生长激素信号通路调节大鼠再生肝的肝细胞增殖北大核心CSCD

生长激素(growth hormone,GH)信号通路对机体生长发育具有重要的调控作用。GH通过与特异性膜表面受体结合,启动下游一系列信号通路反应,进而调控细胞增殖、分化和迁移,防止细胞凋亡等。GH对细胞增殖的调控机制一直以来都是研究的热点,但部分肝切除(partial hepatectomy,PH)后,生长激素相关的信号通路是否会活化,调控相关基因的表达,从而促进肝实质细胞增殖,尚未见报道。本文以percoll密度梯度离心结合磁珠分离的大鼠再生肝的肝细胞为材料,采用Rat Genome 230 2.0芯片与生物信息学相结合的方法,研究GH信号通路对肝再生的调控作用。结果表明,大鼠再生肝的肝细胞中22种基因与GH信号通路相关,其中,Gh1、Jak3、Stat3等14种基因表达上调,Irs3、Ghr、Mras等8种基因表达下调。谱函数(Et)分析基因表达变化预示的细胞增殖活动和信号转导活性表明,GH信号通路的信号传导活性在大鼠肝再生的2~72 h强于对照,所调节的肝细胞增殖活动在6~72 h也强于对照。综上所述,GH信号通路促进大鼠再生肝的肝细胞增殖。     

细胞外基质相关基因在大鼠肝再生中表达模式分析

细胞外基质具有维持细胞极性、调节细胞粘附、增殖、组织器官形态、发生、分化等功能。为了进一步在基因转录水平了解细胞外基质在大鼠肝再生中变化和作用, 用搜集网站资料和查阅相关论文等方法获得细胞外基质基因, 用Rat Genome 230 2.0芯片检测它们在大鼠再生肝中表达情况, 用真、假手术比较方法确定肝再生相关基因。初步证实上述97个基因与肝再生相关。其中, 肝再生启动(部分肝切除(parital hepatectomy, PH)后0.5~4 h)、G0/G1过渡(PH后4~6 h)、细胞增殖(PH后6~66 h)、细胞分化和组织结构功能重建(PH后72~168 h)等4个阶段起始表达的基因数为49、19、73、5, 基因总表达的次数为84、51、369、144, 表明相关基因主要在肝再生启动阶段起始表达, 在不同阶段发挥作用。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类, 涉及38、21、21、10和7个基因, 共上调411次, 下调186次, 分为24种表达模式, 表明肝再生中细胞生理生化活动具有阶段性、多样性和复杂性。根据细胞外基质相关基因在肝再生中表达变化推测, 肝再生前期纤粘连蛋白形成相关基因表达增强, 肝再生中期胶原形成相关基因表达增强。     

基于IPA分析自噬对大鼠肝再生中树突状细胞的调节作用

为探讨自噬对大鼠肝再生中树突状细胞(Dendritic cells, DCs)的调节作用,文章通过Percoll 密度梯度离心结合免疫磁珠分选分离大鼠DCs,Rat Genome 230 2.0芯片检测大鼠肝再生中自噬相关基因表达变化,利用IPA等软件分析自噬在DCs中的生理活动。结果表明,LC3、BECN1、ATG7和SQSTM1等关键基因在部分肝切除后不同恢复时间段有明显表达变化;芯片中对应的自噬相关基因为593个,其中210个基因发生了有意义的变化。比较分析自噬生理活动情况,发现自噬在再生早期和晚期阶段增强,增殖期减弱。与自噬相关的生理活动主要有RNA表达、RNA转录细胞分化和增殖,其中涉及的信号通路主要有PPARα/RXRα激活、急性期反应、TREM1 信号通路、IL-6 信号通路、IL-8 信号通路和IL-1 信号通路等,它们在肝再生阶段发生了不同程度的上调或下调。Cluster 分析还发现,P53和AMPK信号参与调控DCs的自噬活动,在肝再生早期主要是AMPK信号,在肝再生末期P53和AMPK信号共同参与自噬的调节。以上研究结果说明DCs自噬可能在肝再生早期激活细胞免疫反应和后期清除DCs等方面发挥着重要作用。     

大鼠8种肝脏细胞中缺血反应相关基因与肝再生的作用分析

为了解8种肝脏细胞的缺血反应相关基因与大鼠肝再生的相关性, 用percoll密度梯度离心和免疫磁珠方法分离大鼠部分肝切除后不同时间(0, 2, 6, 12, 24, 30, 36, 72, 120和168 h)再生肝中的8种细胞, 用Rat Genome 230 2.0芯片等方法检测上述8种细胞的缺血反应相关基因在大鼠肝再生中表达变化, 用生物学和系统生物学等方法分析上述基因与大鼠肝再生的相关性. 结果显示, 缺血反应主要在肝再生启动阶段及进展阶段前期发挥作用, 且上调占优势, 可刺激il6, tnf等肝再生关键基因表达; 肝星形细胞、树突状细胞中的缺血反应相关基因具有表达的相似性. 缺血反应能推动肝再生的顺利进行, 但胆管上皮细胞的基因表达情况特殊, 值得进一步研究.     

细胞连接相关基因在大鼠肝再生中表达模式

细胞连接是组织、器官形成的基础。为在基因转录水平了解紧密连接、粘附连接、粘着斑和间隙连接相关基因在肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome 230 2.0芯片检测它们在大鼠再生肝中表达情况,将3次检验结果相同或相似、在肝再生中发生有意义表达变化、真手术组和假手术组表达差异显著的基因视为肝再生相关基因。初步证实上述4种细胞连接中79、53、109和53个基因与肝再生相关。其中,肝再生启动(部分肝切除后0.5～4h)、G0/G1过渡(PH后4～6h)、细胞增殖(部分肝切除后6～66h)、细胞分化和组织结构功能重建(部分肝切除后72～168h)等4个阶段起始表达的基因数和基因的总表达次数为124、43、122、10和249、145、957、306。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共上调972次,下调540次,表明肝再生中大多数细胞连接相关基因表达加强,少数基因表达降低。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类,涉及102、38、73、27和16个基因,它们表达的时间相关性分为0.5和1h、2h、4和6h、8和12h、16h、18和48h、24h、30和42h、36h、54和60h、66和72h、96h、120h、144和168h等14组,表明肝再生中细胞生理生化活动具有阶段性。它们的表达模式分为41类,表明肝再生中细胞生理生化活动具有多样性和复杂性。根据肝再生中基因表达变化和表达模式推测,肝再生早期和前期间隙连接形成增强,晚中期和后期间隙连接形成减少;早期、前期和后期粘着斑形成增强;紧密连接和粘附连接的形成贯穿于整个肝再生。     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

脂肪细胞分化相关基因在大鼠再生肝中表达变化

肝脏由多种细胞构成,肝再生与细胞分化密切相关,细胞分化受基因转录水平调控。为在基因转录水平了解脂肪细胞分化基因在大鼠肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome2302.0芯片检测它们在大鼠肝再生(liver regeneration,LR)中表达情况,将三次检验结果相同或相似、在肝再生中表达变化2倍以上、真手术组和假手术组相比差异显著的基因视为肝再生相关基因。初步证实上述基因中75个基因与肝再生相关。肝再生启动(PH后0.5-4h)、G0/G1过渡(PH后4-6h)、细胞增殖(PH后6-66h)、细胞分化和组织结构功能重建(PH后72-168h)等四个阶段起始表达的基因数为44、13、30和1;基因的总表达次数为88、58、302和90。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共表达上调313次、下调167次,分为43种表达方式。表明肝再生中脂肪细胞发生和分化相关基因活动多样和复杂。根据本文研究结果推测,上述基因不仅调节脂肪细胞分化,而且参与肝再生的生理生化活动。     

肌细胞分化基因与大鼠肝再生的相关性分析

肌细胞是组织器官的重要组成部分。为在基因转录水平了解肌细胞分化相关基因在大鼠肝再生中的作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因．用Rat Genome2302．0芯片检测它们在大鼠肝再生（liver regeneration,LR）中表达情况,用比较真、假手术基因表达的差异性方法确定肝再生相关基因。初步证实上述基因中52个基因与肝再生相关。根据肝再生中基因表达的时间相关性将上述基因聚合为0．5-1h;2—12h;16、30、42、96h;18—24、36、48—60h;66—72、120-168h等5类,表达上调和下调的基因数分别为8和10,24和8,21和24,53和64,28和36。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调次数相近等5类,涉及15、10、17、7和3个基因,共上调表达143次、下调136次,分为8类表达方式。表明肌细胞分化相关基因表达变化多样和复杂。根据上述结果推测,肝再生中成肌细胞和平滑肌细胞分化增强：骨骼肌和心肌细胞分化相关基因参与肝再生的生理生化活动。     

NDRG2调控大鼠肝细胞周期的机制

目的:阐明NDRG2(N-Myc downstream-regulated gene2)在大鼠肝再生过程中时肝细胞周期的调控机制.方法:大鼠70%肝切除后收集0 h、8 h、24 h、48 h、72 h、7 d、10 d的再生肝组织,采用Real-time PCR和Western blot方法检测大鼠肝再生过程中NDRG2基因和蛋白的动态变化.流式细胞仪检测腺病毒介导的高表达NDRG2对大鼠正常肝细胞系(BRL)细胞周期的影响.Real.time PCR和Western blot方法检测高表达NDRG2对肝细胞周期调控的分子机制.结果:NDRG2基因和蛋白的表达水平在肝再生达到高峰时明显下降,在肝细胞进入分化期时显著上调;流式细胞仪检测显示BRL细胞中高表达NDRG2 48 h后,G0/G1期细胞百分比从对照组39.30+1.97上升至57.44±2.56,S期从37.66±1.73下降至13.27±2.01,差异有统计学意义(P＜0.05).对周期调控相关分子的检测显示高表达NDRG2对肝细胞周期的影响是通过上调p21,抑制Cyclin E实现的.结论:NDRG2通过影响细胞周期参与调控大鼠肝再生过程.     

肝大部切除后肝再生调节的研究

肝再生调节因子主要有促使肝细胞增殖的刺激因子和抑制因子两大类。前者如ＥＧＦ、ＩＧＦ－α、ＨＳＳ、ＨＰＴＢ、ＨＰＴＡ／ＨＧＦ等,它们是肝细胞增殖的完全促分裂原;后者主要有肝抑素、ＴＧＦ－β等,它们对肝细胞增殖具有负性调节作用。本实验室已提取纯化了大鼠肝抑素并证明了它的生物效应。肝大部切除后的动物在上述两类因子的调控下,肝细胞增殖迅速而有规律,肝再生完成后肝细胞增殖趋于停止。     

Analysis of the role of the integrin signaling pathway in hepatocytes during rat liver regeneration

To explore the role of the integrin signaling pathway in hepatocytes during rat liver regeneration, the integrin signaling pathway-related gene expression profile in hepatocytes of regenerative liver was detected using Rat Genome 230 2.0 array. The chip data showed that 265 genes of the integrin signaling pathway were included by Rat Genome 230 2.0 array and 132 genes showed significant expression changes in hepatocytes of regenerative liver. The numbers of up-, down- and up/down-regulated genes were 110, 15 and 7 respectively. In addition, bioinformatics and systems biology methods were used to analyze the role of the integrin signaling pathway in hepatocytes. The analysis of gene synergy value indicated that paths 1, 8, 12, and 15 promoted hepatocyte proliferation at the priming phase of liver regeneration; paths 1, 3, 8, and 12–15 enhanced hepatocyte proliferation at the progressing phase; paths 11 and 14 promoted hepatocyte proliferation, while paths 12 and 13 reduced hepatocyte proliferation at the terminal phase. Additionally, the other 8 paths (2, 4, 5–7, 9–10, and 16) were not found to be related to liver regeneration. In conclusion, 132 genes and 8 cascades of the integrin signaling pathway participated in regulating hepatocyte proliferation during rat liver regeneration.     

Rat Hepatocytes Weighted Gene Co-Expression Network Analysis Identifies Specific Modules and Hub Genes Related to Liver Regeneration after Partial Hepatectomy

The recovery of liver mass is mainly mediated by proliferation of hepatocytes after 2/3 partial hepatectomy (PH) in rats. Studying the gene expression profiles of hepatocytes after 2/3 PH will be helpful to investigate the molecular mechanisms of liver regeneration (LR). We report here the first application of weighted gene co-expression network analysis (WGCNA) to analyze the biological implications of gene expression changes associated with LR. WGCNA identifies 12 specific gene modules and some hub genes from hepatocytes genome-scale microarray data in rat LR. The results suggest that upregulated MCM5 may promote hepatocytes proliferation during LR; BCL3 may play an important role by activating or inhibiting NF-kB pathway; MAPK9 may play a permissible role in DNA replication by p38 MAPK inactivation in hepatocytes proliferation stage. Thus, WGCNA can provide novel insight into understanding the molecular mechanisms of LR.     

SPINK3: A novel growth factor that promotes rat liver regeneration

Serine peptidase inhibitor, Kazal type 3 (SPINK3) is a trypsin inhibitor, and also a growth factor that has an identical structure to epidermal growth factor (EGF), which could combine with epidermal growth factor receptor (EGFR) to promote cell proliferation. To shed light on the role and regulation mechanism of SPINK3 in rat liver regeneration (LR), Rat Genome 230 2.0 assay was used to detect the expression profiles of LR genes after partial hepatectomy (PH). The results showed that Spink3 was significantly up-regulated at 2–24 h and 72–168 h after PH. In the present study, RT-PCR and immunoblotting were used to validate the assay results. Ingenuity Pathway Analysis 9.0 (IPA) software was used to build the SPINK3 signaling regulating LR and analyze the possible mechanism. And then the expression of cell proliferation-associated gene Ccna2 was examined by RT-PCR in normal rat liver cell line BRL-3A in which Spink3 was overexpressed. The results showed that Ccna2 was significantly up-regulated in BRL-3A in which Spink3 was over-expressed. SPINK3 combining with EGFR accelerated cell proliferation during rat liver regeneration via P38, PKC, JAK-STAT and AKT pathways. Thus, SPINK3 was likely to promote hepatocytes proliferation in LR through P38, PKC, JAK-STAT and AKT pathways.     

Comparative analysis of gene expression profiles of acute hepatic failure and that of liver regeneration in rat

To explore the relevance of rat liver regeneration (LR) to acute hepatic failure (AHF), Rat Genome 230 2.0 Array was used to detect their gene expression profiles in this study, and the reliability of the detection results was confirmed by real-time-PCR. 1012 genes were found to be significantly changed in AHF occurrence and 948 genes in LR. Hierarchical clustering analysis showed that physiological activities of AHF and those of LR had no time correlation. Hierarchical clustering analysis (which is performed to group genes based on the similarity of expression patterns) showed that physiological activities of AHF and those of LR had no time correlation. K-means clustering analysis (which is used to check the difference in the relevant predictor variables between different groups is significant or not) demonstrated that gene expression trend of C1 group (genes relate to categories of stimulus–response and cell apoptosis, etc.) in AHF and in LR was extremely similar, that those of their C2 group (categories of regulation of homeostasis and hormone stimulation, etc.) were contrary, and that those of their C3 (material and energy metabolism and oxidation reduction, etc.), C4 (Cell cycle-related genes) and C5 (cell proliferation-related genes) groups were also similar with the gene expression changes of LR more abundant. GO classifications and functional clustering analysis (which was used to statistics the numbers or composition of proteins or genes at a function level) revealed that cellular processes including immune response, inflammatory reaction, cell migration and adhesion, etc. were increased both in AHF and in LR, whereas material and energy metabolism were decreased. Of them, stimulus response, inflammatory reaction and regulation of apoptosis, etc. were stronger in AHF occurrence than in LR, but ion homeostasis, hormonal response, regulation of cell division and proliferation, etc. were weaker in AHF occurrence. Gene expression changes and physiological activities of AHF and those of LR not only have similarities but also differences.