补体应答基因32在小鼠部分肝切除后肝再生过程中的表达及意义.pdf
《补体应答基因32在小鼠部分肝切除后肝再生过程中的表达及意义.pdf》由会员分享,可在线阅读,更多相关《补体应答基因32在小鼠部分肝切除后肝再生过程中的表达及意义.pdf(10页珍藏版)》请在咨信网上搜索。
1、临床肝胆病杂志第39卷第10期2023年10月 J Clin Hepatol,Vol.39 No.10,Oct.2023补体应答基因32在小鼠部分肝切除后肝再生过程中的表达及意义李兴元1,杨艳芳2,陈琰1,胡文慧1,赵小英3,唐俊明3,孔德营11 遵义医科大学基础医学院生理学教研室,贵州 遵义 563000;2 贵州省人民医院中心实验室,贵阳 550002;3 湖北医药学院基础医学院生理学教研室,湖北 十堰 442000通信作者:孔德营,(ORCID:000000026851955X)摘要:目的探讨补体应答基因32(RGC32)在部分肝切除(PH)术后肝再生过程中的表达及作用。方法42只10周
2、龄雄性C57BL/6小鼠,随机分为对照组 切除小鼠完整的肝脏称重、拍照作为正常对照(sham组),进一步切除肝左叶和肝中叶后称重、拍照作为手术对照(0 d组),sham组和0 d组共用一组小鼠、术后1天组(1 d)、术后2天组(2 d)、术后4天组(4 d)、术后6天组(6 d)、术后8天组(8 d)和术后10天组(10 d),每组6只;PH术建模成功后分别于术后1、2、4、6、8、10天处死小鼠,收集小鼠肝脏,检测肝脏大小变化。HE和油红O染色评估肝组织形态学变化,血清ALT、AST检测评价肝功能变化,免疫组化染色检测增殖细胞核抗原(PCNA)和Ki67表达并分析肝再生过程中细胞增殖变化,实
3、时荧光定量PCR和免疫组化染色技术检测肝再生过程中RGC32的表达及其亚细胞分布。EdU细胞增殖实验分析L02细胞过表达或者敲除RGC32对肝细胞增殖的影响。计量资料多组间比较采用方差分析,进一步两两比较采用LSDt检验;两组间比较采用成组t检验。相关性分析采用Pearson相关分析法。结果PH术后肝脏逐渐增大,第06天为肝体比(肝脏质量/体质量)上升的高峰期,不同时间点比较差异均有统计学意义(P值均0.05);第610天肝脏大小变化不明显。PH术后肝脏脂滴显著增多,随着肝再生,脂滴减少,且呈现门静脉区和中央静脉区差异化(P值均0.05)。与sham组比较,PH术后1天,血清ALT、AST水平
4、明显升高(P值均0.05),随后分别于术后第6天和术后第2天恢复至sham组水平(P值均0.05)。免疫组化染色结果显示,PH术后PCNA和Ki67阳性肝实质细胞数迅速增多,第2天数目最多,分别为865和895,随后逐渐减少;而PCNA和Ki67阳性非实质细胞数逐渐增多,第6天才达到高峰,分别为345和253,随后逐渐减少。PH术后总RGC32表达在第2天升到最高,随后逐渐降低,细胞质RGC32表达变化趋势与之一致;而细胞核RGC32的表达在PH术后第2天降到最低,随后逐渐升高。相关性分析显示,细胞核RGC32的表达与肝实质细胞的增殖呈负相关(R2=0.308 3,P=0.016 7),细胞质
5、RGC32的表达与肝实质细胞的增殖呈正相关(R2=0.808 6,P0.000 1)。细胞实验显示,与对照组相比,RGC32过表达EdU阳性率减少15.6%(P0.01),敲低RGC32表达EdU阳性率增加19.2%(P0.01)。结论肝实质细胞和非实质细胞增殖不同步,共同参与了肝脏再生。肝切除术后肝再生过程中,RGC32表达存在核质分布差异,核RGC32对肝细胞增殖具有抑制作用。关键词:补体应答基因32;肝切除术;肝再生;小鼠,近交C57BL基金项目:国家自然科学基金(31760339);湖北省教育厅科学研究计划资助项目(Q20212107);贵州省科技计划项目(黔科合基础ZK 2022 一
6、般594)Expression and significance of response gene to complement 32 in liver regeneration after partial hepatectomy in miceLI Xingyuan1,YANG Yanfang2,CHEN Yan1,HU Wenhui1,ZHAO Xiaoying3,TANG Junming3,KONG Deying1.(1.Department of Physiology,School of Basic Medical Sciences,Zunyi Medical University,Zu
7、nyi,Guizhou 563000,China;其他肝病DOI:10.3969/j.issn.1001-5256.2023.10.0182396李兴元,等.补体应答基因32在小鼠部分肝切除后肝再生过程中的表达及意义2.Department of Central Laboratory,Guizhou Provincial People s Hospital,Guiyang 550002,China;3.Department of Physiology,School of Basic Medicine,Hubei University of Medicine,Shiyan,Hubei 44200
8、0,China)Corresponding author:KONG Deying,(ORCID:000000026851955X)Abstract:Objective To investigate the expression and role of response gene to complement 32(RGC32)in liver regeneration after partial hepatectomy(PH).MethodsA total of 42 male C57BL/6 mice,aged 10 weeks,were randomly divided into contr
9、ol group,postoperative day 1 group(1d group),postoperative day 2 group(2d group),postoperative day 4 group(4d group),postoperative day 6 group(6d group),postoperative day 8 group(8d group),and postoperative day 10 group(10d group),with 6 mice in each group.In the control group,the complete liver of
10、the mice was resected for weighing and photography as the normal control group(sham group);further,the left and middle lobes of the liver were resected for weighing and photography as the surgical control group(0day group);the sham group and the 0day group shared the same group of mice.After success
11、ful modeling by PH,the mice were sacrificed on days 1,2,4,6,8,and 10 after surgery,and the liver was collected to measure the change in size.HE staining and oil red O staining were used to evaluate liver histomorphological changes;serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST
12、)were measured to evaluate the changes in liver function;immunohistochemical staining was used to measure the expression of proliferating cell nuclear antigen(PCNA)and Ki67 and analyze the change in cell proliferation during liver regeneration;quantitatie realtime PCR and immunohistochemical stainin
13、g were uused to measure the expression and subcellular distribution of RGC32 during liver regeneration;EdU cell proliferation assay was used to analyze the effect of RGC32 overexpression or knocknout on hepatocyte proliferation in L02 cells.For continuous data,comparison between multiple groups was
14、made by analysis of variance,and further pairwise comparisons were conducted using the LSDt test.The independent samples ttest was used for comparison of continuous data between two groups.A Pearson correlation analysis was performed.ResultsThe liver gradually enlarged after PH,and the liver/body we
15、ight ratio rose to the peak from days 0 to 6,with significant differences between different time points(all P0.05),while there was no significant change in liver size from days 6 to 10.The number of liver lipid droplets significantly increased after PH surgery and gradually decreased with liver rege
16、neration,with a significant difference between the portal vein region and the central vein region(all P0.05).Compared with the sham group,the 1d group had significant increases in the serum levels of ALT and AST(all P0.05).Immunohistochemical staining showed that there were rapid increases in the nu
17、mbers of PCNA and Ki67positive liver parenchymal cells after PH surgery,with the highest numbers of 865 and 895,respectively,on day 2,which then gradually decreased;however,there were gradual increases in the numbers of PCNA and Ki67positive nonparenchymal cells,with the peak numbers of 345 and 253,
18、respectively,on day 6,which then gradually decreased.The total expression of RGC32 increased to the highest level on day 2 after PH surgery and then gradually decreased,and the changing trend of RGC32 expression in cytoplasm was consistent with that of total RGC32 expression;however,the expression o
19、f RGC32 in nucleus decreased to the lowest level on day 2 after PH surgery and then increased gradually.The correlation analysis showed that the expression of RGC32 in nucleus was negatively correlated with the proliferation of liver parenchymal cells(R2=0.308 3,P=0.016 7),and the expression of RGC3
20、2 in cytoplasm was positively correlated with the proliferation of liver parenchymal cells(R2=0.808 6,P0.000 1).Cell experiments showed that compared with the control group,the EdUpositive rate was reduced by 15.6%after RGC32 overexpression(P0.01)and was increased by 19.2%after RGC32 knockdown(P0.01
21、).ConclusionLiver parenchymal cells and nonparenchymal cells show asynchronous proliferation and participate in liver regeneration together.During liver regeneration after hepatectomy,there are differences in the expression of RGC32 between nucleus and cytoplasm,and RGC32 in nucleus may inhibit hepa
22、tocyte proliferation.Key words:RGC32;Hepatectomy;Liver Regeneration;Mice,Inbred C57BLResearch funding:National Natural Science Foundation of China(31760339);Scientific Research Program of Education 2397临床肝胆病杂志第39卷第10期2023年10月 J Clin Hepatol,Vol.39 No.10,Oct.2023Department of Hubei Province(Q20212107
23、);Guizhou Provincial Science and Technology Plan Project(Qiankehe FoundationZK 2022 General 594)部分肝切除(partial hepatectomy,PH)术是治疗肝肿瘤、肝内胆管结石等疾病的重要手段,但术后肝衰竭的发生严重影响患者预后,甚至导致患者短期内死亡,无法达到治疗疾病的目的1。PH术后的顺利恢复主要取决于剩余肝脏的迅速再生以及肝功能的迅速恢复2,因此研究肝脏再生的过程及其调控机制十分重要。有研究3表明补体系统在调控两栖类动物肢体再生过程中发挥了重要作用,在一些有尾动物中,其肢体胚芽细胞的再生
24、起始于C3组分的表达。补体应答基因32(response gene to complement 32,RGC32)是一种重要的补体激活基因,补体系统被激活后会促进其表达4。RGC32在肝脏再生过程中是否也发挥重要作用,目前尚不清楚。本研究旨在观察小鼠PH术后肝再生过程中肝脏的结构和功能变化,不同时期细胞增殖情况,以及不同时期RGC32的表达变化,分析PH术后肝再生过程中RGC32对细胞增殖的影响。1材料与方法1.1材料10周龄雄性C57BL/6小鼠42只,由湖北医药学院实验动物中心提供,生产许可证号:SCXK(鄂)20190008,实验动物使用许可证号:SYXK(鄂)20190031。L02细
25、胞来自于湖北省胚胎干细胞重点实验室。增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)(ab29)购自Abcam公司。RGC32(bs9079R)购自北京博奥森生物技术有限公司。ALT、AST检测试剂盒购自南京建成生物工程研究所。实时荧光定量PCR(qRTPCR)试剂购自Toyobo公司。油红O染色液(GP1067)、Ki67(GB121141)、脂肪固定液(G1119)、抗荧光淬灭封片剂(G1401)、柠檬酸修复缓冲液(G1201)、PBS缓冲液(G0002)购自武汉塞维尔生物有限公司。DAB免疫染色试剂(ZLI9018)、苏木素染色液(ZLI9
- 配套讲稿:
如PPT文件的首页显示word图标,表示该PPT已包含配套word讲稿。双击word图标可打开word文档。
- 特殊限制:
部分文档作品中含有的国旗、国徽等图片,仅作为作品整体效果示例展示,禁止商用。设计者仅对作品中独创性部分享有著作权。
- 关 键 词:
- 补体 应答 基因 32 小鼠 部分 切除 再生 过程 中的 表达 意义
1、咨信平台为文档C2C交易模式,即用户上传的文档直接被用户下载,收益归上传人(含作者)所有;本站仅是提供信息存储空间和展示预览,仅对用户上传内容的表现方式做保护处理,对上载内容不做任何修改或编辑。所展示的作品文档包括内容和图片全部来源于网络用户和作者上传投稿,我们不确定上传用户享有完全著作权,根据《信息网络传播权保护条例》,如果侵犯了您的版权、权益或隐私,请联系我们,核实后会尽快下架及时删除,并可随时和客服了解处理情况,尊重保护知识产权我们共同努力。
2、文档的总页数、文档格式和文档大小以系统显示为准(内容中显示的页数不一定正确),网站客服只以系统显示的页数、文件格式、文档大小作为仲裁依据,平台无法对文档的真实性、完整性、权威性、准确性、专业性及其观点立场做任何保证或承诺,下载前须认真查看,确认无误后再购买,务必慎重购买;若有违法违纪将进行移交司法处理,若涉侵权平台将进行基本处罚并下架。
3、本站所有内容均由用户上传,付费前请自行鉴别,如您付费,意味着您已接受本站规则且自行承担风险,本站不进行额外附加服务,虚拟产品一经售出概不退款(未进行购买下载可退充值款),文档一经付费(服务费)、不意味着购买了该文档的版权,仅供个人/单位学习、研究之用,不得用于商业用途,未经授权,严禁复制、发行、汇编、翻译或者网络传播等,侵权必究。
4、如你看到网页展示的文档有www.zixin.com.cn水印,是因预览和防盗链等技术需要对页面进行转换压缩成图而已,我们并不对上传的文档进行任何编辑或修改,文档下载后都不会有水印标识(原文档上传前个别存留的除外),下载后原文更清晰;试题试卷类文档,如果标题没有明确说明有答案则都视为没有答案,请知晓;PPT和DOC文档可被视为“模板”,允许上传人保留章节、目录结构的情况下删减部份的内容;PDF文档不管是原文档转换或图片扫描而得,本站不作要求视为允许,下载前自行私信或留言给上传者【自信****多点】。
5、本文档所展示的图片、画像、字体、音乐的版权可能需版权方额外授权,请谨慎使用;网站提供的党政主题相关内容(国旗、国徽、党徽--等)目的在于配合国家政策宣传,仅限个人学习分享使用,禁止用于任何广告和商用目的。
6、文档遇到问题,请及时私信或留言给本站上传会员【自信****多点】,需本站解决可联系【 微信客服】、【 QQ客服】,若有其他问题请点击或扫码反馈【 服务填表】;文档侵犯商业秘密、侵犯著作权、侵犯人身权等,请点击“【 版权申诉】”(推荐),意见反馈和侵权处理邮箱:1219186828@qq.com;也可以拔打客服电话:4008-655-100;投诉/维权电话:4009-655-100。