基质金属蛋白酶-2在猪胆汁性肝纤维化活性及表达的改变

目的　研究猪胆汁性肝纤维化时基质金属蛋白酶-2（MMP-2）表达及活性改变,分析其在肝纤维化初始阶段的作用。方法　普通猪10头行胆总管结扎术(BDL)前和术后取其肝组织作样本自身对照,用原位杂交法研究MMP-2的mRNA表达改变;用酶图法研究MMP-2活性改变。结果　术后4周原位杂交显示肝星状细胞(HSC)细胞增多、增生、向肝细胞坏死区聚集、胞浆呈强阳性。术前组和术后组平均吸光度分别为0.0246±0.0090和0.0671±0.0091。酶图法表明MMP-2酶原表达是正常的1.68倍（51±6/86±11）,活性升高至2.29倍（21±4/48±3）,差异均有非常显著性（P＜0.01）。结论　猪胆汁性肝纤维化时HSC增生,其胞浆中MMP-2mRNA表达增多,酶活性增强,MMP-2在肝维化的早期参与ECM的重塑和肝小叶的改建。     

组织转谷氨酰胺酶特异性抑制剂预防肝纤维化的实验研究

Objective To observe the effects of cystamine, a tissue transglutaminase (tTG) inhibitor, on the development of rat liver fibrosis induced by carbon tetrachloride. Methods One hundred male SD rats were randomly divided into control group (n=20), hepatic fibrosis group (n=40) and cystamine group (n=40) . Liver fibrosis model was induced by intraperitoneal injection of carbon tetrachloride. Cystamine (112 mg·kg-1·d-1) was administered by intraperitoneal injection 2 days before injection of carbon tetrachloride. The rats were sacrificed at weeks 4 and 8, and the liver tissues and serum specimens were obtained. The mRNA expression of tTG, smooth muscle-alpha (α-SMA), collagen-Ⅰ and tissue inhibitors of metalloproteinase-1 (TIMP-1) were detected by real time PCR. The protein expression of tTG and α-SMA, liver function and content of hydroxyproline in liver tissues were determined by Western blot. Histological changes of the liver was observed under microscope. The fibrosis conditions of rat liver in each group were evaluated according to the semi-quantita-tive scoring system. All the data were analyzed by one-way ANOVA. Results Eight weeks after the injection of carbon tetrachloride, obvious injury of the liver in liver fibrosis group was observed. The levels of alanine trans-aminase (ALT), total bile acid, total bilirubin, hydroxyproline, tTG, α-SMA, collagen-Ⅰ and TIMP-1 were (1313±157)U/L, (99.9±18.5)μmol/L, (10.9±1.6)μmoL/L, (55±12)μg/g, 145.6±51.2, 130.3±44.6, 211.3±75.1 and 162.4±53.5. After administration of cystamine, the levels of ALT, total bile acid, total bilirubin, hydroxyproline, tTG, α-SMA, collagen-Ⅰ and TIMP-1 were (378±87) U/L, (61.0±12.7) μmol/L, (9.8±1.7) μmol/L, (70±14 ) μg/g, 48.6±12.3, 40.7±12.3, 63.9±16.0, 59.2μ23.4. Conclusion Cystamine can alleviate the carbon tetrachloride-induced rat liver fibrosis by inhibiting the tTG pathway.     

结缔组织生长因子通过ERK1/2和PI3K信号通路促进大鼠肝星状细胞迁移和基质金属蛋白酶2表达

目的：研究结缔组织生长因子（CTGF）对大鼠肝星状细胞（HSC）迁移及基质金属蛋白酶2（MMP-2）表达及活化的影响。方法分别应用RT-PCR和Western blotting法检测MMP-2表达,明胶酶谱法检测MMP-2的活性;运用Western blotting法检测ERK1/2和Akt的磷酸化水平。运用Transwell小室检测肝星状细胞迁移能力。结果 CTGF呈剂量依赖性促进大鼠HSC中MMP-2 mRNA和蛋白表达;同时CTGF能够促进HSC迁移,MMP-2特异性抑制剂能够抑制CTGF的促迁移作用。ERK1/2和PI3K抑制剂能够显著抑制CTGF诱导的MMP-2表达以及CTGF促进HSC的迁移作用。结论 MMP-2表达和活性的增强在CTGF促进HSC迁移过程中发挥着重要作用,ERK1/2和PI3K信号通路在CTGF促进MMP-2表达发挥关键作用。     

Cellular and molecular functions of hepatic stellate cells in inflammatory responses and liver immunology

The liver is a central immunological organ. Liver resident macrophages, Kupffer cells (KC), but also sinusoidal endothelial cells, dendritic cells (DC) and other immune cells are involved in balancing immunity and tolerance against pathogens, commensals or food antigens. Hepatic stellate cells (HSCs) have been primarily characterized as the main effector cells in liver fibrosis, due to their capacity to transdifferentiate into collagen-producing myofibroblasts (MFB). More recent studies elucidated the fundamental role of HSC in liver immunology. HSC are not only the major storage site for dietary vitamin A (Vit A) (retinol, retinoic acid), which is essential for proper function of the immune system. This pericyte further represents a versatile source of many soluble immunological active factors including cytokines [e.g., interleukin 17 (IL-17)] and chemokines [C-C motif chemokine (ligand) 2 (CCL2)], may act as an antigen presenting cell (APC), and has autophagy activity. Additionally, it responds to many immunological triggers via toll-like receptors (TLR) (e.g., TLR4, TLR9) and transduces signals through pathways and mediators traditionally found in immune cells, including the Hedgehog (Hh) pathway or inflammasome activation. Overall, HSC promote rather immune-suppressive responses in homeostasis, like induction of regulatory T cells (Treg), T cell apoptosis (via B7-H1, PDL-1) or inhibition of cytotoxic CD8 T cells. In conditions of liver injury, HSC are important sensors of altered tissue integrity and initiators of innate immune cell activation. Vice versa, several immune cell subtypes interact directly or via soluble mediators with HSC. Such interactions include the mutual activation of HSC (towards MFB) and macrophages or pro-apoptotic signals from natural killer (NK), natural killer T (NKT) and gamma-delta T cells (γδ T-cells) on activated HSC. Current directions of research investigate the immune-modulating functions of HSC in the environment of liver tumors, cellular heterogeneity or interactions promoting HSC deactivation during resolution of liver fibrosis. Understanding the role of HSC as central regulators of liver immunology may lead to novel therapeutic strategies for chronic liver diseases.     

红花对肝星状细胞基质分解素-1及其抑制因子基因表达的调节

目的 观察红花对HSC-T6细胞基质分解素-1(MMP3)及其抑制因子基因表达的影响.方法 以不同浓度的红花(1.0、0.5 g/L)作用于培养的肝星状细胞株HSC-T6细胞48 h,收集细胞,提取总RNA;用逆转录-聚合酶链反应(RT-PCR)方法测定MMP3及金属蛋白酶组织抑制因子-1(TIMP-1)的基因表达水平.结果 MMP3基因表达水平在红花1.0、0.5 g/L两组分别为2.80±0.36、2.52±0.32,而空白对照组为2.13 ±0.30.TIMP-1基因表达水平在红花1.0、0.5 g/L两组分别为1.66±0.23、2.04±0.30,而空白对照组为4.03±0.56.结论 红花可增强HSC-T6细胞MMP3的基因表达,并显著抑制HSC-T6细胞TIMP-1的基因表达,且与剂量有关.     

Bilateral sympathectomy improves postinfarction left ventricular remodeling and function

Objectives

To evaluate the influence of bilateral or left sympathectomy on left ventricular remodeling and function after myocardial infarction in rats.

小鼠肝星状细胞分离纯化和体外培养模型的建立

目的 建立小鼠肝星状细胞(m-HSCs)分离纯化的高效稳定方案,并通过原代和传代培养观察其生物学特性.方法 依次应用预灌注液、0.1% Pronase E、0.075% Collagenase NB4G对在体肝脏原位灌注消化.肝脏离体后,在0.02%DNaseI液中磁力搅拌消化10 min,低速离心(25g,5 min)去除残余肝实质细胞,进一步用Optiprep密度梯度液获得纯化的m-HSCs.采用锥虫蓝染色法评估细胞产量及活力;采用自发荧光及油红O染色鉴定细胞纯度;采用光镜、电镜以及Desmin/α-SMA免疫荧光双染色观察细胞形态和生物学特征.结果 分离得到的原代m-HSCs数量为(1.4±0.3)×106/g肝脏,细胞活率＞95%;原代培养24 b后,细胞纯度＞90%,传1代后细胞纯度接近100%.静止期和不同活化阶段的m-HSCs在亚显微结构以及α-SMA表达强度等方面存在差异.结论 建立的m-HSCs分离和纯化方法及体外细胞培养模型具有稳定性和高纯度性及高活率性.

Abstract：

Objective To establish a high-performance and stable model for isolation and purification of mouse hepatic stellate cells, and investigate their biological phenotypes by primary culture and subculture. Methods The liver was digested by in situ perfusion of pre-perfusion solution, 0.1% pronase E and 0.075% collagenase NB4G in turn. The liver was continued to digest using 0.02% DNase Ⅰ liquid with magnetic stirring for 10 min in vitro. After low-speed centrifugation used to remove residual hepatocytes, cells were treated with Optiprep density gradient solution to obtain the purified m-HSCs. Trypan blue staining method was used to calculate cell production and viability. The cell purity was identified by mHSCs autofluorescence and oil red O staining. Light microscopy, electronic transmission microscopy and double immunofluorescence staining of Desmin and α-SMA were done to observe morphological characteristics and biological phenotypes of m-HSCs. Results The yeild rate of m-HSCs was (1.4±0.3)×106/g of liver tissue, the cell viability was more than 95%, the cell purity was more than 90% after 24 h of primary cluture and close to 100% after the first cell passage. The activated m-HSCs had different characteristics of submicroscopic structures and expression level of α-SMA. Conclusion This study established a stable model for isolation, purification and culture of m-HSCs, which obtained the high purity and high-living rate of m-HSCs.     

H2O2诱导的肝星状细胞凋亡后胶原的变化

Objective To explore the changes of collagen (COL) Ⅰ and COLⅢ after apoptosis of HSC induced by H2O2. Methods Different apoptosis rate of HSC-T6 induced by H2O2 ( 100 nmol/L) was evaluated by flow cytometry. RT-PCR was used to detect the gene expression levels of COL Ⅰ and COL Ⅲ. Results Apoptosis of HSC was induced by 100 nmol/L H2O2 with the poptosis rate being 5.86%, 58.55% ,and 71.98% ,respectively. The COL Ⅰ and COLⅢ were highly expressed in activated HSC,and decreased sharply as apoptosis increased. The changes in gene expression of COL Ⅰ were much more obvi-ous than those of COL Ⅲ. Conclusion Inducing apoptosis of HSC may decrease the gene expression of collagens.     

Effects of stellate ganglion block on cerebral haemodynamics as assessed by transcranial Doppler ultrasonography

Background. Stellate ganglion block (SGB) causes vasodilatationin the skin of the head and neck because of regional sympatheticblock. Its effects on cerebral haemodynamics, in health or indisease, are not clear. We evaluated the effects of SGB on ipsilateralmiddle cerebral artery flow velocity (MCAFV), estimated cerebralperfusion pressure (eCPP), zero flow pressure (ZFP), carbondioxide reactivity (CO₂R) and cerebral autoregulation usingtranscranial Doppler ultrasonography (TCD). Methods. Twenty male patients, with pre-existing brachial plexusinjury, and undergoing SGB for the treatment of complex regionalpain syndrome of the upper limb, were studied. For SGB, 10 mlof plain lidocaine 2% was used and the onset of block was confirmedby presence of ipsilateral Horner's syndrome. The MCAFV, eCPP,ZFP, CO₂R, and cerebral autoregulation were assessed beforeand after SGB using established TCD methods. The changes inthese variables were analysed using Wilcoxon's signed rank test. Results. The block caused a significant decrease in MCAFV frommedian (inter-quartile range) value of 61 (53, 67) to 55 (46,60) cm s^–1, a significant increase in eCPP from 59 (51,67) to 70 (60, 78) mm Hg, and a significant decrease in ZFPfrom 32 (26, 39) to 25 (16, 30) mm Hg. There were no significantchanges in CO₂R or cerebral autoregulation. Conclusion. The increase in eCPP, decrease in ZFP, and no changesin CO₂R or cerebral autoregulation suggest that the SGB decreasescerebral vascular tone without affecting the capacity of thevessels to autoregulate. These effects may be of therapeuticadvantage in relieving cerebral vasospasm in certain clinicalsettings.