Effects of interferon-alpha on expression of hepatic stellate cell and transforming growth factor-β1 and α-smooth muscle actin in rats with hepatic fibrosis

AIM: To investigate the effect of interferon-α (IFN-α) onpreventing or reversing hepatic fibrosis in rat experimental model induced by CCl4.METHODS: One hundred and ten Sprague-Dawley rats were divided into five groups: group A (normal controls, n = 18), group B (fibrotic model controls, n = 22), group C (IFN-α prevention, n = 22) initially treated with intramuscular injection of IFN-α in saline daily at the doses of 1×105 U for 6 wk, group D (IFN-α treatment, n = 24) treated with intra-muscular injection of IFN-α in saline daily atthe doses of 1×105 U for 6 wk after the first 6 wk, group E (0.9% sodium chloride treatment control, n = 24) treated with intra-muscular injection of 0.01 mL/kg daily for 6 wk after the first 6 wk. At the end of the experiment, all rats of each group were killed. Samples of the liver obtained by biopsy were subjected to histological, immunohistochemicaland electron microscopic studies for the expressions of transforming growth factor-β1 (TGF- β1) and α-smoothmuscle actin (α-SMA).RESULTS: The expressions of TGF-β1, the number of activated hepatic stellate cells and α-SMA in hepatic tissue of group C were significantly less than those of group B (P＜0.01). The degree of fibrosis score in group B was also significantly less than that of group C under light microscope (P＜0.01).CONCLUSION: IFN-α can inhibit the production of TGF-β1,decrease HSC activation and stimulate its apoptosis.     

前列地尔对肝肾综合征患者血浆TNF-α、NO及内毒素水平的影响

目的观察前列地尔对肝肾综合征患者血浆TNF-α、NO及内毒素水平的影响。方法选择肝硬化合并肝肾综合征患者80例，健康体检者40例。肝硬化合并肝肾综合征组80例随机分为两组：对照组40例，试验组40例。对照组给予常规的保肝、退黄、支持治疗，试验组在对照组治疗基础上每日加用前列地尔20μg，连用2周。检测各组患者血浆TNF-α、NO及内毒素水平。应用ELISA双抗体夹心法检测血浆TNF-α，亚硝酸盐还原酶法检测血浆NO，采用鳖试验检测血浆内毒素。结果对照组、试验组治疗前血浆TNF-α、NO及内毒素水平较健康体检组显著升高（P〈0．05）；对照组、试验组之间无显著性差异（P〉0．05）；试验组治疗前后血浆TNF-α、NO及内毒素水平有显著差异（P〈0．01）；试验组治疗后血浆TNF-α、NO及内毒素水平明显低于对照组治疗后水平，有显著差异（P〈0．05）。结论前列地尔可以显著降低肝硬化合并肝肾综合征患者血浆TNF-α、NO及内毒素水平，是治疗肝硬化合并肝肾综合征的作用机制之一。     

大鼠纤维化肝组织中PTEN的动态表达及其与肝星状细胞增殖和活化的关系

目的 探讨第10号染色体缺失的磷酸酶张力蛋白同源物基因(PTEN)在大鼠纤维化肝组织中的动态表达及其对在体肝星状细胞(HSC)活化、增殖的影响. 方法 采用胆总管结扎法建立大鼠肝纤维化模型;应用免疫组织化学染色、Western blot和实时荧光定量PCR技术测定大鼠肝组织中PTEN的表达;应用免疫荧光双标记共聚焦激光扫描显微术测定大鼠肝组织中活化HSC的PTEN表达;采用免疫组织化学染色检测大鼠肝组织中α-平滑肌肌动蛋白的表达.结果 免疫组织化学染色显示正常大鼠肝组织中PTEN有广泛表达,主要表达于细胞质,随着肝纤维化的发展,PTEN表达逐渐减少(P<0.01),而α-平滑肌肌动蛋白阳性细胞明显增多(P<0.01);造模1、2、3周及4周不同时间大鼠纤维化肝组织中PTEN的mRNA(分别为假手术组的0.66、0.53、0.44和0.37)及蛋白质表达(吸光度比值分别为1.20±0.13,1.07±0.16,0.88±0.08,0.73±0.07)均低于假手术组(P<0.01),并随着肝纤维化的进展逐渐降低(P<0.01);免疫荧光双标记共聚焦激光扫描显微术显示PTEN在活化HSC广泛表达,主要表达于细胞质,随着肝纤维化的进展,表达PTEN的活化HSC占总的活化HSC的比例逐渐减少(P<0.01). 结论 大鼠纤维化肝组织中PTEN的mRNA及蛋白质表达均下调;在体HSC的PTEN表达亦降低;肝组织中PTEN的动态表达与HSC的活化、增殖呈显著负相关.     

结缔组织生长因子短发夹状RNA表达质粒的构建及其对肝星状细胞结缔组织生长因子表达的影响

结缔组织生长因子（CTGF）是一种新发现的促成纤维细胞分裂和胶原沉积的生长因子，在肝纤维化形成中的作用逐渐受到关注。RNA干扰技术是目前最有效的基因沉默技术，能特异性抑制靶基因的转录，进而下调相应蛋白水平及功能。本研究将能在体内自主合成短发夹状RNA（shRNA）的pEGFP-CTGF表达质粒转人大鼠肝星状细胞（HSC-T6），观察其对CTGF mRNA及蛋白表达的影响。     

化学缺氧对肝星状细胞基质金属蛋白酶-2表达及活性的影响

目的探讨化学缺氧对大鼠HSC MMP-2的表达和活性的影响及其调节机制。方法用不同浓度的氯化钴（CoCl2,0、50、100、200μmol/L）造成大鼠HSC化学缺氧,RT-PCR、酶图法分别检测MMP-2 mRNA表达及其酶活性;Western blot检测缺氧诱导因子-1α（HIF-1α）表达;用荧光素酶报告基因分析技术测HIF-1α对MMP-2基因的激活作用。结果随CoCl2浓度从0μmol/L依次递增到200μmol/L,MMP-2 mRNA表达的量（用吸光度值表示）从0.53±0.12依次上调到1.57±0.11,4组间差异有统计学意义（F=34.21,P〈0.01）,其活性下降幅度（用吸光度值表示）从84.49±5.38逐渐下降至53.70±3.42,4组间差异有统计学意义（F=29.54,P〈0.01）,HIF-1α表达量依次增加;核蛋白提取物与MMP-2基因探针（含缺氧反应元件）共浴后,电泳迁移条带产生延迟现象,竞争性序列能部分拮抗其结合。结论化学缺氧能使HSC MMP- 2 mRNA表达上调,酶活性下降。HIF-1α可能参与了缺氧条件下MMP-2的表达调节。     

Role of NF-kappaB activation and nitric oxide expression during PGE protection against d-galactosamine-induced cell death in cultured rat hepatocytes.

Prostaglandin E1 (PGE1) reduces cell death in experimental and clinical liver dysfunction. Nitric oxide (NO) mediates PGE1 protection against D-galactosamine (D-GalN)-induced cell death. Nuclear factor kappa-B (NF-kappaB) plays a protective role in different experimental models of cell death. We investigated if NF-kappaB was responsible for inducible nitric oxide synthase (iNOS) expression and cytoprotection induced by PGE1 against D-GalN cell death in cultured hepatocytes. Rat hepatocytes were isolated following the classical method of collagenase perfusion of liver. A kinetic study of cell death, NF-kappaB activation, mRNA and protein iNOS expression, and NO production was carried in hepatocytes treated with D-GalN (5 mM) in the presence or absence of PGE1 (1 microM) administered 2 h before the hepatotoxin. A proteasome inhibitor was used to evaluate the role of NF-kappaB activation in our experimental conditions. PGE1 protection against D-GalN-induced cell death was associated with its capacity to rapidly enhance NF-kappaB activation, mRNA and protein iNOS expression, and NO production in D-GalN-treated hepatocytes. The inhibition of NF-kappaB activation abolished iNOS expression and cell protection by PGE1 in hepatocytes treated with the hepatotoxin. The present study shows that the cytoprotection by PGE1 against D-GalN-induced apoptosis was related to NF-kappaB-dependent iNOS expression.     

反义及小干扰RNA对大鼠肝星状细胞金属蛋白酶组织抑制因子-1表达的抑制作用

目的观察以腺相关病毒（AAV）为载体，含有针对大鼠金属蛋白酶组织抑制因子（TIMP）-1的反义RNA及小干扰RNA（siRNA）的重组AAV（rAAV／ANTI-TIMP—1／neo及rAAV／siRNA—TIMP—1／neo）感染大鼠肝星状细胞系HSC—T6后，TIMP—1表达的受抑制情况。方法经聚合酶链反应（PCR）扩增及酶切后连接，将针对TIMP—1的反义RNA片段及甄RNA片段分别构建于AAV载体中并测序鉴定，将其包装成重组病毒后感染大鼠肝星状细胞系HSC—T6，同时设空白对照组。经G418以400ug／m1的浓度筛选30d后，分别应用荧光定量PCR技术及Westernblot检测重组病毒感染组及空白对照组HSC-T6 TIMP-1的基因转录及蛋白质表达水平。结果经PCR、酶切及序列测定证实，两种重组AAV载体质粒（pd16-95／ANTI—TIMP-1／neo和pd16—95／siRNA-TIMP-1／neo）克隆成功。将重组质粒包装成病毒感染HSC—T6细胞30d后，通过荧光定量PCR技术及Westernblot分析显示，重组病毒rAAV／siRNA-TIMP-1／neo组与对照组细胞相比，HSC—T6中的TIMP-1基因的转录被抑制（P〈0．01），且表达水平与对照组相比约下降60％。而rAAV／ANTI—TIMP—1／neo组及空载体组与对照组相比TIMP—1基因的转录及表达水平差异无统计学意义（P〉0．05）。结论通过AAV载体技术重组病毒rAAV／siRNA-TIMP—1／neo可有效地抑制TIMP-1基因的表达，而针对TIMP—1基因全长的重组病毒rAAV／ANTI—TIMP—1／neo对体外培养的HSC—T6细胞TIMP—1基因的转录与表达无明显抑制作用。     

The role of endocannabinoids in gonadal function and fertility along the evolutionary axis

Endocannabinoids are natural lipids able to bind to cannabinoid and vanilloid receptors. Their biological actions at the central and peripheral level are under the tight control of the proteins responsible for their synthesis, transport and degradation. In the last few years, several reports have pointed out these lipid mediators as critical signals, together with sex hormones and cytokines, in various aspects of animal and human reproduction. The identification of anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in reproductive cells and tissues of invertebrates, vertebrates and mammals highlights the key role played by these endogenous compounds along the evolutionary axis. Here, we review the main actions of endocannabinoids on female and male reproductive events, and discuss the interplay between them, steroid hormones and cytokines in regulating fertility. In addition, we discuss the involvement of endocannabinoid signalling in ensuring a correct chromatin remodeling, and hence a good DNA quality, in sperm cells.