纯化HBV促进人肝星状细胞系LX-2细胞Smad3表达

目的体外实验观察纯化HBV对人肝星状细胞系LX-2细胞表达Smad3的影响。方法采用蔗糖密度梯度离心法从HBV感染者的高滴度血清中进行病毒纯化浓缩,然后应用MTT法观察纯化HBV对LX-2细胞增殖率的影响;再用纯化HBV培养LX-2细胞24h和48h后,应用实时荧光定量PCR方法检测细胞Smad3mRNA表达的变化,以Western blot方法检测细胞Smad3蛋白表达的变化。结果经蔗糖密度梯度离心可得到高纯度的HBV颗粒,10^3～10^7copies／ml的HBV可逐渐增加LX-2细胞的增殖率,纯化HBV培养LX-2细胞24h和48h后,Smad3mRNA表达分别为阴性对照的1．7倍和4．3倍。纯化HBV培养LX-2细胞24h后阴性对照组和纯化HBV组HSmad3／β-actin吸光度比值分别为0．62、0．91;培养48h后阴性对照组和纯化HBV组Smad3／-actin吸光度比值分别为0．33、1．02。结论纯化HBV可促进人肝星状细胞系LX-2细胞Smad3表达。     

TRAIL体外诱导人肝星形细胞LX-2凋亡作用及调控机制

目的研究肿瘤坏死因子相关凋亡诱导配体（TRAIL）诱导肝星形细胞凋亡情况及调控机制。方法用RT-PCR检测LX-2中α-SMAmRNA和DR5mRNA的表达;MTT比色法、流式细胞术检测外源性TRAIL对LX-2细胞增殖和诱导细胞凋亡的影响;采用Westernblot检测Bax、Caspase3表达。结果培养的LX-2表达α-SMAmRNA和DR5mRNA逐渐增加,TRAIL可以抑制其细胞增殖,与剂量相关（P〈0.05）。与对照组比较,TRAIL诱导活化的LX-2细胞凋亡明显增加（P〈0.05）,Western blot分析显示TRAIL作用下,LX-2线粒体Bax、细胞质Caspase3表达上调。结论外源性TRAIL可以诱导活化的LX-2凋亡,其机制与DR5及线粒体Bax表达上调有关。     

Liver inflammatory cells, apoptosis, regeneration and stellate cell activation in non-alcohol steatohepatitis

Background, The pathogenesis of non-alcoholic steatohepatitis remains unclear from several points of view. Minimal diagnostic criteria are still not defined. Aim. To gather information useful for diagnosis and to improve the understanding of pathogenic mechanisms.

Patients. A series of 14 patients with non-alcoholic steatohepatitis, identified among liver outpatients, were paired for age, sex and alanine amino transferase values with 14 patients with hepatitis C virus infection without steatosis.

Methods. Clinical, biochemical and immunohistological examination, including characterisation of inflammatory cell population, evaluation of type 111 collagen and tenascin deposition, activation of stellate cells, hepatocellular apoptosis and proliferation.

Results. Patients with non-alcoholic steatohepatitis were more frequently obese, had higher triglyceride concentrations and lower gamma-globulins. T lymphocytes outnumbered polymorphonuclear cells, both in hepatitis C and in steatohepatitis, with a larger number of CD8 lymphocytes in patients with viral hepatitis but a comparable number of granulocytes. This resulted in a higher granulocytes to T cells ratio in steatohepatitis, possibly making these cells more easily detectable in spite of similar absolute numbers. Portal fibrosis and piecemeal necrosis were prevalent in hepatitis C virus infection, pericentral fibrosis was similar. Hepatocellular, apoptosis and proliferation as well as stellate cell activation were less relevant in steatohepatitis than in hepatitis C virus infection in spite of similar alanine amino transferase levels.

Conclusions. These data provide a possible explanation for the relatively low tendency to progression of non-alcoholic steatohepatitis in most patients despite increased alanine amino transferase and suggest that non-death-related release of alanine amino transferase might occur in non-alcoholic steatohepatitis. This makes liver biopsy an essential part of the clinical setting supporting diagnosis, evaluation of severity and possibly definition of the evolutionary trend.     

干扰素β对肝星状细胞活化调控机制的影响

以往的研究已经证实干扰素β（IFNβ）不仅具有抗肝炎病毒的作用，还具有抗肝纤维化的作用，但其作用机制尚不明确。本研究旨在探讨能否经过调控转化生长因子β1（TGFβ1）、Smad4、Smad7、血小板衍生生长因子（PDGF）-BB等细胞因子的表达而影响肝星状细胞（HSC）的激活。     

2-methoxyestradiol alters cell motility,migration, and adhesion

The effect of 2-methoxyestradiol, 2ME2, an endogenous metabolite of 17beta-estradiol (E2), on cell growth and cytoskeletal functions in a BCR-ABL-transformed cell line model was investigated. We determined the interaction of 2ME2 with STI571 (Gleevec, imatinib mesylate) in STI571 drug-sensitive and -resistant cell lines. In cells expressing BCR-ABL, STI571 cooperated with 2ME2 in reducing cell growth, and STI571-resistant cells were sensitive to 2ME2 treatment. 2ME2 also inhibited growth of several cancer cell lines by a mechanism independent of BCR-ABL. BCR-ABL transformation leads to altered motility, increased adhesion, and spontaneous migration in different in vitro model systems. 2ME2 was found to specifically inhibit the spontaneous motility of BCRABL-transformed Ba/F3 cells and to change the morphology and volume of treated cells. Cells attached to fibronectin-coated surfaces showed a reduced number of filipodia and lamellipodia. In addition, 2ME2 significantly reduced BCRABL-mediated adhesion to fibronectin. The spontaneous migration of BCR-ABL-transformed cells through a transwell membrane also was found to be significantly decreased by 2ME2. Cytoskeletal changes were accompanied by alteration of tubulin formation, distinct from paclitaxel treatment. These results demonstrate that 2ME2 treatment of transformed cells strongly reduces cytoskeletal functions and may also be useful for the treatment of cancers with high metastatic potential. Combination of 2ME2 with other anticancer drugs may be beneficial to treatment of drug-resistant cancers.     

cGMP-dependent protein kinase I (cGKI) modulates human hepatic stellate cell activation

BackgroundThe activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrosis, however the role of HSCs is less understood in hepatic insulin resistance. Since in the liver cGMP-dependent protein kinase I (cGKI) was detected in HSC but not in hepatocytes, and cGKI-deficient mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice) displayed hepatic insulin resistance, we hypothesized that cGKI modulates HSC activation and insulin sensitivity.Materials and MethodsTo study stellate cell activation in cGKI-SM mice, retinol storage and gene expression were studied. Moreover, in the human stellate cell line LX2, the consequences of cGKI-silencing on gene expression were investigated. Finally, cGKI expression was examined in human liver biopsies covering a wide range of liver fat content.ResultsRetinyl-ester concentrations in the liver of cGKI-SM mice were lower compared to wild-type animals, which was associated with disturbed expression of genes involved in retinol metabolism and inflammation. cGKI-silenced LX2 cells showed an mRNA expression profile of stellate cell activation, altered matrix degradation and activated chemokine expression. On the other hand, activation of LX2 cells suppressed cGKI expression. In accordance with this finding, in human liver biopsies, we observed a negative correlation between cGKI mRNA and liver fat content.ConclusionsThese results suggest that the lack of cGKI possibly leads to stellate cell activation, which stimulates chemokine expression and activates inflammatory processes, which could disturb hepatic insulin sensitivity.     

姜黄素对肝星状细胞增殖与凋亡的影响

目的　观察姜黄素对体外培养肝星状细胞 (HSC)增殖与凋亡的影响。方法　不同浓度姜黄素处理HSC株HSC T6 ,MTT法检测细胞增殖 ,流式细胞仪、透射电镜和琼脂糖凝胶电泳法检测细胞凋亡。结果　在 2 0～ 10 0 μmol/L浓度范围内 ,姜黄素可剂量依赖性地抑制HSC增殖 (P <0 .0 1)。 2 0、4 0、6 0 μmol/L姜黄素处理HSC 2 4h后 ,细胞周期分析发现S期细胞减少 ,G2 /M期细胞显著增加 (P <0 .0 1) ;流式细胞术检测到明显的亚G1峰 ,各组的凋亡指数 (% )分别是 15 .3± 1.9,2 6 .7± 2 .8,37.6± 4 .4 ,与对照组 (1.9± 0 .6 )相比 ,差异有显著性 (P <0 .0 1) ;4 0 μmol/L姜黄素作用 12、2 4、36、4 8h ,凋亡指数 (% )分别是 12 .0± 2 .4、2 6 .7± 3.5、33.8± 1.8和 4 9.3± 1.6 ,与对照组相比 ,差异有显著性 (P <0 .0 1) ;透射电镜观察到细胞皱缩、核染色质浓缩沿核膜排列和凋亡小体形成等 ;琼脂糖凝胶电泳可见到明显的DNA梯带形成。结论　姜黄素可显著抑制HSC增殖 ,使细胞周期停滞于G2 /M期 ,并诱导其凋亡 ,其作用具有时间和剂量依赖性     

芍药甙诱导人肝癌细胞系HepG2凋亡的研究

目的：观察芍药甙诱导人肝癌细胞系HepG2凋亡及凋亡相关基因的变化。方法：用0．5～0．2g／L芍药甙处理人肝癌HepG2细胞，光镜下观察细胞形态；MTT法检测细胞增殖率；流式细胞仪检测凋亡峰；免疫组化法检测P53，bcl-2，bax等凋亡相关基因的表达。结果：0．5～2．0g／L浓度的芍药甙对HepG2有显著抑制作用；可诱导HepG2凋亡，并能上调凋亡相关基因P53和bax表达、下调抗凋亡基因bcl-2表达。结论：0．5～2．0g／L芍药甙可诱导人肝癌细胞HepG2凋亡，其机制可能与凋亡相关基因表达增强和抗凋亡相关基因表达减弱有关。     

奥沙利铂对人肝癌细胞株HepG2体外增殖的影响

奥沙利铂(Oxaliplatin，L-OHP)是第三代铂类化合物，其化学名为左旋反式二氨环己烷草酸铂，国际通用名为草酸铂。临床前研究表明该药对人和鼠多种肿瘤细胞均有抑制作用，且对各种顺铂耐药的肿瘤细胞株无交叉耐药。研究通过抗肿瘤药物奥沙利铂对人肝癌细胞株HepG2体外增殖的影响，为奥沙利铂应用于肝细胞癌的临床治疗提供理论依据。     

SphK1对结肠癌lovo细胞的增殖、迁移和凋亡的影响

目的:探讨鞘胺醇激酶1(SphK1)对结肠癌lovo细胞增殖、迁移和凋亡的影响及其作用机制.方法:培养人结肠癌lovo细胞株,实验分3组:对照组、PMA组和DMS组.PMA组加入佛波醇-12-豆蔻酸酯-13-乙酸酯(PMA,100nmol/L),DMS组加入N,N-二甲基鞘胺醇(DMS,50μmol/L),对照组加入等量的培养基.采用MTT法和克隆形成实验检测细胞生长增殖的变化,流式细胞术检测细胞凋亡,Tranwell小室模型观察细胞迁移能力的变化,RT-PCR检测mRNA的表达,Westernblot检测蛋白的表达.结果:PMA显著促进细胞的增殖、迁移并抑制细胞的凋亡,DMS则显著抑制细胞的增殖、迁移并促进细胞的凋亡(对照组、PMA组和DMS组的细胞增殖活力:0.71±0.03vs1.05±0.05vs0.46±0.04;克隆形成率:1.32%±0.26%vs2.17%±0.17%vs0.73%±0.13%;凋亡率:16.25%vs9.15%vs32.58%;迁移细胞数:72.19±3.36vs98.46±6.25vs40.48±4.27;均P<0.05).PMA显著促进黏着斑激酶(FAK)的活性和表达,相反DMS则抑制FAK的活性和表达[对照组、PMA组和DMS组FAKmRNA的表达强度:0.151±0.008vs0.212±0.014vs0.114±0.021;蛋白:0.332±0.022vs0.374±0.029vs0.296±0.018;磷酸化FAK(p-FAKTyr397)蛋白:0.186±0.032vs0.234±0.017vs0.112±0.023;均P<0.05].结论:SphK1可促进lovo细胞的增殖和迁移能力并抑制细胞的凋亡,其机制可能是通过激活FAK通路而发挥作用.     

Effect of protein kinase C activation and inhibition on rat hepatic stellate cell activation

Protein kinase C (PKC) may play a role in the intracellular signaling pathways responsible for transforming hepatic stellate cells into myofibroblasts. This study examined the effects of inhibitors and activators of PKC on hepatic stellate cell activation. Stellate cells isolated from normal rats were incubated with either 10^–5 M chelerythrine, 10^–7 M bisindolylmaleimide I hydrochloride (BIM), or 10^–6 M staurosporine (PKC inhibitors), or 10^–7 M phorbol myristate acetate (PMA) or 10^–6 M thymeleatoxin (PKC activators). Chelerythrine suppressed -smooth muscle actin expression and proliferation by 49% and 33%, respectively. BIM inhibited -smooth muscle actin expression by 60%, but had no significant effect on proliferation. Staurosporine decreased proliferation by 86% and completely prevented -smooth muscle actin expression. PKC activators had divergent effects on proliferation and -smooth muscle actin expression. PMA and thymeleatoxin caused a 2.8- to 3.2-fold increase in proliferation, while suppressing -smooth muscle actin expression by 50–70%. The demonstration that hepatic stellate cell activation can be suppressed by PKC inhibitors suggests a role for PKC in the regulation of hepatic stellate cell activation.     

Effects of angiotensin Ⅱreceptor antagonist, Losartan on the apoptosis, proliferation and migration of the human pancreatic stellate cells

AIM: To investigate the effects of AT1 (Type 1 angiotensin II receptor) antagonist (Losartan) on the apoptosis, proliferation and migration of the human pancreatic stellate cells (hPSCs). METHODS: hPSCs were isolated from pancreatic sample of patients with pancreatic carcinoma using radioimmunoassay (RIA) technique to detect the concentration of AngII in culture media and cell homogenate. Immunocytochemistry (ICC) and in situ hybridization (ISH) methods were utilized to test AT1 expression in hPSCs. Effects of Losartan on hPSCs proliferation, apoptosis and migration were investigated using BrdU incorporation, TUNEL, flow cytometry (FCM), and phase-contrast microscope separately when cells treated with Losartan. Immunofluorescence and Western blot were applied to quantify the expression of type I collagen in hPSCs. RESULTS: There exists AT1 expression in hPSCs, while no AngII was detected in culture media and cell homogenate. Losartan induces cell apoptosis in a dose- and time-dependent manner (apparently at 10(-5) mol/L), no pro-proliferative effect was observed in the same condition. Corresponding dosage of Losartan can also alleviate the motion capability and type I collagen content of hPSCs compared with AngII treatment and non-treatment control groups. CONCLUSION: These findings suggest that paracrine not autocrine functions of AngII may have effects on hPSCs, which was mediated by AT1 expressed on cells, while Losartan may exert anti-fibrotic effects by inhibiting hPSCs motion and partly by inducing apoptosis.