Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.

Excessive production of collagen type I is a major contributor to hepatic fibrosis. Activated (myofibroblastic), but not quiescent, hepatic stellate cells (lipocytes) have a high level of collagen type I and alpha-smooth muscle actin expression. Therefore, stellate cell activation is a critical step in hepatic fibrosis. Here we show that quiescent stellate cells were activated by the generation of free radicals with ascorbate/FeSO4 and by malondialdehyde, a product of lipid peroxidation. In addition, stellate cell activation by collagen type I matrix and TGF alpha was blocked by antioxidants, such as d-alpha-tocopherol and butylated hydroxytoluene. Moreover, oxidative stress, TGF alpha and collagen type I markedly stimulated stellate cell entry into S-phase, NFkB activity, and c-myb expression, which were prevented by antioxidants. c-myb antisense oligonucleotide blocked the activation and proliferation of stellate cells induced by TGF alpha. Nuclear extracts from activated, but not from quiescent, stellate cells formed a complex with the critical promoter E box of the alpha-smooth muscle actin gene, which was disrupted by c-myb and NFkB65 antibodies, and competed by c-myb and NFkB cognate DNA. c-Myb expression was also stimulated in activated stellate cells in carbon tetrachloride-induced hepatic injury and fibrogenesis. This study indicates that oxidative stress plays an essential role, through the induction of c-myb and NFkB, on stellate cell activation.     

转染IL-16基因对T淋巴瘤细胞的增殖抑制作用

目的 构建IL-16真核表达载体,研究其对T淋巴瘤细胞的增殖抑制作用。方法 应用RT-PCR技术从人外周血单个核细胞中获取IL-16cDNA,构建并鉴定真核表达载体PcDNA3-IL-16转染Karpas T淋巴瘤细胞,采用MTT法和流式细胞术分析IL-16对Karpas T淋巴瘤细胞的增殖抑制。结果 PT-PCR显示转染PcDNA3-IL-16的Karpas T淋巴瘤细胞高表达IL-16mRNA,MTT法检测显示其对Karpas T淋巴瘤细胞具有抑制作用,流式细胞术检测转染PcDNA3-IL-16的Karpas T淋巴瘤细胞高表达CD95分子。结论 成功构建了真核表达载体PcDNA3-IL-16,转染Karpas T淋巴瘤细胞能使其生长受到明显抑制,并且CD95分子表达增高,可能与诱导细胞凋亡有关,说明向肿瘤细胞转染IL-16是一种有用的肿瘤生物治疗方法。     

靶向性血小板衍生生长因子siRNA对肝星状细胞增殖的影响

目的探讨小干扰RNA（siRNA）降低血小板衍生生长因子B链（PDGF-B）基因对肝星状细胞（HSCs）增殖与细胞外基质表达的影响。方法构建PDGF-B靶向siRNA重组表达质粒pSilencer3.l-Hlhygro-PDGF-B siR-NA,将重组质粒转染HSCs细胞,噻唑蓝（MTT）法测定转染后12 h、24 h、36 h、48 h、60 h的增殖抑制率;半定量逆转录聚合酶链反应（RT-PCR）与免疫印迹检测转染后36 h HSCs细胞PDGF-B表达。放免法检测HSCs细胞株上清液中透明质酸和Ⅲ型前胶原的表达。结果经酶切鉴定和测序结果证实PDGF-B靶向siRNA重组表达载体构建成功,转染重组质粒36 h后,PDGF-B siRNA-1、siRNA-2、siRNA-3干预组的HSCs增殖抑制率明显高于对照质粒转染组（34.04%、14.89%、25.53%VS 3.19%;均P〈0.01～0.05）;RT-PCR和免疫印迹显示：各siRNA表达质粒阳性HSCs细胞PDGF-B mRNA和蛋白表达明显降低,与空脂质体组及对照质粒组比较,差异具有统计学意义（均P〈0.05）,放免法显示：siRNAs干预组HSCs细胞透明质酸和Ⅲ型前胶原表达明显降低,与空脂质体组及对照质粒组比较,差异具有统计学意义（均P〈0.05）。结论靶向PDGF-B链的siRNA可降低HSCs的PDGF-B基因表达,具有抑制HSCs增殖和分泌细胞外基质的能力。     

NADPH oxidase signal transduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis

Angiotensin II (Ang II) is a pro-oxidant and fibrogenic cytokine. We investigated the role of NADPH oxidase in Ang II-induced effects in hepatic stellate cells (HSCs), a fibrogenic cell type. Human HSCs express mRNAs of key components of nonphagocytic NADPH oxidase. Ang II phosphorylated p47phox, a regulatory subunit of NADPH oxidase, and induced reactive oxygen species formation via NADPH oxidase activity. Ang II phosphorylated AKT and MAPKs and increased AP-1 DNA binding in a redox-sensitive manner. Ang II stimulated DNA synthesis, cell migration, procollagen alpha1(I) mRNA expression, and secretion of TGF-beta1 and inflammatory cytokines. These effects were attenuated by N-acetylcysteine and diphenylene iodonium, an NADPH oxidase inhibitor. Moreover, Ang II induced upregulation of genes potentially involved in hepatic wound-healing response in a redox-sensitive manner, as assessed by microarray analysis. HSCs isolated from p47phox-/- mice displayed a blunted response to Ang II compared with WT cells. We also assessed the role of NADPH oxidase in experimental liver fibrosis. After bile duct ligation, p47phox-/- mice showed attenuated liver injury and fibrosis compared with WT counterparts. Moreover, expression of smooth muscle alpha-actin and expression of TGF-beta1 were reduced in p47phox-/- mice. Thus, NADPH oxidase mediates the actions of Ang II on HSCs and plays a critical role in liver fibrogenesis.     

Induction of apoptosis in rat hepatic stellate cells by disruption of integrin-mediated cell adhesion.

Cell-matrix adhesion is recognized as a physiologic determinant of cell growth and survival. Integrin occupancy seems to be a primary role. We sought to investigate the signal transduction pathways for integrin effects on cell survival in hepatic stellate cells. Integrin function was antagonized by the soluble integrin recognition sequence pentapeptide Gly-Arg-Gly-Asp-Ser (GRGDS) in primary cultures of rat hepatic stellate cells. Integrin antagonism with GRGDS peptide induced apoptosis. To investigate signal transduction mechanisms for the effect of integrins on cell survival in hepatic stellate cells, the expression of p53, Bcl-2, and Bax was analyzed. Incubation with soluble GRGDS peptide resulted in increased expression of p53 and decreased the Bcl-2/Bax ratio. In conclusion, these findings indicate that the abrogation of cell adhesion with soluble GRGDS peptide plays a critical role in the induction of apoptosis of rat hepatic stellate cells.     

肝星状细胞活化增殖和凋亡及奥曲肽的影响

背景:肝星状细胞的活化增殖在肝硬化的发生发展中起关键作用,临床广泛应用的生长抑素衍生物奥曲肽有多种生物学效应,但它对肝星状细胞的活化增殖及凋亡有何影响尚不清楚。目的:观察奥曲肽对大鼠肝星状细胞增殖和凋亡的影响。方法:实验选用肝星状细胞株HSC-T6的传代细胞。MTT法检测不同浓度(1×10-2,1×10-3,1×10-4,1×10-5,1×10-6,0mmol/L)奥曲肽干预24,48,72h对肝星状细胞增殖的影响;TUNEL凋亡检测试剂盒荧光显微镜方法检测不同浓度(0,1×10-5,1×10-2mmol/L)奥曲肽干预24h对肝星状细胞凋亡的影响。结果与结论:奥曲肽浓度越高、作用时间越长对培养的肝星状细胞增殖抑制越明显,呈现浓度和时间依赖性;奥曲肽对培养的肝星状细胞凋亡随奥曲肽浓度的增加而增加。结果可见奥曲肽对培养的肝星状细胞增殖抑制呈浓度(0～10-2mmol/L)和时间(24,48,72h)依赖性,并能诱发其凋亡。     

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.     

胶原酶门静脉灌注对兔肝硬化及组织中星状细胞激活的影响

目的 CCl4皮下注射建立兔肝硬化动物模型,观察Ⅳ型胶原酶门静脉灌注对肝硬化及肝组织中α-平滑肌动蛋白(α-SMA)表达的影响。方法取雄性新西兰大白兔,采用臀部皮下注射50%CCl4橄榄油0.23ml/kg,每周2次,共12周制作肝硬化动物模型,注射等量橄榄油作为对照。12周后各组动物均建立门静脉给药通路,将已形成肝硬化并门静脉插管成功的33只兔随机分为两组(组1,组2),组1为16只、组2为17只。组1经门静脉给药通路注入0.1%Ⅳ型胶原酶1.5ml,组2注入等量0.9%氯化钠,5次/周,共4周。将造模对照组中门静脉插管成功的30只兔同样随机分为两组(组3,组4),每组15只,处理方法同前。4周后,将各组动物处死后留取肝脏组织,观察其病理学及羟脯氨酸含量变化,标本固定后,行α-SMA免疫组织化学染色,行积分光密度、面密度分析。结果肝硬化动物肝脏α-SMA表达显著增强;门静脉灌注0.1%Ⅳ型胶原酶肝硬化动物肝脏纤维化程度明显降低,羟脯氨酸含量显著降低,但α-SMA表达强度显著增高。结论采用门静脉灌注0.1%Ⅳ型胶原酶可显著降低肝纤维化程度,但α-SMA表达增高,可能与肝脏星状细胞激活有关。     

用Syn标记肝星状细胞观察慢性乙型病毒性肝炎组织160例

目的 探讨肝星状细胞(HSC)在慢性乙型病毒性肝炎(CHB)肝穿组织中的表达及与肝纤维化的关系.方法 应用免疫组化SP法对160例慢性乙型病毒性肝炎和10例正常肝组织标本进行突触素(Syn)标记,观察肝星状细胞表达情况.结果 正常人肝组织中无HSC表达;160例CHB中,轻度(G1-2,S1-2)104例.HSC表达占20.1%(21/104);中～重度(G3-4,S3-4)56例,HSC表达占67.8%(38/56);中、重度HSC表达较轻度显著增强(P<0.01).结论 CHB肝组织中HSC过度表达可以反映肝组织损伤程度,这与肝脏炎症活动及纤维化有关.     

Pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis

Liver fibrosis is a complex pathological process controlled by a variety of cells, mediators and signaling pathways. Hepatic stellate cells play a central role in the development of liver fibrosis. In chronic liver disease, hepatic stellate cells undergo dramatic phenotypic activation and acquire fibrogenic properties. This review focuses on the pathophysiological mechanisms of hepatic stellate cells activation in liver fibrosis. They enter the cell cycle under the influence of various triggers. The “Initiation” phase of hepatic stellate cells activation overlaps and continues with the “Perpetuation” phase, which is characterized by a pronounced inflammatory and fibrogenic reaction. This is followed by a resolution phase if the injury subsides. Knowledge of these pathophysiological mechanisms paved the way for drugs aimed at preventing the development and progression of liver fibrosis. In this respect, impairments in intracellular signaling, epigenetic changes and cellular stress response can be the targets of therapy where the goal is to deactivate hepatic stellate cells. Potential antifibrotic therapy may focus on inducing hepatic stellate cells to return to an inactive state through cellular aging, apoptosis, and/or clearance by immune cells, and serve as potential antifibrotic therapy. It is especially important to prevent the formation of liver cirrhosis since the only radical approach to its treatment is liver transplantation which can be performed in only a limited number of countries.     

Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells.

Hypertension is associated with insulin-resistant states such as diabetes and obesity. Nitric oxide (NO) contributes to regulation of blood pressure. To gain insight into potential mechanisms linking hypertension with insulin resistance we directly measured and characterized NO production from human umbilical vein endothelial cells (HUVEC) in response to insulin using an amperometric NO-selective electrode. Insulin stimulation of HUVEC resulted in rapid, dose-dependent production of NO with a maximal response of approximately 100 nM NO (200,000 cells in 2 ml media; ED50 approximately 500 nM insulin). Although HUVEC have many more IGF-1 receptors than insulin receptors (approximately 400,000, and approximately 40,000 per cell respectively), a maximally stimulating dose of IGF-1 generated a smaller response than insulin (40 nM NO; ED50 approximately 100 nM IGF-1). Stimulation of HUVEC with PDGF did not result in measurable NO production. The effects of insulin and IGF-1 were completely blocked by inhibitors of either tyrosine kinase (genestein) or nitric oxide synthase (L-NAME). Wortmannin (an inhibitor of phosphatidylinositol 3-kinase [PI 3-kinase]) inhibited insulin-stimulated production of NO by approximately 50%. Since PI 3-kinase activity is required for insulin-stimulated glucose transport, our data suggest that NO is a novel effector of insulin signaling pathways that are also involved with glucose metabolism.     

人骨髓间质干细胞对肝星状细胞的体外调控

背景:目前肝纤维化尚没有公认的特效疗法,近年来骨髓间质干细胞应用于肝纤维化的治疗已取得一定效果,但具体机制仍不明确.目的:体外探讨人骨髓间质干细胞对肝星状细胞的调控作用.设计、时间及地点:细胞学体外观察,于2008-06/12在中山大学干细胞与组织工程研究中心、中山大学第三附属医院中心实验室完成.材料:骨髓来源于青年健康志愿者髂骨,入肝星状细胞购自中山大学实验动物中心,人正常肝细胞系L-02购自中山大学实验动物中心.方法:将分离提纯的人骨髓间质干细胞与肝星状细胞在β孔Transwell板上建立上下共培养体系.接种密度均为2×104cells/well.另设L-02代替人骨髓间质干细胞作为阴性对照,单纯培养的肝星状细胞为空白对照,培养72 h.主要观察指标:肝星状细胞形态及免疫细胞化学染色结果,流式细胞仪检测肝星状细胞凋亡情况,Western blot法检测肝星状细胞活化标志α-肌动蛋白的表达.结果:倒置显微镜下活化的肝星状细胞呈扁平状,胞浆内缺乏脂肪滴,α-肌动蛋白位于肝星状细胞胞质内,呈高张力纤维状分布.与L-02+肝星状细胞组、单纯肝星状细胞组比较,骨髓间质干细胞+肝星状细胞共培养组的肝星状细胞凋亡率明显升高(P<0.05),α-肌动蛋白表达水平明显下调.结论:人骨髓间质干细胞可抑制肝星状细胞活化,促进其凋亡,这可能是骨髓间质干细胞抗肝纤维化的作用机制.     

复方鳖甲软肝方对离体肝星形细胞增殖的影响

目的:观察复方鳖甲软肝方药物血清对离体肝星形细胞(hepaticstellatecells,HSC)增殖的影响,以探讨其抗肝纤维化的作用机制。方法:将成年健康雄性SD大鼠75只,随机分为正常对照组、模型组、高、中、低剂量药物组共5组,并制备各组血清。采用大鼠CCl4造模后肝星形细胞分离、培养,药物血清制备,MTT法检测法各组血清对HSC不同时相增殖的影响,酶标仪波长为450nm。结果:培养24h,各组HSC增殖比较,差异无显著性意义(P>0.05);培养48,72h,高、中、低剂量药物组HSCA值(0.47±0.02,0.55±0.07;0.46±0.02,0.58±0.08;0.51±0.03,0.69±0.07)与模型组(0.66±0.05和0.96±0.05)比较,差异有显著性意义(P<0.05);高、中剂量组药物组与对照组比较,差异有显著性意义(P<0.05)。结论:不同剂量的复方鳖甲软肝方药物血清作用48,72h后均能降低或抑制HSC的增殖,尤以高、中剂量作用明显。     

骨髓间质干细胞抑制大鼠肝纤维化形成的可能性受体肝内干细胞存活时间、肝星状细胞活化程度及生存分析的7周观察

目的观察在体外培养条件下,具有强大的增殖和分化为肝细胞能力的骨髓间质干细胞输注治疗肝纤维化模型大鼠对其肝纤维化形成的影响及肝功能和生存分析,并对治疗组、空白对照组和病理对照组7周内的相关情况予以对照比较.方法实验于2003-08/2004-10在中山大学基础医学院病理学于病理生理实验室完成.①动物分组选用雄性6周龄Wistar大鼠5只为供体,供分离骨髓间质干细胞;雌性6周龄Wistar大鼠60只,其中50只被造成肝纤维化模型,在首次用二甲基亚硝胺处理后的第10天,将造模大鼠随机分为2组骨髓间质干细胞治疗组10只(为受体),于治疗39 d后结束实验,取材分析病理对照组40只.另10只作为空白对照组(仅静脉注射生理盐水).②肝纤维化模型制作在实验的当天,给予10g/L二甲基亚硝胺溶液(生理盐水配置)1 mL/kg,腹腔注射,每周连续3次,共6周.③骨髓间质干细胞治疗组每只大鼠输注3×106个骨髓间质干细胞;病理对照组使用等体积生理盐水代替骨髓间质干细胞.④动物的一般生活状态每天观察饮食、活动、二便、体质量、皮毛、呼吸情况等.⑤肝脏的组织细胞形态结构将肝组织以苏木精-伊红染色后观察.⑥肝星形细胞及其活化程度检测前者检测肝脏α-平滑肌肌动蛋白含量,用免疫组织化学染色法,后者以德国IBAS2.5图像分析软件半定量分析.⑦肝脏胶原分布的相对含量检测采用Masson三色染色法,从而反映肝纤维化程度.⑧肝脏羟脯氨酸含量测定采用氯胺T法.⑨血清总胆红素和谷丙转氨酶水平检测采用全自动生化分析仪.⑩血清层粘蛋白和透明质酸水平测定采用放射免疫法.11雄性大鼠骨髓间质干细胞在雌性大鼠肝内的Y染色体性别决定区基因的表达检测采用聚合酶链式反应.12统计学分析两组样本均数比较采用t检验;多组样本均数比较采用方差分析,两两比较采用q检验;生存分析采用K,plan-Meier法.结果雌性大鼠60只进入结果分析.①实验动物生存状况造模6周后,骨髓间质干细胞治疗组生存率为80%,而病理对照组生存率为10%,空白对照组生存率为100%,说明骨髓间质干细胞治疗可能通过阻止肝纤维化发展,从改而善大鼠的生存率.②肝脏组织学显示,骨髓间质干细胞治疗组的肝脏结构明显改善,炎症减轻、假小叶结构减少或消散.相反,病理对照组出现广泛的肝脏结构重塑,包括增厚的纤维间隔形成和明显的桥接坏死.半定量分析胶原染色,骨髓间质干细胞治疗组较病理对照组显著减低(8.12±1.98,15.71±2.13,t=6.11,P＜0.05).③肝脏星状细胞活化的标志α-平滑肌激动蛋白的阳性表达空白对照组仅见血管壁有少量α-平滑肌激动蛋白的阳性表达,而病理对照组和骨髓间质干细胞治疗组可见在纤维间隔、肝窦和汇管区内明显表达.半定量分析α-平滑肌激动蛋白的阳性染色,骨髓间质干细胞治疗组较病理对照组显著减低(11.06±2.03,18.50±3.87,t=4.47,P＜0.05).④Y染色体性别决定区基因的表达经雄性Wistar大鼠(供体)骨髓间质干细胞治疗39 d的雌性大鼠,肝脏DNA经聚合酶链式反应检测结果显示,Y染色体性别决定区基因的表达阳性,而空白对照组和病理对照组则无此阳性信号.说明骨髓间质干细胞移植后,能够在受体肝内存活5周以上.⑤反映肝脏纤维化程度的肝脏羟脯氨酸含量及血清层黏蛋白和透明质酸水平骨髓间质干细胞治疗组明显高于空白对照组(P＜0 05)但明显低于病理对照组(P＜0.05),说明骨髓间质干细胞治疗后能够减少肝脏胶原蛋白的含量,从而减少肝纤维化的形成,使肝纤维化得到控制.⑥反映肝功能的血清总胆红素、谷丙转氨酶水平骨髓间质干细胞治疗组明显低于病理对照组(P＜0.05),接近空白对照组.说明了骨髓间质干细胞治疗有助于改善肝功能状态.结论①二甲基亚硝胺能成功诱导大鼠肝纤维化模型的建立.②骨髓间质干细胞可改善肝纤维化大鼠的生存状况和肝功能.③骨髓间质干细胞能够减少肝脏胶原蛋白的产生,抑制肝纤维化形成.④肝纤维化发生发展过程中活化态的肝星状细胞数量减少可能是骨髓间质干细胞抑制肝纤维化形成的原因之一.⑤雌性大鼠肝脏内存在雄性供体Y染色体性别决定区基因的信号说明骨髓间质干细胞能够在受体肝内存活5周以上.     

大鼠肝星状细胞分离纯化方法的改进

目的 研究肝损伤后细胞基质的沉积及纤维化形成机制,建立一种经济、简便、可靠的分离大鼠肝星状细胞(HSC)的方法.方法 用链霉蛋白酶和胶原酶灌流大鼠肝脏,Optiprep密度梯度离心分离HSC,并进行体外培养.结果 细胞得率为5.0×107/鼠,活率和纯度分别为98%、96%.结论 该方法简便、实用、稳定、可靠,为进一步研究HSC与肝纤维化的关系,尤其是细胞水平及分子生物学水平的研究奠定了基础.     

干扰素α-2a与肝纤维化大鼠肝星状细胞的凋亡

背景：干扰素α-2a改善肝纤维化的机制直到目前仍尚未阐明。目的：进一步验证干扰素α-2a对C14诱导大鼠肝纤维化模型中肝星状细胞凋亡的影响。方法：建立CC14诱导肝纤维化模型,健康SD雌性大鼠50只,采用随机对照原则将SD大鼠分成5组,即生理盐水对照组、纤维化模型组、6×10^4U／kg干扰素α-2a干预组、12×10^4U／kg干扰素α-2a干预组及6×10^4U／kg干扰素α-2a对照组。造模8周时取肝组织标本,分别进行肝纤维化指标检测;RT-PCR分析肝组织bcl-2、bax的表达;免疫组织化学染色用a平滑肌肌动蛋白对活化的肝星状细胞进行标记。结果与结论：肝组织病理形态显示CC14诱导肝纤维化成功建立,表现为纤维化模型组汇管区周围纤维化明显,有芒状纤维和纤维间隔形成,各干扰素α-2a干预组肝纤维化有不同程度缓解。纤维化模型组有大量a平滑肌肌动蛋白阳性表达,6×10^4U／kg干扰素α-2a干预组a平滑肌肌动蛋白阳性表达较纤维化模型组减少,12×10^4U／kg干扰素α-2a干预组更少,6×10^4U／kg干扰素α-2a对照组未见a平滑肌肌动蛋白阳性表达。结果提示干扰素α-2a能下调CC14诱导肝纤维化bcl-2的表达,及上调bax的表达。提示干扰素α-2a阻断CC14诱导肝纤维化机制存在通过调节bcl-2、bax的表达,诱导肝星状细胞凋亡途径,该调节作用可能与干扰素α-2a剂量相关。     

Selective targeting of pentoxifylline to hepatic stellate cells using a novel platinum-based linker technology.

Targeting of antifibrotic drugs to hepatic stellate cells (HSC) is a promising strategy to block fibrotic processes leading to liver cirrhosis. For this purpose, we utilized the neo-glycoprotein mannose-6-phosphate-albumin (M6PHSA) that accumulates efficiently in HSC during liver fibrosis. Pentoxifylline (PTX), an antifibrotic compound that inhibits HSC proliferation and activation in vitro, was conjugated to M6PHSA. We employed a new type of platinum-based linker, which conjugates PTX via coordination chemistry rather than via covalent linkage. When incubated in plasma or in the presence of thiol compounds, free PTX was released from PTX-M6PHSA at a sustained slow rate. PTX-M6PHSA displayed pharmacological activity in cultured HSC as evidenced by changes in cell morphology and reduction of collagen I production. PTX-M6PHSA and platinum coupled PTX did not induce platinum-related toxicity (Alamar Blue viability assay) or apoptosis (caspase activation and TUNEL staining). In vivo distribution studies in fibrotic rats demonstrated specific accumulation of the conjugate in nonparenchymal cells in the fibrotic liver. In conclusion, we have developed PTX-M6PHSA employing a novel type of platinum linker, which allows sustained delivery of the drug to HSC in the fibrotic liver.     

肝纤维化中肝星状细胞和肌纤维母细胞的形态学观察

目的：观察肌纤维母细胞和肝星状细胞在人慢性乙型病毒性肝炎肝纤维化过程中的作用。方法：对69例肝穿刺活检组织标本行HE、Masson三色及Sweet网织纤维的染色，按照2000年新的病毒性肝炎病理分级分期标准，进行炎症活动度和纤维化程度评定，应用免疫组织化学方法，观察平滑肌肌动蛋白（α-SMA）、vimentin、结蛋白（desmin)在肝星状细胞、肌纤维母细胞的表达。结果：在慢性肝炎肝纤维化过程中α－SMA阳性细胞不仅仅是肝星状细胞的活化。结论：在人的慢性肝炎肝纤维化过程中，并不排除肝星状细胞所起的作用，但随着肝组织炎症损伤的修复，以肌纤维母细胞为代表的间叶细胞在纤维化过程中可能起主要作用。     

血吸虫病肝纤维化小鼠中肝星状细胞迁移功能改变的实验研究

目的探讨影响日本血吸虫病肝纤维化小鼠肝组织中肝星状细胞(HSCs)迁移运动功能变化的相关因素。方法 SPF级6⁸周龄Balb/c小鼠16只,随机分为模型组(8只)和对照组(8只),以血吸虫尾蚴腹部贴附法建立感染模型,正常组予以生理盐水代替。于感染后8周末处理小鼠,取部分肝组织石蜡包埋,进行病理学评估,免疫荧光染色检测HSCs(α-SMA,红光)运动蛋白Fascin(绿光)的表达;另取部分肝组织,采用Real-time PCR方法检测迁移诱导因子转化生长因子β1(TGF-β1)、血小板源性生长因子(PDGF)以及单核细胞趋化因子1(MCP-1)的表达以及HSCs运动蛋白α-SMA、Fascin的表达。结果 8周末时,模型组小鼠肝组织中已形成明显肝纤维化。模型组小鼠肝组织中TGF-β1、PDGF以及MCP-1的基因表达水平分别是对照组的30倍、14倍及14倍,差异具有统计学意义(P=0.033、P=0.039以及P=0.037);同时,模型组中HSCs运动相关蛋白α-SMA和Fascin的基因表达水平分别是对照组的9倍和5倍,差异具有统计学意义(P=0.004、P=0.018);荧光共聚焦结果提示,模型组小鼠肝组织中α-SMA(红色)和Fascin(绿色)表达部位一致,集中在虫卵周围肝纤维化区域,较对照组二者表达明显增加,且红绿光分布多重叠。结论诱导HSCs运动迁移的因子表达增加和HSCs自身的运动相关蛋白表达增加均有利于HSCs运动迁移能力增强。     

Intestinal secretion of intravenous talinolol is inhibited by luminal R-verapamil.

OBJECTIVE: To examine the secretion of the beta1-adrenergic receptor antagonist talinolol into the small intestine during its intravenous administration and to show the relevance of the P-glycoprotein-modulating drug verapamil for this secretory transport mechanism in humans. METHODS: In six healthy volunteers the intestinal steady-state perfusion technique (triple lumen tubing system) was used for measuring the appearance of talinolol within the small intestine while the drug was infused intravenously. During four of the seven perfusions performed, the perfusion fluid was changed from a verapamil-free solution and talinolol appearance was measured while a R-verapamil-containing solution (565 micromol/L) was perfused. RESULTS: Talinolol was transported into the intestinal lumen up to a concentration gradient between lumen and blood of about 5.5:1. While perfusing the small intestine with a verapamil-free solution, the intestinal secretion rate of talinolol ranged from 1.94 to 6.62 microg/min per 30 cm length of the intestine (median values). Perfusion of a R-verapamil-containing perfusion fluid resulted in lower secretion rates (0.59 to 3.71 microg/30 cm x min), corresponding to 29% to 56% of the values obtained without verapamil supplied intraluminally. CONCLUSION: Intravenously administered talinolol is actively secreted into the human small intestine. This secretion is reduced by the intraluminal supply of the P-glycoprotein modulating drug R-verapamil. This gives further rationale for P-glycoprotein-mediated intestinal drug secretion as a cause for incomplete oral bioavailability and for drug interactions during intestinal absorption.