miR-29b介导的TGF-β/Smad信号通路对大鼠肝纤维化进程的影响

目的:初步研究微小RNA-29b(mi R-29b)介导的TGF-β/Smad信号通路在肝星状细胞(HSC)活化中的作用及其对大鼠肝纤维化进程的影响。方法:构建肝纤维化大鼠模型并分离其HSC,同时通过体外获取并鉴定正常大鼠HSC。运用RT-qPCR和Western blot检测以上获取细胞中mi R-29b、TGF-β/Smad信号通路相关蛋白和肝纤维化标志蛋白的变化水平,并通过双萤光素酶报告基因检测系统鉴定mi R-29b对TGF-β1的直接靶向结合情况。结果:随着HSC活化加深,mi R-29b的表达量逐渐减少(P 0. 01),而HSC活性标志物I型胶原蛋白和α-平滑肌肌动蛋白的表达量逐渐增加(P 0. 01)。在TGF-β/Smad信号通路中,Smad2/3/4的表达显著增加,而Smad7的表达明显下降(P 0. 01)。双萤光素酶报告基因检测结果显示,mi R-29b可直接结合于TGF-β1 3’UTR的"UCUCUCCGU"序列,表明TGF-β1为mi R-29b的一个下游靶基因。结论:mi R-29b可参与抑制HSC的活化和迁移,进而抑制肝纤维化进程,而其生物学功能可能是通过直接靶向抑制TGF-β1进而调控TGF-β/Smad信号通路实现的。     

The hepatic arterial blood flow response to portal vein occlusion in the dog

The acute effect of portal vein occlusion on hepatic arterial blood flow was studied in a group of nine anaesthetised dogs. The influence of hepatic artery denervation and total liver denervation on the hepatic arterial flow response was determined subsequently. Blood flows in the hepatic artery and portal vein were measured with electromagnetic flowmeters, and hepatic tissue perfusion with⁸⁵Krypton clearance. A side-to-side mesocaval shunt was constructed to provide a drainage channel for the mesenteric venous blood during the periods of portal vein occlusion.Occlusion of the portal vein produced an immediate and significant increase in hepatic arterial flow which was sustained at approximately 80% above control for the 6 min period of observation. Total liver blood flow and hepatic tissue perfusion were both significantly reduced by about 40%. Denervation either of the hepatic artery alone or the entire liver produced no change in the response, and it is concluded that there is no neurogenic component either initiating or modifying the early changes in hepatic arterial flow.     

Plasma catecholamines in hepatic coma and liver cirrhosis: Role of octopamine

Summary Plasma levels of adrenaline, noradrenaline and octopamine were estimated by a radioenzymatic method in nine cirrhotic outpatients with encephalopathy and in ten patients with hepatic coma (coma grade III–IV). In the cirrhotic outpatients normal as well as elevated plasma levels of noradrenaline were found. Octopamine could not be detected in the plasma of these patients as well as of ten healthy volunteers. Elevated noradrenaline levels were present in all patients with hepatic coma. Plasma noradrenaline remained elevated or even further increased during the course of hepatic coma, whereas adrenaline was elevated less frequently. In eight of the ten patients with hepatic coma octopamine was again not detectable in plasma. Only in two patients high levels of octopamine up to 59.5 ng/ml could be found in addition to increased noradrenaline concentrations. The infusion of the branched chain amino acid L-valine had no influence on the plasma level of either noradrenaline or octopamine.The data indicate that the sympathetic nervous system is activated during the course of hepatic coma. An accumulation of octopamine is not a common finding in chronic liver disease and hepatic coma. Since in the two patients with elevated octopamine levels the rise in octopamine occured concomitantly with a rise in noradrenaline, a displacement of noradrenaline by the false neurotransmitter octopamine in the noradrenergic neuron of the peripheral sympathetic nervous system seems unlikely. The results indicate that the development of hypotension in the course of liver cirrhosis and hepatic coma cannot be related to a deficiency of noradrenaline.Deeply moved we have to inform the readers about the sudden death of our colleague and teacher Professor Dr. F. Wewalka     

结缔组织生长因子在血吸虫病肝纤维化鼠肝窦内皮细胞中的表达及意义

目的:动态观察结缔组织生长因子(CTGF)蛋白在血吸虫病肝纤维化鼠肝窦内皮细胞表达水平的时相变化,探讨其与肝窦内皮细胞下基底膜形成的关系。方法:采用腹部敷贴法建立血吸虫病肝纤维化模型,HE、Masson染色和透射电镜观察其病理变化;免疫组化技术检测CTGF、Ⅳ型胶原(ColⅣ)和层粘连蛋白(LN)在小鼠肝脏组织中的表达和分布;并应用彩色图像分析仪进行定量分析。结果:与正常对照组比较,模型组鼠肝窦内皮细胞表达CTGF蛋白阳性或强阳性,肝窦壁LN、ColⅣ表达水平增高,且随着肝纤维化的发展,CTGF和LN、ColⅣ表达逐渐增强,肝窦内皮下基底膜逐渐增厚。图像定量分析两组平均吸光度值、平均灰度值和阳性面积比具有统计学差异;CTGF蛋白与LN、ColⅣ水平呈正相关。结论:血吸虫病肝纤维化时小鼠肝窦内皮细胞通过CTGF蛋白表达上调,调控细胞外基质产生,导致ColⅣ、LN分泌增加,参与肝窦内皮下基底膜形成,从而引起肝内微循环障碍。     

硫化氢对体外大鼠肝星状细胞内Ca2+浓度变化及细胞增殖的影响

目的 研究硫化氢(H₂ S)对大鼠肝星状细胞-T6(HSC-T6) Ca²⁺浓度、细胞增殖的影响及其机制。 方法 活化HSC-T6用含10%小牛血清DMEM培养液制备为1×10⁵个肝星状细胞(HSC)悬液。钙离子荧光探针Fluo-3/AM负载细胞后，在不同刺激条件下，利用激光扫描共焦显微镜动态扫描HSC-T6细胞内Ca²⁺荧光强度（FI）变化，FI表示细胞内Ca²⁺浓度。四唑盐比色法，观察不同浓度H₂S供体——NaSH对HSC-T6细胞增殖的影响。 结果 低浓度H₂S（100μmol/L）明显降低HSC-T6细胞内Ca²⁺浓度（P<0.05)，而细胞增殖增加（增殖率为116%）；K_ATP通道阻断剂——格列本脲可阻断H2S的作用。高浓度H₂S（1mmol/L）刺激HSC-T6细胞内Ca₂₊浓度增加，但细胞增殖无明显变化(P＞0.05)。 结论 低浓度H₂S通过激活HSC-T6细胞K_ATP通道降低细胞内Ca²⁺浓度，可能通过调节细胞氧化应激促进细胞增殖；高浓度H2S刺激HSC-T6细胞内Ca₂₊浓度增加。提示H₂S在肝硬化门脉高压症的发生机制中具有双重作用。     

肝门阻断和再开放对兔胰腺功能的影响

目的：观察肝门阻断（HVO）及再开放（HVR）后胰腺内外分泌功能的改变。方法：选择健康日本大耳白兔25只，体重2.3-3.0 kg，分别于HVO前、HVO 10、20 min及HVR后（10、30、60、120 min）不同时点取血，并在HVO前、HVO 20 min、HVR后120 min 3个时点取胰腺组织，电镜下观察其超微结构的变化。结果：HVO时，血浆葡萄糖、胰岛素水平及一氧化氮代谢产物（NO₂^-/NO₃^-）含量均明显低于阻断前，胰高血糖素/胰岛素及丙二醛（MDA）浓度显著高于阻断前（P<0.05或P<0.01），并随阻断时间延长而加重；血浆淀粉酶、脂肪酶、游离脂肪酸水平则无明显差异（P>0.05）；胰岛细胞的线粒体肿胀、粗面内质网扩张，细胞核形态、结构基本正常，而胰腺腺泡细胞变化则不明显。HVR后上述差异逐渐不明显，至120min才接近阻断前水平。结论：急性肝门阻断和再开放对胰腺内分泌功能有较大的影响。     

精-甘-天冬-丝氨酸4肽对肝星状细胞增殖及凋亡的影响

目的: 探讨精-甘-天冬-丝氨酸(RGDS) 4肽对纤维连接蛋白(FN)刺激的肝星状细胞(HSCs)增殖、凋亡及caspase-3表达的影响。方法: 应用体外HSCs培养技术, 采用[³H]-胸腺嘧啶核苷([³H]-TdR)掺入法测定HSCs增殖;膜联蛋白(Annexin-V)/碘化丙啶(PI)双标记流式细胞术、TUNEL、扫描电镜及透射电镜等方法测定HSCs凋亡;采用甲苯胺兰染色方法测定细胞粘附率;应用流式细胞方法测定caspase-3蛋白表达。结果: ①25 mg·L^-1、50mg·L^-1、100mg·L^-1浓度RGDS 4肽剂量、时间依赖性抑制HSCs增殖, P＜0.01。②RGDS 4肽对HSCs凋亡的诱导作用亦呈剂量和时间依赖关系, P＜0.01。扫描电镜、透射电镜观察, RGDS 4肽组出现典型的凋亡征象。③RGDS 4肽作用于HSCs 2 h, 25 mg·L^-1、50mg·L^-1、100mg·L^-1组粘附抑制率分别是8.82%、29.41%、45.59%, 而RGES 4肽组的粘附抑制率仅为4.41%, P＜0.01。④RGDS 4肽处理组caspase-3表达明显高于FN、RGES 4肽组。结论: RGDS 4肽剂量和时间依赖性抑制HSCs增殖并诱导其凋亡。RGDS 4肽抑制增殖及诱导凋亡效应, 依赖于caspase-3, 也与其抗粘附作用有关。     

Expression of mRNAs related to connective tissue metabolism in rat hepatic stellate cells and myofibroblasts

Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.     

Hepatic angiomyolipoma and hepatic stellate cells share a similar gene expression profile

BACKGROUND AND AIMS: Angiomyolipomas (AMLs) of the liver are rare neoplasms composed of large epithelioid cells with intermixed fat and blood vessels. Hepatic AMLs have no clear normal-cell counterpart in the liver. However, AMLs and stellate cells both are positive for neural crest-derived markers including HMB-45 antigen. METHODS: To further explore the similarities between hepatic AMLs and stellate cells, gene expression of a hepatic AML was studied by cDNA microarray. Real-time polymerase chain reaction was used to confirm gene expression. Hepatic stellate cells can be quiescent, activated, or have a myofibroblastic phenotype depending on their state of activation. Expression of known markers of activated stellate cells was compared between the AML, activated primary mouse stellate cells, and stellate cell lines with activated and myofibroblastic phenotypes. Next, 5 novel genes from the AML were selected because they were not previously known to be markers of stellate cells and mRNA expression measured in the activated mouse stellate cells and in myofibroblastic stellate cell lines. Finally, expression levels of 10 novel genes were determined in 5 cirrhotic and 5 noncirrhotic human livers. RESULTS: Overexpression of known markers of activated stellate cells including transforming growth factor beta (TGF- beta ), smooth muscle actin, and collagen was found in the hepatic AML. Three of 5 novel markers that were identified in the AML, RRAD (Ras-related associated with diabetes), CTSK (cathepsin K), and NIBAN were also found to be overexpressed in activated stellate cells compared with quiescent or myofibroblastic stellate cells. In addition, 9 of 10 novel genes overexpressed in AML were also overexpressed in cirrhotic human livers versus noncirrhotic livers. CONCLUSIONS: Hepatic AMLs share a similar gene expression profile and may differentiate toward activated stellate cells.     

The role of the splanchnic circulation in the regulation of total intravascular volume during alpha adrenergic receptor stimulation

Previous studies have not defined the contribution of the splanchnic circulation to the total intravascular volume change associated with selective alpha adrenergic receptor stimulation. Since the splanchnic circulation is responsible for the total volume changes associated with other types of selective autonomic receptor stimulation, the present study was undertaken to examine the influence of alpha adrenergic receptor stimulation on splanchnic intravascular volume, the hemodynamic mechanism responsible for the splanchnic volume change, and the contribution of the splanchnic volume change to the change in total volume. In 35 anesthetized dogs, blood from the vena cavae was drained into an extracorporeal reservoir and returned to the right atrium at a constant rate so that changes in total intravascular volume could be measured as reciprocal changes in reservoir volume. Phenylephrine infusion (100 g/min) for 20 min in 28 dogs was associated with a decrease in total volume of 64±17 (SEM) ml (P<0.0001). The response was abolished by either alpha adrenergic blockade or evisceration but was not attenuated by beta adrenergic blockade, sinoaortic baroreceptor denervation, ganglionic blockade, or splenectomy. In 5 animals with separate splanchnic perfusion and drainage, total and splanchnic volumes decreased 59±8 ml (P<0.0001) and 317±20 ml (P<0.0001), respectively, while transhepatic vascular resistance increased 17±4 cm H₂O·min/l (P<0.0001). These responses were abolished after alpha adrenergic blockade. Thus, splanchnic volume decreases with alpha adrenergic receptor stimulation, despite an increase in hepatic resistance to splanchnic, venous outflow. The splanchnic volume decrement is entirely responsible for the total volume decrement.The study was supported by NHLBI Grant 1 R23-HL27185, Grant 11-203-812 from the American Heart Association of Greater Hartford, Inc., and the Duberg Cardiovascular Research Fund. Dr. Rutlen was the Duberg Scholar in Cardiovascular Disease when the study was performed.This work was presented in part at the 1982 Scientific Sessions of the American Heart Association (Circ. 66:II-311)