mRNA剪接调控不同物种细胞多能性向全能性转换作用研究

mRNA剪接调控不同物种细胞多能性向全能性转换作用研究摘要：干细胞在再生医学和多种疾病的治疗上有着广阔研究前景。依据细胞的分化潜能可将干细胞分为多能性、全能性和单能性干细胞，其中全能性干细胞因其分化潜能最高可发展为一个完整个体而成为研究的热点。现有研究表明通过抑制剪接因子（如SF3B1、SRSF2）的表达，可以驱使全能性基因表达，调控细胞命运。通过向小鼠多能性的胚胎干细胞中添加剪切体抑制剂普拉地内酯B（PladienolideB,PlaB)能激活小鼠早期逆转录转座子L（MouseEarlyRetrotransposonL,MERVL）表达，并获得全能性囊胚样细胞（Totipotentblastomere-likecells,TBLCs）。然而，mRNA剪接调控细胞全能性是否在物种中保守目前研究的并不多。本研究基于已发表的人、猪和小鼠的早期胚胎转录组数据，揭示了差异表达基因在各物种全能性胚胎时期均与RNA剪接相关。在此基础上结合本实验室建立的全能性报告细胞系MERVL::H2B-tdTomato，通过RT-qPCR、荧光强度和双荧光素报告实验，首先筛选剪切体抑制剂PlaB在细胞多能性向全能性转变过程中的作用，明确了PlaB最佳作用浓度与作用时间确认。随后，进行流式细胞术检测MERVL阳性细胞比例与分选阳性细胞，分别对对照组阴性细胞与分选所得阳性细胞进行RNA测序（RNAsequencing,RNA-seq）。对测序结果进行生物信息学分析。并与2CLCs共同分析，获得关键可变剪切事件，进行PCR验证后，供下一步研究筛选。从而揭示PlaB调控细胞全能性的物种间保守规律。关键词：mRNA剪接；RNA-seq；全能性；多能性StudyontheregulationofmRNAsplicingonthetransitionfrompluripotencytopluripotencyincellsofdifferentspeciesAbstract：Stemcellshavebroadresearchprospectsinregenerativemedicineandthetreatmentofvariousdiseases.Accordingtothedifferentiationpotentialofcells,stemcellscanbeclassifiedintomultipotent,totipotent,andunipotentstemcells.Amongthem,totipotentstemcellshavebecomearesearchhotspotduetotheirhighestdifferentiationpotential,whichcandevelopintoacompleteindividual.ExistingresearchhasshownthatinhibitingtheexpressionofsplicingfactorssuchasSF3B1andSRSF2candrivetheexpressionofpluripotencygenesandregulatecellfate.AddingtheshearbodyinhibitorPladienolideB(PlaB)topluripotentembryonicstemcellsinmicecanactivatetheexpressionofMouseEarlyRetrotransposonL(MERVL)andobtaintotipotentblastocystlikecells(TBLCs).However,thereiscurrentlylimitedresearchonwhethermRNAsplicingregulatescellularpluripotencyinspecies.Thisstudyisbasedonpublishedtranscriptomedataofearlyembryosinhumans,pigs,andmice,revealingthatdifferentiallyexpressedgenesareassociatedwithRNAsplicingduringthepluripotencyembryonicstageinvariousspecies.Onthisbasis,combinedwiththepluripotencyreportercelllineMERVL::H2BtdTomatoestablishedinourlaboratory,RTqPCR,fluorescenceintensity,anddoublefluorescencereporterexperimentswereconductedtofirstscreenfortheroleofthecleavageinhibitorPlaBinthetransitionfromcellularpluripotencytopluripotency,andtoconfirmtheoptimalconcentrationanddurationofactionofPlaB.Subsequently,flowcytometrywasusedtodetecttheproportionofMERVLpositivecellsandtosortpositivecells.RNAsequencingwasperformedonnegativecellsinthecontrolgroupandpositivecellsobtainedfromsorting,respectively.Performbioinformaticsanalysisonsequencingresults.Andanalyzedtogetherwith2CLCstoobtainkeyvariableshearevents,whichwerevalidatedbyPCRforfurtherresearchscreening.ThusrevealingtheinterspeciesconservationpatternofPlaBregulatingcellpluripotency.gKeywords：mRNAsplicing;RNA-seq;Omnipotence;Pluripotency

-7-文献综述细胞全能性一般指细胞（比如植物细胞或者动物受精卵）具有发育成完整个体的潜能。而多能性则指细胞（如胚胎干细胞）能分化为多种组织，但无法独立形成完整个体的分化能力。基因表达的精细调控离不开RNA剪接这一核心过程，其精确性和效能对细胞特性的维持至关重要。作为转录后调控的重要环节，mRNA剪接通过选择性去除内含子并连接外显子来调节基因表达，从而深刻影响细胞命运。目前有研究发现，向小鼠胚胎干细胞（ESCs，EmbryonicStemCells）培养体系添加剪切体抑制剂PladienolideB(PlaB)能诱导细胞从多能性向全能性转变，获得的细胞称为全能性囊胚样细胞（Totipotentblastomere-likecells，TBLCs）ADDINEN.CITE<EndNote><Cite><Author>Hui</Author><Year>2021</Year><RecNum>51</RecNum><DisplayText>(Huietal.2021)</DisplayText><record><rec-number>51</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747452617">51</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>ShenHui</author><author>YangMin</author><author>LiShiyu</author><author>ZhangJing</author><author>PengBing</author><author>WangChunhui</author><author>ChangZai</author><author>OngJennie</author><author>DuPeng</author></authors></contributors><auth-address>Article;MOEKeyLaboratoryofCellProliferationandDifferentiation,SchoolofLifeSciences,PekingUniversity,Beijing100871,China;Peking-TsinghuaCenterforLifeSciences,AcademyforAdvancedInterdisciplinaryStudies,PekingUniversity,Beijing100871,China;SchoolofLifeSciences,TsinghuaUniversity,Beijing100871,China</auth-address><titles><title>Mousetotipotentstemcellscapturedandmaintainedthroughspliceosomalrepression</title><secondary-title>Cell</secondary-title></titles><periodical><full-title>Cell</full-title></periodical><volume>184</volume><number>11</number><keywords><keyword>embryonicstemcell</keyword><keyword>totipotent</keyword><keyword>pluripotent</keyword><keyword>spliceosome</keyword><keyword>splicing</keyword><keyword>pladienolideB</keyword><keyword>transcriptome</keyword><keyword>embryonic</keyword><keyword>extraembryonic</keyword><keyword>chimeras</keyword></keywords><dates><year>2021</year></dates><isbn>0092-8674</isbn><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCcYU_qk3lqooELJHZ9jeqF8qqFyok6iLRGbOGbWeQgYbT3vJoq1UsEDdhfgH331Rtf6JkPpc4AXsuaNBuDZzmumigv2gHlE8Y2dWGBG71oKVKnU03KyR764AfPhhFND5NBSo8B8zo1qVQIYQv61lzlIcHSlxJTZ4e8CQcSK81uIZmM8rJhfdlvjYdPwJUJslnFronpHBotUDBRUaxzVuUj8&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Huietal.2021)，但该调控机制在物种间是否保守尚不明确。解析其普适性对于加深全能性激活机制理解意义重大。干细胞在再生医学和多种疾病的治疗上有着广阔研究前景。依据细胞的分化潜能可将干细胞分为多能性、全能性和单能性干细胞，其中全能性干细胞因其分化潜能最高可发展为一个完整个体而成为研究的热点。现有研究表明剪接因子（如SF3B1、SRSF2）能通过调控关键基因的剪接模式影响细胞命运，驱使全能性基因表达。通过向小鼠多能性的胚胎干细胞中添加剪切体抑制剂普拉地内酯B（PladienolideB,PlaB)能激活小鼠早期逆转录转座子L（MouseEarlyRetrotransposonL,MERVL）表达，并获得全能性囊胚样细胞（Totipotentblastomere-likecells,TBLCs）。然而，mRNA剪接调控细胞全能性是否在物种中保守目前研究的并不多。因此，通过研究mRNA剪接调控不同物种细胞多能性向全能性转换作用，有助于帮助我们进一步揭示多能性干细胞向全能性干细胞的转变机制，促进全能性细胞体外建系和再生医学的发展。干细胞干细胞定义及分类在个体发育过程中，细胞通过分裂以及分化从多能状态转变成分化状态，并形成个体不同胚层的组织器官。干细胞根据分化潜能可分为全能干细胞(Totipotency)，多能干细胞(Pluripotency)，专能干细胞(Multipotent)和单能干细胞(Unipotent)。全能性干细胞一般存在于胚胎发育的早期阶段，比​如受精卵和2-4细胞期胚胎细胞，随后细胞分裂发育一直到囊胚形成均能保持其全能性。全能性干细胞能够分化为胚胎及胚外组织ADDINEN.CITE<EndNote><Cite><Author>J</Author><Year>1977</Year><RecNum>45</RecNum><DisplayText>(Jetal.1977)</DisplayText><record><rec-number>45</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747308786">45</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>KellySJ</author></authors></contributors><titles><title>Studiesofthedevelopmentalpotentialof4-and8-cellstagemouseblastomeres</title><secondary-title>TheJournalofexperimentalzoology</secondary-title></titles><periodical><full-title>TheJournalofexperimentalzoology</full-title></periodical><pages>365-76</pages><volume>200</volume><number>3</number><dates><year>1977</year></dates><isbn>0022-104X</isbn><urls><related-urls><url>/kcms2/article/abstract?v=IVqNFfq6ZnLg3iQsUN3m2gIaH3PqgPDHGAYY0B5vAeZ7q_8Jukf4I1B1zPPAgy8HoZA75p3h7Jy30W6MbWHbU41Rzib-xPQPlH8NxI35MTLru3GA6YjZw7UrfJ1FkNpT4dL5ZlV_pOYXyDZuemAwv7PJ5WvPD8jp36bVRScgEz2yOGdErOPnwLxICs_rtC3Q6bEYI-vyJlg=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Jetal.1977)，形成完整个体ADDINEN.CITE<EndNote><Cite><Author>Seydoux</Author><Year>2006</Year><RecNum>46</RecNum><DisplayText>(SeydouxandBraunetal.2006)</DisplayText><record><rec-number>46</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747311172">46</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>GeraldineSeydoux</author><author>RobertE.Braun</author></authors></contributors><auth-address>DepartmentofMolecularBiologyandGenetics,HowardHughesMedicalInstitute,JohnsHopkinsSchoolofMedicine,725NorthWolfeStreet,Baltimore,MD21205,USA;;DepartmentofGenomeSciences,UniversityofWashingtonSchoolofMedicine,1705N.E.PacificStreet,Seattle,WA98195,USA</auth-address><titles><title>PathwaytoTotipotency:LessonsfromGermCells</title><secondary-title>Cell</secondary-title></titles><periodical><full-title>Cell</full-title></periodical><pages>891-904</pages><volume>127</volume><number>5</number><dates><year>2006</year></dates><isbn>0092-8674</isbn><urls><related-urls><url>/kcms2/article/abstract?v=IVqNFfq6ZnLFCRuSGuKLP5L0PCwMQrHAfJp6S6kwb7gyNd5OiCL_iAtI_QDfDKl7vMlqfCstaiyq0DmhdygfXCVC6Z4Kx1hzzQNlg-TvFaY8GheqTabXDMrc8JJ6cdQ29bl9qlOunelloXjtgYUFHqhSkCrXQKsWrusvYjS3QvaFlb2Dmn0EmIbDJl6cC9pF9XpHAtGlhkyC_B4FVy0BaviLgnUVPru6&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(SeydouxandBraunetal.2006)，而多能性干细胞​（如胚胎干细胞ESCs）仅能形成胚内三胚层组织，缺乏胚外分化能力ADDINEN.CITEADDINEN.CITE.DATA(grid.250671.7etal.2012)，而专能与单能性干细胞​仅能分化为单一谱系。因此全能性干细胞具有最高最完整的发育潜力，研究全能性干细胞成为目前的重要领域。全能性干细胞表观遗传调控(epigeneticregulation)在胚胎发育阶段尤其活跃与关键，它通过各种化学修饰（比如DNA甲基化、组蛋白修饰）或者改变染色质（DNA和蛋白质的复合体）的结构，来控制基因表达ADDINEN.CITE<EndNote><Cite><Author>朱晓玲</Author><Year>2024</Year><RecNum>52</RecNum><DisplayText>(朱晓玲etal.2024)</DisplayText><record><rec-number>52</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747452740">52</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>朱晓玲</author></authors></contributors><auth-address>江苏省射阳县高级中学;</auth-address><titles><title>表观遗传调控机制概述</title><secondary-title>中学生理科应试</secondary-title></titles><periodical><full-title>中学生理科应试</full-title></periodical><pages>108-109</pages><number>Z1</number><dates><year>2024</year></dates><isbn>1005-6491</isbn><call-num>23-1351/G4</call-num><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCfxjHWANwjgZh2iW2gXOYHJ3q_e-SC72mF69OU3FZezkhccvi0ACmxdMgvF6fowv6Zw1NbooykxuPkRFWy6LTmF8zO1abqmx0FR1FBKUuHRfNamW0POyGDKWbtbVuY4r0gjcrEHRnJL1Pr5tVIaxdYHHqLy-f3ZoB-Rjv1pSdAftNyHv0hGMtLguCvSpaH8Qmg=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(朱晓玲etal.2024)。小鼠的全能性干细胞相关转录本的激活正是在表观遗传调控下实现。比如含锌指和SCAN结构域的蛋白质4（ZincFingerandSCANDomainContaining4​,Zscan4）、Eomesodermin转录因子（Eomesodermin,Eomes）等特异性基因，并使转座子小鼠内源性逆转录病毒-L（MurineEndogenousRetrovirus-L,MERVL）ADDINEN.CITE<EndNote><Cite><Author>Yang</Author><Year>2017</Year><RecNum>54</RecNum><DisplayText>(Yangetal.2017)</DisplayText><record><rec-number>54</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747452980">54</key></foreign-keys><ref-typename="ConferenceProceedings">10</ref-type><contributors><authors><author>LeiYang</author><author>LishuangSong</author><author>XuefeiLiu</author><author>LigeBai</author><author>GuangpengLi</author></authors><subsidiary-authors><author>内蒙古自治区实验动物管理委员会办公室,</author></subsidiary-authors></contributors><auth-address>TheKeyLaboratoryoftheNationalEducationMinistryforMammalianReproductiveBiologyandBiotechnology,InnerMongoliaUniversity;</auth-address><titles><title>TheDynamicMechanismofEndogenousViralMuERV-L/MERVLinMouseSomaticCellNuclearTransfer</title><secondary-title>2017年第十五届中国北方实验动物科技年会</secondary-title></titles><pages>1</pages><keywords><keyword>SCNT</keyword><keyword>ESCs</keyword><keyword>MERVL</keyword><keyword>embryo</keyword><keyword>KDM6A</keyword><keyword>KDM6B</keyword><keyword>H3K27me3</keyword></keywords><dates><year>2017</year></dates><pub-location>中国内蒙古呼和浩特</pub-location><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCe-Oy9gLj7hqiBMpeLQpDsAS4nFFoAZf1X8DHEYq_0nRDUJrfHZaK1_RbVBxup8LkhYzBsEbsCxOf_4JL-T18b_-T4PBKchfU_X-8PlBZfQj6qRbkqq76dO2AKIV7ChKkkGBIA69In8O9I7ytcY4FaeRe8z80tk_GxhZdOP9YwxDPR-zyjDJKrLTnZKsWx8agk=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Yangetal.2017)的激活都涉及到组蛋白修饰的改变。MERVL是小鼠ZGA阶段的核心调控元件转座原件（TE）之一，其活性与全能性状态密切相关。Zscan4是小鼠早期2-cell胚胎及全能性干细胞中特异性表达的基因，能够通过调控染色质重塑和转座子活性维持全能性，而其高表达与合子基因组激活（ZygoticGenomeActivation,ZGA）密切相关。ZGA是哺乳动物早期胚胎发育的核心事件，指受精后胚胎从依赖母源RNA和蛋白质转向自主基因组转录的过渡阶段，这是胚胎发育的第一个重要转折。在这个阶段，母源因子(maternalfactors)的依赖不断降低，而自主基因组转录开始不断增加。ZGA通常分为次要ZGA（minorZGA）和主要ZGA（majorZGA）两个阶段。次要ZGA阶段一般发生在受精后早期，也就是全能性干细胞时期（如小鼠1细胞晚期、牛8细胞期），该时期会激活少量基因，包括转录因子（如KLF17、DUXA）和转座元件（TE）。随后开始进行主要ZGA时期（如小鼠2细胞晚期、牛16细胞期），这个时期会驱动大规模基因表达，比如DUX的大量表达从而ADDINEN.CITEADDINEN.CITE.DATA(DivisionofHumanBiologyetal.2017)提高MERVL表达水平并促进全能性干细胞的转换。ZGA的启动依赖于染色质重塑、转录因子招募及三维基因组结构的动态调控，而转座原件（TE）的激活则是其最大的特点ADDINEN.CITEADDINEN.CITE.DATA(Hetal.2002)。在ZGA被激活的TE中，MERVL最为关键。其短暂激活为胚胎发育提供基因组可塑性，而精确的退出机制确保多能性程序的启动，因此MERVL激活可以认为是全能性激活的标志ADDINEN.CITEADDINEN.CITE.DATA(grid.250671.7etal.2012)。小鼠的2-cell胚胎具有分化出完整个体并形成胎外组织的能力ADDINEN.CITE<EndNote><Cite><Author>K</Author><Year>1959</Year><RecNum>48</RecNum><DisplayText>(Ketal.1959)</DisplayText><record><rec-number>48</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747377459">48</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>TARKOWSKIAK</author></authors></contributors><titles><title>Experimentsonthedevelopmentofisolatedblastomersofmouseeggs</title><secondary-title>Nature</secondary-title></titles><periodical><full-title>Nature</full-title></periodical><pages>1286-7</pages><volume>184</volume><keywords><keyword>OVUM</keyword></keywords><dates><year>1959</year></dates><isbn>0028-0836</isbn><urls><related-urls><url>/kcms2/article/abstract?v=IVqNFfq6ZnIJg3LudrItLhB3B2D5ppKMFoGFz1yYS2uS3SCyNRhv7PZUtmJFjTaHVwQdyaWnDo_UHsIGV-lwIinKSszd5YO5BhnzsdyiAAlWAPVYSBK2_RWdx5HyIz6Zrt3-ORlbsA4VjhzdC67XxYkiANFUlKCvFHxUGDlkFl-LtYAHbJZLTob2U_sKBpjlV6Xlsv4Wor8=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Ketal.1959)，而牛和猪则是在4-cell和8-cell胚胎具有形成完整个体的全能性ADDINEN.CITEADDINEN.CITE.DATA(Johnsonetal.1995,SandHetal.1991)。因此，全能性干细胞的时期在物种之间也有不同的差异。可变剪接与细胞全能性可变剪接（AlternativeSplicing,AS）是基因表达调控的核心机制之一，通过选择性保留或排除外显子，从单个前体mRNA生成多种异构体，显著扩展蛋白质组的多样性。其调控依赖于两类元件其中一类是顺式作用元件​，它包括外显子/内含子的剪接增强子（ESE/ISE）和沉默子（ESS/ISS），直接参与剪接位点的识别。另一类是反式作用因子​，它们以以RNA结合蛋白（RBPs）为主ADDINEN.CITEADDINEN.CITE.DATA(黄昕欣等2025)。在早期胚胎，转录因子DUX直接激活内源性逆转录病毒MERVL的LTR区域，驱动邻近基因（如Zscan4、Tcstv1）的剪接激活。MERVL的保留内含子通过“病毒模拟”机制招募染色质重塑复合物，维持染色质开放状态。有关研究报道指出剪接因子LIN28能通过抑制let-7miRNA的成熟，促进多能性基因的剪接异构体表达，维持干细胞自我更新。而相反，剪接因子PTBP1的缺失则导致多能性退出，促进神经分化。随着胚胎发育，H3K27me3和DNA甲基化逐步恢复，抑制全能性相关剪接事件。例如，DUXBL（DUX抑制因子）通过招募TRIM24复合物，沉默MERVL并激活囊胚期特异性剪接程序。而在诱导多能干细胞（iPSCs）中，化学重编程通过调控剪接因子的磷酸化状态，重置剪接模式至胚胎样状态，提高重编程效率。这再一次证实了可变剪接能够调控干细胞全能性向多能性的转变。可变剪接中的mRNA剪接一般指前体mRNA（pre-mRNA）通过剪接体去除内含子并将外显子规范连接，形成单一成熟mRNA的过程ADDINEN.CITE<EndNote><Cite><Author>Huang</Author><Year>2025</Year><RecNum>62</RecNum><DisplayText>(Huangetal.2025)</DisplayText><record><rec-number>62</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747461216">62</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>HanFeiHuang</author><author>YuanFang</author><author>ZhiTaoLi</author><author>SiMingQu</author><author>BoYuan</author><author>KaiGan</author><author>ChengLongYue</author><author>HaiJingLi</author><author>YuBoWen</author><author>ZhongZeng</author></authors></contributors><auth-address>OrganTransplantationCenter,TheFirstAffiliatedHospitalofKunmingMedicalUniversity,295XichangRoad,650032,Kunming,YunnanProvince,PRChina</auth-address><titles><title>SF3B4regulatesproliferationandapoptosisinhepatocellularcarcinomaviaalternativesplicingandinteractionwithTRIM28andSETD5</title><secondary-title>JournalofTranslationalMedicine</secondary-title></titles><periodical><full-title>JournalofTranslationalMedicine</full-title></periodical><pages>441-441</pages><volume>23</volume><number>1</number><keywords><keyword>SF3b4</keyword><keyword>Alternativesplicingevents</keyword><keyword>RNAbindingprotein</keyword><keyword>Apoptosis</keyword><keyword>Cellproliferation</keyword></keywords><dates><year>2025</year></dates><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCc9jijx630e8rbJSj8EodoAiHZlzSLE2dLfXcdkmoLw7mk49P0qirB_u9F5rGyt5ZUXs6mIn8h6o_IUXt7G9Um_oG6g7o5Ho6prCa9cvvKn_4FXUFUV246au4scBv1cgiFtP04cHRk3nqf4fwgX6Tx_m_kAZupwFXpfx0RTbUL_NltSnIS8DkpMczgfQOA4iMY4piWdaYNObxHQkYAFoiUp&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Huangetal.2025)。其剪接过程中每个外显子按固定顺序连接，仅生成一种mRNA亚型，因此一般被用作基础基因的表达来来确保基础蛋白质的稳定合成并维持细胞基础功能。而可变剪接会动态选择剪接位点从而会生成多种mRNA亚型，因此单个基因可产生多个异构体从而显著扩展蛋白质组的复杂性，被广泛应用于生物调控发育、组织特异性、应激响应及疾病发生的活动。它们二者均依赖剪接体识别剪接位点来切除掉内含子并连接外显子从而生成成熟的mRNA。据剪接位点的组合方式，可变剪接可分为外显子跳跃（ExonSkipping）、可变5’/3’剪接位点（Alternative5’/3’SpliceSite）、内含子保留（IntronRetention）、互斥外显子（MutuallyExclusiveExons）、可变启动子（AlternativePromoter）、​​可变终止子（AlternativeTerminator）、​以及外显子延长/缩短（ExonExtension/Truncation）ADDINEN.CITEADDINEN.CITE.DATA(Albrechtetal.2025)。诱导性多能干细胞由于早期胚胎获取困难，严重阻碍了对全能性细胞的研究，同时，由于缺乏可靠的培养体系，由受精卵和2细胞胚胎分离的细胞在体外很难维持，极大限制了胚胎干细胞的应用。因此近些年来研究者们重点展开了对诱导性多能干细胞（inducedpluripotentstemcells，iPSCs）的研究。诱导性多能干细胞是通过对已分化的体细胞（如皮肤细胞、血细胞）进行基因重编程，使其恢复类似胚胎干细胞（ESCs）的多能性状态的细胞ADDINEN.CITE<EndNote><Cite><Author>Wang</Author><Year>2025</Year><RecNum>64</RecNum><DisplayText>(Wangetal.2025)</DisplayText><record><rec-number>64</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747461368">64</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>ShuotingWang</author><author>YashuFeng</author><author>QiXing</author><author>TianchengZhou</author><author>JiajunLiu</author></authors></contributors><auth-address>DepartmentofHematology,TheThirdAffiliatedHospital,SunYat-senUniversity,Guangzhou510630,China.;CASKeyLaboratoryofRegenerativeBiology,SouthChinaInstituteforStemCellBiologyandRegenerativeMedicine,GuangzhouInstitutesofBiomedicineandHealth,ChineseAcademyofSciences,Guangzhou510530,China.;DepartmentofHematology,TheThirdAffiliatedHospital,SunYat-senUniversity,Guangzhou510630,China.Electronicaddress:liujiaj@.</auth-address><titles><title>Generationofananti-CD19CARknock-inhumaninducedpluripotentstemcelllineusingCRISPR/Cas9technology</title><secondary-title>Stemcellresearch</secondary-title></titles><periodical><full-title>Stemcellresearch</full-title></periodical><pages>103721</pages><volume>86</volume><dates><year>2025</year></dates><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCcAz9oacDsngFeoXzW8_DHf81SFnTJFkV0rJLX6EpdrQK_q6S7lLTvwSvZENhTmh4eh0MFnPL-GORnm9Mp2vtQW06gLS1aYKd0OTJnYWfaKsDTs9CjWtlZGKSXDhpl1g6TfC75q2Orx227uDWPXGp89CuJIWteHmnSMOaQSWeKzQ06ev30ihD0kvMnksFk-yLO7aj2W9GTmQTXQ70wn8n7R&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Wangetal.2025)。2006年，日本科学家山中伸弥团队首次通过引入四个转录因子（Oct3/4、Sox2、Klf4、c-Myc）成功将小鼠成纤维细胞转化为iPSCs，这一发现颠覆了传统的细胞分化的认知，并使山中教授荣获2012年诺贝尔生理学或医学奖ADDINEN.CITEADDINEN.CITE.DATA(郭晓强等2011)。iPSCs的​经典重编程方法​一般为通过病毒载体（如逆转录病毒、慢病毒）或非病毒方法（质粒、mRNA、蛋白质）递送关键转录因子（如Yamanaka因子Oct4/Sox2/Klf4/c-Myc），也有使用小分子化合物（如VPA、CHIR99021）替代部分转录因子，降低致瘤风险并提高效率。目前也有研究者通过抑制剪接体活性，改变mRNA剪接模式，促进多能性基因激活得到诱导性多能干细胞。mRNA剪接抑制与多能性转变的关联​​根据上面的内容，剪接体抑制剂（如PlaB）能够通过抑制pre-mRNA剪接，诱导细胞进入类胚胎早期状态，促进多能性基因激活。剪接体抑制通过调控关键基因（如ZSCAN4、DUXA）的可变剪接，维持iPSCs的全能性特征。研究发现通过化学重编程、表观遗传重塑及剪接调控等多种策略，可以实现了多能性干细胞向全能性状态的转变。杜鹏团队（2021）通过抑制剪接体核心亚基SF3B1，利用剪接抑制剂PlaB诱导小鼠胚胎干细胞（mESCs）转化为全能性囊胚样细胞（TBLCs），这些细胞在转录组、表观组和功能上接近2细胞期胚胎，并能在嵌合实验中分化为胚内和胚外组织ADDINEN.CITE<EndNote><Cite><Author>Hui</Author><Year>2021</Year><RecNum>59</RecNum><DisplayText>(Huietal.2021)</DisplayText><record><rec-number>59</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747460276">59</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>ShenHui</author><author>YangMin</author><author>LiShiyu</author><author>ZhangJing</author><author>PengBing</author><author>WangChunhui</author><author>ChangZai</author><author>OngJennie</author><author>DuPeng</author></authors></contributors><auth-address>Article;MOEKeyLaboratoryofCellProliferationandDifferentiation,SchoolofLifeSciences,PekingUniversity,Beijing100871,China;Peking-TsinghuaCenterforLifeSciences,AcademyforAdvancedInterdisciplinaryStudies,PekingUniversity,Beijing100871,China;SchoolofLifeSciences,TsinghuaUniversity,Beijing100871,China</auth-address><titles><title>Mousetotipotentstemcellscapturedandmaintainedthroughspliceosomalrepression</title><secondary-title>Cell</secondary-title></titles><periodical><full-title>Cell</full-title></periodical><volume>184</volume><number>11</number><keywords><keyword>embryonicstemcell</keyword><keyword>totipotent</keyword><keyword>pluripotent</keyword><keyword>spliceosome</keyword><keyword>splicing</keyword><keyword>pladienolideB</keyword><keyword>transcriptome</keyword><keyword>embryonic</keyword><keyword>extraembryonic</keyword><keyword>chimeras</keyword></keywords><dates><year>2021</year></dates><isbn>0092-8674</isbn><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCcUjddWkaclKjdwo1dhddbmk7E61PGiIYwrqzi9qdxrzF_8QhiCHN4mXnsFwApXZctNr19302cUIf1wJDObw-Pj4PtoSy2gLZZdcEYQCTjhQVW9y4dAwIwuRBayEoJN7KB1v4pjO30RleIxtrO0KPu1GehqCMCVnoVniny0l18Wtk7SLebYIy8JwdgHWNcmhkrurnnIaL_WnZZ8T6tKUNKH&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Huietal.2021)。在胚胎发育的合子基因组激活（ZGA）阶段，剪接事件（如外显子跳跃和内含子保留）显著增加，称为“合子剪接激活（ZSA）”，其失败会导致胚胎停滞在2细胞期并引发全能性基因异常激活ADDINEN.CITEADDINEN.CITE.DATA(Zhangetal.2024)。因此，全能性细胞的激活机制是干细胞研究领域的重要突破方向之一，目前已有多种策略被证实mRNA剪接抑制可诱导多能性干细胞向全能性状态转化。mRNA剪接在多能性向全能性转换中的核心作用主要体现在以下两方面，一个是剪接抑制驱动全能性基因优势表达​。剪接体抑制剂（如PlaB）通过靶向SF3B1阻断前体mRNA的正常剪接，导致未剪接RNA积累。。第二个是剪接因子动态调控网络​，在ZGA阶段，剪接因子（如Snrpd2、Snrpb）表达受抑制，降低DNA损伤相关基因的外显子跳跃水平，从而调控细胞周期并维持全能性ADDINEN.CITE<EndNote><Cite><Author>Li</Author><Year>2024</Year><RecNum>61</RecNum><DisplayText>(Lietal.2024)</DisplayText><record><rec-number>61</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747460655">61</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>FanLi</author><author>NajmehKarimi</author><author>SiqiWang</author><author>TianshiPan</author><author>JingxiDong</author><author>XinWang</author><author>SinanMa</author><author>QingtongShan</author><author>ChaoLiu</author><author>YingZhang</author><author>WeiLi</author><author>GuihaiFeng</author></authors></contributors><auth-address>UniversityofChineseAcademyofSciences,Beijing,China.;MedicalSchool,UniversityofChineseAcademyofSciences,Beijing,China.;StateKeyLaboratoryofStemCellandReproductiveBiology,InstituteofZoology,ChineseAcademyofSciences,Beijing,China.;CollegeofLifeSciences,NortheastAgriculturalUniversity,Harbin,China.;BeijingInstituteforStemCellandRegenerativeMedicine,Beijing,China.;KeyLaboratoryofOrganRegenerationandReconstruction,ChineseAcademyofSciences,Beijing,China.</auth-address><titles><title>mRNAisoformswitchesduringmousezygoticgenomeactivation</title><secondary-title>Cellproliferation</secondary-title></titles><periodical><full-title>Cellproliferation</full-title></periodical><pages>e13655-e13655</pages><volume>57</volume><number>7</number><dates><year>2024</year></dates><isbn>0960-7722</isbn><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCeCs_qIqNh6r6Z1qM4sw2k3Uy_3qwasRas8HmXQRsoAjJCAE7i4x-nRGW_PBMmNyToM3PZkdJ9NWbMBVtVzTlxT-LDA8H8KK7ZDjtBGdZVKTHVmYy9Za9PfVUq2sG-jJGLXeu_hbLHjGzf_k4LqS8FbR3IXoRno0GKYAJ-PDvKRCf5Ptj8y79Jfp8zjjtRQDjTjU9kWKMxL8VShAlNig-Vx&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Lietal.2024)。此外，剪接因子与组蛋白修饰（如H3K9me3）协同作用，通过抑制DUX等全能性基因的启动子区域染色质开放状态，限制其异常激活ADDINEN.CITE<EndNote><Cite><Author>Yang</Author><Year>2024</Year><RecNum>60</RecNum><DisplayText>(Yangetal.2024)</DisplayText><record><rec-number>60</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747460479">60</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>JianYang</author><author>LaurynCook</author><author>ZhiyuanChen</author></authors></contributors><auth-address>DepartmentofPediatrics,UniversityofCincinnatiCollegeofMedicine,Cincinnati,OH45267,USA.Electronicaddress:zhiyuan.chen@.;ReproductiveSciencesCenter,DivisionofDevelopmentalBiology,CincinnatiChildren'sHospitalMedicalCenter,Cincinnati,OH45229,USA.;DepartmentofPediatrics,UniversityofCincinnatiCollegeofMedicine,Cincinnati,OH45267,USA.</auth-address><titles><title>SystematicevaluationofretroviralLTRsascis-regulatoryelementsinmouseembryos</title><secondary-title>Cellreports</secondary-title></titles><periodical><full-title>Cellreports</full-title></periodical><pages>113775-113775</pages><volume>43</volume><number>3</number><keywords><keyword>CP:Developmentalbiology</keyword><keyword>MERVL</keyword><keyword>MT2Cmm</keyword><keyword>MT2mm</keyword><keyword>endogenousretroviruses</keyword><keyword>epigenomeediting</keyword><keyword>longterminalrepeats</keyword><keyword>preimplantationembryos</keyword><keyword>zygoticgenomeactivation</keyword></keywords><dates><year>2024</year></dates><isbn>2211-1247</isbn><urls><related-urls><url>/kcms2/article/abstract?v=vG2M3utQQCdFmK_WRHrtR1hpf1rQdMXyXJsIPI7Ud3qERoSH2JgyogRF24sCSoRwB3dlWRnYO_n-mh6nSxaJjCA-_0gmFJoWYJzVpOXqUvPNZZdMin4Xka20CoZHfgEP1-Ya2-kkAP4xKGdoa4QjMnsUjYJ8-QuOFmBu5_AWeC-lE7RwB0FHJAx6-owzdZ1L74helv8uac57er5Qcgph9tswacTXirZS&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>(Yangetal.2024)。这也为我们揭示了剪接事件通过“未剪接前体mRNA选择性翻译”模型驱动细胞命运转变的分子逻辑，为今后科研发展中理解胚胎早期发育提供了新视角​。一些干细胞研究技术与方法实时荧光定量PCR（qPCR）qPCR通过在反应体系中引入荧光探针或荧光染料，实时监测DNA扩增过程中荧光信号的变化，从而定量目标mRNA的起始浓度其核心参数为Ct值（阈值循环数），通过标准曲线计算相对表达量ADDINEN.CITE<EndNote><Cite><Author>Sam</Author><Year>1993</Year><RecNum>66</RecNum><DisplayText>(Sametal.1993)</DisplayText><record><rec-number>66</rec-number><foreign-keys><keyapp="EN"db-id="ze9psazwdwaxaee2zprv5xtk0extdsararxv"timestamp="1747461918">66</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>BabuJohnSam</author><author