Kupffer细胞与肝脏缺血再灌注损害的研究进展

全身80%-90%的巨噬细胞为位于肝血窦内的Kupffer细胞(KC).KC激活后将诱发TNF-α、前列腺素、一氧化氮及氧自由基等各种炎症细胞因子的大量形成,除导致自身功能形态发生改变外,还直接影响邻近的肝细胞,血管内皮细胞以及位于血窦腔内的中性粒细胞等多种细胞.进而启动热缺血或冷缺血后肝脏的缺血再灌注损害(ischemia reperfusion injury,IRI).本文拟就KC与肝脏IRI的关系进行综述,以期为IRI的防治寻求到适宜的治疗靶点及途径.     

SK&F96365对大鼠肝缺血再灌注损伤后Kupffer细胞钙池操纵的钙通道电流的影响

目的:研究缺血再灌注损伤对大鼠肝Kupffer细胞钙池操纵的钙通道电流(ISOC)的影响,并筛选有效的钙通道阻滞剂进行拮抗.方法:建立SD大鼠肝缺血再灌注模型,利用低浓度胶原酶循环灌注、差速离心、选择性贴壁的方法急性分离肝Kupffer细胞,应用全细胞膜片钳记录技术,研究肝缺血再灌注损伤对大鼠肝Kupffer细胞ISOC的影响及钙通道阻滞剂SK&F96365的拮抗作用.结果:大鼠Kupffer细胞的ISOC为:假手术组-78.7±25.2 pA,缺血再灌注组-159.3±27.3pA,两组有显著统计学差异(n=15,P＜0.01).5-50μmol/L的SK&F96365对Kupffer细胞ISOC的抑制呈浓度依赖性增强,ISOC由-152.7±42.5 pA依次降至-81.4±24.2 pA,-56.1±26.4pA,-45.2±21.6 pA,-34.8±17.1 pA,-25.6±13.4 pA,SK&F96365对Kupffer细胞ISOC的抑制率逐渐增加,分别为43.9%±18.1%,59.2%±24.0%,66.3%±23.0%,73.8%±17.8%,80.9%±12.6%,其IC50值为6.53μmol/L.结论:缺血再灌注损伤可以促进S D大鼠Kupffer细胞上SOC的进一步开放,导致ISOC的增大,Kupffer细胞活化,进一步加重缺血再灌注损伤.SK&F96365对Kupffer细胞ISOC的抑制呈浓度依赖性增强,对肝细胞损伤具有保护作用.     

肝移植缺血再灌注损伤与细胞粘附分子

自本世纪80年代以来,伴随着手术技巧的提高、新型免疫抑制剂和UW保存液的相继问世,临床肝移植取得了长足的进步。然而,缺血再灌注损伤依然是困扰着肝移植的研究难点,它是引发术后原发性移植物无功能(primary graftnonfunction)的重要原因。而随着近年来对细胞粘附分子研究的不断深入,表明细胞粘附分子恰恰参与并介导了缺血再灌注损伤过程中的各个步骤。本文综述近年来在此领域的研究进展。 一、细胞粘附分子的种类、结构与功能 细胞粘附分子分布极其广泛,涉及生命活动的许多现象,包括细胞分裂、分化以及细胞凋亡的调控等等。根据结构与功能,粘附分子可初步分为5类,即整合素家族、免疫球蛋白超家族、选择素家族、钙粘附蛋白家族和其它粘附分子。前三者是参与肝移植过程中缺血再灌注等炎症反应和免疫应答的重要家族。     

肝缺血再灌注损伤与细胞凋亡关系的临床研究

目的 通过检测肝缺血再灌注前后肝组织的细胞凋亡情况,探讨临床手术中肝缺血再灌注与细胞凋亡之间的关系,为更好地预防或减轻临床肝脏手术中造成的缺血再灌注损伤(HIRI)提供理论基础.方法 以细胞凋亡测定法(TUNEL 法)测定肝缺血再灌注前后肝细胞的凋亡情况.结果 肝门阻断前与肝门开放时和关腹前肝细胞的凋亡指数各组间的差异有统计学意义(P＜0.01),肝门阻断前肝细胞的凋亡指数高于肝门开放时和关腹前肝细胞的凋亡指数(P＜0.01);肝门开放时肝细胞的凋亡指数高于关腹前肝细胞的凋亡指数(P＜0.01).结论 研究表明肝脏手术中,在肝细胞短时间(15 min左右)缺血后的再灌注损伤中,肝细胞凋亡和缺血再灌注损伤呈负相关,它并不是术后早期肝细胞损伤的一种主要方式.     

缺血预处理对移植肝缺血再灌注损伤中细胞凋亡的影响

目的 探讨细胞凋亡在移植肝缺血再灌注损伤中的作用及缺血预处理对其影响。方法 通过对移植肝进行 因预处理,用全自动生化分析仪检测肝功能、比色法测定移植肝组织的ＭＤＡ、用流式细胞仪结合原位标记技术检测细胞调７亡。结果 移植肝再灌注后血中ＡＳＴ、ＡＬＴ、ＬＤＨ和肝组织中ＭＤＡ均明显升亮,肝细胞调亡明显增加,经缺血预处理后,血中ＡＳＴ,ＡＬＴ,ＬＤＨ和肝组织中ＭＤＡ均降低,肝细胞调亡亦明显减少,结论 缺血     

黄芪对大鼠肝缺血再灌注损伤的影响

目的 探讨黄芪对大鼠肝缺血再灌注损伤的影响,以及与缺血预处理(IP)作用效果的比较.方法 清洁级健康雄性SD大鼠96只,随机分为假手术组、缺血再灌注组(IR组)、缺血预处理组(IP组)、黄芪组,每组又按再灌注时间(1、3、6、24 h)分为4个时相,每组各时相为6只.制作70%肝缺血再灌注模型,测血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、乳酸脱氢酶(LDH)水平;测肝组织髓过氧化物酶(MPO)含量;用免疫组化法检测IL-10、TNF-α表达;以及光镜及电镜观察大鼠肝形态学变化.结果 IR、IP、黄芪组ALT、AST、LDH和MPO含量均明显高于假手术组(P<0.01).与IR组相比,IP、黄芪组各个时相点的值均下降(P<0.05).与IP组比较,黄芪组各个时相点的值均降低(P<0.05).IR、IP、黄芪组肝组织TNF-α、IL-10的阳性细胞表达率均比假手术组高(P<0.05);与IR组比较,IP、黄芪组TNF-α的表达减少,而IL-10的表达增强(P<0.05);与IP组比较,黄芪组TNF-α的表达减少,IL-10表达增强(P<0.05).在光镜及电镜下观察肝形态学变化,可见IR组损伤甚为明显,IP及黄芪组损伤程度较IR组轻,黄芪组更轻,而假手术组肝形态正常.结论 IP和黄芪都可减轻缺血再灌注对肝脏的损伤,且后者较前者效果好.     

不同缺血预处理方案对小鼠肝缺血再灌注损伤的影响

肝脏缺血再灌注（ischemia-reperfusion，I／R）损伤是临床常见的问题，是肝脏手术患者术后恢复慢甚至手术失败的重要原因之一。缺血预处理（ischemic preconditioning，IPC）可减轻肝脏I／R损伤。我们通过比较不同IPC方案对C57BL／6小鼠肝脏I／R损伤的影响，为寻找合理有效的IPC方案提供依据。     

脂肪肝缺血再灌注损伤的研究

随着人民生活水平的提高，脂肪肝的发病率越来越高。在外科手术及肝移植中，脂肪肝的检出率也很高。手术中常因阻断血流而造成缺血再灌注损伤，脂肪肝对缺血再灌注损伤比正常肝脏更为敏感，其机制可能是多方面的，此文主要对脂肪肝缺血再灌注损伤机理作一综述。     

库普弗细胞与肝纤维化的关系

活化的库普弗细胞发挥吞噬、抗原提呈、释放细胞因子、免疫调节等功能，成为机体防御的重要屏障，同时也参与了肝脏的炎症损伤、纤维化的形成和降解等病理过程。     

Protection against hepatic ischemia/reperfusion injury via downregulation of toil-like receptor 2 expression by inhibition of Kupffer cell function

AIM: To elucidate the mechanism of liver protection by inhibition of Kupffer cells (KCs) function.METHODS: All the animals were randomly divided into three groups. Blockade group (gadolinium chloride solution (GdCl3) injection plus ischemia/reperfusion (I/R) injury):GdCl3 solution was injected once every 24 h for 2 d via the tail vein before I/R injury. Non-blockade group (saline solution injection plus I/R injury): saline instead of GdCl3 as a control was injected as in the blockade group. Sham group: saline was injected without I/R injury. Liver samples were collected 4 h after blood inflow restoration. The blockade of the function of KCs was verified by immunostaining with an anti-CD68 mAb. Toll-like receptor 2 (TLR2) was immunostained with a goat antimouse polyclonal anti-TLR2 antibody. Membrane proteins were extracted from the liver samples and TLR2 protein was analyzed by Western blot. Portal vein serum and plasma were taken respectively at the same time point for further detection of the levels of tumor necrosis factor-α (TNF-α) and alanine aminotransferase (ALT), an indicator of liver function.RESULTS: Compared to non-blockade group, CD68+ cells significantly reduced in blockade group (OPTDI, optical density integral): 32.97±10.55 vs 185.65±21.88,P＜0.01)and the liver function impairment was relieved partially (level of ALT: 435.89±178.37 U/L vs890.21±272.91 U/L,P＜0.01). The expression of TLR2 protein in blockade group significantly decreased compared to that in non-blockade group (method of immunohistochemistry, OPDTI: 75.74±17.44vs 170.58±-25.14, P＜0.01; method of Western blot,A value: 125.89±15.49 vs433.91±35.53, P＜0.01). The latter correlated with the variation of CD68 staining (r = 0.745,P＜0.05). Also the level of portal vein TNF-α decreased in blockade group compared to that in non-blockade group (84.45±14.73 ng/L vs112.32±17.56 ng/L, P＜0.05), but was still higher than that in sham group (84.45±14.73 ng/Lvs 6.07±5.33 ng/L, P＜0.01).CONCLUSION: Inhibition of the function of KCs may protect liver against I/R injury via downregulation of the expression of TLR2.     

Influence of Kupffer cells and platelets on ischemia-reperfusion injury in mild steatotic liver

AIM: To investigate the effect of mild steatotic liver on ischemia-reperfusion injury by focusing on Kupffer cells (KCs) and platelets. METHODS: Wistar rats were divided into a normal liver group (N group) and a mild steatotic liver group (S group) induced by feeding a choline-deficient diet for 2 wk. Both groups were subjected to 20 min of warm ischemia followed by 120 min of reperfusion. The number of labeled KCs and platelets in sinusoids and the blood perfusion in sinusoids were observed by intravital microscopy (IVM), which was performed at 30, 60 and 120 min after reperfusion. To evaluate serum alanine aminotransferase as a marker of liver deterioration, blood samples were taken at the same time as IVM.RESULTS: In the S group, the number of platelets adhering to KCs decreased significantly compared with the N group (120 after reperfusion; 2.9±1.1 cells/acinus vs 4.8±1.2 cells/acinus, P<0.01). The number of KCs in sinusoids was significantly less in the S group than in the N group throughout the observation periods (before ischemia, 19.6±3.3 cells/acinus vs 28.2±4.1 cells/acinus, P<0.01 and 120 min after reperfusion, 29.0±4.3 cells/acinus vs 40.2±3.3 cells/acinus, P<0.01). The blood perfusion of sinusoids 120 min after reperfusion was maintained in the S group more than in the N group. Furthermore, elevation of serum alanine aminotransferase was lower in the S group than in the N group 120 min after reperfusion (99.7±19.8 IU/L vs 166.3±61.1 IU/L, P=0.041), and histological impairment of hepatocyte structure was prevented in the S group. CONCLUSION: Ischemia-reperfusion injury in mild steatotic liver was attenuated compared with normal liver due to the decreased number of KCs and the reduction of the KC-platelet interaction.     

Protection against hepatic ischemia/reperfusion injury via downregulation of toll-like receptor 2 expression by inhibition of Kupffer cell function

AIM: To elucidate the mechanism of liver protection by inhibition of Kupffer cells (KCs) function. METHODS: All the animals were randomly divided into three groups. Blockade group (gadolinium chloride solution (GdCl3) injection plus ischemia/reperfusion (I/R) injury): GdCl3 solution was injected once every 24 h for 2 d via the tail vein before I/R injury. Non-blockade group (saline solution injection plus I/R injury): saline instead of GdCl3 as a control was injected as in the blockade group. Sham group: saline was injected without I/R injury. Liver samples were collected 4 h after blood inflow restoration. The blockade of the function of KCs was verified by immunostaining with an anti-CD68 mAb. Toll-like receptor 2 (TLR2) was immunostained with a goat antimouse polyclonal anti-TLR2 antibody. Membrane proteins were extracted from the liver samples and TLR2 protein was analyzed by Western blot. Portal vein serum and plasma were taken respectively at the same time point for further detection of the levels of tumor necrosis factor-α (TNF-α) and alanine aminotransferase (ALT), an indicator of liver function. RESULTS: Compared to non-blockade group, CD68+ cells significantly reduced in blockade group (OPTDI, optical density integral): 32.97±10.55 vs 185.65±21.88, P<0.01) and the liver function impairment was relieved partially (level of ALT: 435.89±178.37 U/L vs 890.21±272.91 U/L, P<0.01).The expression of TLR2 protein in blockade group significantly decreased compared to that in non-blockade group (method of immunohistochemistry, OPDTI: 75.74±17.44 vs 170.58±25.14, P<0.01; method of Western blot, A value: 125.89±15.49 vs 433.91±35.53, P<0.01). The latter correlated with the variation of CD68 staining (r= 0.745, P<0.05). Also the level of portal vein TNF-a decreased in blockade group compared to that in non-blockade group (84.45±14.73 ng/L vs 112.32±17.56 ng/L, P<0.05), but was still higher than that in sham group (84.45±14.73 ng/L vs 6.07±5.33 ng/L, P<0.01). CONCLUSION: Inhibition of the function of KCs may protect liver against I/R injury via downregulation of the expression of TLR2.     

GdCl_3 abates hepatic ischemia-reperfusion injury by inhibiting apoptosis in rats

BACKGROUND:Gadolinium chloride(GdCl3)is a specific inhibitor of Kupffer cells(KCs),which are important promoters of various liver injuries.It is therefore of interest to explore the role of KCs in liver ischemia-reperfusion injury and their relations with apoptosis caused by ischemia-reperfusion injury. METHODS:One hundred male Wistar rats(190-210 g, 6-7 weeks old)were divided into two groups at random, GdCl3 group and control group.Samples were collected at 0.5,1,6,12,and 24 hours from each group after rep...     

库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响

目的 研究库普弗细胞功能状态对肠缺血再灌注小鼠肝细胞凋亡和血清肿瘤坏死因子的影响。方法 封闭和不封闭BALB／c小鼠库普弗细胞(从尾静脉注射GdCl3或生理盐水27ml／kg体重)48h后，夹闭肠系膜上动脉1h后松夹，复制肠缺血再灌注模型。运用流式细胞术和酶联免疫法，分别检测缺血前、缺血60min、再灌注30min、60min肝细胞凋亡情况和血清肿瘤坏死因子的变化。结果 结果表明，肠缺血60min、再灌注30min、60min时，肝细胞凋亡数增多，血清肿瘤坏死因子水平逐渐升高；封闭库普弗细胞后，肝细胞凋亡数增多更显著，血清肿瘤坏死因子水平在同时间点上无显著差异。结论 库普弗细胞功能状态的变化对肠缺血再灌注时肝损伤有重要影响。     

Expression of macrophage inflammatory protein-1α in Kupffer cells following liver ischemia or reperfusion injury in rats

AIM: To explore the expression of macrophage inflammatory protein-1α (MIP-1α) in Kupffer cells (KCs)following liver ischemia/reperfusion injury IRI in rats.METHODS: Forty male SD rats were divided randomly into five groups. A model of partial warm ischemia/reperfusion injury in the rat liver was established. KCs were isolated and incubated one hour, six hours, 12 h,and 24 h after the reperfusion. Tumor necrosis factor alpha (TNF-α) and interleukin-1beta (IL-1β) in the supernatants were measured by ELISA. MIP-1α in KCs was detected by immunocytochemical and RT-PCR.RESULTS: No or few MIP-1α protein and mRNA were expressed in the KCs of the control group. Its expression in the IRI group had a significant increase after the reperfusion (P ＜ 0.05), which was contrary to the control group.CONCLUSION: The active behavior of the MIP-1α gene in KCs following liver ischemia/reperfusion injury is assumed to be one of the major causes for the hepatic ischemia/reperfusion injury.     

库普弗细胞Toll样受体2在小鼠肝脏缺血再灌注损伤中的表达及意义

目的探索库普弗细胞Toll样受体2(TLR2)在肝脏缺血再灌注损伤中的表达变化,分析库普弗细胞TLR2信号通路参与肝脏缺血再灌注损伤的机制。方法动物分假手术对照(SH)组、缺血再灌注(I/R)组及氯化钆处理(Gd)组,复制小鼠肝脏部分缺血1 h再灌注4 h损伤模型,结束再灌注后,取缺血叶肝脏组织及其库普弗细胞,提取总RNA及膜蛋白质分析TLR 2 mRNA及蛋白的表达,同时提取缺血肝叶组织核蛋白分析核因子(NF—κB),并检测门静脉血中肿瘤坏死因子(TNF α)、丙氨酸氨基转移酶(ALT) 及内毒素水平。结果再灌注4 h,(1)I/R组缺血肝叶TLR2 mRNA及蛋白表达水平较SH组高,△Ct值分别为1.06±0.91和5.08±1.32,t=7.80,P<0.01;A值分别为433.91±25.53和102.86±13.58,t=28.04, P<0.01。Gd组缺血肝叶TLR2 mRNA及蛋白表达水平较I/R组下降,△Ct值分别为4.22±0.84和1.06±0.91,t=7.56,P<0.01;A值分别为125.89±15.49和433.91±25.53,t=25.27,P<0.01。(2)I/R组缺血肝叶库普弗细胞中TLR2 mRNA及蛋白表达水平较SH组高,△Ct值分别为0.52±0.23和2.61±0.1 8, t=17.47,P<0.01;A值分别为379.70±34.16和114.98±21.90,t=15.98,P<0.01。Gd组缺血肝叶库普弗细胞中TLR2 mRNA及蛋白表达水平则较I/R组下降,△Ct值分别为1.90±0.14和0.52±0.23,t= 12.     

Isolation of Kupffer cells and their suppressive effects on T lymphocyte growth in rat orthotopic liver transplantation

AIM: To develop a practical method for isolation, purifi cation and culture of hepatic Kupffer cells (KCs) and to observe their suppressive effects on the proliferation of alloreactive T cells. METHODS: Perfusion in situ in vivo combined with density gradient centrifugation was applied in isolation, purifi cation and culture of hepatic KC. The suppression by KCs on the T cell proliferation in mixed lymphocyte reaction (MLR) was observed. RESULTS: This method resulted in a satisfactorily high yield of (1.1 ± 0.2) × 107 KCs per liver, (93.5/ ± 1.8/) viable cells, over 90/ purity and positive for ED-2. After the first 24 h in culture, a great number of KCs which exhibited typical characteristics were observed. Using 3H-TdR incorporation assay, non-irradiated KCs significantly suppressed allo-MLR. The KCs recovered from accepted liver allografts in groups D and E were more effective in suppressing allo-MLR. CONCLUSION: A standardized procedure for isolation of highly purified rat KCs is proposed and KCs have suppressive effects on the proliferation of alloreactive T cells, especially those derived from accepted liver allografts.     

干细胞移植在肝脏损伤修复中作用的研究进展

急慢性肝损伤痛死率高,其治疗始终是较为棘手的问题.干细胞及其"横向分化"特性的发现为各种疾病的治疗提供了一种新的手段.近年来干细胞移植作为一种治疗肝脏损伤的有效方法正得到日益广泛应用.本文旨在对干细胞移植治疗急慢性肝损伤的新进展作一综述.