实验性酒精性肝病时脂多糖结合蛋白和脂多糖受体CD14的表达

目的观察大鼠酒精性肝病时脂多糖结合蛋白(lipopolysaccharide binding protein,LBP)和脂多糖受体CD14的表达及其在酒精性肝损害中的作用.方法随机将Wistar大鼠分为乙醇喂养组和葡萄糖喂养对照组,分剐在饮水中加入乙醇(剂量5-12 g@kg-1@d-1)和相同量的葡萄糖.两组大鼠分别于4周和8周测定其血浆中内毒揪素(LPS)浓度及血清中ALT变化,同时用RT-PCR测定肝组织中LBP和CD14 mRNA的表达,并在光镜和电镜下观察肝脏的形态学改变.结果乙醇喂养组4周和8周时大鼠血浆LPS浓度分别为(129±21)pg/ml和(187±35)Pg/m1,明显高于对照组的(48±9)pg/ml和(53±11)pg/ml(f值分别为11.2和11.6,P＜0.05);乙醇组大鼠血清ALT浓度为(112±15)U/L和(147±22)U/L,也明显高于对照组的(31±12)U/L和(33±9)U/L(t值分别为5.9和20.6,P＜0.05).乙醇组大鼠肝组织中LBP和CD14 mRNA的表达水平明显高于对照组(P＜0.05),其肝组织发生显著的病理变化,主要表现为脂肪变性、炎性细胞浸润及细胞坏死.对照组肝组织中LBP和CD14mRNA无明显表达,其病理变化也不明显.结论乙醇能诱导大鼠血中LPS浓度升高和肝缝织中LBP与CD14 mRNA的表达显著增强,增高的LBP和CD14 mRNA能增加肝脏对LPS的敏感性,可能造成肝脏损害.     

内毒素上调肝细胞表达CD14基因和蛋白的实验研究

目的 观察内毒素血症时肝组织和游离肝细胞中CD14基因和CD14蛋白的表达。方法 经尾静脉注入脂多糖（LPS,E coli O111.B4）建立大鼠急性内毒素血症动物模型,并用原位胶原酶灌注法分离肝细胞。用流式细胞仪（FCM）测定异硫氢酸荧光素（FITC）－CD14阳性肝细胞数及其荧光强度。同时用逆转录－PCR和Western blot法测定肝组织和肝细胞中CD14 mRNA和CD14蛋白的表达。结果 FCM显示：内毒素血症大鼠6h和12hFITC－CD14阳性细胞数明显增多,荧光强度也明显增加。RT－PCR显示,肝组织和肝细胞中CD14 mRNA的表达在3h时明显增强,6h达高峰,24h恢复正常水平;Western blot分析示肝组织和肝细胞中CD14蛋白的表达在6h明显增高,12h达高峰,24h仍有一定表达,各时相点间比较有显著差别（P＜0.01）。结论 内毒素血症时能明显上调肝细胞CD14基因和CD14蛋白的表达。     

LPS和CD14在肠源性内毒素血症中的作用

革兰阴性杆菌内毒素脂多糖（Ｌｉｐｏｐｏｌｙｓａｃｃｈａｒｉｄｅ，ＬＰＳ）常引起内毒素血症，通过单核巨噬细胞系统扩大炎症反应，甚至导致中毒性休克。肝病患者由于肝细胞功能受损及门体分流的存在，来自肠道的内毒素易进入体循环，产生肠源性内毒素血症。近年来，发现脂多糖结合蛋白（ＬＢＰ）／ＣＤ１４是体内增敏ＬＰＳ细胞效应的重要系统，并激活细胞内信号传导，刺激一系列细胞因子（如ＴＮＦ、ＩＬ、ＰＡＦ）合成、释放，加重肝脏损害并可导致多器官功能受损。本文就ＬＢＰ和ＣＤ１４在肠源性内毒素血症中的重要作用作一介绍。一…     

益气化痰解毒中药对脂肪肝大鼠脂多糖结合蛋白CD14表达的作用

[目的]揭示益气化痰解毒中药抗脂肪性肝损伤药效学机制，探寻其辨证施治规律及安全、有效、持久的治疗方法，为中医药治疗脂肪性肝炎提供理论和实验依据。[方法]采用高脂饮食和乙醇灌胃，经8周建立大鼠脂肪肝炎模型，用益气化痰解毒中药（肝脂消颗粒剂）治疗4周，与东宝肝泰对比，观测肝组织脂多糖结合蛋白（LBP）、脂多糖受体CD14表达；血清白细胞介素6（IL-6）、丙氨酸氨基转移酶（ALT）、天冬氨酸转氨酶（AST）水平。[结果]模型组大鼠CD14 mRNA与LBP mRNA的表达明显高于正常组（P〈0．01）；肝脂消组抑制肝组织LBP、CD14 mRNA表达和降低血清IL-6、ALT、AST水平均明显优于东宝肝泰组（P〈0．05，〈0．01）。[结论]益气化痰解毒中药通过抑制肝组织LBP、CD14 mRNA的表达，阻断内毒素血症致病途径，达到有效治疗脂肪性肝炎的作用。     

甘氨酸在肝硬化发生发展过程中对肝组织CD14基因及蛋白表达的影响

目的 观察甘氨酸在肝硬化形成过程中对肝组织表达CD14基因和CD14蛋白的影响。方法 采用复合因素建立肝硬化动物模型的同时，实验组分别用甘氨酸液（1g/d)给大鼠灌胃，或用含5%甘氨酸食物喂养大鼠，并于实验的2周末、4周末和8周末以逆转录多聚酶链反应（RT-PCR）和western blot法检测大鼠肝组织CD14 mRNA和CD14蛋白的表达。结果 在用含甘氨酸食物喂养的脂肪肝和肝硬化大鼠中，其肝组织CD14 mRNA及CD14蛋白的表达较对照组大鼠明显减弱，其中用含甘氨酸食物喂养8周末的肝硬化大鼠肝组织的CD14 mRNA及CD14蛋白表达最弱。结论 甘氨酸可下调肝组织CD14基因和CD14蛋白的表达。     

内毒素受体在肝星状细胞的表达及其作用

目的了解内毒素受体在肝星状细胞活化中的变化和作用。方法分离正常大鼠的肝星状细胞，以逆转录聚合酶链反应法检测其在体外培养过程中内毒素受体（CD14和TLR4）mRNA的表达变化。以细胞免疫染色法检测肝星状细胞内毒素受体CD14的表达。制作肝纤维化和肝硬化的大鼠模型，免疫组织化学法动态检测肝组织内CD14和α平滑肌肌动蛋白的表达变化和定位。结果初分离的肝星状细胞表达低水平的CD14 mRNA，不表达TLR4 mRNA，培养活化的肝星状细胞内毒素受体的表达增强，内毒素可上调这种表达。体外培养10d的肝星状细胞表达CD14蛋白，内毒素作用后CD14表达更明显。在肝纤维化的发展过程中，肝组织内CD14阳性细胞增多，阳性细胞多分布于肝窦周围，晚期CD14阳性细胞聚集在纤维隔内，与α平滑肌肌动蛋白阳性细胞的分布一致。结论肝星状细胞在体内外的活化过程中内毒素受体的表达增强，因此，内毒素受体可能参与肝星状细胞在肝脏炎症和纤维化中的作用。     

内毒素受体CD14在四氯化碳诱导的大鼠肝损伤和纤维化过程中的变化

内毒素是革兰阴性杆菌细胞壁外层的结构成分,内毒素能活化单核/巨噬细胞释放炎症介质,诱导一系列的炎症反应.许多研究提示内毒素是慢性肝损伤中的一个重要的共同因子[1].内毒素血症在肝病时的发生率高,由于无明显的外源性感染,通常称作肠源性内毒素血症.内毒素血症的程度常与肝损的程度相关.     

Toll样受体及内毒素信号传导研究进展

1997年以来 ,《Ｎａｔｕｒｅ》(《自然》杂志 )发表人类Ｔｏｌｌ蛋白[1]和《Ｓｃｉｅｎｃｅ》(《科学》杂志 )发表Ｔｏｌｌ样受体 (Ｔｏｌｌ ｌｉｋｅｒｅｃｅｐｔｏｒ ,ＴＬＲ)及其生物学作用后[2 ] ,ＴＬＲ研究开始成为热点。研究揭示了ＴＬＲ是内毒素信号传导链中致病的关键环节。本文着重介绍ＴＬＲ4及其在内毒素信号传导中的作用 ,简略介绍与ＬＰＳ致病的其它相关分子 ,并对未来研究阻断内毒素致病的方法和途径提出设想。一、脂多糖 (ＬＰＳ)与血液中相关结合蛋白目前认为 ,革兰阴性细菌进行活体繁殖或崩解时可以释放内毒素或ＬＰＳ ,…     

急性前脑缺血致多器官功能障碍综合征动物模型的建立及内毒素受体CD14的表达

目的 探讨建立大鼠急性前脑缺血致多器官功能障碍综合征模型的可能性,研究脑缺血模型内毒素受体CD14 mRNA在肺、肝、肠和肾组织的表达变化规律与导致多器官功能障碍综合征(MODS)的发生机制。 方法 通过阻断双侧颈总动脉30 min建立急性前脑缺血模型,随机将54只大鼠分为正常对照组(6只)、假手术组(8只大鼠)及缺血后5个亚组(12、24、36、48及72 h组,每组8只大鼠),依据全身炎症反应综合征(SIRS)、MODS的诊断标准判断SIRS、MODS的发生率,采用原位杂交技术检测缺血后肺、肝、肠和肾脏器CD14的基因表达。 结果 (1)缺血后12 h肺、肝、肠和肾组织CD14 mRNA表达升高,24-36 h达高峰,48 h后下降,以肺脏变化最显著(P<0.001);(2)缺血后12、24、36、48及72 h时相点动物肺、肝、肠和肾组织均有不同程度的损害,但以24-48 h时脏器病理变化较显著,其中以肺脏改变为著,与CD14在各器官的基因表达峰值一致;(3)大鼠急性前脑缺血后SIRS发生率为100%,MODS发生率为53.1％;(4)正常对照组和假手术组中肺、肝、肠和肾组织CD14 mRNA均有不同程度的表达,其中两组肺脏CD14 mRNA的表达差异有显著意义(P<0.01)。结论 (1)大鼠急性前脑缺血可成功建立脑缺血致MODS的动物模型;(2)脑缺血致MODS动物模型的肺、肝、肠和肾各脏器CD14 mRNA的表达明     

肝窦内皮细胞与肝再生

肝窦内皮细胞(liver sinusoidal endothelial cells,LSECs)是肝脏非实质细胞中数目最多的细胞,约占肝非实质细胞总数的70％,在表型、功能上与普通毛细血管内皮细胞有较大差异.有关LSECs在肝再生过程中作用的研究相对较少.近年来,关于LSECs再生行为及其与肝内其他细胞成分相互作用的研究结果提示其在肝再生和肝衰竭中具有重要作用,但具体机制尚未完全明确.现就LSECs的再生行为及其在肝再生中的作用,以及以LSECs为靶点干预肝再生等进行综述.     

Expression of CD14 protein and its gene in liver sinusoidal endothelial cells during endotoxemia

AIM：To observe expression of CD14 protein and CD14 gene in rat liver sinsoidal endothelial cells(LSECs) during endotoxemia,and the role of CD14 protein in the activation of lipopolysaccharide(LPS)-induced LSECs.METHODS:Wistar rat endotoxemia model was established first by injection of a dose of LPS(5mg/kg,Escherichia coli O111:B4)via the tail vein,then sacrificed after 0h,3h,6h,12h,and 24h,respectively,LSECs were isolated from normal and LPS-injected rats by an in situ colagenase perfusion technique.The isolated LSECs were incubated with rabbit anti-rat CD14 polyclonal antibody,then stained with goat anti rabbit IgG conjgated fluorescein isothiocyanate(FITC) and flow cytometric analysis(FCM) was performed.The percentage and mean fluorescence intensity(MFI) of CD14-positive cells were taken as the indexes.LSECs were collected to measure the expression of CD14 mRNA by in situ hybridization analysis.The isolated LSECs from normal rats were incubated firstly with anti-CD14 antibody,then stimulated with different concentrations of LPS,and the supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)-a and lnterleukin(IL)-6 with ELISA.RESULTS:In rats with endotoxemia,LSECs displayed a strong MFI distinct from that of control rats .CD14 positive cells in rats with endotoxemia were 54.32%,65.83%,85.64%,and 45.65%,at 3h,6h,12h,and 24h respectively there was significant difference when compared to normal group of animals(4.45%)(P&lt;0.01).The expression of CD14 mRNA in isolated LSECs was stronger than that in control rats,In LPS group,the levels of TNF-αand IL-6were 54&#177;6ng.L^-1,85&#177;9ng.L^-1,206&#177;22ng.L^-1,350&#177;41ng.L^-1,366&#177;42ng.L^-1and103&#177;11ng.L^-1,187&#177;20ng.L^-1,244&#177;26ng.L^-1,290&#177;31ng.L^-1,and 299&#177;34ng.L^-1,respectively at different concentration points,In anti-CD14 group,the levels of TNF-αand IL-6 were 56&#177;5ng.L^-1,67&#177;8ng.L^-1,85&#177;10ng.L^-1,113&#177;12ng.L^-1, ,199&#177;22ng.L^-1and104&#177;12ng.L^-1,125&#177;12ng.L^-1,165&#177;19ng.L^-1,185&#177;21ng.L^-1,and222&#177;23ng.L^-1,respectively at different concentration poirts.THere was significant difference between the two groups(P&lt;0.01).CONCLUSION:LSECs can synthesize CD14 protein and espress CD14 gene during endotoxemia .CD14 protein plays an important role in the activation of LPS-induced LSECs.This finding has important implications for the understanding of the mechanisms by which LPS may injure liver sinusoidal endothelial cells during sepsis.     

肝窦内皮细胞调控肝脏微环境影响肝纤维化的研究进展

肝窦内皮细胞不仅是血液和肝细胞进行物质交换的重要中介细胞,也是慢性肝损伤因素导致肝纤维化和肝硬化的重要肝非实质细胞。它主要通过与肝星状细胞、肝细胞、Kupffer细胞的相互作用和介导肝脏硬度、肝脏血管再生从而调控肝脏微循环,参与肝纤维化的发展。阐明这些机制,有助于探索肝纤维化治疗的新靶点和方案。     

Alcohol-Induced Expression of the CD14 Endotoxin Receptor Protein in Rat Kupffer Cells

Gut-derived endotoxins (lipopolysaccharide, LPS) are believed to contribute to alcohol-induced liver disease (ALD) by stimulating Kupffer cells, the resident liver macrophages, to release proinflammatory cytokines. This activation is largely mediated by CD14, a high-affinity membrane-anchored receptor for LPS. We observed, by chemiluminescence-enhanced detection, an increase in immunoreactive CD14 protein in Kupffer cells isolated from rats treated with ethanol for 2 weeks. Immunocytofluorescence experiments confirmed that this increase was confined to the membranes of Kupffer cells from the alcohol-treated rats. The increase was regulated pretranslationally: a 3-fold elevation ( p < 0.01) in the hepatic level of CD14 mRNA was observed. The marked increase in CD14 expression suggests a new mechanism by which alcohol increases the LPS-mediated cytokine signaling by the liver macrophages, thus promoting the interaction between alcohol and endotoxins in the development of liver damage.     

肝窦内皮细胞在肝损伤中的功能研究进展

肝窦内皮细胞（liver sinusoidal endothelial cells, LSECs）位于肝血窦表面,是肝脏与血液接触的第一道防线,也是肝脏中含量最多的非实质细胞。在生理情况下,LSECs通过参与物质运输、代谢废物清除而诱导肝脏免疫耐受,从而维持肝脏稳态;在病理情况下,LSECs通过抗原递呈促进肝脏炎症反应。LSECs在维持肝再生和肝纤维化平衡中发挥了重要的调节作用。本文对LSECs功能、LSECs在肝损伤中的变化、调节LSECs功能相关的信号通路以及LSECs与肝内其他细胞的相互作用等四方面研究进展进行综述,从而进一步明确LSECs的功能及在肝损伤中的作用。     

Physiological role of sinusoidal endothelial cells and Kupffer cells and their implication in the pathogenesis of liver injury

We reviewed the morphological characteristics and physiological functions of hepatic sinusoidal endothelial cells (SECs) and Kupffer cells (KCs), both of which are major components of the hepatic sinusoid, and we showed the implication of these hepatic sinusoidal lining cells in the pathophysiology of the liver, based on our experimental studies. The most outstanding feature of SECs is that they are provided with numerous fenestrae, thereby allowing direct communication between the sinusoidal lumen and the space of Disse. Physiologically, SECs play a role in filtration function, endocytic function, and putative participation in the regulation of sinusoidal blood flow. As for KCs, they account for major portion of fixed macrophages in the entire body, and exhibit vigorous activity for phagocytosis, and produce many kinds of soluble mediators such as cytokines, prostanoids, oxygen radicals, and proteases. To determine whether these cells are implicated in pathophysiological processes in the liver we directed our attention to liver injury associated with sepsis and cold‐preservation injury of liver tissue. In a septic rat model, we found that when KCs that included hepatic macrophages were activated, they released excess tissue‐toxic mediators, probably leading to SEC damage. In the cold‐preserved liver, we demonstrated that KCs were functionally activated and that the morphology of SECs was destroyed. When the liver was reperfused with plasma and a leucocyte suspension, hypercoagulability and increased leucocyte adherence occurred. In both experimental models, we demonstrated that KC blockade ameliorated the liver injury, and this was associated with the morphological improvement of SECs. Thus, we showed the pathogenetic implication of KCs and SECs, due possibly to microcirculatory disturbance in the hepatic sinusoid, and further emphasized the involvement of activated KCs in SEC impairment.     

Structural and functional aspects of the liver and liver sinusoidal cells in relation to colon carcinoma metastasis

Nowadays, liver metastasis remains difficult to cure. When tumor cells escape and arrive in the liver sinusoids, they encounter the local defense mechanism specific to the liver. The sinusoidal cells have been widely described in physiologic conditions and in relation to metastasis during the past 30 years. This paper provides an "overview" of how these cells function in health and in diseases such as liver metastasis.     

肝窦内皮细胞与肝窦毛细血管化研究进展

肝窦内皮细胞具有开放的、没有横膈膜的窗孔,内皮下没有基底膜。这种结构有利于调控肝细胞与肝窦血液的物质交换。肝窦内皮细胞能分泌内皮素-1和一氧化氮,并通过窗孔的变化,对肝脏微循环进行调节,同时可分泌大量的形成基底膜的成分,在肝窦毛细血管化的过程中起主导作用。肝窦内皮细胞表型标志发生变化,Ⅷ因子相关抗原、CD44等表达增强,也是肝窦毛细血管化的重要标志。笔者对肝窦内皮细胞的结构及其在肝窦毛细血管化中的功能和表型变化进行综述。