m6A阅读器YTHDF1调控小鼠心肌纤维化的作用

中文摘要背景及目的伴随社会经济水平的显著提升与人口老龄化态势的日趋凸显，心血管疾病防治工作所面临的挑战持续加剧。心肌纤维化作为多种心血管疾病演进进程中的共性病理特征，也已成为公共卫生领域亟待解决的核心环节。作为全球致死率最高的疾病类别，此类疾患涵盖冠状动脉粥样硬化性心脏病、急性心肌梗死、慢性心力衰竭以及高血压等主要临床类型，其发病率和死亡率均居高不下。现有研究证实，m6ARNA甲基化修饰通过调控基因表达可介导心血管系统疾病的发生发展，进而影响成纤维细胞的活化、增殖及胶原合成等过程。YTHDF1是m6A修饰中最通用且功能强大的阅读蛋白之一，通过识别并调节m6A修饰的RNA表达，进而调控细胞的增殖、迁移、侵袭、免疫和自噬等基因表达。然而，m6A阅读器YTHDF1是否参与调控心肌纤维化尚未阐明。因此，本研究旨在探讨m6A阅读器YTHDF1是否参与调控心肌纤维化病理进程。方法1：构建心肌梗死诱导的心肌纤维化模型，同时设置假手术组。2：使用TGF-β诱导乳鼠原代分离的心肌成纤维细胞构建体外心肌成纤维细胞活化模型。3：采用qRT-PCR技术对各组小鼠的心脏组织及心肌成纤维细胞中各组YTHDF1mRNA的表达变化进行检测。4：运用Westernblot技术对各组小鼠心脏组织及心肌成纤维细胞中各组YTHDF1蛋白的表达变化进行检测。5：使用免疫荧光技术检测小鼠心脏组织及心肌成纤维细胞的荧光强度。结果实验数据显示，相较于对照组，心肌梗死模型小鼠心肌组织及TGF-β诱导激活的成纤维细胞m6A甲基化识别蛋白YTHDF1表达量显著升高（P<0.05）。结论m6A阅读器YTHDF1在心肌纤维化小鼠中表达上调，可能参与小鼠心肌纤维化的病理进程。关键词：心肌纤维化；m6A甲基化修饰；YTHDF1

Theroleofm6AreaderYTHDF1inregulatingmyocardialfibrosisinmiceAbstractBackgroundandObjectivesWiththesignificantimprovementofthesocialandeconomiclevelandtheincreasinglyprominenttrendofpopulationaging,thechallengesfacedbythepreventionandtreatmentofcardiovasculardiseasescontinuetointensify.Myocardialfibrosis,asacommonpathologicalfeatureintheevolutionprocessofvariouscardiovasculardiseases,hasalsobecomeacorelinkthaturgentlyneedstobesolvedinthefieldofpublichealth.Asthediseasecategorywiththehighestglobalfatalityrate,thistypeofdiseasecoversmajorclinicaltypessuchascoronaryatheroscleroticheartdisease,acutemyocardialinfarction,chronicheartfailureandhypertension,anditsincidenceandmortalityratesremainhigh.Existingstudieshaveconfirmedthatm6ARNAmethylationmodificationcanmediatetheoccurrenceanddevelopmentofcardiovascularsystemdiseasesbyregulatinggeneexpression,andtherebyaffectprocessessuchastheactivation,proliferationandcollagensynthesisoffibroblasts.YTHDF1isoneofthemostuniversalandpowerfulreadingproteinsinm6Amodification.ByrecognizingandregulatingtheRNAexpressionofm6Amodification,ittherebyregulatesthegeneexpressionsofcellproliferation,migration,invasion,immunityandautophagy.However,ithasnotbeenclarifiedwhetherthem6AreaderYTHDF1isinvolvedinregulatingmyocardialfibrosis.Therefore,thisstudyaimstoexplorewhetherthem6AreaderYTHDF1isinvolvedinregulatingthepathologicalprocessofmyocardialfibrosis.Method1:Amyocardialfibrosismodelinducedbymyocardialinfarctionwasconstructed,andashamoperationgroupwassetupsimultaneously.2:TheactivationmodelofmyocardialfibroblastsinvitrowasconstructedbyinducingprimaryisolatedmyocardialfibroblastsfromSucklingmicewithTGF-β.3:TheexpressionchangesofYTHDF1mRNAinthecardiactissuesandmyocardialfibroblastsofmiceineachgroupweredetectedbyqRT-PCRtechnology.4:WesternblottechniquewasusedtodetecttheexpressionchangesofYTHDF1proteininthecardiactissuesandmyocardialfibroblastsofmiceineachgroup.5:Thefluorescenceintensityofmousecardiactissueandmyocardialfibroblastswasdetectedbyimmunofluorescencetechnique.ResultExperimentaldatashowedthatcomparedwiththecontrolgroup,theexpressionlevelsofm6AmethylationrecognitionproteinYTHDF1inmyocardialtissuesandTGF-β-inducedactivatedfibroblastsofmyocardialinfarctionmodelmiceweresignificantlyincreased(P<0.05).ConclusionTheexpressionofthem6AreaderYTHDF1isupregulatedinmicewithmyocardialfibrosisandmaybeinvolvedinthepathologicalprocessofmyocardialfibrosisinmice.Keywords:Myocardialfibrosis;m6Amethylationmodification;YTHDF1

1、文献综述1.1心肌纤维化心肌纤维化是多种心脏疾病中普遍存在的重要病理过程，其发生具有广泛性病理特征ADDINEN.CITE<EndNote><Cite><Author>Weber</Author><Year>2013</Year><RecNum>15</RecNum><DisplayText><styleface="superscript">[2]</style></DisplayText><record><rec-number>15</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741616967">15</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Weber,KarlT</author><author>Sun,Yao</author><author>Bhattacharya,SyamalK</author><author>Ahokas,RobertA</author><author>Gerling,IvanC</author></authors></contributors><titles><title>Myofibroblast-mediatedmechanismsofpathologicalremodellingoftheheart</title><secondary-title>NatureReviewsCardiology</secondary-title></titles><periodical><full-title>NatureReviewsCardiology</full-title></periodical><pages>15-26</pages><volume>10</volume><number>1</number><dates><year>2013</year></dates><isbn>1759-5002</isbn><urls></urls></record></Cite></EndNote>[2]，涉及缺血性损伤、机械/容量超负荷、代谢紊乱等多元损伤刺激，这些病理级联反应通过诱导心肌细胞程序性死亡机制，驱动纤维化病理进程。该病理性重塑的核心表现为心肌间质成纤维细胞的异常活化与肌成纤维细胞转分化，伴随细胞外基质（ECM）内胶原代谢稳态失衡，最终导致非功能性瘢痕组织替代性增生ADDINEN.CITE<EndNote><Cite><Author>Weber</Author><Year>1995</Year><RecNum>17</RecNum><DisplayText><styleface="superscript">[3,4]</style></DisplayText><record><rec-number>17</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741617317">17</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Weber,KT</author><author>Sun,Y</author><author>Campbell,SE</author></authors></contributors><titles><title>Structuralremodellingoftheheartbyfibroustissue:roleofcirculatinghormonesandlocallyproducedpeptides</title><secondary-title>Europeanheartjournal</secondary-title></titles><periodical><full-title>Europeanheartjournal</full-title></periodical><pages>12-18</pages><volume>16</volume><number>suppl_N</number><dates><year>1995</year></dates><isbn>1522-9645</isbn><urls></urls></record></Cite><Cite><Author>Porter</Author><Year>2009</Year><RecNum>18</RecNum><record><rec-number>18</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741617640">18</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Porter,KarenE</author><author>Turner,NeilA</author></authors></contributors><titles><title>Cardiacfibroblasts:attheheartofmyocardialremodeling</title><secondary-title>Pharmacology&therapeutics</secondary-title></titles><periodical><full-title>Pharmacology&therapeutics</full-title></periodical><pages>255-278</pages><volume>123</volume><number>2</number><dates><year>2009</year></dates><isbn>0163-7258</isbn><urls></urls></record></Cite></EndNote>[3,4]。这些病理变化不仅导致心肌失去正常的舒缩功能，还可能影响心肌组织的电生理特性ADDINEN.CITE<EndNote><Cite><Author>Uriel</Author><Year>2018</Year><RecNum>19</RecNum><DisplayText><styleface="superscript">[5]</style></DisplayText><record><rec-number>19</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741617733">19</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Uriel,Nir</author><author>Sayer,Gabriel</author><author>Annamalai,Shiva</author><author>Kapur,NavinK</author><author>Burkhoff,Daniel</author></authors></contributors><titles><title>Mechanicalunloadinginheartfailure</title><secondary-title>JournaloftheAmericanCollegeofCardiology</secondary-title></titles><periodical><full-title>JournaloftheAmericanCollegeofCardiology</full-title></periodical><pages>569-580</pages><volume>72</volume><number>5</number><dates><year>2018</year></dates><isbn>0735-1097</isbn><urls></urls></record></Cite></EndNote>[5]。心肌纤维化是心室重构的关键病理枢纽，而心室重构的过程本质上是是心肌梗死、心力衰竭等心血管事件后，心肌细胞表观遗传重编程与非心肌细胞交互网络失调共同介导的动态适应性反应ADDINEN.CITE<EndNote><Cite><Author>Chen</Author><Year>2021</Year><RecNum>14</RecNum><DisplayText><styleface="superscript">[6]</style></DisplayText><record><rec-number>14</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741616809">14</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Chen,Ye-shi</author><author>Ouyang,Xin-ping</author><author>Yu,Xiao-hua</author><author>Novák,Petr</author><author>Zhou,Le</author><author>He,Ping-ping</author><author>Yin,Kai</author></authors></contributors><titles><title>N6-adenosinemethylation(m6A)RNAmodification:anemergingroleincardiovasculardiseases</title><secondary-title>Journalofcardiovasculartranslationalresearch</secondary-title></titles><periodical><full-title>Journalofcardiovasculartranslationalresearch</full-title></periodical><pages>1-16</pages><dates><year>2021</year></dates><isbn>1937-5387</isbn><urls></urls></record></Cite></EndNote>[6]。其分子机制涵盖心肌细胞死亡模式转换（包括坏死性凋亡、焦亡及自噬流障碍）、代偿性肥大反应，以及ECM重构介导的纤维化微环境形成等多维病理事件。因此，基于上述机制，靶向心肌细胞保护与纤维化进程调控的双重干预策略，已成为逆转病理性重构的核心治疗范式。通过精准干预这些关键病理节点，不仅可有效延缓疾病进展，更为改善心血管疾病患者远期预后提供了转化医学研究的重要切入点。1.2m6A甲基化修饰m6A甲基化修饰是指RNA分子中腺嘌呤碱基6号氮位置的甲基化修饰，是真核生物中最常见的RNA修饰之一，广泛存在于信使RNA、转运RNA、核糖体RNA和非编码RNA中。其甲基化修饰主要发生在RRACH核苷酸序列的腺嘌呤上，通常富集于3'非翻译区、蛋白质编码区和终止密码子附近的长外显子ADDINEN.CITE<EndNote><Cite><Author>Boulias</Author><Year>2023</Year><RecNum>22</RecNum><DisplayText><styleface="superscript">[7]</style></DisplayText><record><rec-number>22</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741618057">22</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Boulias,Konstantinos</author><author>Greer,EricLieberman</author></authors></contributors><titles><title>BiologicalrolesofadeninemethylationinRNA</title><secondary-title>NatureReviewsGenetics</secondary-title></titles><periodical><full-title>NatureReviewsGenetics</full-title></periodical><pages>143-160</pages><volume>24</volume><number>3</number><dates><year>2023</year></dates><isbn>1471-0056</isbn><urls></urls></record></Cite></EndNote>[7]。m6A甲基化修饰是一个动态可逆的过程，由甲基转移酶、去甲基化酶和甲基化阅读蛋白共同调控ADDINEN.CITE<EndNote><Cite><Author>Li</Author><Year>2024</Year><RecNum>23</RecNum><DisplayText><styleface="superscript">[8]</style></DisplayText><record><rec-number>23</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741618164">23</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Li,Bin</author><author>Xuan,Hanqin</author><author>Yin,Yuye</author><author>Wu,Shusheng</author><author>Du,Longfei</author></authors></contributors><titles><title>TheN6-methyladenosinemodificationinpathologicangiogenesis</title><secondary-title>LifeSciences</secondary-title></titles><periodical><full-title>LifeSciences</full-title></periodical><pages>122417</pages><volume>339</volume><dates><year>2024</year></dates><isbn>0024-3205</isbn><urls></urls></record></Cite></EndNote>[8]。这种动态可逆性使得m6A修饰能够灵活响应细胞内外的生理和病理变化ADDINEN.CITE<EndNote><Cite><Author>陈雨涵</Author><Year>2024</Year><RecNum>7</RecNum><DisplayText><styleface="superscript">[9]</style></DisplayText><record><rec-number>7</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741615404">7</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>陈雨涵</author><author>胡雪峰</author></authors></contributors><auth-address>福建师范大学生命科学学院/福建省发育与神经生物学重点实验室;</auth-address><titles><title>m6A甲基化修饰及其生物学功能</title><secondary-title>生物学教学</secondary-title></titles><periodical><full-title>生物学教学</full-title></periodical><pages>6-9</pages><volume>49</volume><number>07</number><keywords><keyword>RNA修饰</keyword><keyword>表观遗传</keyword><keyword>生物学功能</keyword></keywords><dates><year>2024</year></dates><isbn>1004-7549</isbn><call-num>31-1009/G4</call-num><urls><related-urls><url>/interlibSSO/goto/11/+jmr9bmjh9mds/kcms2/article/abstract?v=DnpHqYycDUNTwiqCvqRqif1CUBk1tyVginrN5KNi3f7l1yRjvD3O-smnviu8JnJRYyArtNnHWS5WmcipY83gAbXu6lOd7gz1G6B_fGRDj0wST7aLZGpeB-eUbecP-FL7gCaaZqQNTYPJJIZxyLWHT92I4wYqqdyAOeAqHtPls5jjMC6lrfkphC0ywJQ30y0YUVrwzdY2WuA=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[9]。m6A甲基化修饰在心血管疾病的发生和发展中具有重要作用，尤其在心肌纤维化中，其通过调控相关基因的表达，影响成纤维细胞的活化、增殖及胶原合成等过程。研究表明，m6A修饰异常可能导致病理性心脏重塑、心力衰竭、动脉粥样硬化和先天性心脏病等多种心血管疾病ADDINEN.CITE<EndNote><Cite><Author>尹功华</Author><Year>2024</Year><RecNum>6</RecNum><DisplayText><styleface="superscript">[10]</style></DisplayText><record><rec-number>6</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741615301">6</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>尹功华</author><author>徐若瑶</author><author>张丽娟</author><author>张一凡</author><author>齐洁</author><author>张钧</author></authors></contributors><auth-address>上海师范大学体育学院;</auth-address><titles><title>m6A甲基化修饰非编码RNA调控病理性心脏重塑的作用</title><secondary-title>中国组织工程研究</secondary-title></titles><periodical><full-title>中国组织工程研究</full-title></periodical><pages>3252-3258</pages><volume>28</volume><number>20</number><keywords><keyword>m6A甲基化修饰</keyword><keyword>病理性心脏重塑</keyword><keyword>非编码RNA</keyword><keyword>病理性心肌肥大</keyword><keyword>心肌细胞死亡</keyword><keyword>心肌纤维化</keyword><keyword>血管重塑</keyword><keyword>综述</keyword></keywords><dates><year>2024</year></dates><isbn>2095-4344</isbn><call-num>21-1581/R</call-num><urls><related-urls><url>/urlid/21.1581.R.20230728.1032.002</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[10]。因此，深入研究m6A甲基化修饰在心肌纤维化中的调控机制，对于揭示心血管疾病的发病机制及寻找新的治疗靶点具有重要意义。1.3YTHDF1蛋白YT521-B同源性蛋白家族含有YTH结构域，是研究最为深入的m6A阅读蛋白家族之一。该家族主要包括细胞质中的YTHDF1、YTHDF2、YTHDF3、YTHDC2和细胞核中的YTHDC1ADDINEN.CITE<EndNote><Cite><Author>Zhu</Author><Year>2023</Year><RecNum>25</RecNum><DisplayText><styleface="superscript">[11]</style></DisplayText><record><rec-number>25</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741618356">25</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Zhu,Yanan</author><author>Li,Jing</author><author>Yang,Hang</author><author>Yang,Xinyi</author><author>Zhang,Ya</author><author>Yu,Xinchao</author><author>Li,Ying</author><author>Chen,Gangxian</author><author>Yang,Zuozhang</author></authors></contributors><titles><title>Thepotentialroleofm6AreaderYTHDF1asdiagnosticbiomarkerandthesignalingpathwaysintumorigenesisandmetastasisinpan-cancer</title><secondary-title>CellDeathDiscovery</secondary-title></titles><periodical><full-title>CellDeathDiscovery</full-title></periodical><pages>34</pages><volume>9</volume><number>1</number><dates><year>2023</year></dates><isbn>2058-7716</isbn><urls></urls></record></Cite></EndNote>[11]。这些蛋白通过与m6A修饰的RNA结合，调控RNA的剪接、翻译、降解及核质转运，从而影响多种疾病的发生和发展。YTHDF1是m6A修饰中最通用且功能强大的阅读蛋白之一，可通过与真核起始因子eIF3相互作用，促进m6A修饰的mRNA翻译ADDINEN.CITE<EndNote><Cite><Author>朱琳</Author><Year>2023</Year><RecNum>8</RecNum><DisplayText><styleface="superscript">[12]</style></DisplayText><record><rec-number>8</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741615526">8</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>朱琳</author><author>于涛</author><author>郑法康</author><author>龚海东</author></authors></contributors><auth-address>牡丹江医学院附属红旗医院神经外科;</auth-address><titles><title>m6A-RNA甲基化YTHDF1结合蛋白在胶质瘤中作用的研究进展</title><secondary-title>现代肿瘤医学</secondary-title></titles><periodical><full-title>现代肿瘤医学</full-title></periodical><pages>2512-2518</pages><volume>31</volume><number>13</number><keywords><keyword>YTHDF1</keyword><keyword>胶质瘤</keyword><keyword>m6A甲基化</keyword><keyword>肿瘤干细胞</keyword></keywords><dates><year>2023</year></dates><isbn>1672-4992</isbn><call-num>61-1415/R</call-num><urls><related-urls><url>/interlibSSO/goto/11/+jmr9bmjh9mds/kcms2/article/abstract?v=DnpHqYycDUOvNVoFz7Ie2ps0nANBhiNMqUo1ZCtL7_iBdjLhTqgaPFdP_J-W3pP9D2m7q487SetwyZ65mOSo3K1W6D8Sj79Qx9d-oMtied0xI_Z_yIcuy3eq4nTxMQuJbMG95Nff7e6VUm9HdRwXjNoEH2WFEf5as6qfk0bm47q4lUf4h8mx9oAr9yvAK-PnJ0pecxo-CHc=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[12]。此外，YTHDF1还能通过与eIF4G介导的环结构结合，进一步提高翻译效率。因此，深入研究YTHDF1对于理解各类疾病的发病机制具有重要意义，近年来也取得了极大进展，然而在心肌纤维化损伤中的报道仍然寥寥无几，所以大有研究空间。1.3.1YTHDF1蛋白与肿瘤疾病的研究进展研究表明，YTHDF1在宫颈癌组织和细胞中的表达显著升高，通过促进糖酵解、乳酸积累和细胞表面PD-L1表达，降低CD8+T细胞的浸润，从而抑制免疫系统对肿瘤细胞的杀伤作用ADDINEN.CITE<EndNote><Cite><Author>Xiong</Author><Year>2024</Year><RecNum>27</RecNum><DisplayText><styleface="superscript">[13]</style></DisplayText><record><rec-number>27</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741920075">27</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Xiong,Jing</author><author>He,Ling</author><author>Chai,Xiaoshan</author><author>Zhang,Yongjing</author><author>Sun,Shujuan</author></authors></contributors><titles><title>YTHDF1booststhelactateaccumulationtopotentiatecervicalcancercellsimmuneescape</title><secondary-title>CellDeath&Disease</secondary-title></titles><periodical><full-title>CellDeath&Disease</full-title></periodical><pages>843</pages><volume>15</volume><number>11</number><dates><year>2024</year></dates><isbn>2041-4889</isbn><urls></urls></record></Cite></EndNote>[13]；YTHDF1在胃癌中高表达，其表达水平与CD8+T细胞、B细胞等免疫细胞的浸润水平呈显著负相关，同时与调节性T细胞和T细胞耗竭标志物的表达呈负相关，表明其在调节免疫细胞浸润方面发挥重要作用ADDINEN.CITE<EndNote><Cite><Author>Liao</Author><Year>2024</Year><RecNum>28</RecNum><DisplayText><styleface="superscript">[14]</style></DisplayText><record><rec-number>28</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741920287">28</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Liao,Quan</author><author>Xiong,Jianping</author></authors></contributors><titles><title>YTHDF1regulatesimmunecellinfiltrationingastriccancerviainteractionwithp53</title><secondary-title>ExperimentalandTherapeuticMedicine</secondary-title></titles><periodical><full-title>ExperimentalandTherapeuticMedicine</full-title></periodical><pages>255</pages><volume>27</volume><number>6</number><dates><year>2024</year></dates><isbn>1792-0981</isbn><urls></urls></record></Cite></EndNote>[14]；YTHDF1在肝细胞癌中表达异常，通过调控m6A修饰的mRNA的翻译效率，影响肿瘤的发生发展ADDINEN.CITE<EndNote><Cite><Author>Shao</Author><Year>2025</Year><RecNum>29</RecNum><DisplayText><styleface="superscript">[15]</style></DisplayText><record><rec-number>29</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741920467">29</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Shao,Na</author><author>Xi,Lei</author><author>Lv,Yangfan</author><author>Idris,Muhammad</author><author>Zhang,Lin</author><author>Cao,Ya</author><author>Xiang,Jingyi</author><author>Xu,Xi</author><author>Ong,BelindaX</author><author>Zhang,Qiongyi</author></authors></contributors><titles><title>USP5stabilizesYTHDF1tocontrolcancerimmunesurveillancethroughmTORC1-mediatedphosphorylation</title><secondary-title>NatureCommunications</secondary-title></titles><periodical><full-title>NatureCommunications</full-title></periodical><pages>1313</pages><volume>16</volume><number>1</number><dates><year>2025</year></dates><isbn>2041-1723</isbn><urls></urls></record></Cite></EndNote>[15]。1.3.1YTHDF1蛋白在神经系统方面的研究进展YTHDF1在小鼠大脑的海马体中表达上调，对空间学习和记忆具有重要作用，并且在神经元刺激下，通过增强靶向m6A修饰转录本的蛋白质翻译效率，促进学习和记忆；YTHDF1在小脑颗粒细胞中表达下调，而其缺失会显著增加颗粒细胞的轴突生长速率，促进小脑平行纤维的生长和突触形成，以及运动协调能力ADDINEN.CITE<EndNote><Cite><Author>Ren</Author><Year>2023</Year><RecNum>30</RecNum><DisplayText><styleface="superscript">[16]</style></DisplayText><record><rec-number>30</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741920954">30</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>Ren,Wenjun</author><author>Yuan,Yixiao</author><author>Li,Yongwu</author><author>Mutti,Luciano</author><author>Peng,Jun</author><author>Jiang,Xiulin</author></authors></contributors><titles><title>ThefunctionandclinicalimplicationofYTHDF1inthehumansystemdevelopmentandcancer</title><secondary-title>BiomarkerResearch</secondary-title></titles><periodical><full-title>BiomarkerResearch</full-title></periodical><pages>5</pages><volume>11</volume><number>1</number><dates><year>2023</year></dates><isbn>2050-7771</isbn><urls></urls></record></Cite></EndNote>[16]。

2、前言心肌纤维化作为众多心血管疾病发展的重要病理机制，其主要表现为细胞外基质的异常沉积，这一过程将引发心脏结构重塑与功能衰竭。近年来，RNA表观遗传修饰m6A被证实在治疗肿瘤类疾病中有着重要作用。但其在心肌纤维化中的功能却尚未明确。本研究旨在通过构建心肌纤维化小鼠模型，综合运用qRT-PCR等分子生物学手段，探究m6A的阅读器YTHDF1对小鼠心肌成纤维细胞活化及纤维化进程的调控是否有关。本研究的开展对开发基于m6A修饰的靶向治疗策略具有重要意义。

结果4.1YTHDF1在心肌梗死诱导的纤维化小鼠心脏组织中表达上调为了探究m6A阅读器是否参与心肌梗死诱导的心肌纤维化调节，本研究首先检测了心肌梗死小鼠心脏组织中几种常见m6A阅读器蛋白的mRNA表达水平，涉及YTHDF1、YTHDF2、YTHDF3、YTHDC1和YTHDC2（图-1A-1B）。结果显示，YTHDF1在心肌梗死（MI）小鼠中的表达上调最为显著。在心肌纤维化的发生和发展过程中，心肌组织中的多种细胞类型均参与其中，而心肌细胞和心肌成纤维细胞是心脏组织中的两种主要细胞类型ADDINEN.CITE<EndNote><Cite><Author>韩国玲</Author><Year>2024</Year><RecNum>4</RecNum><DisplayText><styleface="superscript">[17]</style></DisplayText><record><rec-number>4</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741614821">4</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>韩国玲</author><author>郝琰琰</author><author>李若朴</author><author>刘维静</author><author>刘俊</author><author>聂宇</author><author>白丽娜</author><author>王玉瑶</author></authors></contributors><auth-address>山西医科大学基础医学院生物化学与分子生物学教研室;北京协和医学院中国医学科学院阜外医院国家心血管病中心心血管疾病国家重点实验室;</auth-address><titles><title>骨髓源性生长因子缺失导致小鼠心肌梗死后纤维化加剧</title><secondary-title>四川大学学报(医学版)</secondary-title></titles><periodical><full-title>四川大学学报(医学版)</full-title></periodical><pages>886-892</pages><volume>55</volume><number>04</number><keywords><keyword>骨髓源性生长因子</keyword><keyword>心肌纤维化</keyword><keyword>心肌梗死</keyword><keyword>肌成纤维细胞</keyword></keywords><dates><year>2024</year></dates><isbn>1672-173X</isbn><call-num>51-1644/R</call-num><urls><related-urls><url>/interlibSSO/goto/11/+jmr9bmjh9mds/kcms2/article/abstract?v=DnpHqYycDUNakSfDB_nMpjctxaoe9SBV5ung3AWxoc6b2PhmqVv6aTO5MjjRFjyfty9aV_iZHzuOfsM7a9POX6poZ_3KAnn1O212atRocWKe1X-01VMZ1xarVi9ONc3WVUi3mguyh_d1SHcjWCcbY9SHfMq3Uj4isr-3KwPuOMVyOYxF6QWGKjcjG2SfYDk6-LbYCaEKAhY=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[17]。鉴于此，为明确YTHDF1的细胞特异性表达，本研究通过分析人类蛋白质ATLAS数据库，并结合qRT-PCR实验进行验证。结果表明，YTHDF1主要在心肌成纤维细胞中表达（图-1C-1D）。图1.YTHDF1在心肌梗死（MI）诱导的小鼠中上调。（A）在心肌梗死（MI）的小鼠中m6A阅读器蛋白的mRNA表达水平。n=3。（B）对小鼠心脏中YTHDF1蛋白表达的代表性Westernblot图像及其定量分析。n=3。（C）人类蛋白质ATLAS数据库中关于YTHDF1在多种心肌细胞中的表达情况。（D）来自新生小鼠的心肌细胞（CMs）和心肌成纤维细胞中YTHDF1的mRNA表达水平。n=3。*P<0.05，**P<0.01vs.假手术组/心肌细胞组。Figure1.YTHDF1isupregulatedinmyocardialinfarction(MI)-inducedmice.(A)mRNAexpressionlevelsofm6Areaderproteininmicewithmyocardialinfarction(MI).n=3.(B)RepresentativeWesternblotimagesofYTHDF1proteinexpressioninmouseheartsandtheirquantitativeanalyses.n=3.(C)HumanProteinATLASdatabaseontheexpressionofYTHDF1inawiderangeofcardiomyocytes.n=3.(D)mRNAexpressionlevelsofYTHDF1incardiomyocytes(CMs)andcardiacfibroblastsfromneonatalmice.n=3.*P<0.05,**P<0.01vs.sham-operatedgroup/cardiomyocytegroup.4.2YTHDF1在活化的心肌成纤维细胞中表达上调在体外实验中，我们分离了乳鼠的心肌成纤维细胞，并采用TGF-β诱导细胞48小时，以此构建体外心肌纤维化模型ADDINEN.CITE<EndNote><Cite><Author>牛丕莲</Author><Year>2022</Year><RecNum>5</RecNum><DisplayText><styleface="superscript">[18]</style></DisplayText><record><rec-number>5</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741615061">5</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>牛丕莲</author><author>范永鑫</author><author>路富瑞</author><author>周学章</author><author>白明生</author></authors></contributors><auth-address>宁夏大学生命科学学院;吉林大学;</auth-address><titles><title>甘草提取物对TGF-β1诱导心肌成纤维细胞纤维化的影响</title><secondary-title>中国临床药理学与治疗学</secondary-title></titles><periodical><full-title>中国临床药理学与治疗学</full-title></periodical><pages>129-135</pages><volume>27</volume><number>02</number><keywords><keyword>心肌纤维化</keyword><keyword>转化生长因子-β1</keyword><keyword>TGF-β1/Smad信号通路</keyword><keyword>甘草提取物</keyword><keyword>心肌成纤维细胞</keyword></keywords><dates><year>2022</year></dates><isbn>1009-2501</isbn><call-num>34-1206/R</call-num><urls><related-urls><url>/interlibSSO/goto/11/+jmr9bmjh9mds/kcms2/article/abstract?v=DnpHqYycDUNdAreMo-nbtJAldW34Yu1T_8GaztdLZLya5Z0_2PhpxDhR-nGAc5Z4V57YGqGNvcFmXILctL52Bez8dg2lDbi6C46h0n76nxY8jWtVeKYZ93UkVchjoDWNkYgzOQ36AUxAoCHKAmEHOyQdGcZw9uQoGc_ZOmihit7Q7cvs4JwAc_9p61iWSeuQgte-Gyp5-mU=&uniplatform=NZKPT&language=CHS</url></related-urls></urls><remote-database-provider>Cnki</remote-database-provider></record></Cite></EndNote>[18]。所得结果与体内实验结果一致，活化的心肌成纤维细胞中YTHDF1的mRNA和蛋白表达水平显著上升。之后，通过免疫荧光染色法检测YTHDF1在成纤维细胞中的表达情况。结果显示（图2C），与阴性对照组（Ctrl组）相比，TGF-β处理显著增强了YTHDF1的荧光强度。此外，我们还分离了成年小鼠的心肌成纤维细胞，并检测到在心肌梗死（MI）小鼠的心肌成纤维细胞中，YTHDF1的mRNA表达水平显著升高（图2D）。这些结果表明，YTHDF1在活化的心肌成纤维细胞中表达上调，且这一现象在体外和体内模型中均得到证实。图2.YTHDF1在TGF-β诱导的心肌成纤维细胞中上调。（A）经TGF-β处理后的新生小鼠心肌成纤维细胞中，YTHDF1的mRNA表达水平显著上调。n=3。(B)经TGF-β处理后的新生小鼠心肌成纤维细胞中，YTHDF1蛋白的表达水平显著上调。n=3。（C）新生小鼠心肌成纤维细胞中YTHDF1蛋白表达水平的代表性图像。比例尺=20µm。n=3。（D）成年雄性C57BL/6小鼠心肌成纤维细胞中YTHDF1的蛋白表达水平。n=3。*P<0.05,**P<0.01vs.阴性对照组。Figure2.YTHDF1isupregulatedinTGF-β-inducedcardiacfibroblasts.(A)ThemRNAexpressionlevelofYTHDF1wassignificantlyup-regulatedinneonatalmousecardiacfibroblaststreatedwithTGF-β.n=3.(B)TheexpressionlevelofYTHDF1proteinshowedasignificantincreaseinneonatalmousecardiacfibroblaststreatedwithTGF-β.n=3(C)TheexpressionlevelofYTHDF1proteininneonatalmousecardiacfibroblastsRepresentativeimagesofYTHDF1proteinexpressioninneonatalmousecardiacfibroblasts.Scalebar=20µm.n=3.(D)ProteinexpressionlevelsofYTHDF1incardiacfibroblastsofadultmaleC57BL/6mice.n=3.*P<0.05,**P<0.01vs.negativecontrols.

5、讨论据统计，城乡居民疾病死亡构成比中，心血管疾病(cardiovasculardiseases，CVD)占首位，2021年农村、城市心血管疾病分别占死因的48.98%和47.35%ADDINEN.CITE<EndNote><Cite><Author>刘明波</Author><Year>2024</Year><RecNum>9</RecNum><DisplayText><styleface="superscript">[19]</style></DisplayText><record><rec-number>9</rec-number><foreign-keys><keyapp="EN"db-id="052t0et5aeetv1et0zkpr0aerzzx5adewr99"timestamp="1741615686">9</key></foreign-keys><ref-typename="JournalArticle">17</ref-type><contributors><authors><author>刘明波</author><author>何新叶</author><author>杨晓红</author><author>王增武</author><author>胡盛寿</author></authors></contributors><auth-address>国家心血管病中心北京协和医学院中国医学科学院阜外医院;</auth-address><titles><title>《中国心血管健康与疾病报告2023》概要（心血管疾病流行及介入诊疗状况）</title><secondary-title>中国介入心脏病学杂志</secondary-title></titles><periodical><full-title>中国介入心脏病学杂志</full-title></periodical><pa