黏着斑激酶酪氨酸磷酸化促大鼠肝纤维化形成及其可能机制

目的探讨在肝纤维化发生中,黏着斑激酶(FAK)酪氨酸磷酸化与肝星状细胞(HSC)增殖的关系,并从细胞周期角度探讨其促HSC增殖的机制。方法采用胆总管结扎(BDL)方法建立大鼠肝纤维化模型;免疫组织化学方法测定α-平滑肌肌动蛋白(α-SMA)表达;Western blot检测p-FAK(Tyr397)、细胞周期蛋白D1(cyclin D1)和细胞周期蛋白依赖激酶4(CDK4)在肝组织中的含量。结果正常大鼠肝组织有少量α-SMA分布,随着肝纤维化发展,α-SMA阳性细胞明显增多;肝组织中p-FAK(Tyr397)、cyclin D1及CDK4蛋白表达逐渐增加,造模4周时达峰值。多元相关分析示α-SMA与p-FAK(Tyr397)正相关(r=0.964,P<0.01);α-SMA与cyclin D1、CDK4正相关(r=0.953,0.906;P<0.01);p-FAK(Tyr397)与cyclin D1、CDK4亦明显正相关(r=0.969,0.893;P<0.01)。结论FAK磷酸化促HSC增殖及肝纤维化形成机制可能与调控细胞周期相关蛋白有关。     

Intercellular communication via gap junctions in activated rat hepatic stellate cells

BACKGROUND AND AIMS: Gap junctional communication was studied in quiescent and activated hepatic stellate cells. METHODS: Connexin expression and intercellular dye transfer were studied in rat hepatic stellate cells in culture and in vivo. RESULTS: Protein expression of connexin 43 was up-regulated in activated hepatic stellate cells in vivo and in vitro and was mainly localized on the cell surface, whereas connexin 26 was found intracellularly. In contrast to hepatocytes, hepatic stellate cells do not express connexin 32. Confluent hepatic stellate cells in culture communicate via gap junctions, resulting in lucifer yellow transfer and propagation of intracellular calcium signals. Phorbol ester induces a protein kinase C-dependent hyperphosphorylation and degradation of connexin 43 and inhibits intercellular communication on a short-term time scale. At the long-term level, vitamin D(3) , lipopolysaccharide, thyroid hormone T(3), dexamethasone, platelet-derived growth factor, endothelin 1, and interleukin 1beta up-regulate connexin 43 protein and messenger RNA expression and enhance intercellular communication. Slight down-regulation of connexin 43 is observed in response to vitamin A. Connexin 43 induction by endothelin 1 is inhibited by both endothelin A and endothelin B receptor antagonists. In coculture systems, hepatic stellate cells communicate with each other, which is suggestive of a syncytial organization, but no communication was found between hepatic stellate cells and other liver cell types. As shown by immunohistochemistry and electron microscopy, gap junctions are formed between activated hepatic stellate cells in vivo. CONCLUSIONS: Gap junctional communication occurs between hepatic stellate cells, is enhanced after activation, and underlies complex regulation by cytokines, hormones, and vitamins.     

sp600125对乙醛刺激的大鼠肝星状细胞增殖及bcl-2、c-myc蛋白表达的影响

肝星状细胞（HSC）的活化与增殖是肝纤维化发生的关键环节。乙醛刺激HSC活化与增殖，是导致酒精性肝纤维化发生的关键因素。研究表明，丝裂原活化蛋白激酶（MAPK），包括细胞外信号调节激酶（ERK）、JNK、P38是HSC活化与增殖导致肝纤维化发生的主要信号传导通路之一。乙醛刺激HSC中JNK磷酸化水平随sp600125（JNK信号传导通路特异性阻断剂）浓度增加而减少。本实验用sp600125处理乙醛刺激的HSC，观察sp600125对HSC增殖以及bcl-2、c-myc蛋白表达的影响，以探讨酒精性肝纤维化的发生机制。     

过氧化物酶体增殖物激活受体γ与肝星状细胞在肝纤维化中的关系

肝纤维化是慢性肝损伤后常见的形态学表现，大多数是由慢性肝脏疾病发展而来。而肝损伤（肝实质炎症、坏死）激活肝星状细胞（HSC）引起大量细胞外基质沉积，是肝纤维化发生机制的中心环节。在正常肝脏中，HSC处于静息状态，细胞质中脂滴丰富，具有合成和分泌少量细胞外基质和胶原酶的能力。在肝损伤及各种慢性肝病时，HSC被激活转化为肌成纤维母样细胞，发生明显的形态和结构变化：细胞质中脂滴减少或消失，增殖迁移活性明显增强，分泌多种细胞因子和黏附分子，合成各种细胞外基质（ECM）的能力明显增强，抑制基质金属蛋白酶（MMPs）的合成和分泌，而上调基质金属蛋白酶抑制剂（TIMPs）的表达，同时发生多种基因表达的改变。     

肝纤维化相关细胞因子双重RNA干扰质粒的构建及转染肝星状细胞研究

目的构建以大鼠CTGF和TIMP-1基因为靶点的双重RNA干扰表达载体质粒,以检测其转染肝星状细胞的效率.方法筛选出对CTGF和TIMP-1基因最有效的RNA干扰靶位,各设计1对含有短发夹结构的RNA 干扰靶点序列,分别克隆到质粒载体psiRNA-DUO-GFPzeo,构建含目的靶基因片段的重组质粒载体psiRNA-GFP-CT-GF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com（含有CTGF和TIMP-1）,进行酶切和测序鉴定.将构建成功的重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染大鼠肝星状细胞,在荧光显微镜下观察质粒转染情况,用流式细胞仪检测转染效率.结果酶切与测序结果提示重组质粒 psiRNA-GFP-CTGF、psiR-NA-GFP-TIMP-1和psiRNA-GFP-Com成功构建;成功将重组质粒psiRNA-GFP-CTGF、psiRNA-GFP-TIMP-1和psiRNA-GFP-Com转染肝星状细胞,在24小时,空质粒组、CTGF组、TIMP-1组和CTGF ＋TIMP-1组转染效率分别为15±2%、13±1%、15±1%和14±2%,均低于质粒psiRNA-GFP-Com转染组（Com组,20±2%,P〈0.05）;在48小时,空质粒组、CTGF组、TIMP-1组和CTGF＋TIMP-1组转染效率分别为10±2%、9±1%、8±2%和10±1%,均低于Com组的15±2%（P〈0.05）.结论成功构建靶向大鼠CTGF和TIMP-1最有效的RNA干扰靶位的双重RNA干扰表达质粒,能转染至肝星状细胞,并且psiRNA-GFP-Com转染效率最高.     

Transforming growth factor‐α attenuates hepatic fibrosis: possible involvement of matrix metalloproteinase‐1

Background: The effect of transforming growth factor (TGF)‐α on fibrosis varies between cell types and the role of TGF‐α in hepatic fibrosis has not been fully elucidated. Methods: We examined the effect of TGF‐α on hepatic fibrosis using TGF‐α‐expressing transgenic mice fed a methionine‐ and choline‐deficient (MCD) diet and human hepatic stellate cells (HSCs) line LX‐2, rat and human primary HSCs. Results: Although the expression levels of the tissue inhibitor of metalloproteinases‐1 and α1(I) collagen mRNA were unchanged, feeding the TGF‐α transgenic mice the MCD diet resulted in greater expression of the murine functional analogue of matrix metalloproteinase‐1 (MMP‐1), MMP‐13 mRNA and protein and attenuated hepatic fibrosis compared with wild‐type mice. TGF‐α overexpression did not affect the extent of the steatosis, oxidative stress and hepatic inflammation in the MCD diet‐fed mice. The effect of TGF‐α on the fibrogenic and anti‐fibrogenic gene expressions varied between cell types in vitro. TGF‐α increased MMP‐1 mRNA expressions that were completely blocked by gefitinib in LX‐2 cells. The extracellular signal‐regulated kinase (ERK) 1/2, c‐Jun N‐terminal kinase and p38 pathways were involved in MMP‐1 mRNA expression in LX‐2 cells. Although TGF‐α increased the phosphorylation of p38, the p38 inhibitor activated the RAS‐ERK pathway and increased TGF‐α‐induced MMP‐1 mRNA expression, which suggested that there may be a crosstalk between the RAS‐ERK and the p38 pathways in LX‐2 cells. Conclusions: The TGF‐α may attenuate hepatic fibrosis in part because of upregulation of the expression of MMP‐1. The balance between fibrogenic and anti‐fibrogenic gene expression and between the activity of the RAS‐ERK and the p38 pathways may be crucial for the fibrotic process.