Expressed genes in regenerating rat liver after partial hepatectomy

AIM: To reveal the liver regeneration (LR) and its control as well as the occurrence of liver disease and to study the gene expression profiles of 551 genes after partial hepatectomy (PH) in regenerating rat livers. METHODS: Five hundred and fifty-one expressed sequence tags screened by suppression subtractive hybridization were made into an in-house cDNA microarray, and the expressive genes and their expressive profiles in regenerating rat livers were analyzed by microarray and bioinformatics. RESULTS: Three hundred of the analyzed 551 genes were up- or downregulated more than twofolds at one or more time points during LR. Most of the genes were up- or downregulated 2-5 folds, but the highest reached 90 folds of the control. One hundred and thirty-nine of them showed upregulation, 135 displayed downregulation, and up or down expression of 26 genes revealed a dependence on regenerating livers. The genes expressed in 24-h regenerating livers were much more than those in the others. Cluster analysis and generalization analysis showed that there were at least six distinct temporal patterns of gene expression in the. regenerating livers, that is, genes were expressed in the immediate early phase, early phase, intermediate phase, early-late phase, late phase, terminal phase. CONCLUSION: In LR, the number of down-regulated genes was almost similar to that of the upregulated genes; the successively altered genes were more than the rapidly transient genes. The temporal patterns of gene expression were similar 2 and 4 h, 12 and 16 h, 48 and 96 h, 72 and 144 h after PH. Microarray combined with suppressive subtractive hybridization can effectively identify the genes related to LR.     

Gene expression differences of regenerating rat liver in a short interval successive partial hepatectomy

AIM: To identify the genes expressed differentially in the regenerating rat liver in a short interval successive partial hepatectomy (SISPH), and to analyze their expression profiles. METHODS: Five hundred and fifty-one elements selected from subtractive cDNA libraries were conformed to a cDNA microarray (cDNA chip). An extensive gene expression analysis following 0-36-72-96-144 h SISPH was performed by microarray. RESULTS: Two hundred and sixteen elements were identified either up- or down-regulated more than 2-fold at one or more time points of SISPH. By cluster analysis and generalization analysis, 8 kinds of ramose gene expression clusters were generated in the SISPH. Of the 216 elements, 111 were up-regulated and 105 down-regulated. Except 99 unreported genes, 117 reported genes were categorized into 22 groups based on their biological functions. Comparison of the gene expression in SISPH with that after partial hepatectomy (PH) disclosed that 56 genes were specially altered in SISPH, and 160 genes were simultaneously up-regulated or down-regulated in SISPH and after PH, but in various amount and at different time points. CONCLUSION: Genes expressed consistently are far less than that intermittently; the genes strikingly increased are much less than that increased only 2-5 fold; the expression trends of most genes in SISPH and in PH are similar, but the expression of 56 genes is specifically altered in SISPH. Microarray combined with suppressive subtractive hybridization can in a large scale effectively identify the genes related to liver regeneration.     

Gene expression profile in the regenerating rat liver after partial hepatectomy

BACKGROUND/AIMS: When a loss of hepatic mass occurs, the expression of a large number of genes is either induced or altered, accompanying hepatocyte proliferation. In the present study, we made an in-house cDNA microarray containing 4608 elements (Liver chip), and analyzed extensively gene expression profiles of the regenerating liver after 70% partial hepatectomy (PHx) in rats. METHODS: RNAs were prepared from three rat livers at each time point (taken at 0, 6, 12, 18, 24, 48, 72 h, and 1 week after PHx). Using the liver chip, we performed large-scale analysis of gene expression during liver regeneration. Elements either up- or down-regulated more than twofold at one or more time points were selected. RESULTS: Among the 4608, 382 were identified. Using cluster analysis, we found great similarity between gene-expression profiles at 12 and 18 h after PHx as well as between 48 and 72 h after PHx. We also found that there are at least six distinct temporal patterns of gene expression in the regenerating rat liver after PHx. CONCLUSIONS: These results indicated that microarray analysis is a powerful approach for monitoring molecular events in the regenerating liver.     

Differentiation of rat bone marrow stem cells in liver after partial hepatectomy

AIM: To investigate the differentiation of rat bone marrow stem cells in liver after partial hepatectomy. METHODS: Bone marrow cells were collected from the tibia of rat with partial hepatectomy, the medial and left hepatic lobes were excised. The bone marrow stem cells (Thy CD3-CD45RA- cells) were enriched from the bone marrow cells by depleting red cells and fluorescence-activated cell sorting. The sorted bone marrow stem cells were labeled by PKH26-GL in vitro and autotransplanted by portal vein injection. After 2 wk, the transplanted bone marrow stem cells in liver were examined by the immunohistochemistry of albumin (hepatocyte-specific marker). RESULTS: The bone marrow stem cells (Thy CD3-CD45RA- cells) accounted for 2.8% of bone marrow cells without red cells. The labeling rate of 10μM PKH26-GL on sorted bone marrow stem cells was about 95%. There were sporadic PKH26-GL-labeled cells among he-patocytes in liver tissue section, and some of the cells expressed albumin. CONCLUSION: Rat bone marrow stem cells can differentiate into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.     

肝细胞生成素及肝部分切除快速诱导肝再生相关基因Tec的表达

目的：肝部分切除术后肝细胞生成素（HPO）迅速增加，该文观察人重组肝细胞生成素（rhHPO）和肝部分切除（PH）迅速诱导瞬时早期反应基因表达情况。方法：将Wister大鼠分为PH组和对照组，每组3只。利用表达性差异显示分析技术和序列分析，检测2／3 PH后1h及原代培养大鼠肝细胞体系的选择性基因表达。结果：EST库中的大部分为瞬时早期反应基因，发现一种新的可能与肝再生调控相关的Tec基因，Northern杂交证实2／3 PH可迅速诱导Tec基因的表达，其表达高峰为术后1－2h。HPO在原代培养大鼠肝细胞体系中可迅速诱导瞬时早期反应基因（c-fos、LRF－1和Tec等）表达。结论：HPO和PH可迅速诱导瞬时早期反应基因表达，首次报道了Tec基因是一种与肝再生调控密切相关的早期反应基因。     

肝细胞生成素及肝部分切除诱导肝再生基因PC3的表达

目的 观察重组人肝细胞生成素（rhHPO)和肝部分切除迅速诱导瞬时早期反应基因表达情况。方法 利用表达性差异显示分析技术和序列分析研究2／3肝部分切除后1h及原代培养大鼠肝细胞体系的选择性基因表达。结果 揭示EST集中的大部分为瞬时早期反应基因，发现一种可能与肝再生调控相关的新基因PC3，Northern杂交证实2／3肝部分切除可迅速诱导PC3基因的表达，其表达高峰为术后1－2h。HPO在原代培养大鼠肝细胞体系中可迅速诱导瞬时早期反应基因（c-fos,LRF-1和PC3等）表达。结论 HPO和肝部分切除可迅速诱导瞬时早期反应基因表达，PC3基因是一种与肝再生调控密切相关的早期反应基因。     

HGF及其受体c-met在肝衰竭与部分肝切除模型中表达差异性研究

目的研究HGF及其受体c-met在肝衰竭和部分肝切除两种动物模型肝组织的表达差异。方法分别采用D-GalN/LPS腹腔注射和70%肝脏外科切除术建立肝衰竭和部分肝切除模型,同时以健康大鼠作为对照组。采用ELISA法检测血清HGF水平,采用Real-time PCR和Western-blot法分别检测肝组织c-met基因和蛋白的表达水平。结果与对照组比,部分肝切除模型大鼠血清HGF及其受体c-met表达水平均有明显升高（P〈0.01）,而在肝衰竭模型大鼠血清HGF水平虽有升高,但c-met蛋白表达水平却明显降低（P〈0.01）。结论肝细胞c-met表达水平的降低是D-GalN/LPS诱导肝衰竭大鼠肝细胞再生障碍的主要原因。     

Proliferating cell nuclear antigen (PCNA) expression in regenerating rat liver after partial hepatectomy

Proliferating cell nuclear antigen (PCNA) is a nuclear protein maximally elevated in the S phase of proliferating and transformed cells and is recognized by the monoclonal antibody PC-10 in paraffin tissue sections. The liver regenerative process after partial hepatectomy in rats was estimated with thein vivo incorporation of [³H]thymidine into liver DNA and the liver thymidine kinase activity. The expression of PCNA in rat liver after partial hepatectomy was performed by immunohistochemical staining with PC-10 in paraffin embedded tissues, at different time intervals up to 240 hr. Proliferating cell nuclear antigen expression, [³H]thymidine incorporation into DNA, and liver thymidine kinase activity exhibited marked oscillations during the liver regenerative process. A close relationship was demonstrated among DNA synthesis, thymidine kinase activity, and PC-10 score. Our results suggest that PC-10 monoclonal antibody may be used as a worthwhile proliferation index in the evaluation of the rate of liver regeneration in rats.     

Cloning and analysizing the up-regulated expression of transthyretin-related gene(LR1) in rat liver regeneration following short interval successive partial hepatectomy

AIM: Cloning and analysizing the up-regulated expressionof transthyretin-related gene following short intervalsuccessive partial hepatectomy (SISPH) to elucidate themechanism of differentiation, division, dedifferentiation andredifferentiation in rat liver regeneration (LR).METHODS: Lobus external sinister and lobus centralissinister, lobus centralis, lobus dexter, lobus candatus wereremoved one by one from rat liver at four different time points4, 36, 36 and 36 hr (total time: 4 hr, 40 hr, 76 hr, 112 hr)respectively. Suppression subtractive hybridization (SSH) wascarried out by using normal rat liver tissue as driver and thetissue following short interval successive partial hepatectomy(SISPH) as tester to construct a highly efficient forward-subtractive cDNA library. After screening, an interested ESTfragment was selected by SSH and primers were designedaccording to the sequence of the EST to clone the full-lengthcDNA fragment using RACE (rapid amplification of cDNAend). Homologous detection was performed between thefull-lenth cDNA and Genbank.RESULTS: Forward suppression subtractive hybridization(FSSH) library between 0 h and 112 h following SISPH wasconstructed and an up-regulated full-length cDNA (namedLR1), which was related with the transthyretin gene, wascloned by rapid amplification of cDNA end. It was suggestedthat the gene is involved in the cellular dedifferentiation inLR following SISPH.CONCLUSION: Some genes were up-regulated in 112 hfollowing SISPH in rat. LR1 is one of these up-regulatedexpression genes which may play an important role in rat LR.     

Cloning and analysizing the up-regulated expression of transthyretin-related gene （LR1） in rat liver regeneration following short interval successive partial hepatectomy

AIM: Cloning and analysing the up-regulated expression of transthyretin-related gene following short interval successive partial hepatectomy (SISPH) to elucidate the mechanism of differentiation, division, dedifferentiation and redifferentiation in rat liver regeneration (LR). METHODS: Lobus external sinister and lobus centralis sinister, lobus centralis, lobus dexter, lobus candatus were removed one by one from rat liver at four different time points 4, 36, 36 and 36 hr (total time: 4 hr, 40 hr, 76 hr, 112 hr) respectively. Suppression subtractive hybridization (SSH) was carried out by using normal rat liver tissue as driver and the tissue following short interval successive partial hepatectomy (SISPH) as tester to construct a highly efficient forward-subtractive cDNA library. After screening, an interested EST fragment was selected by SSH and primers were designed according to the sequence of the EST to clone the full-length cDNA fragment using RACE (rapid amplification of cDNA end). Homologous detection was performed between the full-lenth cDNA and Genbank. RESULTS: Forward suppression subtractive hybridization (FSSH) library between 0 h and 112 h following SISPH was constructed and an up-regulated full-length cDNA (named LR1), which was related with the transthyretin gene, was cloned by rapid amplification of cDNA end. It was suggested that the gene is involved in the cellular dedifferentiation in LR following SISPH. CONCLUSION: Some genes were up-regulated in 112 h following SISPH in rat. LR(1) is one of these up-regulated expression genes which may play an important role in rat LR.     

应用基因芯片技术筛选大鼠再生肝中差异表达基因

目的应用基因芯片技术筛选大鼠再生肝中差异表达基因,为探讨急性肝功能衰竭的发病机制提供基础。方法雄性SD大鼠行95％肝部分切除术,用含1176个基因的大鼠尼龙膜微阵列筛选大鼠再生肝组织中差异表达的基因。RT—PCR随机验证若干差异表达基因在微阵列中的表达。结果再生肝组织有138个基因明显较对照组织表达高,它们主要是生长因子基因、核受体基因、核糖体蛋白基因、应急反应蛋白基因、细胞周期调节基因、神经递质基因等;下调基因共50个,主要为代谢酶基因、激素受体基因及表面抗原基因等。结论cDNA微阵列技术有助于研究急性肝功能衰竭的发病机制,并提供诊断和治疗的潜在靶点。     

Liver structural transformation after partial hepatectomy and repeated partial hepatectomy in rats: A renewed view on liver regeneration

BACKGROUND The phenomenon of liver regeneration after partial hepatectomy(PH) is still a subject of considerable interest due to the increasing frequency of half liver transplantation on the one hand, and on the other hand, new surgical approaches which allow removal of massive space-occupying hepatic tumors, which earlier was considered as inoperable. Interestingly, the mechanisms of liver regeneration are extensively studied after PH but less attention is paid to the architectonics of the regenerated organ. Because of this, the question "How does the structure of regenerated liver differ from normal, regular liver?" has not been fully answered yet. Furthermore, almost without any attention is left the liver's structural transformation after repeated hepatectomy(of the re-regenereted liver).To compare the architectonics of the lobules and circulatory bed of normal, regenerated and re-regenerated livers.METHODS The livers of 40 adult, male, albino Wistar rats were studied. 14 rats were subjected to PH-the 1st study group(SG_1); 10 rats underwent repeated PH – the 2nd study group(SG2); 16 rats were subjected to sham operation-control group(CG); The livers were studied after 9 months from PH, and after 6 months from repeated PH. Cytological(Schiff reaction for the determination of DNA concentration), histological(HE, Masson trichrome, CK8 Immunohistochemical marker, transparent slides after Indian Ink injection,), morphometrical(hepatocytes areas, perimeters and ploidy) and Electron Microscopical(Scanning Electron Microscopy of corrosion casts) methods were used.RESULTS In the SG_1 and SG_2, the area of hepatocytes and their perimeter are increased compared to the CG(P 0.05). However, the areas and perimeters of the hepatocytes of the SG_1 and SG_2 groups reveal a lesser difference. In regenerated(SG_1) and re-regenerated(SG_2) livers, the hepatocytes form the remodeled lobules, which size(300-1200 μm) exceeds the sizes of the lobules from CG(300-600 μm). The remodeled lobules(especially the "mega-lobules" with the sizes 1000-1200 μm) contain the transformed meshworks of the sinusoids, the part of which is dilated asymmetrically. This meshwork might have originated from the several portal venules(interlobular and/or inlet). The boundaries between the adjacent lobules(including mega-lobules) are widened and filled by connective tissue fibers, which gives the liver parenchyma a nodular look. In SG_2 the unevenness of sinusoid diameters, as well as the boundaries between the lobules(including the mega-lobules) are more vividly expressed in comparison with SG_1. The liver tissue of both SG_1 and SG_2 is featured by the slightly expressed ductular reaction.CONCLUSION Regenerated and re-regenerated livers in comparison with normal liver contain hypertrophied hepatocytes with increased ploidy which together with transformed sinusoidal and biliary meshworks form the remodeled lobulli.     

熊脱氧胆酸促进肝脏部分切除后肝细胞再生

目的 探讨熊脱氧胆酸（ｕｒｓｏｄｅｏｘｙｃｈｏｌｉｃ ａｃｉｄ，ＵＤＣＡ）对胆道梗阻肝脏部分切除（ＰＨ）后肝细胞再生的影响。方法Ｗｉｓｔａｒ大鼠随机分为正常７０％肝部分切除组（Ｎ－ＰＨ）、胆道梗阻２周７０％ＰＨ组（ＢＤＯ－ＰＨ）、ＢＤＯ—ＰＨ ＵＤＣＡ治疗组及ＢＤＯ—ＰＨ生理盐水治疗组。观察肝组织学改变，检测７０％ＰＨ后肝细胞ＢｒｄＵ标记、肝内肝细胞生长因子（ＨＧＦ）及其受体Ｍｅｔ ｍＲＮＡ表达。结果 ＵＤＣＡ治疗能促进胆道梗阻后肝功能好转并减轻肝组织学病变；ＵＤＣＡ治疗组大鼠７０％ＰＨ后肝内ＨＧＦ／Ｍｅｔ ｍＲＮＡ高峰表达值均高于ＢＤＯ—ＰＨ组（Ｐ ＜ ０．０５），肝细胞 ＢｒｄＵ高峰标记指数（５９．３９±１０．８２）％高于 ＢＤＯ—ＰＨ组肝细胞 ＢｒｄＵ高峰标记指数（３６．２２±８．３７％（ｔ＝４．１４９，Ｐ＜０．０１），而与Ｎ－ＰＮ组肝细胞ＢｒｄＵ高峰标记指数（６８．６４±１１．２６％）％相比差异无显著性（ｔ＝１．４５１，Ｐ＞０．０５）。结论 ＵＤＣＡ通过缓解胆道梗阻后肝组织损害并上调７０％ＰＨ后肝内ＨＧＦ／Ｍｅｔ ｍＲＮＡ表达，从而促进胆道梗阻肝脏部分切除后肝细胞再生。     

5-羟色胺2A受体阻断剂对大鼠肝脏再生的影响

目的观察5-羟色胺2A受体阻断剂酮色林对大鼠肝部分切除后肝再生的影响,了解5-羟色胺及其受体在肝脏再生中的作用。方法 80只雄性Wistar大鼠随机分为实验组和对照组。采用肝大部分切除术建立肝再生模型,术后16 h分别给予腹腔内注射酮色林（实验组）和生理盐水（对照组）,采用免疫组化及流式细胞技术动态观察并比较两组大鼠术后24、36、48、72 h肝脏Ki67、增殖细胞核抗原的表达情况。结果大鼠肝大部切除术后24、36 h肝脏表达Ki67、增殖细胞核抗原最为活跃,而后表达逐渐下降。实验组大鼠肝脏表达Ki67、增殖细胞核抗原较对照组显著下降（P〈0.05）。结论 5-羟色胺2A受体阻断剂酮色林显著抑制大鼠肝大部切除术后的肝脏再生,说明5-羟色胺具有一定的促进肝再生的作用,2A受体是其重要的信号传导受体之一。     

Expression of tumor necrosis factor-alpha converting enzyme in liver regeneration after partial hepatectomy

AIM：To study the expression of tumor necrosis factor-alpha converting enzyme （TACE） and evaluate its significance in liver regeneration after partial hepatectomy in vivo.
METHODS： Male SD rats underwent 70% partial hepatectomy. The remaining liver and spleen tissue samples were collected at indicated time points after hepatectomy. TACE expression was investigated by Western blotting, immunohistochemistry, and serial section immunostaining.
RESULTS： Expression of TACE in liver and spleen tissues after partial hepatectomy was a time-dependent alteration, reaching a maximal level between 24 and 48 h and remaining elevated for more than 168 h. TACE protein was localized to mononuclear cells （MNC）, which infiltrated the liver from the spleen after hepatectomy. The kinetics of TACE expression was in accordance with the number of TACE-staining MNCs and synchronized with those of transforming growth factor-α （TGFα）. In addition, TACE-staining MNC partially overlapped with CD3^＋ T lymphocytes.
CONCLUSION： TACE may be involved in liver regeneration by pathway mediated with TGFα-EGFR in the cellcycle progressive phase in vivo. TACE production and effect by paracrine may be a pathway of involvement in liver regeneration for the activated CD3^＋ T lymphocytes.     

非酒精性脂肪肝大鼠部分肝切除术后肝再生功能的变化

目的 探讨大鼠非酒精性脂肪肝（NAFLD）部分肝脏切除术后肝再生功能的变化。方法 80只Wistar大鼠，随机分为正常对照组（C组，35只）与NAFLD组（F组，45只），C组给予正常饮食喂养，F组给予高脂饲料喂养。在喂养至第12周时行70％肝切除术，两组动物分别于术后0、1、12、24、36h处死，取出残肝，计算再生肝重比；光镜下计数核分裂肝细胞；透射电镜观察术后肝细胞超微结构的变化；免疫组织化学染色法检测增殖细胞核抗原阳性表达率；半定量逆转录聚合酶链反应检测细胞周期蛋白D1的表达变化。结果 光镜和电镜观察显示F组肝窦狭窄迂曲，细胞质内大量脂滴沉积，细胞核小，细胞器少，能量代谢及细胞增殖均不活跃。F组术后12、24、36h核分裂相计数明显低于C组同时相点（P〈0．01）；F组术后再生肝重比、S期细胞分数及增殖指数也较C组下降，差异有统计学意义（P〈0．01）；F组增殖细胞核抗原阳性率、细胞周期蛋白D1的mRNA表达在术后12、24、36h均明显低于C组同时相点（P〈0．01）。结论 中至重度NAFLD大鼠部分肝切除术后DNA合成高峰滞后，肝再生延迟，再生进程主要被阻滞在细胞周期的G1／S期调控点。