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)     

Kinetics of hepatic transport of 4-methylumbelliferone in rats. Analysis by multiple indicator dilution method

Hepatic elimination of 4-methylumbelliferone (4MU), which has been used as a model compound for conjugative metabolism, was studied by means of a multiple indicator dilution (MID) method in the isolated perfused rat liver. Using this method, three intrinsic hepatic clearances, CL _int,inf , CL _{int,eff, and} CL _{int,seq, which represent the influx, efflux, and sequestration processes, respectively, were obtained. When the dose was increased from a low dose (50 g/rat liver) to a high dose (3000 g/rat liver), the hepatic availability of 4MU increased from 0.11 to 0.73. With increasing dose, the} CL _{int,eff value increased approximately two times, while the} CL _{int,seq value decreased to approximately one-third. The remarkable dose dependence of hepatic availability was due to nonlinearity in both} CL _{int,eff and} CL _{int,seq values. However, the} CL_int,inf value was almost independent of dose. The dose-dependent change in CL_int,seq might be explained by the saturation of conjugative metabolism of 4-MU, while the increase in the CL _{int,eff value with increasing dose might be partly explained by the nonlinear tissue binding of 4-MU, since the tissue unbound fraction determined by an ultrafiltration method using liver homogenate increased approximately 1.5 times at higher concentration of 4-MU compared to that at lower concentrations. In addition, based on a comparison of the individual intrinsic clearances, i.e.}, CL _int,inf , CL _{int,eff, and} CL _{int,seq, the major determining process of the apparent hepatic intrinsic clearance of 4MU is thought to be the sequestration process at the high dose. However, at the low dose, the membrane transport process (influx and efflux processes) as well as the sequestration process also determine the apparent hepatic intrinsic clearance}.     

Artificial Liver: Present and Future

Early in 1956, the first model of a biological artificial liver, using a live dog's liver incorporated in a cross-hemodialyzer, was placed in an experimental animal with portocaval encephalopathy. This "biological artificial liver," a hybrid artificial liver in the present terminology, was the first in the world. In October 1958, the first human patient, a young male patient in hepatic coma due to liver cirrhosis, was placed on the laboratory-made biological artificial liver composed of four parabiotic cross-hemodialyzers connected with four live dogs' livers to which the "hepatic reactors" for ammonium adsorption and acid-base balance were additionally equipped. This first case was very successful, resulting in the patient's recovery from coma. This article introduces the past history of the artificial liver, research of which has mainly been conducted in Japan since the early 1950s by the author, M. Mito, and Y. Nosé. Until recently, little progress has been made in this field through the application of blood purification principles such as hemoadsorption, plasmapheresis, and other modifications and combinations. Accumulation of clinical experiences with such conventional methods has stimulated the third generation of the artificial liver to a return to a hybrid organ applying modern science and technology. A concept of hybrid organs in comparison with organ transplants is introduced. The Japanese national project of developing a new artificial liver system, as conducted by the author as the chairman and his associates, is introduced.     

恩替卡韦联合水飞蓟宾对慢性乙型病毒性肝炎患者肝功能及肝纤维化指标的影响

目的探讨恩替卡韦联合水飞蓟宾对慢性乙型病毒性肝炎患者肝功能及肝纤维化的影响。方法 200例慢性乙型病毒性肝炎患者依据治疗方式的不同分为两组各100例,观察组使用恩替卡韦联合水飞蓟宾治疗,对照组使用恩替卡韦治疗,比较两组的肝功能和肝纤维化指标。结果治疗6个月后,两组的ALT、 TBil、 AST、 HA、 IVC、 LN水平均显著低于治疗前,且观察组显著低于对照组(P <0.05)。结论恩替卡韦联合水飞蓟宾治疗慢性乙型病毒性肝炎患者能够有效缓解肝纤维化损伤,改善肝功能。     

肝包虫病合并HBV感染患者Th17、CD4+细胞表面PD-1/PD-L1的表达水平及意义

目的探讨肝包虫合并乙型肝炎病毒(HBV)感染患者辅助性T细胞17(Th17)、CD4⁺细胞表面程序性死亡分子-1/程序性死亡分子1配体(PD-1/PD-L1)的表达水平及其临床意义。方法选取2014年9月一2019年9月新疆医科大学第一附属医院收治的肝包虫病患者作为研究对象﹐其中100例肝包虫病合并HBV感染患者为合并HBV感染组,肝包虫病无HBV感染患者88例为非HBV感染组﹐采用流式细胞仪检测外周血Th17,CD4⁺细胞表面PD-1,PD-L1表达水平。结果合并HBV感染组患者乙型肝炎家族史,B~C Child分级占比均高于非HBV感染组(P<0.05);肝功能指标[谷丙转氨酶(ALT)、谷草转氨酶(AST),γ-谷氨酰转肽酶(γ-GT)]水平以及Th17 PD-1、Th17 PD-L1,CD,PD-1,CD4⁺PD-L1表达水平均高于非HBV感染组(P<0.05);Child分级A级肝包虫病患者的Th17 PD-1,Th17 PD-L1,CD4⁺PD-1,CD4⁺PD-L1表达水平均低于Child分级B~C级患者(P<0.05)。结论HBV感染会影响肝包虫病患者的肝功能及外周血Th17,CD4⁺细胞表面PD-1,PD-L1表达水平,肝功能严重程度可能与外周血Th17,CD4⁺细胞PD-1,PD-L1表达存在关系。