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…     

蛋白质代谢、折叠、运输、定位、装配相关基因在大鼠肝再生中表达变化

为在基因转录水平了解蛋白质代谢、折叠、运输、定位、装配相关基因在大鼠肝再生中表达情况和作用,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 2302.0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。初步证实上述基因中1147个基因与肝再生相关。其中,参与蛋白质代谢、折叠、运输、定位和装配的基因以上调表达为主;参与蛋白质代谢的基因主要在部分肝切除（partial hepatectomy,PH）后0.5-1h和16-30h起始表达;0.5-12h表达的促进蛋白降解基因数多于促进蛋白积累基因数,而16-48h表达的促进蛋白质积累基因数显著多于促进蛋白质降解基因数;蛋白质合成相关基因在肝再生的16、24、42和66h表达上调较多,在42h最多;几乎在整个肝再生中蛋白质降解相关基因表达上调,在早、前期较多,在后期较少;蛋白质折叠相关基因在2、16-24、42、66、72和168h表达上调较多,在66h最多;蛋白质运输和定位相关基因在整个肝再生中表达上调,在66h表达上调最多;蛋白质装配相关基因在96h前均表达上调,其中,12h表达上调基因最多。根据上述结果推测,在肝再生中期蛋白质合成旺盛,几乎整个肝再生中蛋白质降解、折叠、运输定位和装配活动活跃。     

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.     

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     

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。     

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。     

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。     

Identification of differentially expressed genes contributing to radioresistance in lung cancer cells using microarray analysis

Radiotherapy has played a key role in the control of tumor growth in many cancer patients. It is usually difficult to determine what fraction of the tumor cell population is radioresistant after a course of radiotherapy. The response of tumor cells to radiation is believed to be accompanied by complex changes in the gene expression pattern. It may be possible to use these to sensitize radioresistant tumor cells and improve radiocurability. Based on the biological effects of ionizing radiation, in the present study, we developed one oligonucleotide microarray to analyze the expression of 143 genes in cells of two lung cancer cell lines with different radiosensitivities. Compared to NCI-H446 cells, expression of 18 genes significantly increased the basal levels in the radioresistant A549 cells, in which eight genes were up-regulated and 10 genes were down-regulated. In A549 cells irradiated with 5 Gy, 22 (19 up-regulated and three down-regulated) and 26 (eight up-regulated and 18 down-regulated) differentially expressed genes were found 6 and 24 h after irradiation, respectively. In NCI-H446 cells, the expression of 17 (nine up-regulated and eight down-regulated) and 18 (six up-regulated and 12 down-regulated) genes was altered 6 and 24 h after irradiation, respectively. RT-PCR was performed, and we found that MDM2, BCL2, PKCZ and PIM2 expression levels were increased in A549 cells and decreased in NCI-H446 cells after irradiation. Genes involved in DNA repair, such as XRCC5, ERCC5, ERCC1, RAD9A, ERCC4 and the gene encoding DNA-PK, were found to be increased to a higher level in A549 cells than in NCI-H446 cells. Antisense suppression of MDM2 resulted in increased radiosensitivity of A549 cells. Taken together, these results demonstrate the possibility that a group of genes involved in DNA repair, regulation of the cell cycle, cell proliferation and apoptosis is responsible for the different radioresistance of these two lung cancer cells. This list of genes may be useful in attempts to sensitize the radioresistant lung cancer cells.     

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。     

Subproteomic analysis of the mitochondrial proteins in rats 24 h after partial hepatectomy

A 70% partial hepatectomy (70%PHx) induces cell proliferation until the original mass of the liver is restored. Mitochondria are involved directly in the process of liver regeneration (LR); however, their role in the early phase of LR is not clear. In an attempt to identify mitochondrial proteins that are correlated with the early phase of LR, we obtained a mitochondrial fraction via Nycodenz(R) density gradient centrifugation and subcellular proteomic analysis was performed. The mitochondrial proteins were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Compared to the sham-operation control group, 3 proteins were up-regulated and 22 proteins were down-regulated at 24 h after 70%PHx. We identified 22 differentially expressed proteins that were associated with carbohydrate metabolism, lipid metabolism, the respiratory chain and oxidation-phosphorylation, biotransformation and other metabolic pathways. Prohibitin, a proliferation-regulating protein that was down-regulated at 24 h after PHx, was analyzed by applying RNAi (PHBi) with BRL-3A. This demonstrated a decreased mitochondrial membrane potential, implying a potential role in maintaining mitochondrial integrity. These results indicated that hepatic mitochondrial adaptations to LR after 70%PHx affect various cellular metabolic pathways, which advances our knowledge of the role of mitochondria in the early phase of LR.     

大鼠部分肝切除前后热激处理对过氧化物酶基因表达的影响

用过氧化物酶原位复性电泳（SDS－POD－PAGE）技术分析了部分肝切除（partial hepatectomy,PH)、部分肝切除后再热激（partial hepatectomy following heat shock,PH-HS)和先热激再部分肝切除（heat shock following partial hepatectomy,HS-PH)后肝再生期间过氧化物酶（POD）基因表达差异,结果表明,PH中表达的POD基因种类（7个）＞PH－HS（5个）＞HS－PH（3个）;三个模型的POD总活性为：HS－PH＞PH－HS。根据实验结果推测,POD在肝再生和肝损伤恢复中起一定作用。