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     

重组人肝细胞生长因子抗四氯化碳染毒小鼠肝的保护效应

为检测重组人肝细胞生长因子（ｒ－ｈＨＧＦ）的保肝作用,本工作观察到ｒ－ｈＨＧＦ降低ＣＣｌ４染毒小鼠血清ＡＬＴ和ＡＳＴ升高的幅度,减轻肝组织受损的程度和防止肝细胞器的破坏,而且,ｒ－ｈＨＧＦ的这种保肝效应所需剂量极小,为ｎｇ级。根据上述资料推测,ｒ－ｈＨＧＦ为一极有效的保护小鼠肝抗ＣＣｌ４损伤的生长因子     

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。     

血小板第四因子保护造血前体细胞及其作用机制的研究

我们曾报道血小板第四因子（ＰＦ４）是巨核细胞形成的有效的抑制因子,它可以保护干细胞免遭５－ＦＵ的毒性作用。本研究中试比较ＰＦ４和ＴＧＦ－β１对人脐带血ＣＤ３４＾＋来源的ＭＫ的作用。ＰＦ４（５ｕｇ／ｍｌ）和ＴＧＦ－β（１ｎｇ／ｍｌ）在半固态凝块培养和悬浮培养中明显抑制人脐带血ＣＤ３４＾＋来源的ＭＫ的生长。用ＰＦ４处理的ＣＤ３４＾＋细胞在撒去ＰＥ４后仍能再生成集落,说明ＰＦ４的抑制作用的可逆性的。相反     

肝细胞生长因子诱导CCL—64细胞迁移及其受TGF—β1的影响

目的：观察肝细胞生长因子（ＨＧＦ）诱导ＣＣＬ６４ 细胞发生迁移运动及转化生长因子β１（ＴＧＦβ１）对ＨＧＦ诱导的细胞迁移运动的影响。方法：细胞迁移检测。结果：ＨＧＦ不仅具有特异逆转ＴＧＦβ１ 抑制ＣＣＬ６４ 细胞增殖的活性,还具有诱导ＣＣＬ６４ 细胞发生迁移运动的效应。当ＴＧＦβ１ 浓度为５００ ｐｇ／ｍｌ 时,ｒｈＨＧＦ以剂量效应曲线方式诱导细胞迁移,而且低浓度的ＴＧＦβ１（＜１００ ｐｇ／ｍｌ）也可显著增强ｒｈＨＧＦ诱导的ＣＣＬ６４ 细胞迁移。结论：在一定的剂量范围内,ＨＧＦ和ＴＧＦβ１对ＣＣＬ６４ 细胞迁移运动具协同增强作用     

氨对脑细胞胞浆游离钙含量的影响

目的与方法：采用Ｆｕｒａ－２／ＡＭ探针技术观察ＮＨ４Ｃｌ对离体急性分离之Ｗｉｓｔａｒ乳鼠大脑细胞胞头游离钙「Ｃａ＾２＋」ｉ含量的影响。结果：ＮＨ４＾＋浓度为２．５ｍｍｏｌ／Ｌ时脑细胞内「Ｃａ＾２＋」ｉ含量升高。在一定范围内,随着ＮＨ４＾＋浓度的加大,细胞内Ｃａ＾２＋持续升高。ＮＨ４＾＋的升钙作用主要被Ｎｉｃａｒｄｉｐｉｎｅ所阴断,其变化特征类以ＫＣｌ。结论：ＮＨ４＾＋主要通过影响电压依赖性钙离子通     

G蛋白在亮啡肽诱导心肌细胞内钙释放中的作用

本实验采用分离的ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２ＡＭ荧光指示剂负载,检测心肌细胞内游离钙浓度（Ｃａ＾２＋）变化。探讨亮啡肽（ＬＥＫ）对（Ｃａ＾２＋）的作用及其机制。实验结果：ＬＥＫ（６０μｍｏｌ／Ｌ）能升高（Ｃａ＾２＋）移去细胞外液钙此效应仍能出现,用ｃａｆｆｅｉｎｅ （５ｍｍｏｌ／Ｌ）耗竭细胞内钙池的钙,该效应消失,纳洛酮（１００μｍｏｌ／Ｌ）,百日咳毒素（２００ｎｇ／Ｌ）处理８－１０ｈ及ｐｒ     

刺激大鼠视上核后电针“足三里”对蓝斑灌流液中去甲肾上腺素含量的影响

运用行为测痛结合蓝斑（ＬＣ）灌流液去甲肾上腺素（ＮＥ）的高压液相（ＨＰＬＣ）测定；观察 大鼠痛阈（ＰＴ）与ＬＣ灌流液中去甲肾上腺素含量变化间的相互关系，结果表明：（ｌ）视上核 （ＳＯＮ）内注射 １０μｍＬ－谷氨酸（Ｌ－ｇｌｕｔａｍｉｃａｃｉｄ， Ｌ－Ｇｌｕ）后 ３０分钟，大鼠ＰＴ较注射前增加１３３． ２± ２１．４％，此时ＬＣ 灌流液中ＮＥ含量从注射前的４３７．３±２０．４ｎｇ／ｍｌ降到２２９．２±１１．９ｎｇ／ｍｌ，注射 后６０分钟ＰＴ仍比注射前高８３．９±１４．７％，而灌流液中ＮＥ的含量为３２８．６±２８．０ｎｇ／ｍｌ，与人工 脑脊液（ＡＣＳＦ）对照组相比有非常明显的差别（Ｐ＜０．０５～０．００１）。（２） ＳＯＮ注射Ｌ－Ｇｌｕ后，电 针足三里３０分钟（Ｌ－ＧＩＵ＋ＥＡ组）增加到注射前的１８８．２±２３．９％，同ＡＣＳＦ电针组（ＡＣＳＦ＋ ＥＡ）的 ９４．９±７．１％相比有明显差异（Ｐ＜０．０１）。此时ＬＣ灌流液中ＮＥ的含量虽较注射前都明显 降低、分别为１３７．６±７．５ｎｇ／ｍｌ和 １５１，１±１１．５ｎｇ／ｍｌ，但两者相比无明显差异。停针后３０分钟Ｌ－ Ｇｌｕ＋ＥＡ组的ＰＴ仍比注射前高１３３．８±２７．９％，明显高于     

牛磺酸对血管紧张素Ⅱ改变培养心肌细胞内游离Ca^2＋深度作？…

目的和方法：用Ｆｕｒａ－２／ＡＭ荧光显示测定细胞内游离Ｃａ＾２＋浓度（〖Ｃａ＾２＋〗ｉ）的方法,我们研究了牛磺酸（Ｔａｕ）对血管紧张素Ⅱ（ＡｎｇⅡ）引起的培养心肌细胞（〖Ｃａ＾２＋〗ｉ）变化的影响。结果：在有Ｃａ＾２＋和无Ｃａ＾２＋的缓冲液中,ＡｎｇⅡ（１,１０,１００,１０００ｎｍｏｌ／Ｌ）引起的〖Ｃａ＾２＋）ｉ和蔼同。在含Ｃａ＾２＋的缓冲液中,Ｔａｕ（１０,２０ｍｏｌ／Ｌ）可隽浓度地抑制Ａｎｇ     

粒细胞巨噬细胞集落刺激因子诱导的巨噬细胞外向K+电流的特性

以粒细胞巨噬细胞集落刺激因子（ＧＭ－ＣＳＦ,１６ｎｇ／ｍｌ）长期（０．５－６ｄ）刺激小鼠腹腔渗了的巨噬细胞,采用全细胞膜片箝技术研究在ＧＭ－ＣＳＦ刺激过程中细胞膜电流的变化,观察到一种ＧＭ－ＣＳＦ诱导的瞬间失活的外向Ｋ电流,该电流在生理电压范围内可发生稳态失活,且当施加０．５ＨＺ的去极化脉冲刺激时其失活具有频率依赖性。该电流对胞外４－ＡＰ高度敏感,胞内Ｃａ＾２＋浓度「Ｃａ＾２＋」升主可抑制其幅度,     

人肝刺激因子对CCl4损伤大鼠离体肝细胞内钙和钾离子稳态的影响

本工作从自愿流产孕妇的胎儿取肝,按照LaBrecque法提取人肝刺激因子(human hepaticstimulator substance,hHSS)。用荧光探针Fura-2/AM测定离体肝细胞内游离钙,用离子分析仪测细胞染毒(四氯化碳CCl_4)前后基质中钾离子含量,观察hHSS对染毒肝细胞内Ca~(2+)和K~+稳态的影响,并测定肝细胞存活率和细胞内转氨酶(ALT)的漏出作为佐证。结果表明,人胎肝中含有hHSS,hHSS能提高离体肝细胞的存活率,维持肝细胞内游离钙的相对恒定,减少细胞内钾离子和ALT的漏出。这些结果提示,hHSS可保护肝细胞内钙,钾离子稳态和肝细胞膜的稳定,从而加强大鼠离体肝细胞抗CCl_4的损伤。     

Integral role of the EGF receptor in HGF-mediated hepatocyte proliferation.

Hepatocyte growth factor (HGF), insulin, and TGF-alpha stimulate DNA synthesis in cultured hepatocytes. Each ligand activates a distinct tyrosine kinase receptor, although receptor cross-talk modulates signaling. In rat hepatocytes, HGF can stimulate TGF-alpha production while TGF-alpha antibodies or antisense oligonucleotides suppress HGF-stimulated DNA synthesis. We report that the epidermal growth factor receptor (EGFR) kinase inhibitor PKI166 blocked both basal and ligand-induced tyrosine phosphorylation of the EGFR (IC(50) = 60 nM), but not of the insulin receptor or c-met. Pharmacologic inhibition of the EGFR kinase abolished the proliferative actions of HGF and EGF, but not insulin, whereas PI-3 kinase inhibition blocked both EGF and insulin actions. We conclude that in cultured hepatocytes (i) PI-3 kinase is required for EGF- and insulin-induced proliferation and (ii) EGFR mediates both the basal rate of DNA synthesis and that induced by EGF and HGF, but not insulin. The mitogenic effect of HGF may be secondary to increased synthesis or processing of EGFR ligands such as TGF-alpha.     

Suppression of acute hepatic injury by a synthetic prostacyclin agonist through hepatocyte growth factor expression

Previous studies have demonstrated that mice disrupted with the cyclooxygenase-2 gene showed much more severe liver damage compared with wild-type mice after liver injury, and prostaglandins (PGs) such as PGE(1/2) and PGI(2) have decreased hepatic injury, but the mechanisms by which prostaglandins exhibit protective action on the liver have yet to be addressed. In the present study, we investigated the mechanism of the protective action of PGI(2) using the synthetic IP receptor agonist ONO-1301. In primary cultures of hepatocytes and nonparenchymal liver cells, ONO-1301 did not show protective action directly on hepatocytes, whereas it stimulated expression of hepatocyte growth factor (HGF) in nonparenchymal liver cells. In mice, peroral administration of ONO-1301 increased hepatic gene expression and protein levels of HGF. Injections of CCl4 induced acute liver injury in mice, but the onset of acute liver injury was strongly suppressed by administration of ONO-1301. The increases in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) by CCl4 were suppressed by 10 mg/kg ONO-1301 to 39.4 and 33.6%, respectively. When neutralizing antibody against HGF was administered with ONO-1301 and CCl4, the decreases by ONO-1301 in serum ALT and AST, apoptotic liver cells, and expansion of necrotic areas in liver tissue were strongly reversed by neutralization of endogenous HGF. These results indicate that ONO-1301 increases expression of HGF and that hepatoprotective action of ONO-1301 in CCl4-induced liver injury may be attributable to its activity to induce expression of HGF, at least in part. The potential for involvement of HGF-Met-mediated signaling in the hepatotrophic action of endogenous prostaglandins generated by injury-dependent cyclooxygenase-2 induction is considerable.     

肝细胞生长因子对氧糖剥夺/再灌注神经元的保护作用

本文旨在探讨肝细胞生长因子（hepatocyte growth factor,HGF）对神经元氧糖剥夺/再灌注损伤的影响。取原代培养12d的Sprague-Dawley大鼠大脑皮层神经元,无糖、无氧（95%N2＋5%CO2）孵育2h后,换含25mmol/L葡萄糖的培养液、常氧培养0-24h,以MTT比色法检测细胞活力、乳酸脱氢酶（lactate dehydrogenase,LDH）漏出率作为细胞损伤指标,建立体外氧糖剥夺/再灌注损伤细胞模型;用流式细胞仪和Hoechst33258染色分析细胞凋亡率;用RT-PCR和Western blot分别检测大鼠脑皮层神经元HGF受体c-Met mRNA和蛋白的表达。于氧糖剥夺2h/再灌注24h处理前2h,加入不同终浓度（5-120ng/mL）的HGF,观察HGF对皮层神经元的影响。结果显示,c-Met表达于皮层神经元,氧糖剥夺2h/再灌注24h后,c-Met mRNA和蛋白表达均显著上调,神经元细胞活力明显降低,LDH漏出率和细胞凋亡率显著增高。HGF预处理明显促进氧糖剥夺/再灌注损伤神经元的存活,降低LDH漏出率,最大效应剂量为80ng/mL。流式细胞术和Hoechst33258染色结果均显示,HGF（80ng/mL）显著降低氧糖剥夺/再灌注神经元的细胞凋亡率。此外,c-Met抑制剂SU11274（5μmol/L）完全阻断HGF的神经保护作用。结果表明,HGF对皮层神经元氧糖剥夺/再灌注损伤具有直接的保护作用,呈一定的剂量依赖关系,并能有效对抗神经元凋亡。     

Intravenous injection of an adenovirus encoding hepatocyte growth factor results in liver growth and has a protective effect against apoptosis

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine with mitogenic, motogenic and morphogenic effects for a wide variety of cells. Previous studies have reported that the in vivo infusion in normal, untreated mice of recombinant HGF results in low levels of DNA synthesis and liver proliferation. In this study, we examined whether liver regeneration could be obtained by the in vivo injection of a recombinant adenoviral vector encoding human HGF (Ad.CMV.rhHGF) in normal, intact mice. MATERIALS AND METHODS: C57BL/6 mice were infused intravenously with doses increasing from 1 to 4 x 1011 particles of the recombinant human HGF (rhHGF) adenoviral vector or with a control virus encoding Escherichia coli beta-galactosidase (Ad.CMV.lacZ). At day 5, mice were sacrificed and evaluated for the presence of hepatocyte mitogenesis and liver regeneration (5-bromo-2''-deoxyuridine (BrdU) assays and liver weight determination) and for the presence of liver damage (serum alanine amino-transferase (ALT) measurements and TUNEL assays). RESULTS: In vivo administration of rhHGF stimulated DNA synthesis of hepatocytes and liver weight in a dose-dependent fashion. The maximal effect was seen after the infusion of 3 x 1011 particles which resulted at day 5 in >130% increase in relative liver mass with little cytopathic effect. In contrast, administration of the lacZ adenoviral vector caused little hepatocyte replication, but induced high levels of serum ALT (approximately 3 times higher than the rhHGF vector) and significant apoptotic cell death. CONCLUSIONS: This study shows that a single injection of Ad.CMV.rhHGF alone is able to induce in vivo and in a very short period of time, robust DNA synthesis and liver proliferation in normal mice without liver injury or partial hepatectomy. This recombinant adenoviral vector has a lower toxicity than the control lacZ adenovirus. This suggests that HGF may have a protective effect against adenovirus-induced pathology.     

Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloride-treated rats.

A hepatocyte growth factor (HGF)-like substance that strongly stimulated DNA synthesis of adult rat hepatocytes in primary culture was found to increase markedly in liver of rats treated with carbon tetrachloride (CCl4). This increase of HGF-like activity was time- and dose-dependent, and 36 h after a dose of CCl4 of 0.2 ml per 100 g body weight the activity was about 20-times the normal level. The extent of induction of HGF-like factor correlated well with the extent of liver damage. The HGF-like factor was purified to homogeneity from the liver of CCl4-treated rats by a four-step procedure. The purified HGF-like factor had a molecular weight of 82-85 kDa, as estimated by SDS-PAGE, and was a heterodimer composed of a large subunit of about 69 kDa and a small subunit of 34 kDa linked by disulfide bridges. This factor had similar biological and chemical properties to HGF purified from rat platelets. Moreover, the N-terminal amino acid sequence of its 34-kDa subunit was identical to that of the small subunit of rat HGF. These findings indicate that the HGF-like factor in damaged liver of CCl4-treated rats is HGF and that liver itself can produce HGF when injured.     

Change in hepatocyte growth factor concentration promote mesenchymal stem cell-mediated osteogenic regeneration

Mesenchymal stem cells (MSCs) play a crucial role in tissue repair by secretion of tissue nutrient factors such as hepatocyte growth factor (HGF). However, studies examining the effects of HGF on the proliferation and differentiation of MSCs used different concentrations of HGF and reported conflicting conclusions. This study aimed to determine the mechanisms by which different concentrations of HGF regulate MSC proliferation and osteogenic differentiation, and validate the mechanism in an animal model of early stage avascular necrosis of femoral head (ANFH). Our results demonstrate that a low concentration of HGF (20 ng/ml) preferentially promotes MSC osteogenic differentiation through increased c-Met expression and phosphorylation, Akt pathway activation, and increased expression of p27, Runx2 and Osterix. In contrast, a high concentration of HGF (100 ng/ml) strongly induced proliferation by inducing strong activation of the ERK1/2 signalling pathway. As validated by animal experiments, high localized expression of HGF achieved by transplantation of HGF transgenic MSCs into ANFH rabbits increased the number of MSCs. Subsequently, 2 weeks after transplantation, HGF levels decreased and MSCs differentiated into osteoblasts and resulted in efficient tissue repair. Our results demonstrate that sequential concentration changes in HGF control the proliferation and osteogenic differentiation of MSCs in vivo. This phenomenon can be exploited therapeutically to induce bone regeneration and, in turn, improve the efficacy of pharmacological intervention for ANFH treatment.     

Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).