肝细胞生长因子对四氯化碳损伤原代培养大鼠肝细胞的保护作用

本工作采用无血清原代培养大鼠肝细胞法,观察了重组人肝细胞生长因子（ｒ－ｈＨＧＦ）对四氯化碳（ＣＣｌ４）致大鼠肝细胞损伤的保护作用。结果表明,ｒ－ｈＨＧＦ对ＣＣｌ４染毒肝细胞有明显的保护作用。ｒ－ｈＨＧＦ保护组较ＣＣｌ４染毒组细胞存活率显著升高,细胞内丙氨酸转氨酶、钾离子漏出明显降低。结果提示,ｒ－ｈＨＧＦ可减轻ＣＣｌ４对肝细胞膜的损伤,提高细胞膜的结构完整性     

肝细胞生长因子对四氯化碳损伤原代培养大鼠肝细胞的保护…

本工作采用无血清原供培养大鼠肝细胞法,观察了重组人肝细胞生长因子对四氯化碳致大鼠肝细胞损伤的保护作用。结果表明,ｒ－ｈＨＧＦ对ＣＣｌ４染毒肝细胞有明显的保护作用。ｒ－ｈＨＧＦ保护组较ＣＣｌ４染毒组细胞存活率显著升高,细胞内丙氨酸转氨酶,钾离子漏出明显降低。     

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（５ｎｇ／ｍｌ）预自理后可显著提高ＣＣｌ４（１５ｍｍｏｌ／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（５ｎｇ／ｍｌ）合用预处理肝细胞,ＣＣｌ４染毒后细胞内ＡＬＴ、Ｋ＾＋漏出较ｒｈＨＧＦ和     

人肝刺激因子对大鼠实验性慢性肝损伤的保护作用

从健康孕妇水囊引产４─６个月龄的胎儿取肝,采用ＬａＢｒｅｃｑｕｅ方法提取人肝刺激因子（ｈＨＳＳ）。经３Ｈ－胸腺嘧啶核苷参入肝ＤＮＡ法测定其生物活性。表明此ｈＨＳＳ可刺激肝细胞ＤＮＡ合成。采用皮下注射ＣＣｌ４和饮用１０％乙醇来制备慢性肝损伤动物模型,观察了ｈＨＳＳ的保护肝脏作用。结果表明：ｈＨＳＳ可使ＣＣｌ４－乙醇所致慢性肝损伤大鼠的死亡率、血清谷丙转氨酶水平、肝组织中羟脯氨酸含量的升高以及肝组织中丙二醛的含量降低。肝组织切片表明：ｈＨＳＳ能减轻肝组织的损伤程度,促进肝细胞再生,并能明显防止肝纤维化的形成和发展。可见,ｈＨＳＳ对ＣＣｌ４－乙醇所致的慢性肝损伤大鼠有明显的保护作用,其机制可能与促进肝细胞再生及抑制肝细胞膜的脂质过氧化有关。     

肝细胞生长因子刺激大鼠离体肝细胞DNA合成的剂量—与时间—效应

本工作采用３ＨＴｄＲ掺入ＤＮＡ法观察重组人肝细胞生长因子（ｒｈＨＧＦ）刺激大鼠离体肝细胞ＤＮＡ合成的剂量与时间效应。实验结果表明：ｒｈＨＧＦ是最强的促肝细胞分裂剂,在一定剂量范围内,ｒｈＨＧＦ与肝细胞ＤＮＡ合成有明显的量效关系。１ｎｇ／ｍｌｒｈＨＧＦ即可引起３ＨＴｄＲ掺入显著增加（Ｐ＜０．０１）,随剂量增加,刺激ＤＮＡ合成的效应也随之增强;１０ｎｇ／ｍｌ时３ＨＴｄＲ掺入量最大,较对照组高７倍（Ｐ＜０．００１）,剂量再增加即出现抑制效应;ｒｈＨＧＦ刺激肝细胞ＤＮＡ合成存在时间效应关系,表现为ｒｈＨＧＦ作用２４ｈ,ＤＮＡ合成量明显高于对照组（Ｐ＜０．０１）,４８ｈ作用达最高（Ｐ＜０．００１）,随后开始下降,至９６ｈ下降到相当于２４ｈ的水平。     

15—Mt—PGF2α对CCL4所致大鼠离体肝细胞损伤的保护作用

采用大鼠离体肝细胞原代培养２４ｈ,并利用四氯化碳ＣＣｌ４造成急性肝细胞损伤模型,检定１５－甲基－前列腺素Ｆ２α（１５－Ｍｔ－ＰＧＦ２α）对肝细胞损伤的影响。结果表明：（１）１５－Ｍｔ－ＰＧＦ２α可显著降低中毒肝细胞脂质过氧化物水平,抑制肝细胞脂质过氧化,并降低谷丙转氨酶（ＧＰＴ）和谷草转氨酶（ＧＯＴ）水平,稳定脂质膜。（２）显著促进中毒肝细胞ＲＮＡ和ＤＮＡ的合成。（３）超微结构证实１５－Ｍｔ－ＰＧＦ２α能减轻ＣＣｌ４对肝细胞脂质膜,染色质,线粒体,内质网和核蛋白体的损害。     

肝细胞生长因子mRNA在成年小鼠和人胎儿不同器官中的表达

本实验从成年小鼠和胎龄４－５月的人胎儿不同器官中分离总ＲＮＡ。经斑点印迹分析显示,肝细胞生长因子（ＨＧＦ）ｍＲＮＡ在成年ＫＭ小鼠多种器官中表达,其表达水平由高到低依次为：肺、肝、肾、卵巢、睾丸、大脑和胃;在脾、心、骨髓、小肠和骨骼肌组织中以ＨＧＦｍＲＮＡ。在胎龄４－５月的人胎儿中,ＨＧＦｍＲＮＡ表达水平由高到低依次为：大脑、肝、腮腺、胃、小肠、肾、心和骨骼肌;肺和脾组织为阴性。由此可见,ＨＧＦ在成     

四氯化碳损伤成年大鼠肝细胞后原癌基因（c－fos／c－jun）表达的观察

用四氯化碳（ＣＣｌ４）损伤正常大鼠后,采用Ｗｅｓｔｅｒｎ印迹法和免疫组化法观察肝细胞原癌基因（ｃ－ｆｏｓ／ｃ－ｊｕｎ）的表达。Ｗｅｓｔｅｒｎ印迹法表明,当成年大鼠的静息期肝细胞受到ＣＣｌ４损伤性刺激后,ｃ－ｆｏｓ／ｃ－ｊｕｎ产物（Ｆｏｓ和Ｊｕｎ）水平升高,在ＣＣｌ４处理后３０ｍｉｎ开始升高,在４ｈ时消失。８ｈ后Ｆｏｓ／Ｊｕｎ再度出现,并持续２４ｈ以上。ＩＣＣ法表明,Ｊｕｎ阳性细胞为靠近肝中央静脉区的肝实质细胞。根据上述资料推测,肝受ＣＣｌ４损伤后肝细胞的原癌基因ｃ－ｆｏｓ／ｃ－ｊｕｎ出现即时的与滞后的两次表达,这与肝细胞进入细胞周期有关,这种基因表达也许可作为肝再生过程中识别特殊体液因子的标志。     

二甲基苯蒽对小鼠皮肤表皮鸟氨酸脱羧酶和转化生长因子βmRNA水平…

本文利用Ｎｏｒｔｈｅｒｎ印迹分析技术研究了完全致癌物二甲基苯蒽（ＤＭＢＡ）对小鼠表皮鸟氨酸脱羧酶（ＯＤＣ）ｍＲＮＡ和转化生长因子β（ＴＧＦ－β）ｍＲＮＡ水平的影响。ＤＭＢＡ在１５０μｇ和９００μｇ剂量下一次涂用于小鼠肝肤,可使表皮ＯＤＣ ｍＲＮＡ和ＴＧＦ－βｍＲＮＡ表达增加。ＯＤＣ ｍＲＮＡ为单一的２．０ｋｂ大小的条带,其表达在在３６ｈ时最为明显。而ＴＧＦ－βｍＲＮＡ大小为２．５ｋｂ和１．９ｋｂ,     

四氯化碳损伤成年大鼠肝细胞后原癌基因（c—fos／c—jun）表…

用四氯化碳（ＣＣｌ４）损伤正常大鼠后,采用Ｗｅｓｔｅｒｎ印迹法和免疫组化法观察肝细胞原癌基因（ｃ－ｆｏｓ／ｃ－ｊｕｎ）的表达。Ｗｅｓｔｅｒｎ印迹法表明,当成年大鼠的静息期肝细胞受到ＣＣｌ４损伤性刺激后,ｃ－ｆｏｓ／ｃ－ｊｕｎ产物（Ｆｏｓ和Ｊｕｎ）水平升高,在ＣＣｌ４处理后３０ｍｉｎ开始升高,在４ｈ时消失。８ｈ后Ｆｏｓ／Ｊｕｎ再度出现,并持续２４ｈ以上。ＩＣＣ法表明,Ｊｕｎ阳性细胞为靠近肝中央静脉区     

Hepatotrophic growth factor in blood of mice treated with carbon tetrachloride

The presence of a human hepatocyte growth factor (hHGF)-like DNA-synthesis promoter in platelet-poor serum of mice with liver injury was examined. Activity of the serum for stimulating DNA synthesis in cultured rat hepatocytes was low in untreated or vehicle-treated mice, but markedly increased 24 h after carbon tetrachloride administration and then dropped to normal levels prior to the peak of liver DNA synthesis. The effect of the serum was additive with the maximal effects of mouse and human epidermal growth factors, but not with that of hHGF. The growth-stimulating factor in the mouse serum, like hHGF, had affinity for heparin and was heat-labile. These results indicate that the level of a serum hHGF-like hepatocyte growth factor increased in mice treated with carbon tetrachloride prior to liver regeneration.     

A single administration of adenoviral-mediated HGF cDNA permits survival of mice from acute hepatic failure

Heptatocyte growth factor (HGF) having a variety of biological activity was suggested as a protective agent against acute toxic hepatic injury or a potentially therapeutic agent. For the efficient in vivo application of this factor, we employed adenoviral-mediated HGF gene delivery system. In this study, we constructed E1-deleted recombinant adenovirus carrying cDNA of human HGF (Ad.hHGF) and elucidated that HGF was efficiently expressed in the liver of C57/BL mice. A mouse model of acute hepatic failure was induced by high dose (1000mg/kg) of thioacetamide (TA) administration. Mice infected with Ad.hHGF showed a dramatic resistance to TA-induced acute hepatic injury. Serum ALT was increased transiently and then the level was normalized in Ad.hHGF-infected mice with TA administration. Furthermore, the survival rate was remarkably enhanced in the mice infected with Ad.hHGF. In the histological examination, massive hepatic necrosis induced by TA was almost completely protected by HGF produced by Ad.hHGF. Our results indicate that a single dose of HGF-encoding adenoviral vector maintained liver function and prevented the progression of liver necrosis in a mouse model of acute hepatic failure.     

人胎肝中肝细胞生长因子生物活性的研究

人胎肝细胞裂解液经膜超滤,在分子量10～30kD组分中可检测出人肝细胞生长因子(hHGF)活性。hHGF为一热稳定的蛋白质或多肽类物质。它可特异地刺激肝来源细胞~3H-TdR掺入的增加,并且存在量效依赖关系,而对非肝来源细胞的DNA合成无刺激作用。hHGF的生物活性及理化性质与某些已知因子,如胰岛素、胰高血糖素、血小板来源的生长因子、表皮生长因子及增殖刺激因子等有所不同。     

重组人肝细胞生长因子真核表达的定性及定量检测

将人肝细胞生长因子（human hepatocyte growth factor hHGF)全长cDNA重组入pEE14真稳定表达质粒,用lipofectin脂质体将pEE14/rhHGF转染入CHO－K1细胞,蛋氨酸亚氨基代砜（methionine sulfoximine,MSX)筛选出阳性细胞克隆,利用RT－PCR检测rhHGF mRNA的表达通过ELISA法测定rhHGF的蛋白表达,3H掺入法检测培养上清液对大鼠原代培养肝细胞DNA合成的影响,结果表明转染pEE14/rhHGF的细胞可扩增出hHGF特异的396bp RT-PCR片段,培养上清液明显促进大鼠肝细胞DNA的合成,ELISA法测出上清液中,rhHGF的含量在8ug/L以上,显示rhHGF在CHO细胞中以活性形式得到表达。     

Human Umbilical Cord Mesenchymal Stem Cells and Derived Hepatocyte-Like Cells Exhibit Similar Therapeutic Effects on an Acute Liver Failure Mouse Model

Mesenchymal stem cells (MSCs) have exhibited therapeutic effects in multiple animal models so that are promising liver substitute for transplantation treatment of end-stage liver diseases. However, it has been shown that over-manipulation of these cells increased their tumorigenic potential, and that reducing the in vitro culture time could minimize the risk. In this study, we used a D-galactosamine plus lipopolysaccharide (Gal/LPS)-induced acute liver failure mouse model, which caused death of about 50% of the mice with necrosis of more than 50% hepatocytes, to compare the therapeutic effects of human umbilical cord MSCs (hUCMSCs) before and after induction of differentiation into hepatocyte (i-Heps). Induction of hUCMSCs to become i-Heps was achieved by treatment of the cells with a group of growth factors within 4 weeks. The resulted i-Heps exhibited a panel of human hepatocyte biomarkers including cytokeratin (hCK-18), α-fetoprotein (hAFP), albumin (hALB), and hepatocyte-specific functions glycogen storage and urea metabolism. We demonstrated that transplantation of both cell types through tail vein injection rescued almost all of the Gal/LPS-intoxicated mice. Although both cell types exhibited similar ability in homing at the mouse livers, the populations of the hUCMSCs-derived cells, as judged by expressing hAFP, hCK-18 and human hepatocyte growth factor (hHGF), were small. These observations let us to conclude that the hUCMSCs was as effective as the i-Heps in treatment of the mouse acute liver failure, and that the therapeutic effects of hUCMSCs were mediated largely via stimulation of host hepatocyte regeneration, and that delivery of the cells through intravenous injection was effective.     

Identification and partial characterization of two classes of receptors for human hepatocyte growth factor on adult rat hepatocytes in primary culture.

To characterize the receptor(s) for human hepatocyte growth factor (hHGF), a physiological hepatotrophic factor involved in liver regeneration following hepatic injury, recombinant hHGF (rhHGF) was radioiodinated. The labeled rhHGF retained its full biological activity on adult rat hepatocytes in primary culture. The specific binding of [125I]iodo-rhHGF to hepatocytes reached a plateau within 240 min at 4 degrees C. Scatchard plot analysis of the binding data suggested the presence of two classes of high affinity binding sites for [125I]iodo-rhHGF. One of the sites had a dissociation constant (Kd) of about 4.6 pM with 300 sites/cell and the other has a Kd of about 275 pM with 15,160 sites/cell. Unlabeled rhHGF displaced cell surface-bound [125I]iodo-rhHGF in a dose-dependent manner as did native hHGF purified from plasma of patients with fulminant hepatic failure. However, other growth factors to rat hepatocytes in primary culture such as insulin and human epidermal growth factor, and proteins which have high amino acid sequence-homology to hHGF such as plasminogen and prothrombin, did not compete with [125I]iodo-rhHGF in the binding, which suggests the binding was specific to hHGF. Covalent cross-linking experiment of [125I]iodo-rhHGF to cell surface receptor(s) on hepatocytes showed there were two macromolecular species with apparent molecular weights of 330,000 and 230,000. Unlabeled rhHGF and native hHGF competed for the binding of [125I]iodo-rhHGF to the two macromolecular species, but insulin, human epidermal growth factor, plasminogen, and prothrombin did not. Based upon our estimated molecular weight of rhHGF = 84,000, these results suggest that hHGF specifically binds to two polypeptides of 246,000 and 146,000 daltons which are likely to represent the hHGF receptors of primary cultured rat hepatocytes.     

TGF-beta is a potent inhibitor of hepatocyte growth factor secretion by human fibroblasts.

Transforming growth factor-beta 1 (TGF-beta 1) inhibited secretion of human hepatocyte growth factor (hHGF), which is also known as scatter factor or fibroblast-derived tumor cytotoxic factor, by MRC-5 cells. The effect was detectable at as little as 10 pg/ml and was more potent than that of dexamethasone. Complete inhibition was observed after 12 h in the presence of 5 ng/ml of TGF-beta 1. Phorbol 12-myristate 13-acetate-induced secretion of hHGF from human skin fibroblasts was also suppressed by TGF-beta 1. TGF-beta 2 inhibited hHGF secretion by MRC-5 cells to the same extent as TGF-beta 1, but other growth factors such as epidermal growth factor and acidic and basic fibroblast growth factors had only a slight or null inhibitory effect.     

Effects of protein kinase inhibitors on the mitogenic activity of human hepatocyte growth factor on rat hepatocytes in primary culture.

To evaluate the role of protein phosphorylation reactions in signal transduction of human hepatocyte growth factor (hHGF), now known to be the same protein as the scatter factor and tumor cytotoxic factor, we examined the effects of various inhibitors of protein kinases on the mitogenic activity of hHGF on rat hepatocytes in primary culture. Genistein, a specific inhibitor of tyrosine kinase, dose-dependently inhibited the effect of hHGF in stimulating DNA synthesis of hepatocytes. By contrast, 1-(5-isoquinolinesulfonyl)-2- methylpiperazine (H7), a specific inhibitor of protein kinase C, potentiated the stimulatory effect of hHGF on DNA synthesis of hepatocytes. H7 was effective at over 2 micrograms/ml and potentiated the effect of hHGF over 2-fold at 20 micrograms/ml. On the other hand, an inhibitor of Ca++/calmodulin-dependent protein kinase inhibited both the basal and hHGF-stimulated DNA synthesis in the cells, whereas an inhibitor of cyclic nucleotide-dependent protein kinases had little effect on the action of hHGF. These results suggest that tyrosine phosphorylation is required for stimulation of hepatocyte DNA synthesis by hHGF and that the action of hHGF is negatively regulated by protein kinase C activation.