大鼠再生肝中hsbp1、hsf1、hsf2、hsp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因(hsbp1)全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料,按Higgens等方法建立大鼠部分肝切除(PH)模型;用原位杂交等方法分析hsbp1在肝再生中表达变化;用基因表达谱芯片分析hsbp1、hsf1、hsf2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明,PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,hsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0.5-24h,hsp70表达发生了有意义上调。假手术(只打开腹腔和翻动肝叶,但不进行部分肝切除)后0.5-2h,hsbp1表达发生了有意义下调;8-16h,hsf1表达发生了有意义上调;0-144h,hsf2未发生有意义表达变化;0.5-30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量,促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;(假)手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。     

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。     

大鼠再生肝中hsbp1、hsf1、hsf2、shp70表达水平改变的分析

在克隆了大鼠热休克因子结合蛋白1基因（hsbp1）全长cDNA基础上,进一步分析它在肝再生中作用。用SD纯系大鼠为材料。按Higgens等方法建立大鼠部分肝切除（PH）模型;用原位杂交等方法分析凤6pJ在肝再生中表达变化;用基因表达谱芯片分析凤如1、hsf1、如,2和hsp70在肝再生中表达变化。原位杂交和基因表达谱芯片分析表明。PH后6h和66-144h,hsbp1表达发生了有意义上调;8-16h,nsf1表达发生了有意义上调;2-16h,hsf2表达发生了有意义上调;0．5—24h,hsp70表达发生了有意义上调。假手术（只打开腹腔和翻动肝叶。但不进行部分肝切除）后0．5-2h,hsbp1表达发生了有意义下调;8—16h,hsf1表达发生了有意义上调;0—144h,hsf2未发生有意义表达变化;0．5—30h,hsp70表达发生了有意义上调。根据实验结果推测,PH后hsbp1表达上调可增加细胞内HSBP1量。促进生长、发育、分化相关基因表达和再生肝的组织结构功能重建;（假）手术后hsbp1表达下调可减少细胞内HSBP1量,有利于HSF1上调hsp70表达,提高机体和肝脏抗损伤能力。     

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。     

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。     

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。     

大规模鉴定大鼠 0, 4, 36, 72, 96 h 短间隔连续部分肝切除中再生肝差异表达的基因

用抑制性消减杂交方法(SSH)构建了短间隔连续部分肝切除(SISPH)再生肝的消减cDNA文库, 从中筛选出了551个与肝再生相关的基因, 把这些基因制成cDNA 微阵列(cDNA芯片), 分析它们在0 h正常肝及 4, 36, 72, 96 h再生肝中的动态变化发现, 185个基因至少在肝再生的一个时间点表达变化达2倍以上; 185个基因中的86个属未报道的基因, 99个为已报道的基因, 但在此之前尚不知道它们与肝再生有关; 185个基因中的103个在肝再生中表现上调表达, 82个表现下调表达. 用GeneMath软件和GeneSpring方法对这些基因在肝再生中的表达轮廓进行聚类分析表明, 基因的表达模式可分为8组, 即早期诱导、中期诱导、晚期诱导、持续诱导、早期抑制、中期抑制、晚期抑制和持续抑制. 与一次性部分肝切除(PH)相比, 41个基因在SISPH中特异性表达, 其他基因在两个模型中的表达趋势相同, 但在各时间点的表达丰度有差异. 综合分析可见, 抑制性消减杂交技术与基因芯片技术相结合是研究再生肝差异表达基因的有效方法; 肝再生中上调表达的基因多于下调表达的基因; 早期诱导的基因多于晚期诱导的基因; 诱导表达幅度大的基因少于诱导表达幅度小的基因.     

大鼠再生肝8种细胞的丝氨酸族氨基酸代谢相关基因的转录谱

为了解大鼠肝再生中8种肝脏细胞的丝氨酸族氨基酸代谢相关基因转录谱, 文章用Percoll密度梯度离心结合免疫磁珠分选分离大鼠的8种再生肝细胞, 用Rat Genome 230 2.0芯片等检测它们中丝氨酸族氨基酸代谢相关基因的表达变化, 用Cluster和Treeview等软件分析上述基因在肝再生中表达模式, 用生物信息学和系统生物学等方法分析上述细胞中丝氨酸族氨基酸代谢活动。结果表明, 在27个发生有意义表达变化的基因中, 肝细胞、胆管上皮细胞、卵圆细胞、肝星形细胞、窦内皮细胞、库普弗细胞、陷窝细胞、树突状细胞的基因数分别为13、16、11、14、13、11、12、14, 相应细胞的上调、下调和上/下调的基因数分别为7、6和0, 2、10和4, 2、8和1, 8、3和3, 6、5和2, 4、6和1, 2、10和0, 6、6和2。总的来看, 肝再生中各细胞的表达下调基因占优势, 但在肝再生启动阶段, 肝星形细胞和窦内皮细胞的表达上调基因占优势。上述丝氨酸族氨基酸代谢相关基因转录谱预示丝氨酸族氨基酸的合成主要在肝再生启动阶段的肝细胞、肝星形细胞、窦内皮细胞和库普弗细胞中增强, 它们的降解主要在肝再生进展阶段的肝细胞、胆管上皮细胞、陷窝细胞和树突状细胞中进行。     

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.     

大鼠2/3肝切除72 h后再生肝脏质膜比较蛋白质组学研究

大鼠2/3肝切除模型为研究肝细胞增殖和生理性血管生成提供了一个很好的活体内模型.为了揭示肝再生过程中与肝细胞增殖终止相关及与血管生成启动相关的质膜蛋白质,本研究对大鼠肝2/3部分切除72 h后的肝脏质膜进行了研究：利用两步蔗糖密度梯度离心法对切除组和假手术组的肝脏质膜进行纯化;然后通过双向电泳和质谱技术对肝切除样品进行了比较分析并对几个关键蛋白程序性凋亡相关蛋白-6和丝蛋白-A进行了免疫印迹验证.相对于假手术对照组(Sham组),21种蛋白质在切除后72 h的肝脏中上调,15种蛋白质下调.所鉴定的差异表达蛋白参与了血管生成、细胞分裂增殖和凋亡、细胞分化调控、肝脏组织重新构建、代谢及应急反应.本研究为肝脏再生及其血管生成的研究提供了理论依据.     

Expression profiles of the genes associated with metabolism and transport of amino acids and their derivatives in rat liver regeneration

Summary. Amino acids (AA) are components of protein and precursors of many important biological molecules. To address effects of the genes associated with metabolism and transport of AA and their derivatives during rat liver regeneration (LR), we firstly obtained the above genes by collecting databases data and retrieving related thesis, and then analyzed their expression profiles during LR using Rat Genome 230 2.0 array. The LR-associated genes were identified by comparing the gene expression difference between partial hepatectomy (PH) and sham-operation (SO) rat livers. It was approved that 134 genes associated with metabolism of AA and their derivatives and 26 genes involved in transport of them were LR-associated. The initially and totally expressing number of these genes occurring in initial phase of LR (0.5–4 h after PH), G0/G1 (4–6 h after PH), cell proliferation (6–66 h after PH), cell differentiation and structure-function reconstruction of liver tissue (72–168 h after PH) were respectively 76, 17, 79, 5 and 162, 89, 564, 195, illustrating that these LR-associated genes were initially expressed mainly in initial stage, and functioned in different phases. Frequencies of up-regulation and down-regulation of them being separately 564 and 357 demonstrated that genes up-regulated outnumbered those down-regulated. Categorization of their expression patterns into 22 types implied the diversity of cell physiological and biochemical activities. According to expression changes and patterns of the above-mentioned genes in LR, it was presumed that histidine biosynthesis in the metaphase and anaphase, valine metabolism in the anaphase, and metabolism of glutamate, glutamine, asparate, asparagine, methionine, alanine, leucine and aromatic amino acid almost were enhanced in the whole LR; as for amino acid derivatives, transport of neutral amino acids, urea, γ-aminobutyric acid, betaine and taurine, metabolism of dopamine, heme, S-adenosylmethionine, thyroxine, and biosynthesis of hydroxyproline, nitric oxide, orinithine, polyamine, carnitine, selenocysteine were augmented during the entire liver restoration. Above results showed that metabolism and transport of AA and their derivates were necessary in liver regeneration. Authors’ address: Prof. Dr. C. S. Xu, College of Life Science, No. 46, Jianshe RD, Henan, Xinxiang 453007, China     

Identification and characterization of 177 unreported genes associated with liver regeneration

The mammalian liver has a very strong regeneration capacity after partial hepatectomy (PH). To further learn the genes participating in the liver regeneration (LR), 551 cDNAs selected from subtracted cDNA libraries of the regenerating rat liver were screened by microarray, and their expression profiles were studied by cluster and generalization analyses. Among them, 177 genes were identified unreported and up-or down-regulated more than twofold at one or more time points after PH, of which 62 genes were down-regulated to less than 0.5; 99 genes were up-regulated to 2-10 folds, and 16 genes were either up- or down-regulated at different time points during LR. By using BLAST and GENSCAN, these genes were located on responsible chromosomes with 131 genes on the long arms of the chromosomes. The cluster and generalization analyses showed that the gene expression profiles are similar in 2 and 4, 12 and 16, 96 and 144 h respectively after PH, suggesting that the actions of the genes expressed in the same profiles are similar, and those expressed in different profiles have less similarity. However, the types,characteristics and functions of the 177 genes remain to be further studied.     

Large scale of identification of differentially expressed genes in the regenerating rat liver after SISPH

After partial hepatectomy (PH), the remnant paren-chyma can completely recover lost liver mass and function in about one week[1,2]. Although adult hepa-tocytes are normally quiescent, they are readily primed to pass from G0 to G1 phase within 2―6 h after PH. The first peak of DNA synthesis appears 24 h after PH, while cell division peaks at 36 h. The liver cells then enter a second cell cycle, and redifferentiation and reconstruction of structure and function[3―6] take place. A great nu…     

Large scale of identification of differentially expressed genes in the regenerating rat liver after SISPH

Extensive gene expression analysis was carried out after a 0, 4, 36, 72, 96 h short interval successive partial hepatectomy (SISPH) was performed. A total of 185 elements were identified as differing by more than two-fold in their expression levels at one or more time points. Of these 185 elements, 103 were up-regulated, 82 were down-regulated and 86 elements were unreported genes. Quite a few genes were previously unknown to be involved in liver regeneration (LR). Using cluster and general analysis, we found that the genes at five time points of the SISPH share eight different types of different expression profiles and eight distinct temporal induction or suppression patterns. A comparison of the gene expression in SISPH with that after PH found that 41 genes were specifically altered in SISPH, and 144 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but they were present in different amounts at the different time points. The conclusions are that (i) microarrays combined with suppressive subtractive hybridization (SSH) can effectively identify genes involved in LR on a large scale; (ii) more genes were up-regulated than down-regulated; (iii) there are fewer abundantly expressed genes than those with increased levels of 2–5 fold.