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     

睾酮对大鼠再生肝细胞核仁组织区影响的初步研究

以银染核仁组织区(argymphil nucleolar organiting regions,AgNORs)阳性颗粒数为指标,结合放射免疫技术,研究了大鼠部分肝切除(partial hepatectomy,PH)后,睾酮对余留肝细胞核仁组织区的影响。结果显示,pH后0～24h,各种方式(假手术、皮下注射芝麻油或睾酮)处理组,AgNORs数都下降,随后持续升高。在PH后48h和72h,低剂量(0．5ms／kg体重)睾酮对AgNORs数影响最显著,都明显高于其它两个睾酮处理组;中等剂量(2．5ms／kg体重)和高剂量睾酮(5ms／kg体重)处理对AgNORs数的影响与芝麻油处理组相比都无显著差异,但均高于假手术组;假手术后72h的AgNORs恢复到术前水平。血清睾酮含量测定显示,芝麻油处理组PH后0～48h睾酮持续下降,48h后不再下降,48h和72h时的睾酮浓度显著低于假手术组;低剂量处理,血清睾酮浓度在PH后0～24h下降,然后持续上升,在48h、72h达到假手术组的2～3倍;中等剂量和高剂量处理组,血清睾酮在PH后0～72h持续升高,是假手术组的6～7倍。以上结果初步表明,睾酮对PH后肝细胞中AgNORs的影响有两种情况：(1)在血清中的浓度是生理水平的2～3倍时,起促进作用;(2)6～7倍于生理水平时,基本无作用。     

皮质酮对大鼠再生肝鸟氨酸脱羧酶及抗酶基因表达的影响

以部分肝切除诱导的雄性SD大鼠(180～200 g)早期再生肝为材料,用RT-PCR法分析皮质酮处理后再生肝鸟氨酸脱羧酶及抗酶mRNA水平变化.结果表明,皮质酮对鸟氨酸脱羧酶mRNA水平具有剂量依赖性抑制作用;10、20 mg/kg体重皮质酮对抗酶mRNA水平影响不大,40 mg/kg处理可促进其转录.     

肾上腺皮质激素对心房钠尿肽基因表达的促进作用

给完整的及切除肾上腺的雌性 Wistar 大鼠分別注射地塞米松、去氧皮质酮或地塞米松加去氧皮质酮;冷酚法提取心房总 RNA,用α-~(32P)标记的大鼠心房肽 cDNA 探针与之杂交。完整大鼠接受地塞米松和切除肾上腺后接受地塞米松加去氧皮质酮的大鼠,心房肽基因转录产物增加2倍,其余组无显著变化。结果提示糖皮质激素可促进心房肽基因表达,但此作用依赖于盐皮质激素的同时存在,单纯盐皮质激素不能增强该基因的表达。     

大鼠再生肝抗CCl4损伤与其线粒体呼吸活性变化的关系

本工作观察了肝部分切除(68%)后96h 大鼠再生肝的抗 CCl_4损伤作用,并用氧电极法测定了再生肝线粒体的呼吸活性。结果如下: (1)CCl_4(50%,10ml/kg)引起的动物死亡率,肝切除组大鼠较假手术组明显降低;(2)CCl_4(50%,5 ml/kg)损伤后,肝切除组大鼠血清胆红素、血清谷丙转氨酶(sGPT)均明显低于假手术组,组织学检查损伤程度也明显减轻;(3)无论是否伴有 CCI_1损伤,肝切除组大鼠肝线粒体的呼吸活性均强于假手术组,且肝线粒体呼吸活性的变化与血清胆红素、sGPT 及肝组织损伤程度的改善是一致的。上述结果提示:再生肝线粒体呼吸活性增高,同时不易受 CCl_4损伤,可能在再生肝抗 CCl_4损伤机制中起一定作用。     

大鼠再生肝细胞的分离和培养

建立大鼠2/3肝切除模型,分别于术后恢复0 h、6 h、12 h、24 h、48 h、72 h、120 h、168 h等时间点,采用原位胶原酶消化和密度梯度离心法分离、纯化再生肝细胞,获得的再生肝细胞活性95%以上,纯度96%以上。用DMEM培养液添加其他物质后培养再生肝细胞,在培养的96 h内,肝细胞生长状态良好,具有分泌白蛋白和合成尿素的功能。细胞的DNA合成主要在培养24 h以后,72 h时达到高峰。     

大鼠部分肝切后血清对体外培养肝细胞的刺激作用

目的探讨大鼠部分肝切后血清对体外培养肝细胞生长状况的影响。方法对Sprague-Dawley大鼠进行肝部分切除,分别在术后第12、24、36h于心腔内穿刺取血,制备刺激血清。采用酶消化法分离获取Sprague-Dawley乳鼠的肝细胞,在加有10％上述刺激血清的DMEM培养基中进行肝细胞体外培养。倒置显微镜下观察培养细胞的生长状态,采用免疫细胞化学方法检测肝细胞中白蛋白和纤维蛋白原的表达情况。结果发现在肝切后血清刺激作用下,原代培养的肝细胞生长加快,存活时间增长。细胞传代培养后仍用制备血清加以刺激,可产生胶原样细胞外基质,并且在基质上粘附的肝细胞呈现克隆样生长状态,其胞浆内白蛋白和纤维蛋白原均呈阳性表达。结论研究初步表明,体外培养乳鼠肝细胞时加入大鼠部分肝切后血清,可以有效刺激细胞的生长,促进细胞外基质的产生,从而利于肝细胞在体外较长时间存活、增殖和功能保持,同时此种肝细胞体外培养方式还为肝脏细胞生物学研究增添了新的实验途径。     

地塞米松对大鼠再生肝细胞亚精胺合成酶基因转录的影响

采用原位杂交技术研究地塞米松对大鼠再生肝细胞亚精胺合成酶(spermidine syathase,SPDS)基因表达的影响.结果显示,部分肝切除(partial hepatectomy,PH)后,再生肝细胞mRNA表达量呈现先升高后降低的变化趋势,峰值均出现在PH后9 h.PH 去肾上腺使mRNA水平升高,主要表现在PH后6～12 h;地塞米松处理组SPDS基因表达量明显下降,并且随着地塞米松剂量升高,mRNA表达水平降低.结果表明,地塞米松对早期再生肝细胞SPDS基因表达具有抑制作用.     

病理性应激时大鼠肝胞液糖皮质激素受体的变化

以[~3H]地塞米松为配体,用交换法测定了对照和烫伤性应激大鼠肝胞液的糖皮质激素受体的结合容量(R_0)和表面上的解离常数(K_d)。烫伤大鼠肝胞液的 R_0减少。烫伤后1、12和24小时的凡分别为对照组的76 4±15.2、70.6±22.0和79.7±5.8%(均值±标准差)。烫伤后1小时 K_d 略为增大。为了确定 R_0是否真的减少,用去肾上腺大鼠的肝胞液加6nM 皮质酮后,再测定[~3H]地塞米松特异结合的 R_0和 K_d,结果表明,在我们所用的交换测定条件下,内源性皮质酮浓度在6nM 以下时,对测定结果没有明显的影响。而对照组和烫伤组肝胞液的皮质酮浓度平均都不超过6nM。因此可以认为烫伤大鼠肝胞液糖皮质激素受体的 R_0和 K_d 的变化不是由于内源性皮质酮占据受体的结果。其机制有待进一步的研究。     

肝细胞生长因子对四氯化碳损伤肝细胞的保护作用及相关基因表达

利用无血清原代培养大鼠肝细胞,观察重组人肝细胞生长因子(rhHGF)对CCl4染毒肝细胞的保护作用. 结果表明: (1) rhHGF (5 ng/ml)预处理后可显著提高CCl4 (15 mmol/L)染毒肝细胞存活率,降低细胞内丙氨酸氨基转移酶(ALT)、 K+的漏出;(2) 表皮生长因子(EGF,50ng/ml)和rhHGF (5 ng/ml)合用预处理肝细胞,CCl4染毒后细胞内ALT、 K+漏出较rhHGF和EGF单独保护组进一步降低;(3)大鼠肝部分切除和CCl4 (50%,2.5 ml/kg bw)染毒后,再生肝内HGF基因及其受体基因/c-met的表达分别较假手术和盐水对照组显著升高. 结果提示,rhHGF对CCl4染毒大鼠肝细胞具有保护作用;EGF和rhHGF有协同保护作用;HGF及其受体的表达在肝脏再生及修复中可能有重要作用.     

AgNOR numbers in rat pituitary corticotrophs following adrenalectomy or corticotrophin releasing factor administration

Using a combined silver staining/immunoalkaline phosphatase technique, nucleolar organiser regions (AgNORs) were visualised and quantified in rat anterior and intermediate lobe pituitary corticotrophs following bilateral adrenalectomy or sham surgery. Compared to sham operated animals, the mean number of AgNORs was increased in anterior lobe corticotrophs in adrenalectomized rats and there was a shift to the right in the distribution. At 2 weeks after adrenalectomy, AgNOR numbers were greater than at 6 weeks. AgNOR numbers were also quantified in anterior lobe corticotrophs of intact rats receiving daily intraperitoneal injections of ovine CRF-41 at 50 micrograms/kg, which has been shown to stimulate ACTH release and to produce morphological evidence of increased corticotroph stimulation. CRF-41 did not produce an increase in AgNOR numbers, compared to saline injected controls.     

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.     

Contribution of cyclooxygenase 2 to liver regeneration after partial hepatectomy.

Partial hepatectomy (PH) triggers a rapid regenerative response in the remaining tissue to reinstate the organ function and the cell numbers. Among the molecules that change in the course of regeneration is an accumulation of prostaglandin E2 in the sera of rats with PH. Analysis of the cyclooxygenase (COX) isoenzymes in the remnant liver showed the preferential expression of COX-2 in hepatocytes. Cultured regenerating hepatocytes expressed significant levels of COX-2, a process that was not observed in the sham counterparts. Maximal expression of COX-2 was detected 16 h after PH with increased levels present even at 96 h. Pharmacological inhibition of COX-2 activity with NS398 shunted the up-regulation of cell proliferation after PH, which suggests a positive interaction of prostaglandins with the progression of the cell cycle. Similar results were obtained after PH of mice lacking the COX-2 gene. The expression of COX-2 in regenerating liver was concomitant with a decrease in CCAAT-enhancer binding protein (C/EBP-a) level and an increase in the expression of C/EBP-b and C/EBP-d. These results suggest a contribution of the enhanced synthesis of prostaglandins to liver regeneration observed after PH.     

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.     

Glucocorticoid regulation of hepatic cytosolic glucocorticoid receptors in vivo and its relationship to induction of tyrosine aminotransferase

Regulation of rat hepatic cytosolic glucocorticoid receptors was studied using our newly developed exchange assay. Injecting 1 mg of dexamethasone or corticosterone into 150-250 g adrenalectomized rats caused a rapid decline in glucocorticoid receptor binding. Glucocorticoid receptor levels were depressed 80-90% in less than 15 min after hormone treatment, and remained low for about 24-48 h after glucocorticoid administration. 80-90% of glucocorticoid receptor binding was regenerated by 48 h, and complete binding was recovered by 72 h. Regenerated glucocorticoid receptor binding (48-72 h after first hormone injection) could be re-depressed by a second injection of the hormone. Similar results were obtained using normal (intact) rats. Optimum induction of tyrosine aminotransferase activity was obtained within 2 h following the first hormonal injection. Induction of tyrosine aminotransferase activity (measured 2 h after a second injection of the glucocorticoid) correlated with glucocorticoid receptor levels. Thus, 1 mg of dexamethasone or corticosterone greatly enhanced the liver tyrosine aminotransferase activity in the adrenalectomized rats (not previously hormone treated) and in adrenalectomized rats previously injected (48-72 h) with 1 mg of the glucocorticoid hormone. Enhancement of tyrosine aminotransferase activity was lowest 16-24 h after the first hormone injection (when receptor levels were extremely low). These results indicate that the induction of liver tyrosine aminotransferase activity by glucocorticoid hormones is correlated with cytosolic glucocorticoid receptor levels.     

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.     

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.     

Polyribosome distribution in regenerating livers of irradiated adrenalectomized rats.

The effect of gamma-radiation (1800 rad) on polyribosome distribution in the regenerating livers of intact and adrenalectomized rats was studied both before and after partial hepatectomy. The animals were divided into four sub-groups: (1) control; (2) irradiated only; (3) partially hepatectomized; and (4) irradiated partially hepatectomized. The relative distribution of lighter oligosomes to heavier polyribosomes was analysed by sucrose density gradient centrifugation (10-40 per cent). Partial hepatectomy by itself increased the proportion of heavier polyribosomes in both intact and adrenalectomized rats. However, when gamma-rays were delivered 2 hours before or 2 hours after partial-hepatectomy, the formation of heavy polyribosomes was decreased for at least 24 hours after hepatectomy in the adrenal intact rats. This depression was not maintained until later times (48 and 72 hours after hepatectomy) when an increase in the proportion of heavy polyribosomes relative to light oligosomes was observed. In addition, irradiation did not measurably affect the distribution of polyribosomes in regenerating hepatocytes of adrenalectomized rats at any time after partial hepatectomy.