CDE饮食诱导慢性肝损伤小鼠肝组织YAP信号对肝祖细胞增殖的影响

∗基金项目:国家新药开发重大科技专项基金资助项目(编号: 2018ZX09201016)
作者单位:200080上海市上海交通大学附属第一人民医院消化科
第一作者:沈镇扬,男,25岁,硕士研究生㊂主要从事肝再生与非酒精性脂肪性肝病相关研究㊂E-mail:szycheerup@ 通讯作者:蔡晓波,E-mail:caixiaobo19790719@ ㊃实验性肝炎㊃
CDE饮食诱导慢性肝损伤小鼠肝组织YAP信号
对肝祖细胞增殖的影响∗
沈镇扬,王俊俊,陆伦根,蔡晓波
㊀㊀ʌ摘要ɔ㊀目的㊀探讨Yes相关蛋白(YAP)信号对胆碱缺乏/乙硫氨酸补充(CDE)饮食诱导慢性肝损伤模型小鼠肝
组织胆管反应的影响以及体外对肝祖细胞增殖的影响㊂方法㊀采用CDE饮食喂养雄性野生型C57BL/6J小鼠3w,采用Western blot和qPCR法检测肝组织YAP蛋白表达和基因水平;采用免疫组化法检测肝组织CK19表达,观察胆管反应程度;采用免疫荧光评估胆管反应细胞过程中YAP表达;采用两步灌注法分离原代肝祖细胞,以慢病毒感染肝祖细胞,将细胞分为YAP过表达(YAP-OE组)㊁过表达空载体(OE-control组)㊁YAP敲减(shYAP组)和敲减空载体(sh-control组),采用Western blot法检测肝组织YAP表达,采用CCK8和EdU实验评估YAP对肝祖细胞增殖能力的影响㊂结果㊀CDE造模组肝组织YAP蛋白表达比对照组增强,其YAP mRNA水平比对照组提高了2.45倍(P<0.01);CDE造模组肝组织胆管反应程度和肝祖细胞YAP表达显著高于对照组;EdU检测发现在相同培养条件下,YAP-OE组EdU阳性细胞比例较OE-control组显著增加ʌ分别为(0.41ʃ0.05)和(0.25ʃ0.06),P<0.01ɔ,CCK8检测提示在培养72h时YAP-OE组细胞增殖活性比OE-control组提高了1.78倍(P<0.01),而在相同培养条件下,EdU检测发现shYAP组EdU阳性细胞比例较sh-control组显著减少ʌ分别为(0.25ʃ0.04)和(0.13ʃ0.02),P<0.01ɔ,在培养72h时CCK8检测提示shYAP组细胞增殖活性较sh-control组降低了1.92倍(P<0.01)㊂结论㊀CDE饮食诱导小鼠慢性肝损伤导致肝组织胆管反应活跃的祖细胞YAP 表达增强,YAP在体外能促进肝祖细胞增殖㊂
㊀㊀ʌ关键词ɔ㊀肝祖细胞;Yes相关蛋白;胆管反应;细胞增殖;小鼠
㊀㊀DOI:10.3969/j.issn.1672-5069.2021.06.007
㊀㊀Impact of YAP signal on proliferation of liver progenitor cells in mice with CDE diet-induced chronic liver injury㊀Shen Zhenyang,Wang Junjun,Lu Lungen,et al.Department of Gastroenterology,General Hospital,Jiaotong University School of Medicine,Shanghai200080,China
㊀㊀ʌAbstractɔ㊀Objective㊀The aim of this experiment was to investigate the impact of yes-associated protein(YAP)signal on hepatic ductular reaction in mice with choline-deficient and ethionine-supplemented(CDE)diet-induced chronic liver injury (CLI)and YAP s effect on liver progenitor cell proliferation in vitro.Methods㊀The male wild-type C57BL/6J mice were fed with CDE diet for3weeks.The Western blot and qPCR were applied to detect YAP protein and gene levels in liver tissue.The immunohistochemistry were used to detect hepatic ductular reaction,the immunofluorescence to evaluate the YAP expression in ductular reaction cells,and the two-step perfusion method was used to isolate primary liver progenitor cells.The liver progenitor cells were infected by lentivirus and were divided into YAP overexpression(YAP-OE group),overexpression empty vector(OE-control group),YAP knockdown(shYAP group),and knockdown empty vector(sh-control group).The infection efficacy was evaluated by Western K8and EdU experiments were applied evaluate the effects of YAP on the liver progenitor cell proliferation.Results㊀The expression of YAP protein inliver tissues in the CDE-induced model increased greatly compared to in the control group and the YAP mRNA level increased by2.45times than that in the control group(P<0.01);the degree of hepatic ductular response in the CDE model and the YAP mRNA level in liver progenitor cells were higher than in the control group;the
EdU test found that under the same culture conditions,the
percentage of EdU positive cells in the YAP-OE group was
significantly increased compared to in the OE-control group
[(0.41ʃ0.05)vs.(0.25ʃ0.06),respectively,P<0.01];
the CCK8detection showed that the proliferation activity of cells
in the YAP-OE group was1.78times higher than that in the OE
-control group at72hours of culture(P<0.01);the EdU test
found that the proportion of EdU positive cells in the shYAP
group was significantly lower than that in the sh-control group[(0.25ʃ0.04)vs.(0.13ʃ0.02),respectively,P<0.01];the CCK8detection indicated that the proliferation activity of cells in the shYAP group was1.92times lower than that in the sh-control group after72hours of culture(P<0.01).Conclusion㊀The CDE diet could induce chronic liver injury in mice,which results in the increase of YAP expression in progenitor cells with active ductual response.The YAP might promote the proliferation of liver progenitor cells in vitro.
㊀㊀ʌKey wordsɔ㊀Liver progenitor cells;Yes-associated protein;Ductular reaction;Proliferation;Mice
㊀㊀慢性肝病时,肝祖细胞的增殖形式主要表现为胆管反应[1-3]㊂Hippo信号通路与细胞增殖密切相关,Yes相关蛋白(Yes-associated protein,YAP)作为其核心效应分子在其中起到了重要的作用[4-9]㊂本研究构建了胆碱缺乏㊁乙硫氨酸补充(choline-defi-cient㊁ethionine-supplemented,CDE)饮食小鼠模型,并采用免疫组化染色观察到模型组肝组织胆管反应增多,经Western blot和qPCR方法检测发现模型组肝组织YAP表达增高,免疫荧光染色进一步确定模型组肝祖细胞YAP表达增强㊂本研究在体外通过YAP过表达和敲减慢病毒感染肝祖细胞,并采用EdU和CCK8检测证明肝祖细胞YAP过表达能促进细胞增殖,而敲减YAP后细胞增殖减弱,以上研究为慢性肝病肝再生的调控机制和治疗研究提供了新的思路㊂
1㊀材料与方法
1.1动物㊁病毒与试剂㊀6~8周龄雄性野生型C57BL/6J小鼠购自维通利华实验动物公司,饲养在上海市第一人民医院实验动物中心;过表达慢病毒和敲减慢病毒购自上海吉凯基因公司;William E培养基㊁胎牛血清㊁青霉素和链霉素等细胞培养液均购自美国Gibico公司;蛋白提取试剂,BCA蛋白浓度测定试剂等购自上海雅酶生物公司;RNA提取试剂㊁逆转录试剂和qPCR试剂均购自日本TAKARA公司;抗YAP抗体㊁荧光标记的二抗购自美国CST公司;抗GAPDH抗体和辣根过氧化物酶标记的二抗购自上海生工生物公司;抗CK19抗体购自武汉塞维尔公司;细胞计数试剂盒8-CCK8(日本Dojinto公司); EdU增殖检测试剂盒(广州锐博生物公司);免疫组化试剂盒(上海基因科技公司);HE染色试剂盒(武汉赛维尔公司)㊂
1.2CDE饮食小鼠模型制备㊀将15只小鼠随机分成3组,每组5只㊂给予第一组小鼠正常饮食和饮水(wild type,WT组);给予第二组CDE饮食(CDE 组);给予第三组相同CDE饮食,用于提取原代肝祖细胞(原代细胞提取组)㊂在造模3w后,处死各组小鼠,取出肝脏,用于后续实验㊂
1.3原代肝祖细胞分离与培养㊀参照先前文献报道的方法[10],在原代细胞提取组小鼠,取肝脏,采用两步灌注法和密度梯度离心法分离肝祖细胞㊂以1ˑ106cell/ml密度接种于含William s E完全培养基含10%胎牛血清㊁100U/ml青霉素-链霉素溶液(美国Gibico公司)㊁10μg/ml胰岛素(上海翊圣公司)㊁30 ng/ml胰岛素样生长因子II和20ng/ml表皮生长因子(美国PeproTech公司)]的培养皿中,放入37ħ㊁5%CO2的培养箱中培养㊂5d后,用克隆环挑取单克隆细胞集落,传代培养3代,获得稳定的肝祖细胞,用于后续实验㊂
1.4肝组织蛋白表达检测㊀采用Western blot法,在肝组织中加入小钢珠和含PMSF的RIPA裂解液,在组织振荡裂解仪和超声裂解仪裂解组织,离心取上清,采用BCA法测定蛋白含量并调整浓度使其均一㊂在100ħ金属浴煮沸10min,充分变性,上样蛋白并行SDS-PAGE电泳㊂然后,将蛋白转移到PVDF膜上,加5%脱脂牛奶室温封闭1h,用TBST 洗涤5min,3次㊂加抗YAP抗体或抗GAPDH抗体,4ħ孵育过夜;用TBST洗涤5min,3次;再加山羊抗兔辣根过氧化物酶标记的二抗,室温孵育1h;用TBST洗涤5min,3次;用超敏ECL化学发光液显影㊁拍照㊂
1.5肝组织基因水平检测㊀采用实时荧光定量PCR 法,用Trizol法提取组织RNA,测定各组RNA含量㊂取RNA1000ng,在37ħ15min和85ħ5s行逆转录,采用PCR法检测YAP mRNA水平,扩增所用的引物由上海生工生物公司合成,其序列见表1㊂1.6肝组织蛋白表达检测㊀采用免疫组化和免疫荧光法,取石蜡包埋组织切片,脱蜡㊁水化,用柠檬酸抗原修复液进行抗原修复,加内源性过氧化物酶阻断剂(武汉博士德公司)阻断15min,加5%BSA室温封闭1h,加抗CK19抗体,4ħ过夜,再加生物素标记的二抗,室温孵育1h,加DAB显色剂显色,苏木素复染核,封片后在光镜下拍照;在免疫荧光检测前,脱蜡水化抗原修复同前,加5%BSA室温封闭1h,加抗CK19或抗YAP,4ħ过夜,加荧光标记的二抗,室温避光孵育1h,加DAPI染色10min,用抗荧光淬灭封片剂(上海翊圣公司)封片,在荧光显微镜下观察㊂
表1㊀qPCR引物序列
基因正向引物反向引物
YAP CAAATACAGCTGCAG-
CAGTTAC AGAGCTAATTCCTGC-CGAAATA
GAPDH AGGTCGGTGTGAACG-
GATTTG TGTAGACCATGTAGT-TGAGGTCA
1.7慢病毒转染㊀将生长良好的肝祖细胞接种到24孔板,调整细胞密度为2ˑ105/mL,37ħ㊁5%CO2培养箱中培养,待细胞培养至50%融合度时,换新培养基㊂随机将细胞分为四组:分别加入YAP过表达慢病毒(YAP-overexpression,YAP-OE)㊁过表达空载体慢病毒(overexpression-control,OE-control)㊁YAP敲减慢病毒(short hairpin RNA-YAP,shYAP)和敲减空载体慢病毒(short hairpin RNA-control,sh-control)原液各10μl,混匀后,在培养箱中继续培养3d㊂在荧光显微镜下观察荧光强度㊂转染成功的细胞表达绿色荧光蛋白㊂对转染的细胞进行5μg/ml的嘌呤霉素筛选,筛选完成后收集各组蛋白和RNA,进行后续Western blot和qPCR验证㊂
1.8EdU细胞增殖检测㊀将各组肝祖细胞以5ˑ104/mL 密度接种在24孔板中,24h后,根据制造商说明书要求处理细胞,在荧光显微镜下拍照观察,并计算各组细胞增殖率㊂
1.9细胞活力测定㊀将各组肝祖细胞以
2.5ˑ104/mL 密度接种在96孔板中,使用细CCK-8试剂盒,根据制造商说明书在12h㊁24h㊁48h和72h,用酶标仪测定450nm波长处的吸光度(OD值),即细胞活力检测㊂
1.10统计学方法㊀应用SPSS v.23.0统计学软件包分析㊂计量资料以(xʃs)表示,采用t检验和one-way ANOVA检验㊂P<0.05表示差异有统计学意义㊂
2㊀结果
2.1各组小鼠肝组织病理学表现㊀野生型小鼠经CDE饮食喂养3w后,肝组织炎症和胆管样增生反应较对照组明显;免疫组化检测显示,CDE组肝组织胆管反应增强(图1)㊂
2.2各组小鼠肝脏YAP表达比较㊀在CDE造模3w 后,经Western blot和qPCR检测发现,CDE造模组小鼠肝组织YAP蛋白表达水平较对照组增强㊂与此同时,CDE造模组YAP mRNA水平较对照组提高了2.45倍(P<0.01)㊂免疫荧光检测提示,CDE造模组肝组织CK19和YAP共表达的细胞数较对照组增加(图
2)㊂
图1㊀各组肝组织病理学表现
A:WT组(HE,100ˑ);B:CDE组(HE,100ˑ);C:WT 组(CK19染色,100ˑ);D:WT组(CK19染色,400ˑ);E: CDE组(CK19染色,100ˑ);F:CDE组(CK19染色,400ˑ
)
图2㊀各组小鼠肝组织YAP水平比较
A:两组蛋白表达;B:两组Yap mRNA水平,与WT组
比,∗∗P<0.05;C:WT组CK19和YAP荧光表达水平(400ˑ);D:CDE组CK19和YAP荧光表达水平(400ˑ) 2.3各组肝祖细胞增殖能力比较㊀肝祖细胞稳定过表达YAP后,EdU检测发现在相同培养条件下, YAP-OE组EdU阳性细胞比例较OE-control组显著增加[分别为(0.41ʃ0.05)和(0.25ʃ0.06),P< 0.01];CCK8检测提示随着培养时间的延长,YAP-OE组细胞增殖活性较OE-control组明显提高,在培养72h时较对照组提高了1.78倍(P<0.01);在敲减YAP后,EdU检测发现在相同培养条件下,shYAP 组EdU阳性细胞比例较sh-control组显著减少[分别为(0.25ʃ0.04)和(0.13ʃ0.02),P<0.01],CCK8检测提示随着培养时间的延长,shYAP组细胞增殖活性较sh-control组明显降低,在培养72h时较对照组降低了1.92倍(P<0.01)㊂综合以上可知,肝祖细胞YAP过表达后其增殖能力显著高于YAP敲减组(图3)㊂
3㊀讨论
肝祖细胞是一种卵圆形㊁高核质比的细胞,位于肝实质细胞与汇管区交界的Hering s管,从形态和体积上类似于胆管上皮细胞㊂肝祖细胞具有肝细胞㊁胆管细胞双向分化潜能,同时表达胆管上皮细胞标志物CK7㊁CK19和肝细胞标志物白蛋白㊁CK8㊁
图3㊀各组肝祖细胞增殖能力比较
A:各组YAP蛋白水平验证;B:EdU检测各组细胞增殖能力变化(100ˑ);C:各组EdU阳性细胞比例差异;D: CCK8检测各组细胞增殖能力变化(与对照组比,∗∗P< 0.05,∗∗∗P<0.01)
AFP,也表达干细胞标志物CD133㊁OV6和NCAM 等㊂最近一个对人胚胎肝脏单细胞分析的研究进一步确认其存在[5,11]㊂在慢性肝损伤状态下,当正常肝细胞衰老或增殖被抑制时,肝祖细胞可分化成为成熟的肝细胞㊂由于在病理而非生理条件下参与肝脏的更新,因此被称为肝脏再生的次要机制或病理性再生[4,12]㊂因此,在慢性肝损伤时,肝祖细胞的有效增殖和分化或许是决定这些肝病患者预后的关键㊂
多种信号通路参与肝祖细胞增殖和分化的调控,如HGF/c-Met㊁FGF17㊁Hedgehog等[4]㊂Hippo信号通过调控细胞增殖㊁凋亡和干细胞自我更新来控制器官组织再生,该通路失调会导致器官持续性增生和肿瘤的发生等重大疾病㊂YAP转录共激活因子是Hippo信号通路关键效应分子,在该通路被激活时,YAP发生磷酸化修饰而滞留在胞质无法行使转录激活功能㊂相反,该通路被抑制后,非磷酸化修饰的YAP能够进入细胞核与转录因子TEAD等结合进而启动下游一系列促增殖㊁抗凋亡和细胞干性相关基因的表达㊂近年来,越来越多的研究显示YAP 也参与肝脏炎症㊁纤维化和再生㊂在非酒精性脂肪性肝病,YAP活化的活性胆管细胞数量与肝纤维化进展相关㊂在化学损伤诱导的小鼠模型,肝细胞中上调的YAP水平被认为能促进肝脏炎症和纤维化㊂使用MST1/MST2抑制剂激活YAP,能增强对乙酰氨基酚和四氯化碳中毒或胆管结扎引起的急慢性损伤后的肝脏修复,并减轻肝纤维化㊂另一项研究表明,成年小鼠肝细胞活化的YAP过表达可触发其转分化为祖细胞样细胞,该细胞可能分化为胆管上皮细胞或再分化为肝细胞㊂总之,YAP的激活可能通过两种不同的机制来促进肝脏再生:肝细胞增殖和转分化为祖细胞样细胞㊂
为进一步明确YAP对肝祖细胞的增殖是否有影响,本研究构建了慢性肝损伤模型来观察损伤情况下肝脏胆管反应程度与YAP表达之间的关系,研究结果显示损伤组肝脏YAP蛋白和基因水平明显增加,肝祖细胞YAP表达也显著高于对照组,表明肝组织特别是肝祖细胞YAP表达与慢性肝损伤时胆管反应相关㊂在体外,我们分离了原代肝祖细胞,并构建过表达和敲减YAP慢病毒载体感染的肝祖细胞,结果发现肝祖细胞过表达YAP后细胞增殖能力得到了显著的提升,而敲减YAP后肝祖细胞的增殖受到了显著的抑制㊂
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(收稿:2020-11-10)
(本文编辑:陈从新)。