热休克蛋白、蛋白水解酶和磷酸酶在大鼠肝再生中的含量和活性变化

本文以２／３肝切除（ｐａｒｔｉａｌ ｈｅｐａｔｅｃｔｏｍｙ,ＰＨ）大鼠为模型,探讨了ＰＨ后酸性和碱性磷酸酶（ａｃｉｄ ａｎｄ ａｌｋａｌｉｎｅ ｐｈｏｓｐｈａｔａｓｅｓ,ＡＣＰ和ＡＫＰ）,构成性热休克蛋白７０／诱导性热休克蛋白６８（ＨＳＣ７／ＨＳＰ６８）,酸性和中性蛋白水解酶在肝再生期间（０－１４４ｈ）的动态变化。结果显示,在肝切除后的肝再生期间;（１）ＡＣＰ和ＡＫＰ均出现两个活性高峰（４和４８ｈ     

大鼠再生肝刺激因子抗四氯化碳损伤的研究

我们以往的研究证明,大鼠再生肝具有抗四氯化碳损伤的能力。本工作进一步研究其机制,首先从部分（６８％）肝切除后不同的再生肝的取肝刺激因子,用＾３Ｈ胸腺嘧喧核苷测定ｒＨＳＳ的生物活性,结果表明部分切除肝后７２ｈ的ｒＨＳＳ活性较对照组约增加７．７倍。然后将ｒＨＳＳ注射给小鼠,观察其抗ＣＣｌ４损伤肝的效应,具体表现如下：ｒＨＳＳ能减少ＣＣｌ４中毒小鼠的死亡率和降低ＣＣｌ４所增高的血清谷丙转氨酶和谷草转氨酶     

肝部分切除后肝再生增强因子信使核糖核酸（mRNA）表达增强

肝再生增强因子（ＡＬＲ）是一种新的肝增殖刺激因子。本研究选择７０％肝部分切除（ＰＨ）大鼠为模型,观察了ＰＨ后残存肝组织胞浆液促肝细胞增殖活性与ＡＬＲ特异ｍＲＮＡ表达动态变化的关系。发现正常肝组织几无ＡＬＲ ｍＲＮＡ表达,其肝胞质液也无促肝细胞增殖活性;７０％ＰＨ后１２ｈ肝组织ＡＬＲ ｍＲＮＡ表达明显增加,并于术后２４ｈ达高峰,肝胞质液活性也于术后２４ｈ内达高峰,这种ｍＲＮＡ表达－效应的时间关系提示     

大鼠反复肝切除后再生肝组织的实验形态学研究

９０只雄性ＳＤ大鼠随机分为三组,每组各３０只,以第一次切肝量５０％（Ａ组）,７０％（Ｂ组）,８０％（Ｃ组）为分组标准,均作反复四次肝切除。动态观察其再生肝组织的酶组织化学,组织学及超微结构变化。结果显示：Ａ、Ｂ组三次术后累计存活率（８５％）明显高于Ｃ组（５７％）,Ｐ＜０．０１。Ａ、Ｂ、Ｃ各组三次术后累计切肝率各为１１４．２８％,１２０．７２％,１２５．８１％。Ａ、Ｂ组ＳＤＨ、ＡＴＰａｓｅ、ＬＤＨ活性改变相近,优于Ｃ组。各组ＣＨＥ活性均处于低水平。ＡＣＰ活性随切量,切次增加而增强。肝再生的方式既有小叶数量增多,面积增大,又有细胞肥大,细胞数量增加。电镜显示了增生活跃的肝细胞的核发裂像。所示线粒体结构受损与ＳＤＨ活性下降相对应。研究证明;大鼠反复肝切除是可行的,第一次切肝量,切次多少是影响大鼠耐受反复肝切除的重要因素。     

血源性和胞源性肝细胞生长因子对肝再生的调控

肝再生过程中,血源性肝细胞生长因子（ＨＧＦ－ｈ）和胞源性肝细胞生长因子（ＨＧＦ－ｃ）起重要的调控作用。本文通过分析二者与肝再生时相、细胞周期调控基因表达之间的关系,认为它们对肝再生的调控具有时序性。ＨＧＦ－ｈ作为早期信号,启动肝细胞分裂;而ＨＧＦ－ｃ仅作用于感受态细胞,促进其分裂。本文还从促再生作用的器官特异性,重症肝炎的临床治疗以及二者的来源等方面对这一推测加以佐证。     

肝再生的调控及原癌基因表达

肝脏再生过程中受到多种体液因素的调控。肝细胞生长因子（ＨＧＦ）、肝刺激因子（ＨＳＳ）等对肝细胞有促分裂作用;转化生长因子β１（ＴＧＦβ１）等则具有抑制作用。此外,肝再生还需要去甲肾上腺素（ＮＥ）、胰岛素等辅助分裂原的存在,共同调节肝再生。肝细胞分裂增殖与原癌基因表达密切相关。肝细胞从静息期进入细胞周期,以及在整个细胞周期中,某些原癌基因有特征性的表达。     

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。     

肝再生增强因子超家族研究进展

从断奶大鼠的肝脏中纯化得到了肝刺激物质（ＨＳＳ）的有效成份,即肝再生增强因子（ＡＬＲ）的蛋白质,酶解并对其多肽末端测序,据此推导出简并核苷酸序列,合成探针,对大鼠肝脏来源的ｃＤＮＡ文库进行筛选,首选获得了大鼠ＡＬＲ的ｃＤＮＡ克隆,随后又分别克隆了人和小鼠的ＡＬＲ的ｃＤＮＡ。与此同时,从酵母细胞中克隆了与线粒体氧化－－磷酸化功能密切相关的ＥＲＶ１基因,然后克隆了人的ＥＲＶ１同源基因,从功能上证实人     

肝再生增强因子的cDNA克隆,表达及表达产物的生物活性研究

以肝部分切除后再生肝组织为起始材料,利用ＲＴ－ＰＣＲ扩增出大夺再生增强因子（ＡＬＲ）,亚克隆子于ＰＧＥＭ－Ｔ载体,核苷酸序列测定证实为大鼠ＡＬＲ;将ＡＬＰＣＤＮＡ亚克隆于ＰＢＶ２２０质粒,构建了原核表达载体,并获高效表达菌株,特异表达蛋白占细菌总蛋白的１５％,原核表达的ＡＬＲ在体外缺乏促进大鼠原代培养肝细胞及ＳＭＭＣ－７７２１肝癌细胞ＤＮＡ合成的活性,但天体内１／３肝部分切除模型中可刺激肝细胞ＤＮ     

肝再生增强因子

肝脏是机体具有强大再生能力的脏器 ,目前已知的肝再生相关因子如肝细胞生长因子 (ＨＧＦ)、转化生长因子 α(ＴＧＦ α)、表皮生长因子 (ＥＧＦ)等 ,均难以解释具有器官特异性的肝再生调控机制 ,因此寻找新型肝再生调控因子一直是该领域的热点[1] 。1994年 8月 ,Ｈａｇｉｙａ等[2 ] 从初断乳大鼠肝组织中克隆到一种新型的促肝细胞增殖因子 ,称为肝再生增强因子 (ａｕｇｍｅｎｔｅｒｏｆｌｉｖｅｒｒｅｇｅｎｅｒａｔｉｏｎ ,ＡＬＲ)。近来研究发现 ,ＡＬＲ是一种特殊的促肝细胞分裂原 ,在肝损伤修复过程中发挥重要作用。1.ＡＬＲ基因大鼠…     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

肝再生刺激因子对小鼠实验性急性肝损伤的保护机制

我们前文证明肝再生刺激因子(HSS)对小鼠实验性肝损伤有保护作用。本文进一步探讨其机制并获得如下结果:(1)HSS显著提高由CCl_4所降低肝细胞膜、线粒体膜和微粒体膜的流动性,使其上升到对照水平。(2)HSS使CCl_4所致的肝组织丙二醛升高幅度降低。(3)HSS使CCl_4所致的肝组织谷胱甘肽降低的含量回升。(4)HSS能刺激受CCl_4损伤的肝再生,促进肝细胞合成DNA和~3H-TdR掺入肝细胞DNA。这些结果提示,HSS具有抗氧化作用,能抗CCl_4所产生的自由基对膜脂质的过氧化。此外还加强肝细胞本身抗氧化能力和促进受损肝脏再生。这些保肝机制可能相互联系。     

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。     

脊髓TrkC mRNA的表达及其坐骨神经损伤后的变化

目的和方法：本文利用原位杂交和点杂交的方法,对ＴｒｋＣ ｍＲＮＡ大鼠脊髓组织中的分布与坐骨神经损伤后的表达变化进行了研究。结果：ＴｒｋＣ ｍＲＮＡ几乎存在于所有脊髓神经元与胶质细胞中,在坐骨神经损伤后１ｄ表达增加,以后降到较低水平,到１４ｄ又再次升高,但第二峰较第一峰为低。     

Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

Background

Although 70% (or 2/3) partial hepatectomy (PH) is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH) has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s) under this milder but clinically more relevant condition.

Results

Proteins from sham-operated mouse livers and livers regenerating for 24 h after 50% PH were separated by SDS-PAGE and analyzed by nanoUPLC-Q-Tof mass spectrometry. Compared to sham-operated group, there were totally 87 differentially expressed proteins (with 50 up-regulated and 37 down-regulated ones) identified in the regenerating mouse livers, most of which have not been previously related to liver regeneration. Remarkably, over 25 differentially expressed proteins were located at mitochondria. Several of the mitochondria-resident proteins which play important roles in citric acid cycle, oxidative phosphorylation and ATP production were found to be down-regulated, consistent with the recently-proposed model in which the reduction of ATP content in the remnant liver gives rise to early stress signals that contribute to the onset of liver regeneration. Pathway analysis revealed a central role of c-Myc in the regulation of liver regeneration.

Conclusions

Our study provides novel evidence for mitochondria as a pivotal organelle that is connected to liver regeneration, and lays the foundation for further studies on key factors and pathways involved in liver regeneration following 50% PH, a condition frequently used for partial liver transplantation and conservative liver resection.     

皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶活性及其基因表达的影响

采用高效液相色谱和原位杂交技术研究了皮质酮对大鼠再生肝细胞鸟氨酸脱羧酶 (ODC)活性及ODCmRNA表达的影响。结果显示 ,大鼠完整肝脏中ODC水平较低 ,2 / 3肝切除 (PH)后 3h ,不同处理组ODC活性开始升高 ,6h达到最高值 ,其中 ,去肾上腺 NaCl组和糖皮质激素受体拮抗剂RU4 86处理组的酶活性高于对照组 (去肾上腺假手术组 ) ,而去肾上腺 皮质酮处理组的酶活性低于对照组 ,36h恢复到肝切除前水平 ;完整肝脏的ODCmRNA水平极低 ,PH后表达量迅速增加 ,5h达到最大值 ,不同处理组mRNA水平的高低顺序与酶活性一致 ,12h降至肝切除前水平 ;在PH前 12h给大鼠注射RU4 86 (10mg/kg体重 ) ,取得了与去肾上腺 NaCl处理鼠相似的结果。以上结果表明 ,在PH诱导的再生肝细胞中 ,ODCmRNA表达量的增加和 /或减少是造成ODC活性改变的原因之一 ,皮质酮对ODC活性及其mRNA的表达具有抑制作用 ,主要表现在肝再生的早期 ,该作用可能是通过受体实现的     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.