MAPK信号通路在FGF1刺激大鼠原代肝细胞增殖中的作用

目的观察MEK／MAPK信号通路在FGF1刺激体外培养原代大鼠肝细胞增殖中的作用。方法体外培养原代大鼠肝细胞分三组，第二组培养基中加入FGF1，第三组加FGF1和MEK1阻滞剂PD98059，一定时间后检测并分析不同组肝细胞增殖情况。结果FGF1组与FGF1＋PD98059组肝细胞进入S期无差别，但与对照组却有差别。结论本实验条件下Src／Raf／MEK／MAPK通路组分MEK1的特异阻滞剂PD98059不能阻断FGF1促进体外培养的原代大鼠肝细胞增值的作用。     

二甲双胍对正常饮食大鼠血清成纤维细胞生长因子19、21及胰腺成纤维细胞生长因子受体1的影响

目的探讨二甲双胍(METF)对血清成纤维细胞生长因子(FGF)19和FGF21及胰腺成纤维细胞生长因子受体(FGFR)1和Akt通路上分子的影响。方法给予8周龄健康雄性Wistar大鼠(40只)标准饮食,随机分为正常对照(Con)组和METF(500 mg·kg~(-1)·d~(-1))。在24 w末检测血清生化学指标及FGF19和FGF21水平。蛋白免疫印迹检测胰腺组织FGFR1,Akt、磷酸化的Akt(p-Akt),FoxO1、磷酸化的FoxO1(p-FoxO1)的表达水平。结果 METF干预可以减轻大鼠的体重,降低大鼠空腹血糖、低密度脂蛋白和胆汁酸水平。METF组大鼠血清FGF21较Con组明显升高(P<0.05),而FGF19较Con组明显减低(P<0.05)。此外,胰腺FGFR1的表达水平及Akt及FoxO1的磷酸化水平在METF干预后明显升高(均P<0.05)。结论 METF可以降低血清FGF19、升高血清FGF21水平,对两者产生相反的作用。此外,METF可能通过增加FGFR1的表达、Akt及FoxO1的磷酸化对胰腺功能产生影响。     

成纤维细胞生长因子9及其受体对骨骼的作用

成纤维细胞生长因子-9(FGF-9)最初发现于人类神经胶质瘤细胞,具有三叶草核心结构,而且必须与肝素结合才能活化受体。FGF受体(FGFR)均为单链酪氨酸激酶受体,FGF-9主要与FGFR-2和FGFR-3结合。FGF-9分布广泛,具有多种生理功能。在骨组织,FGFR-2主要表达于成骨细胞系,FGFR-3主要表达于软骨细胞系,FGF-9可能通过活化FGFR-2促进膜内成骨,通过活化FGFR-3抑制软骨内成骨。     

成纤维细胞生长因子9及其受体对骨骼的作用

成纤维细胞生长因子-9（FGF-9）最初发现于人类神经胶质瘤细胞，具有三叶草核心结构，而且必须与肝素结合才能活化受体。FGF受体（FGFR）均为单链酪氨酸激酶受体，FGF-9主要与FGFR-2和FGFR-3结合。FGF-9分布广泛，具有多种生理功能。在骨组织，FGFR-2主要表达于成骨细胞系，FGFR-3主要表达于软骨细胞系，FGF-9可能通过活化FGFR-2促进膜内成骨，通过活化FGFR-3抑制软骨内成骨。     

FGFR2:进展期胃癌的关键分子

成纤维细胞生长因子受体2(fibroblast growth factor receptor2,FGFR2)作为人成纤维细胞生长因子受体酪氨酸激酶受体家族成员之一,是进展期胃癌侵袭特性的基础,与胃癌的病理类型、临床分期、淋巴结及远处转移密切相关.围绕FGFR2分子进行的大量研究表明,针对FGFR2的单克隆抗体对FGFR2高表达或活化的胃癌细胞具有明显的抑制作用,联合化疗应用对胃癌的抑制具有协同作用,显示出以FGFR2为治疗靶点治疗进展期胃癌具有良好的应用前景.     

内质网应激在成纤维细胞生长因子21抑制大鼠血管钙化过程中的作用

目的研究内质网应激(ERS)在成纤维细胞生长因子21(FGF21)抑制大鼠血管钙化过程中的作用。方法成年雄性SD大鼠随机分为对照组、模型组、FGF21组、FGF21+衣霉素(TM)组,后3组采用维生素D3联合尼古丁的方式建立血管钙化模型,FGF21组给予1 mg/(kg·d)的FGF21腹腔注射,FGF21+TM组给予1 mg/(kg·d)的FGF21腹腔注射及4.5 mg/(kg·w)的TM腹腔注射,连续28天。比较各组大鼠胸主动脉茜素红染色、钙含量、碱性磷酸酶(ALP)活性、TUNEL染色、成骨标志基因及ERS基因表达水平的差异。结果 FGF21组大鼠胸主动脉的茜素红染色明显减弱,胸主动脉中钙含量、ALP活性、TUNEL染色阳性率及Runt相关转录因子2(RUNX2)、骨形态发生蛋白2(BMP2)、BMP4、骨保护素(OPG)、葡萄糖调节蛋白78(GRP78)、CCAAT/增强子结合蛋白同源蛋白(CHOP)、含半胱氨酸的天冬氨酸蛋白水解酶12(Caspase-12)的蛋白表达水平均明显低于模型组(P0.05)。FGF21+TM组大鼠胸主动脉的TUNEL染色阳性率及RUNX2、BMP2、BMP4、OPG、GRP78、CHOP、Caspase-12的蛋白表达水平均明显高于FGF21组(P0.05)。结论 FGF21对维生素D3和尼古丁诱导的血管钙化具有抑制作用,阻断ERS所介导的细胞凋亡及成骨分化可能是其抑制血管钙化的分子机制。     

成纤维细胞生长因子21治疗肥胖症及其相关代谢性疾病的研究进展

成纤维细胞生长因子(FGF)21是一类新近发现的代谢调节因子, 主要由肝脏分泌, 与靶组织的FGF受体(FGFR)及共受体β-klotho形成的受体复合物结合, 发挥减重、降血糖、改善脂代谢以及减少组织炎性反应等作用。实验证实, 外源性给予FGF21能够通过诱导能量消耗、促进脂肪分解、减少脂质合成、促进白色脂肪组织棕色化、增加脂联素水平和改善瘦素抵抗等发挥减重、改善糖脂代谢、保护胰岛和心肌细胞的作用。目前, 基于FGF21的药物研发成为当前的研究热点, 主要包括人FGF21类似物和FGF21受体激动剂两种, 已在动物和人体试验中应用于改善肥胖症及非酒精性脂肪性肝炎、糖尿病等代谢异常相关疾病, 有望成为治疗这些疾病的新药。     

慢性炎症状态下成纤维细胞生长因子23与终末期肾病血管钙化的相关性

目的探讨炎症状态下成纤维细胞生长因子(FGF)23与终末期肾病(ESRD)血管钙化的关系。方法成年雄性SD大鼠随机分为对照组(Ctr)组、慢性肾功能不全(CRF)组和炎症伴慢性肾功能不全(CRF+I)组。Ctr组用生理盐水灌胃持续6 w,CRF组用腺嘌呤灌胃持续6 w构建大鼠CRF的动物模型,CRF+I组每天用腺嘌呤灌胃持续6 w并隔日皮下注射10%酪蛋白(Casein,1.2 g/kg)构建大鼠炎症伴CRF动物模型。检测各组血清肌酐、尿素氮、磷以及血管组织的钙含量、α-平滑肌肌动蛋白(α-SMA)蛋白表达量、FGF23 mRNA表达水平等指标。结果α-SMA蛋白表达量,Ctr组明显高于CRF组和CRF+I组(P0.05),CRF组明显高于CRF+I组(P0.05);组织钙含量,CRF组和CRF+I组明显高于Ctr组(P0.05),而CRF+I组明显高于CRF组(P0.05);FGF23 mRNA表达,CRF+I组的相对表达量明显高于CRF和Ctr组(P0.05),Ctr组和CRF组FGF23 mRNA的相对表达量无差异(P0.05)。结论炎症除了可以直接引起ESRD血管钙化外还可以通过升高FGF23加剧ESRD血管钙化。     

成纤维细胞生长因子23对磷代谢的调控及其临床意义

成纤维细胞生长因子23（FGF230）是最近发现的一种新型内分泌激素,主要在骨细胞和成骨细胞中表达。FGF23需要与FGFR-α-Klotho复合物结合发挥其生理功能。FGF23通过下调肾脏近曲小管上皮细胞内NPT2a和NPT2c表达降低磷从原尿的重吸收,下调1-羟基酶（Cyp27b1）在肾脏近曲小管的表达降低活性1,25（OH）₂D的合成,进而减少小肠对磷的吸收。磷代谢紊乱常见于先天性代谢疾病和慢性肾病（CKD）。明确FGF23在其中的病理作用将为上述疾病的诊断和治疗提供新思路和目标。     

高脂喂养apoE~(-/-)小鼠组织成纤维细胞生长因子21及其受体的改变

目的 探讨在环境和遗传因素共同诱导的胰岛素抵抗状态下组织成纤维细胞生长因子21(FGF-21)及其受体(FGFR)的变化.方法 雄性apoE~(-/-)小鼠,随机分为普通喂养组(NF组,n=20)和高脂喂养组(HF组,n=20),喂养16周.以3-[~3H]-葡萄糖为示踪剂的扩展胰岛素钳夹技术评价小鼠胰岛素敏感性和糖代谢变化.用实时荧光定量PCR法检测肝脏和脂肪组织FGF-21、FGFR1、FGFR2、FGFR3和FGFR4以及β-klotho mRNA表达,用Western印迹法测定血浆和组织FGF-21蛋白水平.结果 HF组小鼠空腹血糖、血浆胰岛素、甘油三酯、游离脂肪酸和胆固醇水平明显升高(均P<0.01).在钳夹稳态时,HF组血浆胰岛素水平明显高于NF组(P<0.01),HF组葡萄糖输注率(GIR)明显低于NF组(P<0.01).在钳夹结束时,HF组葡萄糖清除率(G_(Rd))明显低于NF组(P<0.01).钳夹稳态期,NF和HF组肝糖输出率(HGP)分别被抑制约70%和51%(均P<0.01).HF组肝和脂肪组织FGF-21和β-klotho mRNA水平均明显高于NF组(均P<0.01).HF组肝和脂肪组织FGFR1和FGFR3 mRNA的表达明显较NF组显著增高(P<0.01和P<0.05).FGFR4 mRNA水平在HF组肝组织中的表达明显高于NF组(P<0.05).HF组血浆FGF-21蛋白水平明显高于NF组(P<0.01),肝和脂肪组织FGF-21蛋白水平也显著升高(均P<0.05).结论 高脂喂养apoE~(-/-)小鼠FGF-21、β-Klotho及FGFR1和FGFR3水平显著升高,它们可能是FGF-21调节糖脂代谢的主要作用靶点.     

一种改良的胶原酶灌流高效分离肝细胞技术的建立

目的 探讨改良、简化肝细胞灌注、分离的方法,以提高细胞产量,降低制作成本.方法 建立一种改良胶原酶灌注分离技术,改变灌注方式,分离的小鼠肝细胞采用Percoil离心,台盼蓝染色检测成活率,比较胶原酶剪切消化法和和改良方法在细胞产量、成活率的差异.结果 采用改良的分离方法可获得大量的肝细胞,细胞产量为( 1.74±0.35)×107/L,细胞成活率为90.1％±1.9％;而对照组胶原酶剪切消化法的细胞产量为(0.44 ±0.06)×107/L,细胞成活率为35.3％±1.4％,P＜0.01,两组差异有统计学意义.结论 该方法要求设备简单、容易操作、细胞产量及成活率较高.     

成人原代肝细胞的分离、培养及冻存

目的:建立一种稳定的成人原代肝细胞分离、培养、冻存方法,为肝细胞移植、生物人工肝支持系统治疗急慢性肝病以及肝细胞体外应用模型提供潜在的肝细胞资源.方法:20例供肝采用离体两步胶原酶灌注技术分离成人原代肝细胞.选择7个不同的预培养时间点(2、6、12、24、36、48和72h),分离所获肝细胞按上述不同预培养时间在4℃人无血清培养基(HepatoZYME-SFM)中培养,然后收集预培养肝细胞转移到含100mL/L胎牛血清和DMSO的HepatoZYME-SFM中,再立即放入-80℃异丙醇冷冻盒过夜,次日投入液氮.分析比较各肝细胞在解冻后细胞活力率、贴壁率、白蛋白分泌及尿素合成.结果:在部分肝叶切除后使用离体两步胶原酶灌流技术分离所得肝细胞活率和贴壁率分别是75.0%±4.6%和72.0%±6.0%.4℃预培养12或24h被证明是最适预培养时间,这两个时间点的白蛋白分泌高于其他时间点(P<0.05).与立即冷冻组相比较,预培养12或24h肝细胞解冻后活力(61.4%±4.8%,62.0%±5.6%vs53.4%±4.2%)、贴壁率(63.2%±5.8%,62.6%±3.6%vs55.2%±4.6%)、白蛋白分泌及...     

The isolation of functionally heterogeneous hepatocytes of the proximal and distal half of the liver acinus in the rat

The objective of this study was to isolate hepatocytes of the proximal half (Zones 1 and 2) or distal half (Zones 2 and 3) of the liver acinus. The zonal origin of the isolated hepatocytes was recognized by: the presence in hepatocytes of a fluorescent marker, acridine orange, selectively delivered to either the proximal or the distal half of the acinus by in situ perfusion prior to cell isolation and the measurement of the induction of cytochrome P-450 by phenobarbital, an induction known to occur predominantly in the distal half of the acinus. Following the selective labeling of the acinus with acridine orange, livers were perfused with collagenase in either the portal to hepatic vein direction (anterograde) or in the retrograde direction. Hepatocytes isolated by either an anterograde or a retrograde perfusion were separated by centrifugation in a Percoll density gradient. This procedure isolated populations of proximal or distal hepatocytes, respectively, which were intact and 90% fluorescent. In an effort of assessing the heterogeneity of the separated proximal and distal hepatocytes, each population was further fractionated by centrifugal elutriation. This resulted in the arbitrary separation of proximal or distal hepatocytes into five fractions. Total cytochrome P-450 was determined spectrophotometrically in each of the fractions isolated from controls and after 3 days of the in vivo administration of phenobarbital. On the basis of the pattern of fluorescence in isolated hepatocytes and on the cytochrome P-450 inductive response to phenobarbital administration, it is proposed that: the anterograde or retrograde perfusion of the liver with collagenase separated hepatocytes predominantly of the proximal or distal half of the liver acinus, respectively and that hepatocytes of the distal half of the liver acinus responded to phenobarbital administration with the highest level of cytochrome P-450 induction, indicating that the isolated hepatocytes conserved the functional heterogeneity observed in vivo.     

Essential role of fibroblast growth factor signaling in preadipoctye differentiation

We have examined the expression and role of autocrine fibroblast growth factors (FGFs) in human preadipocytes through their differentiation in vitro. A high-molecular weight form of FGF-2 was initially strongly expressed, but 6-9 d after induction of differentiation, its expression decreased markedly. This coincided with the first appearance of visible lipid droplets within the cells. FGF-2 (18 kDa) was not found. FGF receptor (FGFR) 1 was detected as a single band of 125 kDa that also decreased with differentiation. Its decrease preceded that of FGF-2. Despite the decrease in cell-associated FGF-2 with differentiation, secreted FGF-2 was 2.5-fold higher in the differentiated preadipocytes. To determine whether FGF-2 had an autocrine role, FGFR signaling was inhibited using recombinant adenovirus expressing dominant negative FGFR1 (RAdDN-FGFR1) and a specific inhibitor of FGFR1 signaling, PD166866. Preadipocytes transduced with RAdDN-FGFR1 expressed a truncated, 79-kDa FGFR1. Differentiation, assessed by lipid droplet formation, was completely prevented by RAdDN-FGFR1 and by PD166866. The protein content in the cell layer and glucose uptake were significantly reduced by both agents. The insulin-sensitizing drug, rosiglitazone, did not prevent the actions of RAdDN-FGFR1 or PD166866. Controlling adipose tissue growth by limiting FGF actions may provide a means to combat obesity.     

库普弗细胞条件培养基对原代培养肝细胞的细胞毒性

目的 探讨内毒素血症时库普弗细胞激活对肝细胞损伤的作用机制.方法 用链霉蛋白酶和胶原酶原位灌流,Percoll密度梯度离心分离、纯化SD大鼠肝细胞和库普弗细胞,制备内毒素(LPS)刺激的库普弗细胞的条件培养基(KCCM)并作用于肝细胞,检测肝细胞培养基丙氨酸转氨酶(ALT)、天门冬氨酸转氨酶(AST)、乳酸脱氢酶(LDH)的含量,四甲基偶氮唑蓝(MTT)法检测KCCM对大鼠原代肝细胞活性的影响.结果 KCCM作用于肝细胞2 h后,肝细胞培养基中ALT、AST及LDH含量均明显升高(P＜0.05),在2～4 h内随时间延长ALT、AST及LDH含量均逐渐增加,呈明显的时间-效应关系;MTT结果显示,KCCM作用后12 h、24 h时间点对肝细胞具有明显损伤作用(P＜0.05).结论 内毒素诱导库普弗细胞释放的可溶性因子作用一定时间后可直接对肝细胞造成损伤.     

猪骨髓间充质干细胞对共培养肝细胞的作用及其机制

目的 探索细胞外基质(ECM)在猪肝细胞与骨髓间充质干细胞(MSCs)体外共培养中的表达与分布规律. 方法自中华实验猪髂前上棘抽取骨髓,采用密度梯度离心法分离单个核细胞,贴壁传代培养至第3代;原位两步胶原酶法分离猪肝细胞后与MSCs随机混合培养,观察肝细胞形态和功能的变化情况.用免疫细胞化学染色法观察ECM的表达和分布情况.RNA干扰MSCs后继续观察共培养肝细胞的功能变化情况.多组间比较采用单因素方差分析,两两比较采用LSD法.结果 第3代MSCs纯度>90%;肝细胞活率>95%,纯度>99%.共培养组肝细胞迅速黏附于MSCs表面,呈肝细胞岛样分布.共培养组肝细胞白蛋白分泌水平和尿素合成能力自共培养第1天起均明显高于单纯肝细胞组(P值均<0.01),并在第2天达到高峰,分别为(457.71士22.62)ng和(69.05±2.12)μg.免疫细胞化学检测结果显示共培养组MSCs表达多种ECMl RNA干扰实验进一步证实ECM的存在与共培养中肝细胞的白蛋白分泌和尿素合成功能有关.结论 骨髓MSCs通过分泌多种ECM改善共培养肝细胞的形态与功能.     

Binding sites for endotoxic lipopolysaccharide on the plasma membrane of isolated rabbit hepatocytes.

The in vitro fixation of bacterial lipopolysaccharide (LPS) on the plasma membrane of mechanically or enzymatically isolated hepatocytes from rabbits was studied by immunofluorescence technique. Antisera against LPS from E. coli 026:B6 and 0111:B4 were induced in rabbits. Antibody titers up to 1:1024 were determined by the passive hemagglutination test. There was no immunologic cross reactivity between the two antisera. IgG and IgM were prepared from anti-LPS as well as from normal rabbit serum and conjugated with fluorescein-isothiocyanate. The antibody activity against LPS was localized in the IgM fraction. Hepatocytes were isolated by a perfusion technique without enzymes and with collagenase. LPS binding to the hepatocellular plasma membrane increased proportionally with the LPS concentration in a range between 0.01 and 1.0 mg per ml. The fluorescence pattern of the membrane fixed IgM anti-LPS-antibody at the surface of LPS coated hepatocytes was coarse granular. The in vitro reaction of LPS with hepatocytes was not influenced by the presence of complement. The demonstration of binding sites for LPS on the hepatocellular plasma membrane supports the hypothesis that not only Kupffer cells but also parenchymal liver cells are involved in the hepatic clearance activity for endotoxin.     

Altered transmembrane ionic flux in hepatocytes isolated from cirrhotic rats

BACKGROUND/AIMS: Although cirrhosis is known to be associated with many hepatocyte abnormalities, there is no well-established model to study the cellular drug uptake process independent of hemodynamic effects. The purpose of the present study was to test the following hypothesis: hepatocytes isolated from cirrhotic animals may be used as a model to study the cellular abnormalities associated with cirrhosis. Our hypothesis was tested by comparing the membrane potential (PD) of hepatocytes in anesthetized healthy and cirrhotic animals, and the PD and [3H]palmitic acid clearance rate of hepatocytes isolated from healthy and cirrhotic animals. METHODS: Mild to moderate cirrhosis was induced in female Sprague-Dawley rats by CCl4 administration. PD was recorded in anesthetized animals using intracellular microelectrodes. Hepatocytes from those livers were subsequently isolated by collagenase perfusion for further determinations of PD and [3H]palmitic acid uptake. RESULTS: The mean (+/-SEM) hepatocyte PD from intact rat livers was 38+/-1 mV (control) and -32+/-1 mV (cirrhosis; n=6/group, p<0.01). The PD (mean+/-SEM) in isolated hepatocytes was -21+/-1 mV (control) and -15+/-1 mV (cirrhosis, n=13/group, p<0.01). The clearance rate of [3H]palmitic acid was lower in hepatocytes isolated from cirrhotic animals (26%) than in those isolated from healthy control animals (p<0.01). CONCLUSION: The results of this study indicate that hepatocytes isolated from cirrhotic animals may be used to study the cellular abnormalities associated with cirrhosis.