Expression of hepatic thrombopoietin mRNA in primary cultured hepatocytes and in rats with acute liver injury or bone marrow suppression with or without cirrhosis

BACKGROUND AND AIMS: The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. The mechanisms by which levels of TPO mRNA are regulated in cirrhosis have not been elucidated. In this study, we investigated some possible mechanisms. METHODS: We used three experimental models: bone marrow suppression, acute liver injury and primary cultured hepatocytes. We used northern blots to assess the kinetics of TPO mRNA expression in the livers of irradiated rats (with and without cirrhosis) in acute liver injury and in primary cultured hepatocytes treated with hepatotoxin or cytokines. RESULTS: Although the bone marrow was hypocellular, there was no apparent enhancement of TPO mRNA expression in the irradiated rats with cirrhotic livers compared with the unirradiated rats with cirrhotic livers. There were no conspicuous changes in hepatic TPO mRNA expression between the livers of the control rats and the three models of acute liver injury. There were no conspicuous changes in the levels of TPO mRNA between control hepatocytes and hepatocytes treated with hepatotoxin or cytokines. CONCLUSIONS: Our results suggest that bone marrow is not a regulator of hepatic TPO production in cirrhosis. The reduced TPO mRNA expression found in cirrhotic rats may not result merely from serious cellular damage; it may be associated with cirrhosis-specific regulatory mechanisms for the expression of the TPO gene. Further studies are needed to search for other factors that may induce reduced TPO expression.     

Expressions of molecules associated with hepatocyte growth factor activation after hepatectomy in liver cirrhosis

BACKGROUND/AIMS: The activation pathway of hepatocyte growth factor (HGF), including HGF activator (HGFA) and HGFA inhibitor-1, 2 (HAI-1, 2), has recently been clarified. The present study examined mRNA expressions of HGF, HGFA and HAI-1 following partial hepatectomy in normal and cirrhotic rats. METHODOLOGY: Liver cirrhosis was induced by intraperitoneal injections of dimethylnitrosamine. Two weeks after, the cirrhotic and normal rats underwent 70% hepatectomy and the liver regeneration rate, DNA synthesis of hepatocytes, plasma HGF level, and mRNA expressions of HGF, HGFA, and HAI-1 in the liver, spleen, and lung were examined at different times. RESULTS: Liver regeneration in the cirrhotic rats was deteriorated with a later peak of hepatocellular DNA synthesis. Hepatic HGF mRNA and splenic HAI-1 mRNA were upregulated and liver HGFA mRNA was downregulated in the cirrhotic rats. CONCLUSIONS: Insufficient HGF activation both by a reduced expression of hepatic HGFA and an increased expression of splenic HAI-1 may be one of the reasons for the impaired liver regeneration in cirrhosis.     

Hepatocyte growth factor promotes liver regeneration and protein synthesis after hepatectomy in cirrhotic rats

BACKGROUND/AIMS: Hepatocyte growth factor, a potent mitogen for hepatocytes has been reported to be a hepatrophic factor in normal livers. In this study, the effect of exogenous hepatocyte growth factor on liver regeneration in cirrhotic rats was investigated, in vitro and in vivo. METHODOLOGY: Liver cirrhosis was induced by intraperitoneal injections of an emulsion, carbon tetrachloride and olive oil, twice weekly for 10 weeks. In vitro, various amounts of exogenous hepatocyte growth factor; 0, 0.5, 1, 2.5, 5, and 10 ng/mL; were added to the hepatocytes isolated using in situ perfusion method. In vivo, partial hepatectomy (Hx), according to the procedure described by Higgins and Anderson, was performed on cirrhotic rats. Saline solution (control group) or 3 micrograms/kg of exogenous hepatocyte growth factor (HGF group) was then injected through the tail vein at intervals 12 hours after Hx. RESULTS: In vitro, DNA synthesis in hepatocytes obtained from cirrhotic livers increased following exogenous hepatocyte growth factor in dose-dependent fashion. In vivo, the labeling index of 5-bromo-2'-deoxyuridine at 24 hours after Hx was markedly increased by exogenous hepatocyte growth factor (control, 10.0 +/- 3.1%; hepatocyte growth factor, 25.8 +/- 9.8%; P < 0.01). Furthermore, serum albumin at 24 and 72 hours and a normotest at 24 hours after Hx, were significantly higher in the HGF group than in the control group. CONCLUSIONS: These results indicate that exogenous hepatocyte growth factor may promote DNA synthesis and protein synthesis during liver regeneration after Hx with cirrhosis.     

Simultaneous transfer of vascular endothelial growth factor and hepatocyte growth factor genes effectively promotes liver regeneration after hepatectomy in cirrhotic rats

BACKGROUND/AIMS: Liver regeneration in a cirrhotic liver is unsatisfactory. In the course of liver regeneration, non-parenchymal cells such as sinusoidal endothelial cells as well as hepatocytes increase in number while the liver structure and physiological functions are maintained. The aim of this study was to examine whether sufficient liver regeneration could be obtained by the simultaneous, preoperative injection of recombinant adenoviral vectors encoding human vascular endothelial growth factor (VEGF), a potent mitogen for sinusoidal endothelial cells, (pAxCAVEGF) and rat hepatocyte growth factor (HGF), a potent mitogen for hepatocytes, (pAxCAHGF) in 70% hepatectomized cirrhotic rats. METHODOLOGY: Forty-eight hours before 70% hepatectomy, dimethylnitrosamine-induced cirrhotic rats were infused intravenously with pAxCAVEGF or with pAxCAVEGF and pAxCAHGF, or with a control virus encoding Escherichia coli beta-galactosidase (pAxCALacZ). RESULTS: Strong VEGF mRNA expressions were shown in the livers of VEGF and VEGF/HGF-treated animals. The plasma HGF concentrations in the VEGF/HGF-treated rats were elevated compared with the other groups. Proliferating cell nuclear antigen immunostaining showed increased labeling indices of hepatocytes in the VEGF/HGF-treated rats at 24 and 48 h after hepatectomy. PCNA labeling indices of SECs were increased in the VEGF and VEGF/HGF-treated rats compared with the control animals at 24 and 48 h after hepatectomy. Moreover, the hepatic regeneration rate after hepatectomy was significantly augmented by the VEGF and VEGF/HGF treatment. CONCLUSIONS: Simultaneous preoperative injection of recombinant adenoviral vectors encoding VEGF and HGF effectively stimulates liver regeneration in cirrhotic rats.     

Expression of growth hormone receptor and its mRNA in hepatic cirrhosis

AIM: To investigate the expression of growth hormone receptor (GHR) and mRNA of GHR in cirrhotic livers of rats with the intension to find the basis for application of recombinant human growth hormone (rhGH) to patients with liver cirrhosis.METHODS: Hepatic cirrhosis was induced in SpragueDawley rats by administration of thioacetamide intraperitoneally for 9-12 weeks. Collagenase Ⅳ was perfused in situ for isolation of hepatocytes. The expression of GHR and its mRNA in cirrhotic livers was studied with radio-ligand binding assay, RT-PCR and digital image analysis.RESULTS: One class of specific growth hormone-binding site, GHR, was detected in hepatocytes and hepatic tissue of cirrhotic livers. The binding capacity of GHR (RT, fmol/mg protein) in rat cirrhotic liver tissue (30.8±1.9) was significantly lower than that in normal control (74.9±3.9) at the time point of the ninth week after initiation of induction of cirrhosis (n=10, P＜0.05), and it decreased gradually along with the accumulation of collagen in the process of formation and development of liver cirrhosis (P＜0.05). The number of binding sites (×10 4/cell) of GHR on rat cirrhotic hepatocytes (0.86±0.16) was significantly lower than that (1.28±0.24)in control (n= 10, P＜0.05). The binding affinity of GHR among liver tissue, hepatocytes of various groups had no significant difference (P＞0.05). The expression of GHR mRNA (riOD,pixel) in rat cirrhotic hepatic tissues (23.3±3.1) was also significantly lower than that (29.3±3.4) in normal control (n=10, P＜0.05).CONCLUSION: The growth hormone receptor was expressed in a reduced level in liver tissue of cirrhotic rats,and lesser expression of growth hormone receptors was found in a later stage of cirrhosis. The reduced expression of growth hormone receptor was partly due to its decreased expression on cirrhotic hepatocytes and the reduced expression of its mRNA in cirrhotic liver tissue.     

肝炎肝硬化患者骨髓TPO、GM—CSF表达与外周血细胞的关系

目的探讨骨髓内环境造血因子血小板生成素（TPO）、粒细胞-巨噬细胞集落刺激因子（GM-CSF）在肝炎肝硬化患者骨髓液中的表达及其与外周血细胞的关系。方法选取31例肝炎肝硬化患者和15例健康对照组,晨起空腹采血、检测血常规、肝脏功能、肝炎病毒标志物,并行骨髓穿刺术,取骨髓液、离心分离,用双抗体夹心ELISA法检测骨髓液中TPO、GM-CSF浓度。结果肝炎肝硬化患者骨髓液TPO浓度与外周血血小板计数呈负相关（r=-0.496,P〈0.05）。骨髓液TPO的浓度在肝炎肝硬化组和对照组分别为（90.756±30.92）pg/ml、（118.414±49.232）pg/ml,二者之间有差异,但差异无统计学意义。肝脏功能按照Child-Pugh分为A、B、C级,分别与对照组比较,发现随肝脏损害加重,TPO浓度呈下降趋势,但差异无统计学意义。GM-CSF的浓度在肝硬化组和对照组之间差异无统计学意义。结论肝炎肝硬化患者骨髓内环境TPO浓度降低可能是外周血血小板计数减少的原因之一。肝脏功能状态及GM-CSF与外周血细胞计数变化的关系还有待进一步研究。     

Synthesis of platelet-activating factor and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis

AIM： To determine the platelet-activating factor （PAF） synthesis and its receptor expression in Kupffer cells in rat carbon tetrachloride-induced cirrhosis.
METHODS： Kupffer cells, isolated from the livers of control and CCl4-induced cirrhotic rats, were placed in serum-free medium overnight. PAF saturation binding, ET-1 saturation and competition binding were assayed. ET-1 induced PAF synthesis, mRNA expression of PAF, preproendothelin-1, endothelin A （ETA） and endothelin B （ETB） receptors were also determined.
RESULTS： A two-fold increase of PAF synthesis （1.42 ± 0.14 vs 0.66 ± 0.04 pg/μg DNA） and a 1.48-fold increase of membrane-bound PAF （1.02 ± 0.06 vs 0.69 ± 0.07 pg/μg DNA） were observed in activated Kupffer cells of cirrhotic rats. The application of ET-1 to Kupffer cells induced PAF synthesis in a concentration-dependent manner in both cirrhotic and normal rats via ETB receptor, but PAF synthesis in the activated Kupffer cells was more effective than that in the normal Kupffer cells. In activated Kupffer cells, PAF receptor expression and PAF binding capacity were markedly enhanced. Activated Kupffer cells raised the [^125I]-ET-1 binding capacity, but changed neither the affinity of the receptors, nor the expression of ETA receptor.
CONCLUSION： Kupffer cells in the course of CCl4- induced cirrhosis are the main source of increased PAF. ET-1 is involved endogenously in stimulating the PAF synthesis in activated Kupffer cells via ETB receptor by paracrine. ETA receptor did not appear in activated Kupffer cells, which may exacerbate the hepatic and extrahepatic complications of cirrhosis.     

Differentiation of hematopoietic progenitor cells towards the myeloid and B-lymphoid lineage by hepatocyte growth factor (HGF) and thrombopoietin (TPO) together with early acting cytokines

OBJECTIVES: The effect of stem cell factor (SCF), flt3-ligand (FL), and interleukin (IL)-3 (SF3) in combination with hepatocyte growth factor (HGF), thrombopoietin (TPO), and Hyper-IL-6 on maintenance and differentiation of early human peripheral blood-derived progenitor cells was investigated. METHODS: Single sorted CD34(+) 38(-) cells were cultured with various combinations of these growth factors in order to identify the most effective cytokine combination. Then, lineage-depleted cells were stimulated for 7 d in bulk culture before they were assessed by flow cytometry and in functional assays. RESULTS: The highest number of clones in the single-cell assay was obtained after culture with SF3 + TPO + HGF. Cell expansion with SF3 + TPO + HGF yielded an increase of the total cell number (11-fold), the number of CD34(+) cells (sevenfold), colony forming cells (CFC; 13-fold), granulocytes (CD15/66b(+); 45-fold) and B-cells (CD19/20(+); 55-fold). However, the number of long-term culture initiating cells (LTC-IC) decreased from 779 +/- 338 per 1 x 10(5) CD34(+) cells on day 0 to 253 +/- 115 on day 7. In parallel, the number of pluripotent mouse repopulating cells decreased by the factor 11, and no significant change in the proportion of human myeloid or lymphoid cells found in the mouse bone marrow was noted. CONCLUSION: The observation that mature cells of different lineages are generated and that transplantable multipotent hematopoietic cells are lost during culture suggests the differentiation of early hematopoietic progenitors toward lineage committed cells by the tested cytokines. The detection of cells expressing B-lymphoid markers after culture indicates a possible role in the propagation of B-cells.     

Interleukin-6 induces proliferation of rat hepatocytes in vivo

Background/Aims: The aim of this study was to assess the effect of interleukin-6 (IL-6) on the proliferation of hepatocytes and to study the interaction between IL-6 and hepatocyte growth factor (HGF) in vivo.Methods: IL-6 was injected at a dose of 200 μg/mg subcutaneously into rats every day for 14 days. Liver and blood samples were obtained at 1, 3, 7 and 14 days during IL-6 administration. Hepatocyte proliferative activity of sera was measured using ³H-thymidine incorporation into cultured rat hepatocytes. To evaluate the proliferative activity of the hepatocytes in tissue sections, hepatic DNA content and immunostaining of the liver tissue sections for proliferating cell nuclear antigen (PCNA) were performed. Plasma HGF levels were measured using specific EIA. In addition, total RNA was extracted from the liver and expression of HGF mRNA was detected by RT-PCR.Results: The DNA contents of liver taken from IL-6-treated rats were increased during IL-6 administration compared with untreated rats. Sera taken from IL-6-treated rats at various intervals during administration also significantly increased ³H-thymidine incorporation by cultured rat hepatocytes compared with sera from untreated rats, suppressing ³H-thymidine incorporation at day 1 and 3 by anti-HGF antibody. IL-6 itself did not increase ³H-thymidine incorporation. Increased expression of PCNA in these hepatocytes was noted from 1 day after IL-6 administration, and at 14 days, the number of PCNA-positive cells was sevenfold greater than in the livers of untreated rats. However, plasma HGF levels showed a peak at day 1, decreased gradually from day 3, and became undetectable by day 14. HGF mRNA expression in livers of IL-6-treated rats was suppressed from day 3 to day 14 of IL-6 administration.Conclusions: These data show that IL-6 induces an early phase of liver cell growth in vivo and suggest that an increase level of HGF mediates this effect.     

Hepatocyte growth factor gene therapy accelerates regeneration in cirrhotic mouse livers after hepatectomy

BACKGROUND: Impaired regeneration and dysfunction of the cirrhotic liver following partial hepatectomy (PHx) are the most serious risk factors for postoperative liver failure. AIMS: Using naked hepatocyte growth factor (HGF) plasmid by the electroporation (EP) in vivo method, we investigated HGF for its role and mechanism of proliferation and restoration of liver mass in cirrhotic mice following PHx. ANIMALS: Eight week old female mice were used. METHODS: HGF plasmid 50 micro g was injected intramuscularly and transferred by EP in vivo once a week for three weeks. After establishment of carbon tetrachloride induced cirrhosis, mice underwent PHx. The HGF treated group was given naked HGF plasmid four days before PHx, and additional HGF was given once a week until they were killed, while a control group was given only empty plasmid. Mice were killed 2, 4, 10, and 14 days after PHx. Morphological and functional restoration of the liver were examined, as well as activation of mitogen activated protein kinase (MAPK) and mRNA levels of HGF activator (HGFA). RESULTS: The HGF treated group demonstrated a continuous threefold increase in HGF levels in plasma. Therapy with HGF in cirrhotic PHx resulted in effective liver regeneration via restoration of HGFA and activation of MAPK p44/p42, accelerated normalisation of liver function, and increased collagen degradation. CONCLUSIONS: HGF gene therapy by in vivo EP may be useful for hepatic resection in cirrhotic livers by stimulating liver proliferative and collagenolytic capacities, as well as accelerating functional recovery.     

部分肝切除后的肝损伤血清和肝细胞生长因子促进大鼠骨髓细胞表达甲胎蛋白和白蛋白

目的 检测大鼠骨髓中是否存在肝脏干细胞，并用肝损伤血清和肝细胞生长因子(HGF)刺激骨髓细胞向肝细胞转化。方法 SD大鼠骨髓单个核细胞分3组进行培养：(1)单纯培养基对照组；(2)肝损伤血清组(15％，血清来自于2-AAF 75％肝切除大鼠)；(3)HGF(20ng／ml)组。用甲胎蛋白(AFP)和白蛋白作为细胞标志，用免疫组织化学、逆转录聚合酶链反应(PCR)、巢式PCR和western blot方法，观察大鼠肝损伤血清和HGF对骨髓细胞转化的促进作用。结果 肝损伤血清组和HGF组培养后10d和20d AFP免疫组织化学和western blot染色阳性；RT-PCR AFP mRNA阳性，新鲜骨髓细胞和单纯IMDM／F12培养基组AFP蛋白和mRNA均阴性。新鲜骨髓细胞存在白蛋白mRNA表达，在肝损伤血清组和HGF组培养后10d和20d，白蛋白mRNA表达增强。结论 大鼠肝损伤血清和HGF可促使体外培养骨髓细胞表达AFP mRNA和AFP。骨髓中可能存在骨髓源性肝干细胞，并微弱表达白蛋白mRNA；肝损伤血清和HGF可以促进骨髓细胞表达白蛋白mRNA。     

导入外源肝细胞生长因子基因对肝细胞增殖的影响

目的 利用重组腺病毒载体将外源人肝细胞生长因子(HGF)基因导入原代培养的大鼠肝细胞, 观察HGF表达对肝细胞增殖特性的影响。方法 同源重组构建表达HGF的复制缺陷型重组腺病毒AdHGF,用 其感染原代培养的肝细胞。逆转录聚合酶链反应检测肝细胞HGF和c—met(HGF受体)mRNA的表达;酶联免 疫吸附实验测定培养上清液中HGF水平。MTS测定感染前后细胞增殖情况;流式细胞仪测定细胞周期的变化; 细胞免疫荧光法检测HGF基因导入后增殖细胞核抗原(PCNA)表达。结果 同源重组获得约4×10~(10)efu/ml 滴度的AdHGF。AdHGF感染原代培养肝细胞后,肝细胞HGF和c-met mRNA表达均明显上调;细胞上清液 中HGF分泌水平显著增加,达到(5 939.00±414.39)pg/ml(F=13.661,P<0.01)。细胞增殖能力增强(F ≥15.158,P<0.01),细胞周期由G_0/G_1期向S期转化(X~2=41.616,P<0.01);细胞免疫荧光法提示HGF 基因导入后PCNA指数显著提高(F=42.122,P<0.01)。结论 通过重组腺病毒载体将外源HGF基因 导入肝细胞后可维持HGF高效表达并能促进肝细胞增殖,是基因修饰供体肝细胞、增强肝细胞移植治疗效 果的有效方法。