人脐带间充质干细胞（hUC-MSC）对肝纤维化小鼠模型的治疗作用及其机制分析.docx

肝纤维化及肝硬化DOI：10.12449/JCH240315人脐带间充质干细胞(hC-MSC)对肝纤维化小鼠模型的治疗作用及其机制分析刘平箕，姚黎超，胡雪，王铮，熊芷玉，江应安武汉大学人民医院感染科，武汉430060通三f:海安,jiangya_cn(ORCID:00-03-4912-7494)摘要：目的探讨人脐带间充质干细胞(hC-MSC)对肝纤维化小鼠的治疗作用及其机制。方法选取18只SPF级6周龄C57BU6小鼠，使用随机数字表法三l分为3组，分别为对照组、CCL模型组(Ca组)和hUGMSC治疗组(MSC组),每§6只。Cd,组和MSC组腹腔注射CCL溶液构建小鼠肝纤维化模型，对照组同时注射相同剂量玉米油，MSC组在注射CCI,的过程经尾静脉注射hC-MSC于第8周末采取小鼠血清，处死小鼠，取小鼠肝脏固定。使用酶联免疫吸附法测定炎症因子水平，自动生化检测仪检测肝功能相关指标，HE染色、MaSSon染色、天狼星红染色及-SMA免疫荧光用于评估肝纤维化情况；TGF-刺激的肝星状细胞(HSC)分另!三有无几丁,3样蛋白1(CHI3L1)添加的培养基中与hUGMSC共培养，WesternBlot试验检测蛋白表达水平。计量资料多组间比较采用单因素方差分析，进一步两两比较采用DLlnnett-检验结果Masson染色、天狼星红染色提示CCL组小鼠纤维化较对照组明显(产值均005)zMSC组小鼠纤婚淡Cd组减轻(P值均0.05).CCI4组IL/。、IL6AST、ALT和ALP水平较对照组明显升高(户值均0.05),MSC组IL-6、AST、ALT及ALP水平较CCI,组明显降低(尸值均005).CC组CHI3L1和-SMA的表达均高于对照组和MSC组(尸值均0.05).细胞培养实验显示，MSC+HSC组Bax表达高于HSC组和MSC+CHI3L1组(产值均0.05),提示CHI3L1逆转了MSC对活化的HSC的促凋亡作用.结论hUC-MSC治疗可以改善小鼠肝纤维化，其作用机制可能与抑制CHI3L1从而促进HSC的凋亡相关。关键词：肝纤维化；间充质干细胞；壳多糖酶3样第白质1；小鼠，近交C57BL基金项目：武汉大学教育发展基金会抗衰老研究中心专款(2002330)；中央高校基本科研业务费专项资金(2042022kf1115)Effectofhumanumbilicalcordmesenchymalstemcellsintreatmentofmicewithliverfibrosisanditsmechanism1.IUPingji,YAOLichao,HUXue,WANGZheng,XIONGZhiyu,JIANGYingan.(DepartmentOflnfectiousDiseaseslRenminHospitaIofWuhanUniversity,Wuhan430060,China)Correspondingauthor:JIANGYingan,jiangya_cn(ORCID:0000-0003-4912-7494)Abstract:ObjectiveToinvestigatetheeffectofhumanumbilicalrdmesenchymalstemcells(hllCMSCs)inthetreatmentofmicewithliverfibrosisanditsmechanism.MethodsAtotalof18specificpathogen-freeC57BL6micefaged6weeksrwereselectedanddividedintocontrolgroup(n=6),carbontetrachloride(CCI4)modelgroup(CCI4group,n=6),andhllCMSCstreatmentgroup(MSCgroup,n=6)usingarandomnumbertable.ThemiceintheCCI4groupandtheMSCgroupweregivenintraperitonealinjectionofCCI4solutiontoestablishamousemodelofliverfibrosis,whilethoseinthentrolgroupwereinjectedwiththesamedoseofcornoil,andthemiceintheMSCgroupwereinjectedwithhUCMSCsviathecaudalveinduringtheinjectionofCCI4.Attheendofweek8,mouseserumwasllected,andthemiceweresacrificedtollectandfixtheliver.Enzyme-Iinkedimmunosorbentassaywasusedtomeasurethelevelsofinflammatoryfactors;anautomaticbiochemicaldetectorwasusedtomeasureliverfunctionparameters;HEstaining,Massonstaining,SiriusRedstaining,and-SMAimmunofluorescenceassaywereusedtoevaluateliverfibrosis.Hepaticstellatecells(HSCs)stimulatedbyTGF-wereco-cultredwithhllCMSCsinthemediumwithorwithtchitinase-3like-protein-1(CHI3L1),andWesternblotwasusedtomeasuretheexpressionlevelsofproteinsAOnewayanalysisOfvariancewasusedforcomparisonofcontinuousdatabetweenmultiplegups,andtheDunnett'SAtestwasusedforfurthercomparisonbetweentwogroups.ResultsMassonstainingandSiriusRedstainingshowedthattheCG4grouphadasignificantlyhigherdegreeoffibrosisthanthecontrolgroup(bothP<0,05),andtheMSCgrphadsignificantalleviationoffibrosiscomparedwiththeCG4group(bothP<0,05).Comparedwiththecontrolgroup,theGJ4grouphadsignificantincreasesinthelevelsofinterieukin-l,interleukin-6(L-6),aspartateaminotransferase(AST),alanineaminotransferase(ALT),andalkalinephosphatase(ALP)(allP<0.05),andcomparedwiththeCQgroup,theMSCghadsignificantreductionsinthelevelsofIL-6,AST,ALT,andALP(all尸<0.05).TheCCI4grouphadsignificantlyhigherexpressionlevelsofCHBLLand-SMAthanthecontrolgroupandtheMSCgroup(allP<0.05),TecellcultureexperimentshowedthattheMSC+HSCgrouphadasignificantlyhighere×pressilevelofBaxthantheHSCgroupandtheMSC+CHBL1group(bothP<0.05),suggestingthatCHBLlreversedthepro-apoptoticeffectofMSCOnactivatedHSCs.ConclusiThisstudyshowsthathUCMSCscanimproveIiverfibrosisinmice,possiblybyinhibitingCHBLltopromotetheapoptosisofHSCs.Keywords:HepaticFibrosis;MesenchymalStemCells;Chitinase-S-LikeProtein1;Mice,InbredC57BLResearchfunding:TheGrantFromtheAnti-AgingResearchCenter,WuhanUniversityEducation&DevelopmentFoundation(2002330);TheFundamentalResearchFundsfortheCentralUniversities(2042022kflll5)肝纤维化表现为细胞外基质蛋白（如I型胶原蛋白和纤维连接蛋白）的过度沉积，是慢性病毒感染、酒精性脂肪性肝炎和非酒精性脂肪性肝炎等慢性肝损伤的常见后遗症，严重时可能导致肝硬化伴门静脉高压tl-2j肝纤维化的特征是肝星状细胞（hepaticstellatecell,HSC）的活化和增殖，HSC是产生基质的肌成纤维细胞的主要来源o静态和储存维生素A的HSC的纤维化激活可由来自肝细胞、肝窦内皮细胞、自然杀伤细胞和巨噬细胞等细胞的细胞外信号介导【2.活化的肝星状细胞（aHSC）在肝纤维化过程中发挥关键作用，诱导细胞凋亡以消耗aHSC的抗纤维化策略被寄予厚望，但目前仍缺乏被认可的临床证据.近几十年来，间充质干细胞（mesenchymalstemcell,MSC）治疗已成为慢性肝病最有希望的治疗方法之一1旬MSC可来源于骨髓、脐带和脂肪组织，临床和实验证据表明，MSC可通过自分泌或旁分泌多种免疫抑制因子、营养因子和外泌体，甚至分化为功能性肝细胞，发挥抗纤维化作用，并促进肝再生几丁质酶3样蛋白1（chitinase-3like-protein-1,CHBLl）是糖基水解酶家族成员，肝脏中高度富含CHBLl,巨噬细胞、中性粒细胞和HSC等均可以表达CHBLl161e研究0】表明，血清CHBLl可作为代偿期肝硬化患者发生首次失代偿事件风险的有效预测因子，且在联合Child-Pugh分级后具有更高的预测价值.但CHI3L1在肝纤维化中的具体作用及其是否与人脐带MSC（humanumbilicalcordderivedmesenchymalstemcell,hUC-MSC）关尚不清楚，本实验将对此研究探讨。1材料与方法1. 1瞬18只SPF级6周龄C57BL/6小鼠，小鼠由武汉大学人民医院中心试验室提供（生产许可证号:SCXK2019-04,蛹许可证号:SYXK2015-27）并饲养于该实验室，适应性饲养1周后使用随机数字表法随机分为3组，分别为对照组、CCI4睡组（CCI4组）和hC-MSC治疗组（MSC组）,每组6只。以ImgZkgCQl腹腔注射构建小鼠肝纤维化模型，CCI4以1:9的比例溶于玉米油溶液中CCl4组和MSC组每周腹腔注射10%CCLt溶液2次，连续注射8周，对照组同时注射相同剂量玉米油，MSc组在注射CCl,溶液的第7、8周经尾静脉注射hUC-MSC（IX吩个/只），每周注射2次。于第8周末采取小鼠血清，处死小鼠，取小鼠肝脏固定，每组选择4只小鼠检测血清，MSC组1只小鼠血清样本不合格。1.2 实验试剂CCI4和玉米油购于上海Sigma