CN111549068B - 一种在体细胞中诱导多线染色体的方法 - Google Patents

一种在体细胞中诱导多线染色体的方法 Download PDF

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CN111549068B
CN111549068B CN202010443507.XA CN202010443507A CN111549068B CN 111549068 B CN111549068 B CN 111549068B CN 202010443507 A CN202010443507 A CN 202010443507A CN 111549068 B CN111549068 B CN 111549068B
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亚历山大·斯特轮尼科夫·弗拉基米罗维奇
刘剑
吴琼芳
梁杰荣
阿卜杜拉林·布卡巴
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Abstract

本发明提供了一种在体细胞中诱导多线染色体的方法，所述方法包括在体细胞中表达减数分裂黏连蛋白复合物的步骤；所述减数分裂黏连蛋白复合物包括RAD21L、SMC1B和STAG3的组合。本发明通过在体细胞中诱导表达SMC1B‑STAG3‑RAD21L减数分裂黏连蛋白复合物，促进并维持体细胞中染色体的配对，诱导体细胞形成独特的多线染色体表型，构建的重组体细胞可以作为研究染色体多线化和减数分裂突触机制的细胞模型工具，实现了在人类生殖腺外研究减数分裂染色体突触的重要突破。

Description

一种在体细胞中诱导多线染色体的方法
技术领域
本发明属于表观遗传学技术领域，涉及一种在体细胞中诱导多线染色体的方法，尤其涉及一种诱导体细胞多线染色体的蛋白组合物、在体细胞中诱导多线染色体的方法及其在研究多线染色体和/或减数分裂突触中的应用。
背景技术
黏连蛋白复合物是有丝分裂和减数分裂过程中染色体分离的关键因素。减数分裂黏连蛋白亚基经常在癌细胞中以癌-睾丸(Cancer-Testis，CT)基因的形式表达，可能与肿瘤发生有关。
减数分裂的基因表达过程极其复杂，涉及到多个相互独立作用的调控因子。例如，减数分裂黏连蛋白复合物REC8/SMC1B/SMC3/STAG3和RAD21L/SMC1B/SMC3/STAG3在减数分裂染色体重组和分离过程中具有重要作用，但是SMC1B和STAG3的诱导受到减数分裂转录因子STRA8的调控，SMC3在体细胞和生殖细胞中均有组成性表达，而REC8和RAD21L是由生殖细胞中的独立机制诱导表达的，具体的机制尚不清楚。传统的遗传学方法，如小鼠遗传学，在研究生殖细胞的特异性基因方面存在一定的局限性。该局限性在研究黏连蛋白复合物中的RAD21L和REC8时尤其明显，黏连蛋白复合物中的RAD21L和REC8同源基因的冗余和特化程度不能通过小鼠敲除实验来阐明，因为RAD21L和REC8是通过粗线期的关键。
减数分裂的关键步骤之一是同源染色体联会、分离和染色体数目减半，因此，有必要建立一种体细胞模型模拟减数分裂的染色体配对过程，为研究生殖系统发育过程提供体外途径。
发明内容
针对现有技术的不足和实际需求，本发明提供了一种在体细胞中诱导多线染色体的方法，通过在体细胞中重构SMC1B(即SMC1β)、STAG3、RAD21L和SMC3减数分裂黏连蛋白复合物，实现了在体细胞内形成多线染色体的效果。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供了一种在体细胞中诱导多线染色体的方法，所述方法包括在体细胞中表达减数分裂黏连蛋白复合物的步骤。
黏连蛋白复合物是有丝分裂和减数分裂过程中染色体分离的关键因素，本发明中在体细胞中重构减数分裂黏连蛋白复合物，与染色质上的众多位点结合并诱导染色体浓缩，在减数分裂过程中组装成类似轴向元件黏连蛋白核心的细丝，促进并维持体细胞中染色体的配对，诱导细胞周期停滞和多线染色体形成。
优选地，所述减数分裂黏连蛋白复合物包括RAD21L、SMC1B和STAG3的组合。
由于SMC3在体细胞存在组成性表达，本发明在体细胞中表达RAD21L、SMC1B和STAG3，便可以与SMC3形成4个核心亚基组成的减数分裂黏连蛋白复合物，发挥诱导染色体浓缩、促进体细胞中染色体配对、形成多线染色体的功能。
优选地，所述RAD21L包括SEQ ID NO:1所示的氨基酸序列；
SEQ ID NO:1：
MFYTHVLMSKRGPLAKIWLAAHWEKKLTKAHVFECNLEITIEKILSPKVKIALRTSGHLLLGVVRIYNRKAKYLLADCSEAFLKMKMTFCPGLVDLPKENFEASYNAITLPEEFHDFDTQNMNAIDVSEHFTQNQSRPEEITLRENFDNDLIFQAESFGEESEILRRHSFFDDNILLNSSGPLIEHSSGSLTGERSLFYDSGDGFGDEGAAGEMIDNLLQDDQNILLEDMHLNREISLPSEPPNSLAVEPDNSECICVPENEKMNETILLSTEEEGFTLDPIDISDIAEKRKGKKRRLLIDPIKELSSKVIHKQLTSFADTLMVLELAPPTQRLMMWKKRGGVHTLLSTAAQDLIHAELKMLFTKCFLSSGFKLGRKMIQKESVREEVGNQNIVETSMMQEPNYQQELSKPQTWKDVIGGSQHSSHEDTNKNINSEQDIVEMVSLAAEESSLMNDLFAQEIEYSPVELESLSNEENIETERWNGRILQMLNRLRESNKMGMQSFSLMKLCRNSDRKQAAAKFYSFLVLKKQLAIELSQSAPYADIIATMGPMFYNI。
优选地，所述SMC1B包括SEQ ID NO:2所示的氨基酸序列；
SEQ ID NO:2：
MAHLELLLVENFKSWRGRQVIGPFRRFTCIIGPNGSGKSNVMDALSFVMGEKIANLRVKNIQELIHGAHIGKPISSSASVKIIYVEESGEEKTFARIIRGGCSEFRFNDNLVSRSVYIAELEKIGIIVKAQNCLVFQGTVESISVKKPKERTQFFEEISTSGELIGEYEEKKRKLQKAEEDAQFNFNKKKNIAAERRQAKLEKEEAERYQSLLEELKMNKIQLQLFQLYHNEKKIHLLNTKLEHVNRDLSVKRESLSHHENIVKARKKEHGMLTRQLQQTEKELKSVETLLNQKRPQYIKAKENTSHHLKKLDVAKKSIKDSEKQCSKQEDDIKALETELADLDAAWRSFEKQIEEEILHKKRDIELEASQLDRYKELKEQVRKKVATMTQQLEKLQWEQKTDEERLAFEKRRHGEVQGNLKQIKEQIEDHKKRIEKLEEYTKTCMDCLKEKKQQEETLVDEIEKTKSRMSEFNEELNLIRSELQNAGIDTHEGKRQQKRAEVLEHLKRLYPDSVFGRLFDLCHPIHKKYQLAVTKVFGRFITAIVVASEKVAKDCIRFLKEERAEPETFLALDYLDIKPINERLRELKGCKMVIDVIKTQFPQLKKVIQFVCGNGLVCETMEEARHIALSGPERQKTVALDGTLFLKSGVISGGSSDLKYKARCWDEKELKNLRDRRSQKIQELKGLMKTLRKETDLKQIQTLIQGTQTRLKYSQNELEMIKKKHLVAFYQEQSQLQSELLNIESQCIMLSEGIKERQRRIKEFQEKIDKVEDDIFQHFCEEIGVENIREFENKHVKRQQEIDQKRLEFEKQKTRLNVQLEYSRSHLKKKLNKINTLKETIQKGSEDIDHLKKAEENCLQTVNELMAKQQQLKDIRVTQNSSAEKVQTQIEEERKKFLAVDREVGKLQKEVVSIQTSLEQKRLEKHNLLLDCKVQDIEIILLSGSLDDIIEVEMGTEAESTQATIDIYEKEEAFEIDYSSLKEDLKALQSDQEIEAHLRLLLQQVASQEDILLKTAAPNLRALENLKTVRDKFQESTDAFEASRKEARLCRQEFEQVKKRRYDLFTQCFEHVSISIDQIYKKLCRNNSAQAFLSPENPEEPYLEGISYNCVAPGKRFMPMDNLSGGEKCVAALALLFAVHSFRPAPFFVLDEVDAALDNTNIGKVSSYIKEQTQDQFQMIVISLKEEFYSRADALIGIYPEYDDCMFSRVLTLDLSQYPDTEGQESSKRHGESR。
优选地，所述STAG3包括SEQ ID NO:3所示的氨基酸序列；
SEQ ID NO:3：
MSSPLQRAVGDTKRALSASSSSSASLPFDDRDSNHTSEGNGDSLLADEDTDFEDSLNRNVKKRAAKRPPKTTPVAKHPKKGSRVVHRHSRKQSEPPANDLFNAVKAAKSDMQSLVDEWLDSYKQDQDAGFLELVNFFIQSCGCKGIVTPEMFKKMSNSEIIQHLTEQFNEDSGDYPLIAPGPSWKKFQGSFCEFVRTLVCQCQYSLLYDGFPMDDLISLLTGLSDSQVRAFRHTSTLAAMKLMTSLVKVALQLSVHQDNNQRQYEAERNKGPGQRAPERLESLLEKRKELQEHQEEIEGMMNALFRGVFVHRYRDVLPEIRAICIEEIGCWMQSYSTSFLTDSYLKYIGWTLHDKHREVRLKCVKALKGLYGNRDLTTRLELFTSRFKDRMVSMVMDREYDVAVEAVRLLILILKNMEGVLTDADCESVYPVVYASHRGLASAAGEFLYWKLFYPECEIRMMGGREQRQSPGAQRTFFQLLLSFFVESELHDHAAYLVDSLWDCAGARLKDWEGLTSLLLEKDQNLGDVQESTLIEILVSSARQASEGHPPVGRVTGRKGLTSKERKTQADDRVKLTEHLIPLLPQLLAKFSADAEKVTPLLQLLSCFDLHIYCTGRLEKHLELFLQQLQEVVVKHAEPAVLEAGAHALYLLCNPEFTFFSRADFARSQLVDLLTDRFQQELEELLQSSFLDEDEVYNLAATLKRLSAFYNTHDLTRWELYEPCCQLLQKAVDTGEVPHQVILPALTLVYFSILWTLTHISKSDASQKQLSSLRDRMVAFCELCQSCLSDVDTEIQEQAFVLLSDLLLIFSPQMIVGGRDFLRPLVFFPEATLQSELASFLMDHVFIQPGDLGSGDSQEDHLQIERLHQRRRLLAGFCKLLLYGVLEMDAASDVFKHYNKFYNDYGDIIKETLTRARQIDRSHCSRILLLSLKQLYTELLQEHGPQGLNELPAFIEMRDLARRFALSFGPQQLQNRDLVVMLHKEGIQFSLSELPPAGSSNQPPNLAFLELLSEFSPRLFHQDKQLLLSYLEKCLQHVSQAPGHPWGPVTTYCHSLSPVENTAETSPQVLPSSKRRRVEGPAKPNREDVSSSQEESLQLNSIPPTPTLTSTAVKSRQPLWGLKEMEEEDGSELDFAQGQPVAGTERSRFLGPQYFQTPHNPSGPGLGNQLMRLSLMEEDEEEELEIQDESNEERQDTDMQASSYSSTSERGLDLLDSTELDIEDF。
优选地，所述方法包括：
(1)采用包含SMC1B基因和STAG3基因的慢病毒共感染体细胞后，筛选共表达SMC1B和STAG3的阳性克隆细胞株；
(2)将筛选的阳性克隆细胞株采用包含RAD21L基因的慢病毒感染后，筛选共表达SMC1B、STAG3和RAD21L的阳性克隆细胞株，在体细胞中表达减数分裂黏连蛋白复合物。
作为优选技术方案，本发明将DLD-1(ATCC CCL-221)和K562(ATCC CCL-243)两种细胞系培养于含10％或20％四环素调控系统tet血清的DMEM(Hyclone)或IMDM(Hyclone)培养基中进行研究，并将HEK293(ATCC CRL-1573)细胞系培养于含10％血清的DMEM(Hyclone)培养基中进行研究；根据制造商的建议，本发明采用罗氏的DNA转染试剂X-tremeGENE 9进行质粒转染；
慢病毒转染前，首先将包装质粒psPAX2和Pmd2.G与含有SMC1B基因、STAG3基因、RAD21L基因或REC8基因的目标质粒共转染到HEK293T/17细胞，进行慢病毒包装，72小时后收集慢病毒；
随后采用8μg/mL聚凝胺和500μL慢病毒原液感染融合率为40％～50％的靶细胞，感染12小时后根据不同的抗生素筛选标记将培养基换成对应含有600μg/mL G418、300μg/mLzeocin(ThermoFisher)、1μg/mL puromycin(MDBIO)或200μg/mL hygromycin B(MDBIO)的细胞培养液；
使用抗生素筛选至少4周，经200ng/mL强力霉素(doxcycline，Dox)诱导，进行减数分裂黏连蛋白复合物在体细胞中的表达，并进行免疫蛋白印迹分析。
优选地，所述方法还包括对体细胞进行强力霉素诱导的步骤。
第二方面，本发明提供了一种诱导体细胞多线染色体的蛋白组合物，所述蛋白组合物包括RAD21L、SMC1B和STAG3的组合。
优选地，所述RAD21L包括SEQ ID NO:1所示的氨基酸序列。
优选地，所述SMC1B包括SEQ ID NO:2所示的氨基酸序列。
优选地，所述STAG3包括SEQ ID NO:3所示的氨基酸序列。
第三方面，本发明提供了一种重组体细胞，所述重组体细胞中表达有减数分裂黏连蛋白复合物。
优选地，所述减数分裂黏连蛋白复合物包括RAD21L、SMC1B和STAG3的组合。
优选地，所述RAD21L包括SEQ ID NO:1所示的氨基酸序列。
优选地，所述SMC1B包括SEQ ID NO:2所示的氨基酸序列。
优选地，所述STAG3包括SEQ ID NO:3所示的氨基酸序列。
优选地，所述减数分裂黏连蛋白复合物结合在体细胞的完整染色体上，所述体细胞形成多线染色体。
本发明中，在体细胞中重组表达的SMC1B-STAG3-RAD21L复合物结合在从着丝粒到端粒的整个染色体上。
第四方面，本发明提供了一种第三方面所述的重组体细胞的构建方法，所述方法包括：
(1)采用包含SMC1B基因和STAG3基因的慢病毒共感染体细胞后，筛选共表达SMC1B和STAG3的阳性克隆细胞株；
(2)将筛选的阳性克隆细胞株采用包含RAD21L基因的慢病毒感染后，筛选共表达SMC1B、STAG3和RAD21L的阳性克隆细胞株，得到所述重组体细胞。
第五方面，本发明提供了一种第二方面所述的蛋白组合物在制备体细胞多线染色体诱导剂、体细胞增殖阻滞剂或细胞染色体配对诱导剂中的应用。
第六方面，本发明提供了一种第三方面所述的重组体细胞在研究多线染色体和/或减数分裂突触中的应用。
与现有技术相比，本发明具有如下有益效果：
(1)本发明通过在体细胞中诱导表达SMC1B-STAG3-RAD21L减数分裂黏连蛋白复合物，发现SMC1B-STAG3-RAD21L复合物以一种不规则的非有丝分裂方式浓缩染色体，在减数分裂过程中组装成类似轴向元件黏连蛋白核心的细丝，促进并维持体细胞中染色体的配对，阐明了与人类癌症中以CT基因形式表达的其他减数分裂黏连蛋白复合物编码基因相比，RAD21L在肿瘤中激活率较低的技术问题；
(2)本发明构建的表达SMC1B-STAG3-RAD21L复合物的体细胞具有独特的多线染色体表型，可以作为研究染色体多线化和减数分裂突触机制的细胞模型工具，实现了在人类生殖腺外研究减数分裂染色体突触的重要突破。
附图说明
图1A为体细胞黏连蛋白复合物(Somatic cohesin complexes)与哺乳动物细胞生殖特异性黏连蛋白复合物(Germline-specific cohesin complexes)的对比示意图，图1B为TCGARNA-seq数据库中人类mei-CC亚基之间表达对比图，图1C为TCGA RNA-seq样本中人类mei-CC亚基表达的相关性和多变量统计，图1D为Mei-CC基因共表达的维恩示意图；
图2为K562细胞表达可诱导的mei-CC亚基染色质分离的蛋白免疫印迹结果图，RAD21L亚基在体细胞中不与染色质结合；
图3A为DOX诱导96h后，DLD-1SMC1B-STAG3-RAD21L细胞的核染色质的免疫荧光图，*表示抗SMC1B抗体检测到的蛋白降解条带，图3B为对诱导96h的DLD-1SMC1B-STAG3-RAD21L细胞中的mei-CC进行ChIP-seq分析，获得的两两相关的热点图；
图4A为未同步化的DLD-1SMC1B-STAG3-RAD21L细胞经诱导后RAD21L的免疫荧光图，RAD21L、STAG3和SMC1B分别以未加标记蛋白标签(左)和附加标记蛋白标签(右)的形式表达，箭头为DLD-1SMC1B-STAG3-RAD21L细胞中的一簇微核(左上角)和一个杂乱无章的有丝分裂，比例尺为10μm，图4B为K562SMC1B-STAG3-RAD21L细胞诱导72h后的DAPI染色结果图，图4C为DLD-1SMC1B-STAG3-RAD21L和DLD-1SMC1B-STAG3-REC8单克隆细胞在添加或不添加DOX诱导下的生长曲线图；
图5A为DLD-1SMC1B-STAG3-RAD21L细胞中组蛋白抗原表位phospho-S10-H3(pS10H3)抗体的免疫荧光图，图5B为DLD-1SMC1B-STAG3-RAD21L细胞中核仁染色质(UBTF)、着丝粒(CENPA)和端粒(TERF)蛋白标记物的免疫荧光图，比例尺为10μm；
图6A为第一次胸腺嘧啶阻滞期间DOX诱导的DLD-1(control)和DLD-1SMC1B -STAG3-RAD21L细胞的流式细胞图(细胞计数与碘化丙啶荧光信号的关系)，0h表示在第二次胸腺嘧啶阻滞后进入细胞释放，图6B为采用抗细胞周期蛋白B1抗体(Cyclin-B，CCNB1)和内参蛋白(Histon，H3)对图6A中各时间点的蛋白提取物进行蛋白免疫印迹的结果图，H3为上样对照；
图7A为第二次胸腺嘧啶阻滞后释放细胞的0h时间点，DLD-1SMC1B-STAG3-RAD21L细胞的免疫荧光图，图7B为第二次胸腺嘧啶阻滞后释放细胞的2h时间点，DLD-1SMC1B-STAG3-RAD21L细胞的免疫荧光图，图7C为DLD-1SMC1B-STAG3-RAD21L细胞在诱导72h后的DAPI染色图，图7D为DLD-1SMC1B-STAG3-RAD21L细胞在诱导72h后，利用着丝粒(CENPA)和着丝粒蛋白(CREST)抗体的免疫荧光图；
图8A为四种细胞系的RNA-seq转录组分析结果，图8B为50个受影响最深基因的折叠表达变化热图；
图9为表达SMC1B-STAG3-RAD21L复合物的体细胞中染色体多线化的示意图。
具体实施方式
为进一步阐述本发明所采取的技术手段及其效果，以下结合实施例和附图对本发明作进一步地说明。可以理解的是，此处所描述的具体实施方式仅仅用于解释本发明，而非对本发明的限定。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。
实施例1
图1A为体细胞黏连蛋白复合物(Somatic cohesin complexes)与哺乳动物细胞生殖特异性黏连蛋白复合物(Germline-specific cohesin complexes)的对比示意图。
根据TCGA源数据，本实施例对代表不同来源肿瘤的9121个数据集样本进行基于RNA-seq转录组分析的STAG3、SMC1B、RAD21L和REC8表达的多变量统计分析，结果如图1B所示，红线对应FPKM100。
进一步分析发现，如图1C所示，每个基因旁边为相应表达的平均值和标准差，SMC1B-STAG3和SMC1B-REC8两组亚基分别显著相关(P<0.0001)；图1D为Mei-CC基因共表达的维恩示意图，显示了mei-CC亚基在肿瘤样本中的稳健表达(FPKM≥10，数值表示每个类别中独立肿瘤的数量，以超表达(FPKM≥100)作为参考。
实施例2
在K562细胞中单独表达减数分裂黏连蛋白复合物(mei-CC)的RAD21L亚基、REC8亚基、STAG3亚基或SMC1B亚基，Tet诱导96小时后，用相应的抗体对可溶性蛋白(S)、核可溶性蛋白(Sn)和染色质颗粒沉淀蛋白(P)进行免疫印迹分析。
如图2所示，RAD21L、REC8和STAG3均不与染色质发生强烈的结合，只有SMC1B与染色质紧密结合(*)，其中，RAD21L不与染色质发生强烈结合的原因可能是由于在没有其他mei-CC亚基(如SMC1B和STAG3)协助的情况下，RAD21L亚基无法与合适的染色质位点建立合适的结合关系。
为了验证以上推断，本实施例构建了能够同时表达三个减数分裂黏连蛋白(mei-cohesin)亚基的细胞系，即DLD-1SMC1B-STAG3-RAD21L、DLD-1SMC1B-STAG3-REC8、K562SMC1B-STAG3-RAD21L和K562SMC1B-STAG3-REC8，RAD21L的氨基酸序列如SEQ ID NO:1所示，SMC1B的氨基酸序列如SEQ IDNO:2所示，STAG3的氨基酸序列如SEQ ID NO:3所示，步骤如下：
首先采用rtTA慢病毒(pwpxLD-rtTA)感染DLD-1或K562细胞系；
对于共表达不带标签的三基因的细胞系，采用pKD-tetO-SMC1B-PGK-Puro和pKD-tetO-STAG3-PGK-Hyg慢病毒共感染DLD-1或K562细胞，进行筛选和表达检测后，将阳性稳定细胞株采用pKD-tetO-RAD21Lwt-PGK-Zeo或pKD-tetO-REC8-PGK-Zeo慢病毒进行感染，最后检测筛选得到稳定表达SMC1B-STAG3-RAD21L或SMC1B-STAG3-REC8的细胞株；
对于共表达带标签的三基因的细胞系，采用pKD-tetO-3Flag-SMC1B-PGK-Puro和pKD-tetO-3Myc-STAG3-PGK-Hyg慢病毒共感染DLD-1或K562细胞，进行筛选和表达检测后，将阳性细胞株采用pKD-tetO-3Flag-RAD21Lwt-PGK-Zeo或pKD-tetO-3Myc-REC8-PGK-Zeo慢病毒进行感染，最后检测筛选得到稳定表达SMC1B-STAG3-RAD21L或SMC1B-STAG3-REC8的细胞株。
对DLD-1SMC1B-STAG3-RAD21L进行DOX诱导96h后，进行核染色质分离，如图3A所示，在该细胞系中，与染色质结合的RAD21L的表达量得到显著提高，与STAG3相当，但是仍然不能替代体细胞RAD21；通过ChIP-seq进一步检测mei-CC亚基间的结合作用，并进行两两相关性分析，如图3B所示，SMC1B-STAG3-RAD21L复合物间存在很强的相关性，采用10291个RAD21L峰值为中心的400bp窗口来计算标准化的标签密度(41×106reads for eachdataset)，RAD21L主要反映了SMC1B和STAG3间的结合。这也初步证实了完整的mei-CC4个核心亚基是mei-CC在体细胞中与染色质结合的决定性因素。
本实施例进一步探讨了mei-CC与染色质结合后产生的表型变化。初步的实验检测发现，如图4A所示，DLD-1SMC1B-STAG3-RAD21L细胞呈现出显著的形态学表型变化，大部分细胞核都产生浓缩的染色体，RAD21L优先与细胞核DNA共定位；同时，DLD-1SMC1B-STAG3-RAD21L细胞也表现出不同程度的染色体浓缩，RAD21L阳性的有丝分裂细胞极少，染色体排列明显紊乱。如图4B所示，这种表型变化对DLD-1细胞没有特异性，在K562细胞系中出现了类似的染色体早凝现象。如图4C所示，采用DOX诱导DLD-1SMC1B-STAG3-RAD21L 48小时后，与DLD-1SMC1B-STAG3-REC8相比，细胞表现出显著的增殖停滞。
如图5A所示，DLD-1SMC1B-STAG3-RAD21L细胞的染色体浓缩不是受有丝分裂诱导形成的，因为它的信号与Phospho-S10-H3(pS10H3)信号无关；如图5B所示，在DLD-1SMC1B-STAG3-RAD21L细胞中，沿着浓缩的染色体组装的RAD21L细丝跨越了从着丝粒(CENPA)到端粒(TERF)的整个染色体长度，但排除了rDNA染色质。
实施例3
为了阐明SMC1B-STAG3-RAD21L复合物的功能，本实施例对同步化后的转基因细胞进行了研究。第一次胸腺嘧啶阻滞诱导mei-CC后，细胞被释放，并且在下一个S期(DNA合成期，已经存在mei-CC)再次被阻滞。如图6A的流式细胞图所示，对照组DLD-1、DLD-1SMC1B -STAG3-RAD21L和DLD-1SMC1B-STAG3-REC8细胞都同步通过S期，说明SMC1B-STAG3-RAD21L复合物与DNA复制是兼容的，但是有丝分裂的通路失败；如图6B所示，Cyclin B1(细胞周期蛋白B1)的动态变化与DLD-1SMC1B-STAG3-RAD21L细胞的有丝分裂通路缺陷相一致。
实施例4
DLD-1SMC1B-STAG3-RAD21L细胞的染色体结构变化是多方面的，基本过程是SMC1B-STAG3-RAD21L复合物将染色体和染色单体连接在一起形成如图7A所示的交联的着丝粒(箭头)，以及如图7B、图7C和图7D所示的明显较厚且具有多染色单体结构的染色体，其中，图7C展示了多股紧密整齐排列的染色质，图7D显示，浓缩的染色体排列在一起，且在许多情况下，四个独立的着丝粒有区别地并列在一起。这类细胞的出现频率为1％，但是它们的出现证明了体细胞中发生了染色体配对。由于这些细胞在第二次诱导阻滞复制期间没有出现，而在细胞释放后立即出现，说明它们是通过线性化而非配对机制出现的。因此，SMC1B-STAG3-RAD21L复合物能够保持多条染色单体排列的惊人能力表明该复合物具有维持染色体配对的能力。
实施例5
DLD-1SMC1B-STAG3-RAD21L细胞通过组装的减数分裂黏连蛋白复合物将染色单体维持在一起是独特的体细胞过程，而不是由某些减数分裂特异性蛋白(如联会复合体组分)诱导产生的。如图8A和图8B所示，采用RNA-seq对四种细胞系DLD-1(Control)、DLD-1SMC1B-STAG3、DLD-1SMC1B-STAG3-RAD21L和DLD-1SMC1B-STAG3-REC8进行转录组分析发现，DLD-1SMC1B-STAG3-RAD21L细胞存在大量的(210个)差异表达基因，但是上调基因中不包括任何联会复合体组分基因。
事实上，没有已知的联会复合体组份被诱导，这表明SMC1B-STAG3-RAD21L复合物可能以一种不规则的非有丝分裂方式浓缩染色体，在减数分裂过程中组装成类似轴向元件黏连蛋白核心的细丝，促进并维持体细胞中染色体的配对，示意图如图9所示，左图代表一个体细胞，RAD21黏连蛋白复合物(RAD21 CC)在姐妹染色单体黏连蛋白(sisterchromatidscohesin，SCC)形成过程中是必不可少的，并通过染色质的3D结构在基因调控中起重要的作用；右图为RAD21L黏连蛋白复合物(RAD21L CC)在体细胞中异位表达的效果图，RAD21L mei-CC至少有三个不同的结构活性：可能导致非有丝分裂压缩的侧链内染色单体的连接活性(lateral chromatid links)，或者形成染色体桥(染色体在细胞分裂后期形成的桥)和多线化染色体间的连接活性(inter chromosomal links)，或者SCC活性。
采用表观遗传药物对细胞进行进一步诱导，诱导前接种合适的细胞密度，过夜培养，24小时后更换添加了强力霉素(doxcycline，Dox)的新鲜培养基，72小时后收集细胞，检测细胞中多线染色体的表达情况，发现DLD-1SMC1B-STAG3-RAD21L中多线性染色体的概率增加到4％。
综上所述，本发明通过在体细胞中诱导表达SMC1B-STAG3-RAD21L减数分裂黏连蛋白复合物，促进并维持体细胞中染色体的配对，诱导体细胞形成独特的多线染色体表型，构建的重组体细胞可以作为研究染色体多线化和减数分裂突触机制的细胞模型工具，实现了在人类生殖腺外研究减数分裂染色体突触的重要突破。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 中国科学院广州生物医药与健康研究院
<120> 一种在体细胞中诱导多线染色体的方法
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355 360 365
Ala Ser Gln Leu Asp Arg Tyr Lys Glu Leu Lys Glu Gln Val Arg Lys
370 375 380
Lys Val Ala Thr Met Thr Gln Gln Leu Glu Lys Leu Gln Trp Glu Gln
385 390 395 400
Lys Thr Asp Glu Glu Arg Leu Ala Phe Glu Lys Arg Arg His Gly Glu
405 410 415
Val Gln Gly Asn Leu Lys Gln Ile Lys Glu Gln Ile Glu Asp His Lys
420 425 430
Lys Arg Ile Glu Lys Leu Glu Glu Tyr Thr Lys Thr Cys Met Asp Cys
435 440 445
Leu Lys Glu Lys Lys Gln Gln Glu Glu Thr Leu Val Asp Glu Ile Glu
450 455 460
Lys Thr Lys Ser Arg Met Ser Glu Phe Asn Glu Glu Leu Asn Leu Ile
465 470 475 480
Arg Ser Glu Leu Gln Asn Ala Gly Ile Asp Thr His Glu Gly Lys Arg
485 490 495
Gln Gln Lys Arg Ala Glu Val Leu Glu His Leu Lys Arg Leu Tyr Pro
500 505 510
Asp Ser Val Phe Gly Arg Leu Phe Asp Leu Cys His Pro Ile His Lys
515 520 525
Lys Tyr Gln Leu Ala Val Thr Lys Val Phe Gly Arg Phe Ile Thr Ala
530 535 540
Ile Val Val Ala Ser Glu Lys Val Ala Lys Asp Cys Ile Arg Phe Leu
545 550 555 560
Lys Glu Glu Arg Ala Glu Pro Glu Thr Phe Leu Ala Leu Asp Tyr Leu
565 570 575
Asp Ile Lys Pro Ile Asn Glu Arg Leu Arg Glu Leu Lys Gly Cys Lys
580 585 590
Met Val Ile Asp Val Ile Lys Thr Gln Phe Pro Gln Leu Lys Lys Val
595 600 605
Ile Gln Phe Val Cys Gly Asn Gly Leu Val Cys Glu Thr Met Glu Glu
610 615 620
Ala Arg His Ile Ala Leu Ser Gly Pro Glu Arg Gln Lys Thr Val Ala
625 630 635 640
Leu Asp Gly Thr Leu Phe Leu Lys Ser Gly Val Ile Ser Gly Gly Ser
645 650 655
Ser Asp Leu Lys Tyr Lys Ala Arg Cys Trp Asp Glu Lys Glu Leu Lys
660 665 670
Asn Leu Arg Asp Arg Arg Ser Gln Lys Ile Gln Glu Leu Lys Gly Leu
675 680 685
Met Lys Thr Leu Arg Lys Glu Thr Asp Leu Lys Gln Ile Gln Thr Leu
690 695 700
Ile Gln Gly Thr Gln Thr Arg Leu Lys Tyr Ser Gln Asn Glu Leu Glu
705 710 715 720
Met Ile Lys Lys Lys His Leu Val Ala Phe Tyr Gln Glu Gln Ser Gln
725 730 735
Leu Gln Ser Glu Leu Leu Asn Ile Glu Ser Gln Cys Ile Met Leu Ser
740 745 750
Glu Gly Ile Lys Glu Arg Gln Arg Arg Ile Lys Glu Phe Gln Glu Lys
755 760 765
Ile Asp Lys Val Glu Asp Asp Ile Phe Gln His Phe Cys Glu Glu Ile
770 775 780
Gly Val Glu Asn Ile Arg Glu Phe Glu Asn Lys His Val Lys Arg Gln
785 790 795 800
Gln Glu Ile Asp Gln Lys Arg Leu Glu Phe Glu Lys Gln Lys Thr Arg
805 810 815
Leu Asn Val Gln Leu Glu Tyr Ser Arg Ser His Leu Lys Lys Lys Leu
820 825 830
Asn Lys Ile Asn Thr Leu Lys Glu Thr Ile Gln Lys Gly Ser Glu Asp
835 840 845
Ile Asp His Leu Lys Lys Ala Glu Glu Asn Cys Leu Gln Thr Val Asn
850 855 860
Glu Leu Met Ala Lys Gln Gln Gln Leu Lys Asp Ile Arg Val Thr Gln
865 870 875 880
Asn Ser Ser Ala Glu Lys Val Gln Thr Gln Ile Glu Glu Glu Arg Lys
885 890 895
Lys Phe Leu Ala Val Asp Arg Glu Val Gly Lys Leu Gln Lys Glu Val
900 905 910
Val Ser Ile Gln Thr Ser Leu Glu Gln Lys Arg Leu Glu Lys His Asn
915 920 925
Leu Leu Leu Asp Cys Lys Val Gln Asp Ile Glu Ile Ile Leu Leu Ser
930 935 940
Gly Ser Leu Asp Asp Ile Ile Glu Val Glu Met Gly Thr Glu Ala Glu
945 950 955 960
Ser Thr Gln Ala Thr Ile Asp Ile Tyr Glu Lys Glu Glu Ala Phe Glu
965 970 975
Ile Asp Tyr Ser Ser Leu Lys Glu Asp Leu Lys Ala Leu Gln Ser Asp
980 985 990
Gln Glu Ile Glu Ala His Leu Arg Leu Leu Leu Gln Gln Val Ala Ser
995 1000 1005
Gln Glu Asp Ile Leu Leu Lys Thr Ala Ala Pro Asn Leu Arg Ala
1010 1015 1020
Leu Glu Asn Leu Lys Thr Val Arg Asp Lys Phe Gln Glu Ser Thr
1025 1030 1035
Asp Ala Phe Glu Ala Ser Arg Lys Glu Ala Arg Leu Cys Arg Gln
1040 1045 1050
Glu Phe Glu Gln Val Lys Lys Arg Arg Tyr Asp Leu Phe Thr Gln
1055 1060 1065
Cys Phe Glu His Val Ser Ile Ser Ile Asp Gln Ile Tyr Lys Lys
1070 1075 1080
Leu Cys Arg Asn Asn Ser Ala Gln Ala Phe Leu Ser Pro Glu Asn
1085 1090 1095
Pro Glu Glu Pro Tyr Leu Glu Gly Ile Ser Tyr Asn Cys Val Ala
1100 1105 1110
Pro Gly Lys Arg Phe Met Pro Met Asp Asn Leu Ser Gly Gly Glu
1115 1120 1125
Lys Cys Val Ala Ala Leu Ala Leu Leu Phe Ala Val His Ser Phe
1130 1135 1140
Arg Pro Ala Pro Phe Phe Val Leu Asp Glu Val Asp Ala Ala Leu
1145 1150 1155
Asp Asn Thr Asn Ile Gly Lys Val Ser Ser Tyr Ile Lys Glu Gln
1160 1165 1170
Thr Gln Asp Gln Phe Gln Met Ile Val Ile Ser Leu Lys Glu Glu
1175 1180 1185
Phe Tyr Ser Arg Ala Asp Ala Leu Ile Gly Ile Tyr Pro Glu Tyr
1190 1195 1200
Asp Asp Cys Met Phe Ser Arg Val Leu Thr Leu Asp Leu Ser Gln
1205 1210 1215
Tyr Pro Asp Thr Glu Gly Gln Glu Ser Ser Lys Arg His Gly Glu
1220 1225 1230
Ser Arg
1235
<210> 3
<211> 1225
<212> PRT
<213> 人工序列
<400> 3
Met Ser Ser Pro Leu Gln Arg Ala Val Gly Asp Thr Lys Arg Ala Leu
1 5 10 15
Ser Ala Ser Ser Ser Ser Ser Ala Ser Leu Pro Phe Asp Asp Arg Asp
20 25 30
Ser Asn His Thr Ser Glu Gly Asn Gly Asp Ser Leu Leu Ala Asp Glu
35 40 45
Asp Thr Asp Phe Glu Asp Ser Leu Asn Arg Asn Val Lys Lys Arg Ala
50 55 60
Ala Lys Arg Pro Pro Lys Thr Thr Pro Val Ala Lys His Pro Lys Lys
65 70 75 80
Gly Ser Arg Val Val His Arg His Ser Arg Lys Gln Ser Glu Pro Pro
85 90 95
Ala Asn Asp Leu Phe Asn Ala Val Lys Ala Ala Lys Ser Asp Met Gln
100 105 110
Ser Leu Val Asp Glu Trp Leu Asp Ser Tyr Lys Gln Asp Gln Asp Ala
115 120 125
Gly Phe Leu Glu Leu Val Asn Phe Phe Ile Gln Ser Cys Gly Cys Lys
130 135 140
Gly Ile Val Thr Pro Glu Met Phe Lys Lys Met Ser Asn Ser Glu Ile
145 150 155 160
Ile Gln His Leu Thr Glu Gln Phe Asn Glu Asp Ser Gly Asp Tyr Pro
165 170 175
Leu Ile Ala Pro Gly Pro Ser Trp Lys Lys Phe Gln Gly Ser Phe Cys
180 185 190
Glu Phe Val Arg Thr Leu Val Cys Gln Cys Gln Tyr Ser Leu Leu Tyr
195 200 205
Asp Gly Phe Pro Met Asp Asp Leu Ile Ser Leu Leu Thr Gly Leu Ser
210 215 220
Asp Ser Gln Val Arg Ala Phe Arg His Thr Ser Thr Leu Ala Ala Met
225 230 235 240
Lys Leu Met Thr Ser Leu Val Lys Val Ala Leu Gln Leu Ser Val His
245 250 255
Gln Asp Asn Asn Gln Arg Gln Tyr Glu Ala Glu Arg Asn Lys Gly Pro
260 265 270
Gly Gln Arg Ala Pro Glu Arg Leu Glu Ser Leu Leu Glu Lys Arg Lys
275 280 285
Glu Leu Gln Glu His Gln Glu Glu Ile Glu Gly Met Met Asn Ala Leu
290 295 300
Phe Arg Gly Val Phe Val His Arg Tyr Arg Asp Val Leu Pro Glu Ile
305 310 315 320
Arg Ala Ile Cys Ile Glu Glu Ile Gly Cys Trp Met Gln Ser Tyr Ser
325 330 335
Thr Ser Phe Leu Thr Asp Ser Tyr Leu Lys Tyr Ile Gly Trp Thr Leu
340 345 350
His Asp Lys His Arg Glu Val Arg Leu Lys Cys Val Lys Ala Leu Lys
355 360 365
Gly Leu Tyr Gly Asn Arg Asp Leu Thr Thr Arg Leu Glu Leu Phe Thr
370 375 380
Ser Arg Phe Lys Asp Arg Met Val Ser Met Val Met Asp Arg Glu Tyr
385 390 395 400
Asp Val Ala Val Glu Ala Val Arg Leu Leu Ile Leu Ile Leu Lys Asn
405 410 415
Met Glu Gly Val Leu Thr Asp Ala Asp Cys Glu Ser Val Tyr Pro Val
420 425 430
Val Tyr Ala Ser His Arg Gly Leu Ala Ser Ala Ala Gly Glu Phe Leu
435 440 445
Tyr Trp Lys Leu Phe Tyr Pro Glu Cys Glu Ile Arg Met Met Gly Gly
450 455 460
Arg Glu Gln Arg Gln Ser Pro Gly Ala Gln Arg Thr Phe Phe Gln Leu
465 470 475 480
Leu Leu Ser Phe Phe Val Glu Ser Glu Leu His Asp His Ala Ala Tyr
485 490 495
Leu Val Asp Ser Leu Trp Asp Cys Ala Gly Ala Arg Leu Lys Asp Trp
500 505 510
Glu Gly Leu Thr Ser Leu Leu Leu Glu Lys Asp Gln Asn Leu Gly Asp
515 520 525
Val Gln Glu Ser Thr Leu Ile Glu Ile Leu Val Ser Ser Ala Arg Gln
530 535 540
Ala Ser Glu Gly His Pro Pro Val Gly Arg Val Thr Gly Arg Lys Gly
545 550 555 560
Leu Thr Ser Lys Glu Arg Lys Thr Gln Ala Asp Asp Arg Val Lys Leu
565 570 575
Thr Glu His Leu Ile Pro Leu Leu Pro Gln Leu Leu Ala Lys Phe Ser
580 585 590
Ala Asp Ala Glu Lys Val Thr Pro Leu Leu Gln Leu Leu Ser Cys Phe
595 600 605
Asp Leu His Ile Tyr Cys Thr Gly Arg Leu Glu Lys His Leu Glu Leu
610 615 620
Phe Leu Gln Gln Leu Gln Glu Val Val Val Lys His Ala Glu Pro Ala
625 630 635 640
Val Leu Glu Ala Gly Ala His Ala Leu Tyr Leu Leu Cys Asn Pro Glu
645 650 655
Phe Thr Phe Phe Ser Arg Ala Asp Phe Ala Arg Ser Gln Leu Val Asp
660 665 670
Leu Leu Thr Asp Arg Phe Gln Gln Glu Leu Glu Glu Leu Leu Gln Ser
675 680 685
Ser Phe Leu Asp Glu Asp Glu Val Tyr Asn Leu Ala Ala Thr Leu Lys
690 695 700
Arg Leu Ser Ala Phe Tyr Asn Thr His Asp Leu Thr Arg Trp Glu Leu
705 710 715 720
Tyr Glu Pro Cys Cys Gln Leu Leu Gln Lys Ala Val Asp Thr Gly Glu
725 730 735
Val Pro His Gln Val Ile Leu Pro Ala Leu Thr Leu Val Tyr Phe Ser
740 745 750
Ile Leu Trp Thr Leu Thr His Ile Ser Lys Ser Asp Ala Ser Gln Lys
755 760 765
Gln Leu Ser Ser Leu Arg Asp Arg Met Val Ala Phe Cys Glu Leu Cys
770 775 780
Gln Ser Cys Leu Ser Asp Val Asp Thr Glu Ile Gln Glu Gln Ala Phe
785 790 795 800
Val Leu Leu Ser Asp Leu Leu Leu Ile Phe Ser Pro Gln Met Ile Val
805 810 815
Gly Gly Arg Asp Phe Leu Arg Pro Leu Val Phe Phe Pro Glu Ala Thr
820 825 830
Leu Gln Ser Glu Leu Ala Ser Phe Leu Met Asp His Val Phe Ile Gln
835 840 845
Pro Gly Asp Leu Gly Ser Gly Asp Ser Gln Glu Asp His Leu Gln Ile
850 855 860
Glu Arg Leu His Gln Arg Arg Arg Leu Leu Ala Gly Phe Cys Lys Leu
865 870 875 880
Leu Leu Tyr Gly Val Leu Glu Met Asp Ala Ala Ser Asp Val Phe Lys
885 890 895
His Tyr Asn Lys Phe Tyr Asn Asp Tyr Gly Asp Ile Ile Lys Glu Thr
900 905 910
Leu Thr Arg Ala Arg Gln Ile Asp Arg Ser His Cys Ser Arg Ile Leu
915 920 925
Leu Leu Ser Leu Lys Gln Leu Tyr Thr Glu Leu Leu Gln Glu His Gly
930 935 940
Pro Gln Gly Leu Asn Glu Leu Pro Ala Phe Ile Glu Met Arg Asp Leu
945 950 955 960
Ala Arg Arg Phe Ala Leu Ser Phe Gly Pro Gln Gln Leu Gln Asn Arg
965 970 975
Asp Leu Val Val Met Leu His Lys Glu Gly Ile Gln Phe Ser Leu Ser
980 985 990
Glu Leu Pro Pro Ala Gly Ser Ser Asn Gln Pro Pro Asn Leu Ala Phe
995 1000 1005
Leu Glu Leu Leu Ser Glu Phe Ser Pro Arg Leu Phe His Gln Asp
1010 1015 1020
Lys Gln Leu Leu Leu Ser Tyr Leu Glu Lys Cys Leu Gln His Val
1025 1030 1035
Ser Gln Ala Pro Gly His Pro Trp Gly Pro Val Thr Thr Tyr Cys
1040 1045 1050
His Ser Leu Ser Pro Val Glu Asn Thr Ala Glu Thr Ser Pro Gln
1055 1060 1065
Val Leu Pro Ser Ser Lys Arg Arg Arg Val Glu Gly Pro Ala Lys
1070 1075 1080
Pro Asn Arg Glu Asp Val Ser Ser Ser Gln Glu Glu Ser Leu Gln
1085 1090 1095
Leu Asn Ser Ile Pro Pro Thr Pro Thr Leu Thr Ser Thr Ala Val
1100 1105 1110
Lys Ser Arg Gln Pro Leu Trp Gly Leu Lys Glu Met Glu Glu Glu
1115 1120 1125
Asp Gly Ser Glu Leu Asp Phe Ala Gln Gly Gln Pro Val Ala Gly
1130 1135 1140
Thr Glu Arg Ser Arg Phe Leu Gly Pro Gln Tyr Phe Gln Thr Pro
1145 1150 1155
His Asn Pro Ser Gly Pro Gly Leu Gly Asn Gln Leu Met Arg Leu
1160 1165 1170
Ser Leu Met Glu Glu Asp Glu Glu Glu Glu Leu Glu Ile Gln Asp
1175 1180 1185
Glu Ser Asn Glu Glu Arg Gln Asp Thr Asp Met Gln Ala Ser Ser
1190 1195 1200
Tyr Ser Ser Thr Ser Glu Arg Gly Leu Asp Leu Leu Asp Ser Thr
1205 1210 1215
Glu Leu Asp Ile Glu Asp Phe
1220 1225

Claims (7)

1.一种在体细胞中诱导多线染色体的方法，其特征在于，所述方法包括在体细胞中表达减数分裂黏连蛋白复合物的步骤；
所述减数分裂黏连蛋白复合物包括RAD21L、SMC1B和STAG3的组合；
所述RAD21L的氨基酸序列如SEQ ID NO:1所示；
所述SMC1B的氨基酸序列如SEQ ID NO:2所示；
所述STAG3的氨基酸序列如SEQ ID NO:3所示。
2.根据权利要求1所述的方法，其特征在于，所述方法包括：
(1)采用包含SMC1B基因和STAG3基因的慢病毒共感染体细胞后，筛选共表达SMC1B和STAG3的阳性克隆细胞株；
(2)将筛选的阳性克隆细胞株采用包含RAD21L基因的慢病毒感染后，筛选共表达SMC1B、STAG3和RAD21L的阳性克隆细胞株，在体细胞中表达减数分裂黏连蛋白复合物。
3.根据权利要求1-2任一项所述的方法，其特征在于，所述方法还包括对表达减数分裂黏连蛋白复合物的体细胞进行强力霉素诱导的步骤。
4.一种重组体细胞，其特征在于，所述重组体细胞中表达有减数分裂黏连蛋白复合物；
所述减数分裂黏连蛋白复合物包括RAD21L、SMC1B和STAG3的组合；
所述RAD21L的氨基酸序列如SEQ ID NO:1所示；
所述SMC1B的氨基酸序列如SEQ ID NO:2所示；
所述STAG3的氨基酸序列如SEQ ID NO:3所示；
所述减数分裂黏连蛋白复合物结合在体细胞的完整染色体上，所述体细胞形成多线染色体。
5.一种权利要求4所述的重组体细胞的构建方法，其特征在于，所述方法包括：
(1)采用包含SMC1B基因和STAG3基因的慢病毒共感染体细胞后，筛选共表达SMC1B和STAG3的阳性克隆细胞株；
(2)将筛选的阳性克隆细胞株采用包含RAD21L基因的慢病毒感染后，筛选共表达SMC1B、STAG3和RAD21L的阳性克隆细胞株，得到所述重组体细胞。
6.一种蛋白组合物在制备体细胞多线染色体诱导剂、体细胞增殖阻滞剂或细胞染色体配对诱导剂中的应用，
其中，所述蛋白组合物包括RAD21L、SMC1B和STAG3的组合；
所述RAD21L的氨基酸序列如SEQ ID NO:1所示；
所述SMC1B的氨基酸序列如SEQ ID NO:2所示；
所述STAG3的氨基酸序列如SEQ ID NO:3所示。
7.一种权利要求4所述的重组体细胞在制备研究染色体多线化和/或减数分裂突触机制的细胞模型中的应用。
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