中分离小胶质细胞。在从脑组织中分离人小胶质细胞的情况下,可以将脑组织在适当的缓冲液(例如,磷酸缓冲生理盐水等)中悬浮并均质化,然后实施密度梯度离心法。
作为人小胶质细胞特异性标志物,可举出例如HLA-DR、Iba1、CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE等,但不限于这些。
通过本方式的制造方法得到的人小胶质细胞可用于人小胶质细胞的功能阐明、以人小胶质细胞作为靶标的病毒感染机制的阐明、以人小胶质细胞作为靶标的药剂的评价等。
实施例
基于实施例来对本发明进行说明。但是,本发明的实施方案不限于这些实施例的记载。虽然下面的实施例使用小鼠作为非人动物,但本发明的实施方式不限于小鼠。对于其他的材料和方法,也不限于下面的实施例中的记载。
[方法]
<NOG-hIL-34小鼠的制备>
使用NOG(NOD Cg-PrkdcscidIl2gtmlSug/Jic)和NOD/ShiJcl(NOD)。NOG小鼠在实验动物中央研究所(CIEA)中于不存在特定病原体的条件下进行饲育。NOD/ShiJcl小鼠购自日本CREA株式会社(东京,日本)。
为了制备人IL-34表达转基因NOG小鼠,将包含CMV-启动子的控制下的人IL-34(hIL-34)cDNA(Origene Technologies Inc.,Rockville,MD,USA)的线状DNA载体(pCMV6-XL4)显微注射至通过将NOG小鼠与NOD小鼠进行交配而得到的受精卵。得到的26只断奶幼崽之中,3只(#11、#13和#24)基于扩增hIL-34cDNA的聚合酶链式反应(PCR)为阳性。
<NOG-hIL-34Tg小鼠的表征>
(基于ELISA的人IL-34的定量)
根据制造商的说明书,使用人IL-34ELISA定量套装(R&Dsystems,MN,USA),通过定量小鼠血浆(1:10稀释)中的人IL-34,评价人IL-34的转基因表达。使用SpectraMax M3(Molecular Devices,USA)测定450nm的吸光度。
(人IL-34转录产物的RT-PCR)
为了从脾脏、肺、肝脏、肾脏、肠、皮肤和脑组织分离RNA,将各组织使用QiagenTissue Lyzer II(Valencia,CA)在Trizol溶液中进行均质化,通过苯酚-氯仿法提取RNA。自RNA的cDNA合成使用Verso cDNA Synthesis Kit(Thermo Scientific,Vilnius,Lithuania)根据制造商的说明书实施,使用TaqMan detection chemistry用ABI Step OnePlus实时PCR装置(Applied Biosystems,MA,USA)实施cDNA的扩增。将人源化NOG-hIL-34Tg小鼠的样品中的人IL-34(Hs01050926_m1)的表达与人源化小鼠的样品进行比较。使用人GAPDH(Hs03929097_g1)作为管家基因。实时PCR的设定如下所述:50℃下2分钟、95℃下10分钟、95℃下15秒的循环40轮;和60℃下1分钟。求出人源化NOG-hIL-34Tg小鼠组(转基因组)与人源化小鼠组(对照组)之间的、相对于GAPDH而言的各靶基因mRNA的相对量的差异倍数。使用了阈值循环(CT)和2-ΔΔCT法(ΔCT=CTIL-34-CTGAPDH,Δ(ΔCT)=ΔCT(转基因组)-ΔCT(对照组))。
<人CD34+HSC的分离>
在获得双亲基于书面的知情同意书和内布拉斯加大学医疗中心的机构内审查委员会(UNMC的妇科和产科)的同意之后,从健康的足月新生儿的脐带血得到人CD34+造血干细胞。将白细胞分离培养基(MP Biomedicals,Santa,ANA,CA,USA)中的脐带血,以300g密度梯度离心分离35分钟,然后根据制造商的说明书(CD34+selection kit;Miltenyi BiotecInc.,Auburn,CA)使用免疫磁珠,采集软膜以浓缩CD34+细胞。通过流式细胞术评价分离的CD34+细胞的纯度。对于CD34+HSC而言,将其直接使用,或使用含有50%牛血清白蛋白(Sigma-Aldrich,St Louis,MO,USA)、40%Iscove的改良Dulbecco培养基(GIBCO,Lifetechnologies,Carlsbad,CA,USA)、和10%二甲亚砜(DMSO;Sigma-Aldrich St Louis,MO,USA)的冷冻培养基保存于液氮中。
<人CD34+HSC移植>
将NOG-hIL-34Tg小鼠饲育在内布拉斯加大学医学中心(UNMC)的无特定病原体的机构(SPF)中。向新生幼崽(出生后第0~1天)照射1Gy(RS 2000X射线照射器,Rad SourceTechnologies,Inc.,Suwanee,GA,USA)4小时,然后向幼鼠肝脏内注射1×105个人CD34+HSC。将共计19只人细胞重建动物用于以下试验。通过以流式细胞术分析来自植入后12周后的面静脉的血液样品,调查人白细胞的植入。
<流式细胞术>
从面静脉或通过安乐死后的直接心脏穿刺,用含有乙二胺四乙酸(EDTA)的管(BDMicrotainer,Franklin Lakes,NJ,USA)采集血液样品,以1800rpm离心分离8分钟。将脾脏组织均质化,使用40u的滤网过滤,从而采集脾细胞。将血细胞和脾细胞在FACS缓冲液(含有2%FBS的磷酸缓冲生理盐水)中重新悬浮,使用针对人免疫细胞标志物的抗体的混合物(cocktail)(CD45+异硫氰酸荧光素(FITC,BD Biosciences,USA);CD3+Alexa Fluor 700(BD,BDBiosciences,USA),CD19+Brilliant Violet 650(BD Biosciences,USA);CD4+别藻蓝蛋白(APC,BD Biosciences,USA);CD8+Brilliant Violet TM421(BV421,BDBiosciences,USA);CD14+PE(BD Biosciences,USA),于4℃孵育30分钟。RBC利用FACS溶解溶液(BD biosciences,USA)进行溶解。用FACS缓冲液洗涤染色细胞,用2%多聚甲醛进行固定。数据收集使用安装于BD LSR2流式细胞仪的收集软件FACS Diva v6(BD Biosciences,USA)实施,使用FLOWJO分析软件v10.2(Tree Star,USA;www.flowjo.com)分析数据。根据适当的对照群设门。
<HIV-1的感染>
使巨噬细胞嗜性HIV-1ADA株腹腔内感染(n=12)至重建人的血液-淋巴系统、且IL-34表达为阳性的小鼠(~6-8月龄),在感染确立的6周后使其安乐死。
<血浆、脾脏、脑中的HIV-1的测定>
在感染开始3周后和6周后,使用COBAS Amplicor System v1.5试剂盒(RocheMolecular Diagnostics,Pleasanton,CA,USA),求得小鼠血浆中的病毒RNA拷贝数。使用TaqMan detection chemistry,用ABI Step One Plus实时PCR装置(Applied Biosystems,MA,USA)以上述方式对脑内的HIV-1型特异性抗原(gag)RNA的表达进行分析。第2次PCR中使用的引物和探针如下所述:反义:5’-ATCTGGCCTGGTGCAATAGG-3’(序列号3);正义:5’-ACATCAAGCAGCCATGCAAAAT-3’(序列号4)(Invitrogen,Life technologies,Pittsburgh,PA,USA);和TaqMan探针:FAM-CATCAATGAGGAAGCTGCAGAATGGGATAGA-TAMRA(序列号5)(Applied Biosystems,Foster City,CA,USA)。使用内源性小鼠GAPDH(Mm9999991515_g1)转录物而得的总RNA表达的归一化之后,使用ΔΔCT法,计算RNA表达的对数变化。
基于脾脏和脑组织中的HIV RNA拷贝的形态学检测的目的,根据制造商的指示实施RNAScope(Advanced Cell Diagnostics,Hayward,CA)。使用通道(channel)1反义HIV-1Clade B靶标探针,其包含以HIV-1的碱基对854~8291作为靶标的78个探针对。通过感染细胞中的褐色点的存在,示出表达的阳性。对于组织免疫组织化学检查而言,针对HIV-1p24(1:20;Dako、Carpenteria,CA,USA)、CD4(1:100;Abcam,Cambridge,MA,USA)、CD8(1:100;Abcam,Cambridge,MA,USA)、和HLA-DR(1:100;Novus Biologicals,Littleton,CO,USA),根据制造商的说明书使用EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHCKit(Abcam,Cambridge,MA,USA)实施。
<免疫组织化学分析>
将组织(脾脏、肺、肝脏、肾脏、心脏、皮肤和脑的左半球)于室温用4%多聚甲醛固定24小时,然后,包埋于石蜡中。根据制造商的说明书,使用Declere/trilogy Solution(Sigma-Aldrich,St Louis,MO,USA)实施石蜡包埋5μm厚组织切片的抗原修复。根据制造商的说明书,使用EXPOSE Mouse and Rabbit Specific HRP/DAB Detection IHC Kit(Abcam,Cambridge,MA,USA)实施免疫组织化学检查。使用的一抗如下所述:HLA-DR(1:100;Novus Biologicals、Littleton,CO,USA)、CD14(1:500;Abcam,Cambridge,MA,USA)、CD68(1:100;人特异性,Dako、Carpenteria,CA,USA)、CD163(1:100;Thermoscientific,Rockford,IL,USA)、CD68(1:100;小鼠特异性,LifeSpan BioSciences,Inc.,Seattle,WA,USA)、和Iba-1(1:500;Wako life sciences,Richmond,VA,USA)。将核用Mayer’s苏木精进行对比染色,在Nuance Multispectral Tissue Imaging system(CRi,Wobum,MA)中使用20倍和40倍的物镜,拍摄明视野图像。为了定量,对HLA-DR染色切片使用高分辨率扫描仪(Ventana Medical Systems、Inc.,Oro Valley,AZ,USA)进行扫描。使用DEFINIENS TissueStudio(注册商标)软件(Definiens AG,Munich,Germany;www.definiens.com/),分析HLA-DR染色的脑切片。
<RNAScope>
为了检测人IL-34,实施了RNAScope(Advanced Cell Diagnostics,Hayward,CA)。将包含以人IL-34的38~1774位作为靶标的20个探针对的通道1反义Hs-IL-34-No-XMm用于单一显色分析。简而言之,对5μm厚的经过脱石蜡化和脱水的福尔马林固定石蜡包埋(FFPE)脑切片进行如下预处理:在HybEZ杂交炉内进行下述前处理:用过氧化氢室温处理10分钟、用靶标回收液于100℃处理8分钟、用蛋白酶IV于40℃处理15分钟。与靶探针的杂交、预扩增、扩增、和使用DAB的显色检测在40℃的HybEZ炉内根据制造商的说明书实施。阳性表达通过细胞内的棕色点的存在示出。
<免疫荧光染色>
为了进行石蜡包埋组织的免疫荧光染色,对切片进行处理,用包含10%正常山羊血清的、含0.5%吐温的1×Tris缓冲生理盐水抑制抗体的非特异性吸附。一抗如下所述:小鼠(Ms)抗人HLA-DR(1:100;Novus Biologicals,Littleton,CO,USA);HIV-1p24(1:20;Dako,Carpenteria,CA,USA);抗突触素(1:800;H.Abcam Cambridge,MA,USA);兔(Rb)抗MAP2(1:500;Millipore,Burlington,MA,USA);Rb抗神经丝(Neurofilament)H(1:400;Millipore,Burlington,MA,USA);多克隆Rb抗胶质细胞纤维性酸性蛋白(1:1000;Dako,Carpenteria,CA,USA);和Rb抗Iba1(1:500;Wako life sciences,Richmond,VA,USA)。二抗如下所述:Alexa Fluor 488缀合山羊抗Rb IgG(1:200;Invitrogen,Grand Island,NY,USA)、和Alexa Fluor 594缀合山羊抗Ms IgG(1:200;Invitrogen)。将Zeiss LSM710共聚焦系统(Carl Zeiss Microscopy,Jena,Germany)用于免疫荧光成像,以63倍拍摄图像。
为了定量人小胶质细胞的数量,使用来自实施了Iba1和HLA-DR的双重免疫染色的小鼠(n=3)的典型的矢状切片。使用Nuance Multispectral Tissue Imaging system(CRi,Wobum,MA),以400倍的倍率在相同脑区域的最少2~4个选择视野,针对Iba+HLADR+(人小胶质细胞)和Iba1+(小鼠小胶质细胞)进行计数。
<下一代测序>
为了测序,将脑组织的前额皮质(4只未感染人源化转基因小鼠、4只HIV-1感染转基因小鼠和4只NOG非人源化对照小鼠)在液氮中快速冷冻,于-80℃保存。对于组织RNA,施以使用RNeasy小量柱(mini column)的RNA纯化(clean up),使用RNase-free DNase套装(Qiagen、CA、USA)除去DNase。对核酸的完整性进行分析之后,使用Illumina HiSeq2500Sequence Analyzer(Illumina、Inc.,San Diego,CA,USA),以100bp/读段、≤4000万读段/样品,对样品进行深度测序。使用fqtrim(ccb.jhu.edu/software/fqtrim/)软件对读段进行修剪,从读段中除去不明确的碱基。在修剪前后,使用FASTQC针对各样品进行质量评价,对于读段而言,针对小鼠参考基因组GRCm38.p3(https://uswest.ensembl.org/index.html),使用默认参数利用STAR-2.5.3a(https://github.com/alexdobin/STAR)进行比对,然后,使用Ensemble注释,利用RSEM 1.2.21(deweylab.github.io/RSEM/)进行定量化。基因和转录产物的存在量作为Transcripts Per Kilobase Million(TPM)值进行测定。将基因的长度归一化之后,为了更容易地比较每个样品中映射至基因的读段的比例,TPM的计算考虑了测序的深度(sequence depth)。使用相同的流水线(pipeline),针对人源化NOG-hIL-34样品将未被映射的读段进一步与人类参考基因组GrCh37(uswest.ensembl.org/index.html)进行比对,在非感染样品与HV感染样品之间进行比较分析。对于与人不一致的读段而言,使用STAR进一步与HIV基因组进行比对,通过RSEM定量化。为了排除未编码蛋白质的对象,过滤计数和表达数据。将该经过滤的基因的子集用于:(1)使用Bioconductor的软件包(package)的R统计软件环境中的样品组间的各种基因的差异表达分析,(2)使用Ingenuity pathway分析(www.qiagenbioinformatics.com)的通路鉴定,和(3)使用Gene Analytics(ga.genecards.org/)的生物过程和分子功能的分析。
<统计分析>
数据使用GraphPad prism 7(Graphpad,USA)进行分析和制图,表示为平均值±平均值标准误差(sem)。为了分析转录组,得到的数据表示为各组的平均值±标准偏差。Student t检验使用R/Bioconductor的软件包实施。调整基于多重检验的错误发现率(FDR),计算Benjamini-Hochberg(BH)调整p值。P值<0.05被认为表示统计学上的显著差异。
[结果]
通过将含有CMV启动子控制下的hIL-34导入基因(Tg)的载体(NOD.Cg-PrkdcscidIl2rgtm1Sug)导入NOG小鼠,研发了NOGCIEA背景的人IL-34转基因小鼠(NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic)。NOG-hIL-34小鼠是通过用耳DNA的PCR分析扩增hIL-34(358bp)转录物来鉴定的。通过基于RT-PCR、ELISA和RNAscope的分析,确认了包含脑的小鼠组织中的IL-34表达(图1A~D、图2)。NOG-hIL-34小鼠的人源化遵循将人CD34+HSC在出生时(CD34—NSG)进行肝内移植的标准方法。
CD34-NOG-hIL-34小鼠中,与CD34-NSG同样,实现了包含人淋巴细胞和骨髓细胞的人免疫系统的稳定的植入(图3)。令人惊讶的是,确认到CD34-NOG-hIL-34小鼠的脑中,HLA-DR、CD14、CD163、CD68和P2RY12为阳性的、具有了小胶质细胞的形态的人细胞较之这样的细胞几乎不存在的人CD34-NSG小鼠而言,明显可见显著的数量增加(图4A、图4B、图5)。人小胶质细胞样细胞(数据示出了6月龄的对象)自4月龄开始可见。人小胶质细胞广泛地分布于整个小鼠脑区域(图4C、图5)。虽然发现了不同类型的人小胶质细胞,但其大部分是分枝状的,一些是未成熟而致密的阿米巴样的形态的小胶质细胞(图4D)。通过对HLA-DR+/Iba1+双阳性的人小胶质细胞和Iba1+的小鼠小胶质细胞进行计数,算出总小胶质细胞群中的人小胶质细胞的比例,人小胶质细胞在某些脑区域中为全部小胶质细胞的多至80%(图4E、图4F、图6)。HLA-DR+/Iba1+阳性细胞在嗅球(OB、59.3±15.4%)、皮质区域(CTX)、纹状体(STR)、和海马体(HC、48.3±34.2%)中多,在脑干((BS、28.4±12.5%))和中脑(MB、29.5±15.7%)中少。小鼠CNS与人小胶质细胞的相互作用显示出正常的星状细胞的行为和神经元的整合性(图7)。
然后,在向腹腔内注射1000TCID50病毒使小鼠感染HIV-1的情况下,在末梢血中达到了~106RNA拷贝/mL的病毒量(图8A~D)。在脑内容易地检测到对人小胶质细胞的强感染(图9A、图10A~C、图11)。在若干小鼠脑区域观察到感染细胞,在OB、HC、和CTX观察到最多的感染细胞。通过RNAscope技术,可以清楚地观察到感染人细胞、和自感染细胞释放的细胞外病毒粒子。在HIV感染小胶质细胞或其附近容易地检测到反应性的星状细胞(图9)。CD34-NOG-hIL-34小鼠的脑较之在末梢具有相同程度的病毒量的人免疫系统重建而得的CD34-NSG模型而言,具有高3~4log10倍(106对比102)的HIV病毒量(图9C、表1)。
[表1]
同样,从感染的CD34-NOG-hIL-34小鼠得到的脑RNA的深度测序显示出在CD34-NSG小鼠的脑中未被检测到的gag、nef和env等与HIV-1相关的基因(Colby,D.等,HIV RNAREBOUND POSTINTERRUPTION IN PERSONS SUPPRESSED IN FIEBIG I ACUTE HIV.inConference on Retroviruses and Opportunistic Infections(IAS-USA,Seattle,Washington,2017).)的显著增加(图9D)。如“[方法]”中所记载的,将已测序的所有读段与小鼠和人的参考基因组进行了比对。共计82个与人骨髓/单核细胞/巨噬细胞/小胶质细胞相关的基因在CD34-NOG-hIL-34的人细胞中表达。表达最高的为MHC II型(CD74)和I型(B2M)(图12A)。可观察到AIF1(IBA1)、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、和TMEM119等典型的巨噬细胞/小胶质细胞标志物的表达。存在CCL2、TNF、HGF(IL6)、CXC18(IL8)、IL-10、IL1A、CXC110等由小胶质细胞分泌的各种细胞因子。还确认到对于小胶质细胞的维持和功能而言重要的转录因子PU.1(SPI 1)、ETV5和APOE(表2)。HIV感染CD34-NOG-hIL-34小鼠与非感染小鼠之间的比较中显示了不同的差异表达的人特异性基因(687个基因)中,大部分的基因(426个基因)在HIV感染小鼠中显示出显著的下调(图13A、B)。被下调的基因的大部分涉及EIF2信号传导和氧化磷酸化。另一方面,被上调的基因涉及干扰素信号传导、模式识别受体、Toll样受体信号转导和细胞死亡受体信号传导(deathreceptor signaling)(图12B~E)。
上述发现支持如下事实:人IL-34在来源于人骨髓的单核细胞在出生后的小鼠脑中的迁移和向小胶质细胞的分化中发挥了重要的作用。
表2A~2H:由小胶质细胞表达的基因的列表
[表2A]
基因 | TPM | Log2(TPM) |
B2M | 17240.7025 | 14.0735309 |
MT-CO1 | 11927.96 | 13.5420597 |
MT-CO2 | 11092.525 | 13.4373002 |
HLA-DRB1 | 5978.9075 | 12.5456662 |
APOE | 4766.6875 | 12.2187713 |
C1QA | 2387.045 | 11.22101 |
TYROBP | 2290.3425 | 11.1613476 |
CTSD | 2085.585 | 11.0262364 |
FCGR3A | 1955.7825 | 10.9335302 |
STAB1 | 1723.205 | 10.7508786 |
AIF1 | 1613.7525 | 10.6562036 |
CD14 | 1081.4125 | 10.0787012 |
CSF1R | 1034.5725 | 10.014819 |
CD68 | 963.7575 | 9.91252637 |
GRN | 930.5725 | 9.86197474 |
CX3CR1 | 900.0075 | 9.81379321 |
IGHM | 862.4175 | 9.75224264 |
CTSH | 755.695 | 9.56166027 |
TREM2 | 747.2575 | 9.54546166 |
ITGB2 | 723.2775 | 9.49840546 |
RGS10 | 706.555 | 9.46465806 |
SLCO2B1 | 677.38 | 9.40382158 |
ALOX5AP | 606.6725 | 9.24477411 |
TNFAIP2 | 536.9825 | 9.06873126 |
P2RY12 | 526.465 | 9.04019381 |
S100A9 | 474.6525 | 8.89072787 |
OLFML3 | 441.1 | 8.78496195 |
CALR | 435.9675 | 8.76807678 |
TGA× | 422.0725 | 8.72134702 |
SG15 | 389.355 | 8.60494234 |
CD37 | 385.3125 | 8.58988518 |
CYBB | 383.24 | 8.58210434 |
SPI1 | 348.0175 | 8.44301604 |
FCGR2A | 291.7375 | 8.18852703 |
NCF4 | 284.915 | 8.15438777 |
FUS | 275.245 | 8.10457255 |
CXCL10 | 272.61 | 8.09069467 |
GAS6 | 259.35 | 8.01875656 |
OLR1 | 259.15 | 8.01764358 |
LAIR1 | 256.285 | 8.00160523 |
CD163 | 255.3425 | 7.99628987 |
[表2B]
基因 | TPM | Log2(TPM) |
TSPO | 245.8775 | 7.94179591 |
GPR34 | 234.75 | 7.87498135 |
HE×B | 233.1925 | 7.86537758 |
MX1 | 232.575 | 7.86155222 |
MERTK | 228.3775 | 7.83527671 |
TMEM173 | 227.92 | 7.83238372 |
CASP4 | 212.845 | 7.73365939 |
CCL2 | 201.3475 | 7.65354375 |
GAL3ST4 | 195.725 | 7.61268423 |
IL18 | 194.38 | 7.60273598 |
BHLHE41 | 193.9175 | 7.59929919 |
SLC2A5 | 193.68 | 7.59753117 |
IRF8 | 182.91 | 7.51499014 |
CPVL | 179.2625 | 7.48592991 |
HCK | 176.9875 | 7.46750366 |
ITGAL | 171.3725 | 7.42099181 |
ANXA11 | 168.15 | 7.39360497 |
P2RY13 | 161.0875 | 7.33170074 |
ITGAM | 154.3525 | 7.27008504 |
PILRA | 150.885 | 7.23730558 |
TMEM119 | 148.245 | 7.21183964 |
BLNK | 142.8825 | 7.15868542 |
TNFRSF1B | 135.825 | 7.08560524 |
HAVCR2 | 134.61 | 7.07264178 |
PTAFR | 134.5925 | 7.07245421 |
FPR1 | 133.11 | 7.05647515 |
ATP6V0A1 | 132.21 | 7.04668749 |
GBP2 | 131.925 | 7.04357417 |
TGFBR1 | 131.4275 | 7.03812337 |
ACP5 | 131.0275 | 7.03372583 |
SLC11A1 | 129.4425 | 7.01616757 |
SOD1 | 128.7525 | 7.00845664 |
EBI3 | 128.75 | 7.00842862 |
PTGS1 | 127.04 | 6.98913901 |
PTOV1 | 125.205 | 6.96814837 |
TLR2 | 117.9125 | 6.88157286 |
PTPRC | 116.335 | 6.86214139 |
PFKFB3 | 112.3975 | 6.81246614 |
MMP14 | 111.02 | 6.79467579 |
CCND1 | 108.9075 | 6.7669595 |
DOK3 | 106.0925 | 6.72917886 |
[表2C]
基因 | TPM | Log2(TPM) |
NFKBIA | 99.81 | 6.64111246 |
IL10RA | 98.44 | 6.62117275 |
RAB3IL1 | 98.055 | 6.61551929 |
SSBP1 | 97.6925 | 6.6101759 |
ITGB5 | 96.5875 | 6.59376459 |
SLA | 94.455 | 6.56155526 |
CD86 | 85.8425 | 6.42362019 |
PFDN1 | 84.91 | 6.40786257 |
IL1B | 84.065 | 6.39343336 |
PTGES2 | 82.8225 | 6.37195085 |
BCL2L1 | 80.74 | 6.33521168 |
ENTPD1 | 80.0475 | 6.32278444 |
CD40 | 79.7175 | 6.31682456 |
CD33 | 77.4675 | 6.27551928 |
M×2 | 77.445 | 6.27510019 |
IFIT1 | 76.23 | 6.25228697 |
TIMP1 | 76.0125 | 6.24816478 |
PIK3AP1 | 75.8725 | 6.24550517 |
CFB | 75.225 | 6.2331403 |
GBP3 | 75.205 | 6.23275668 |
ETV5 | 74.4575 | 6.21834527 |
VCP | 73.4475 | 6.19864148 |
GBP5 | 72.7575 | 6.18502407 |
VPS13C | 68.7675 | 6.10365499 |
PPARD | 65.8475 | 6.04105676 |
ACSL1 | 65.5675 | 6.03490898 |
IFIT3 | 63.87 | 5.99706655 |
IGFBP4 | 60.8375 | 5.92688896 |
PLAUR | 60.29 | 5.91384682 |
PPFIBP2 | 59.7 | 5.89965903 |
CEBPB | 59.1525 | 5.88636724 |
PABPN1 | 57.535 | 5.84636795 |
PTPN7 | 57.0725 | 5.83472386 |
SLAMF7 | 55.8325 | 5.80303325 |
DKC1 | 55.4 | 5.79181407 |
PLAU | 54.55 | 5.76950729 |
AGTRAP | 54.4375 | 5.76652891 |
TLR1 | 53.77 | 5.74872957 |
ABCC5 | 52.8425 | 5.72362682 |
MSRA | 52.185 | 5.70556327 |
GPR84 | 50.4775 | 5.65756855 |
[表2D]
基因 | TPM | Log2(TPM) |
TARDBP | 49.1525 | 5.61919289 |
POLA2 | 49.1175 | 5.61816523 |
CAMK1 | 48.56 | 5.60169652 |
AGPAT1 | 48.325 | 5.59469783 |
SPN | 47.8275 | 5.57976848 |
RCBTB2 | 47.1175 | 5.55819109 |
SLC7A8 | 46.82 | 5.54905303 |
RILPL2 | 46.1825 | 5.52927437 |
ENPP2 | 45.835 | 5.51837777 |
PIM1 | 45.4575 | 5.50644644 |
APBB3 | 45.0175 | 5.49241404 |
EML1 | 44.26 | 5.46793155 |
NLRP3 | 43.3325 | 5.43737757 |
IVNS1ABP | 41.5575 | 5.37703696 |
CCR5 | 41.1875 | 5.36413466 |
FZR1 | 39.992S | 5.32165756 |
MTSS1 | 39.745 | 5.31270147 |
RUN×3 | 39.235 | 5.2940693 |
PSTPIP2 | 38.9475 | 5.28345882 |
RASGRP3 | 38.945 | 5.28336621 |
RSAD2 | 36.235 | 5.17931199 |
SLC31A2 | 35.5 | 5.14974712 |
SESN1 | 35.4625 | 5.14822234 |
PROCR | 35.3175 | 5.14231132 |
ANG | 35.2025 | 5.13760598 |
ARHGEF7 | 35.2025 | 5.13760598 |
IFIT2 | 34.7825 | 5.12028973 |
CCL8 | 33.2775 | 5.05647515 |
NPEPPS | 32.49 | 5.02192384 |
MMP9 | 32.125 | 5.00562455 |
ABCA7 | 31.3925 | 4.97234802 |
LAG3 | 31.29 | 4.96762975 |
APP | 31.0925 | 4.95849472 |
UBE2E2 | 30.8825 | 4.94871764 |
SLC37A2 | 30.54 | 4.93262816 |
IL10 | 28.63 | 4.83945577 |
DNAJC9 | 28.5775 | 4.83680781 |
MCM3 | 28.325 | 4.82400405 |
HK3 | 28.2775 | 4.82158267 |
RUN×1 | 27.4 | 4.77610399 |
ARHGAP18 | 27.065 | 4.75835648 |
[表2E]
基因 | TPM | Log2(TPM) |
BATF | 27.0175 | 4.75582228 |
CRYBB1 | 26.7625 | 4.74214099 |
RAPGEF2 | 25.5175 | 4.67341509 |
IGF1 | 25.4775 | 4.67115181 |
UPP1 | 25.0775 | 4.64832163 |
F13A1 | 24.7325 | 4.62833617 |
JAK2 | 24.6875 | 4.62570884 |
ZDHHC14 | 24.3075 | 4.60332962 |
GPD2 | 24.2825 | 4.60184506 |
GADD45B | 23.7875 | 4.57213175 |
AGER | 23.7425 | 4.56939995 |
ADRB2 | 22.8375 | 4.51333282 |
CD69 | 22.815 | 4.51191075 |
TNF | 22.81 | 4.51159454 |
GCH1 | 22.705 | 4.50493813 |
MCM4 | 22.3825 | 4.48429928 |
CCRL2 | 22.1975 | 4.4723253 |
SUO× | 21.75 | 4.4429435 |
PDGFC | 21.71 | 4.44028782 |
CCL20 | 21.035 | 4.39471991 |
CD274 | 20.58 | 4.36317108 |
SPINT1 | 20.3675 | 4.348197 |
CREM | 20.2 | 4.33628339 |
CD180 | 19.4075 | 4.27854238 |
PROS1 | 18.995 | 4.24754781 |
TRAF1 | 18.885 | 4.23916888 |
GYS1 | 17.935 | 4.16470584 |
PMEPA1 | 17.57 | 4.13504229 |
AKAP10 | 17.45 | 4.12515513 |
SMAD7 | 17.3775 | 4.11914864 |
SNAPC2 | 17.1475 | 4.09992635 |
F11R | 17.045 | 4.09127669 |
NAMPT | 16.79 | 4.06953033 |
IL1RN | 16.7425 | 4.06544306 |
PRIM1 | 16.6475 | 4.05723364 |
NFKBIZ | 15.5275 | 3.95675366 |
PHYH | 15.455 | 3.95000175 |
LRRK2 | 15.4125 | 3.94602899 |
MCM6 | 14.7175 | 3.87946072 |
FAM102B | 14.24 | 3.83187724 |
CABLES1 | 13.75 | 3.78135971 |
[表2G]
基因 | TPM | Log2(TPM) |
PDE4B | 13.515 | 3.75648961 |
ARAP2 | 13.425 | 3.74685018 |
RBL1 | 13.075 | 3.70873904 |
CASP9 | 13 | 3.70043972 |
IL1A | 12.9025 | 3.68957873 |
SLC6A12 | 12.57 | 3.65191274 |
BIRC3 | 12.54 | 3.64846544 |
EPAS1 | 12.215 | 3.61058196 |
IRAK3 | 11.9725 | 3.58165253 |
GK | 11.94 | 3.57773093 |
RGS2 | 11.855 | 3.56742376 |
CABLES2 | 11.5975 | 3.53574194 |
CSF1 | 11.1675 | 3.48123435 |
CYSLTR1 | 10.9875 | 3.45779126 |
RAB11FIP1 | 10.675 | 3.41616416 |
MALT1 | 10.6125 | 3.40769265 |
FGD4 | 10.28 | 3.36176836 |
SDC2 | 10.15 | 3.34340782 |
NFRKB | 9.51 | 3.24944534 |
DUSP16 | 9.3425 | 3.22380866 |
FN1 | 9.28 | 3.21412481 |
FCGR2B | 9.12 | 3.18903382 |
GCNT1 | 8.1525 | 3.02724254 |
FLNB | 7.89 | 2.9800253 |
SLC36A1 | 7.7825 | 2.96023367 |
TMEM154 | 7.285 | 2.86492897 |
CD80 | 6.7825 | 2.76181714 |
LAMP3 | 6.5525 | 2.71204545 |
TGM2 | 5.7175 | 2.51538446 |
HELLS | 5.54 | 2.46988598 |
TET2 | 4.82 | 2.26903315 |
CD300E | 4.64 | 2.21412481 |
CXCL8/IL8 | 4.4525 | 2.15461561 |
ENC1 | 4.3725 | 2.12845838 |
ZBED4 | 4.09 | 2.03210084 |
RMI1 | 3.7975 | 1.92504996 |
BNIP3 | 3.4675 | 1.79389588 |
MSH2 | 3.3725 | 1.75381844 |
CCR2 | 3.285 | 1.71589337 |
WDHD1 | 2.8275 | 1.49952702 |
POLA1 | 2.8025 | 1.48671437 |
[表2H]
基因 | TPM | Log2(TPM) |
ZC3H12C | 2.62 | 1.38956681 |
×YLT1 | 2.5 | 1.32192809 |
IFT57 | 2.3225 | 1.2156786 |
SOCS3 | 2.1875 | 1.12928302 |
GOLM1 | 2.0575 | 1.04089243 |
IRF4 | 1.765 | 0.81966818 |
EXT1 | 1.675 | 0.7441611 |
DCBLD2 | 1.55 | 0.63226822 |
DKK1 | 1.4425 | 0.52857132 |
HGF | 1.415 | 0.50080205 |
HGF/IL6 | 1.415 | 0.50080205 |
OPHN1 | 0.87 | -0.2009127 |
ABCD2 | 0.5925 | -0.7551129 |
SLC7A11 | 0.4425 | -1.1762506 |
DGKH | 0.4375 | -1.1926451 |
产业上的可利用性
根据本发明,可提供人小胶质细胞的保持量多的非人动物、及其制备方法。另外,还可提供所述非人动物的利用方法。
Claims (13)
1.非人动物,其在体内具有人白细胞介素34(interleukin 34:IL-34)。
2.如权利要求1所述的非人动物,其被移植有人CD34阳性造血干细胞。
3.如权利要求1或2所述的非人动物,其脑内存在人小胶质细胞。
4.如权利要求3所述的非人动物,其中,所述人小胶质细胞表达选自由CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE组成的组中的至少1种基因。
5.如权利要求3或4所述的非人动物,其中,所述人小胶质细胞分泌选自由CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a和CXCL10组成的组中的至少1种细胞因子。
6.如权利要求1~5中任一项所述的非人动物,其感染有人免疫缺陷病毒(Humanimmunodeficiency Virus:HIV)。
7.人小胶质细胞的制造方法,其包括自权利要求3~6中任一项所述的非人动物中得到人小胶质细胞。
8.具有人小胶质细胞的非人动物的制备方法,其包括向在体内具有人IL-34的非人动物移植人CD34阳性造血干细胞。
9.如权利要求8所述的具有人小胶质细胞的非人动物的制备方法,其中,所述在体内具有人IL-34的非人动物为免疫缺陷非人动物。
10.如权利要求8或9所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞存在于脑内。
11.如权利要求8~10中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞表达选自由CD74、b2m、AIF1、CD14、CD68、CSF1R、ITGAM(CD11b)、P2RY12、CX3CR1、TREM2、TMEM119、CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a、CXCL10、PU.1(SPI1)、ETV5和APOE组成的组中的至少1种基因。
12.如权利要求8~11中任一项所述的具有人小胶质细胞的非人动物的制备方法,其中,所述人小胶质细胞分泌选自由CCL2、TNF、HGH(IL-6)、CXCL8、IL-10、IL-1a和CXCL10组成的组中的至少1种细胞因子。
13.HIV感染非人动物的制备方法,其包括:
通过权利要求8~12中任一项所述的具有人小胶质细胞的非人动物的制备方法制备具有人小胶质细胞的非人动物,然后使HIV感染所述具有人小胶质细胞的非人动物。
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