一种简易分离大鼠肝脏库普弗细胞的方法

目的建立一种经济、简单、稳定的大鼠肝脏库普弗细胞(KCs)分离方法。方法采用前灌流液(D-Hanks)和胰酶消化液两步法原位灌注大鼠肝脏,低速离心去除肝细胞,Percoll分离液不连续密度梯度离心法和选择性贴壁法纯化KCs,将培养7 d的KCs采用吞噬latex-beads实验及ED1免疫细胞化学法鉴定并检测其纯度。结果每只大鼠肝脏KCs的得率为(1.2～1.8)×107个,细胞存活率达95%,KCs纯度近100%。结论此新方法分离获得的KCs细胞得率和纯度较高且较稳定,同时较传统方法胶原酶的使用相比,此方法更经济实用。     

离心淘洗技术分离纯化大鼠肝库普弗细胞

目的通过制备肝非实质细胞悬液并离心淘洗的方法,分离纯化大鼠肝库普弗细胞.方法采用原位肝脏酶灌注、肝非实质细胞悬液的准备、离心淘洗和原代培养等方法进行分离.结果在经过胶原酶和链酶蛋白酶消化、Nycodenz分离和离心淘洗后,获得大鼠肝库普弗细胞产量约为(28±6)×106/鼠肝,细胞纯度为95%,细胞活力大于90%.结论应用离心淘洗技术能有效地分离纯化大鼠肝库普弗细胞,为体外进一步研究提供了高纯度和活力的库普弗细胞群.     

四氯化碳诱发大鼠肝纤维化自发逆转中库普弗细胞与星状细胞的分布

目的:探讨四氯化碳(CCl_4)诱发大鼠肝纤维化自发逆转过程中肝库普弗细胞与星状细胞的分布和意义.方法:500 mL/L CCl_4腹腔注射8 wk诱发大鼠肝纤维化模型,实验第8,12周末检测血清生化指标,观察肝组织的病理变化,采用免疫组化SP法观察单核巨噬细胞抗原(ED1),α-平滑肌动蛋白(α-SMA)阳性表达的肝库普弗细胞(kupffer cell,KC)和肝星状细胞(hepatic stellate cells,HSC)的分布结果:第8周末模型组大鼠与对照组比较,血清ALT和AST活性(568.18±630.46 nkat/L vs 472.26±167.37 nkat/L.P<0.05:5845.84±1353.27 nkat/L vs 1698.51±663.30 nkat/L.P<0.01),肝/体质比(3.90±0.85 vs 2.56±0.24,P<0.001)及胶原纤维面密度(5.87±1.13 vs 0.52±0.30,P<0.001)明显增高;大量ED1和α-SMA阳性的KC和HSC主要分布在汇管区增生的纤维组织及纤维间隔内.第12周末模型组与第8周比较大鼠血清ALT活性(1020.70±306.73 nkat/L vs 376.74±304.06 nkat/L.P<0.05)仍较高外,胶原纤维面密度减少,汇管区增生的纤维组织及纤维间隔内ED1阳性的KC减少,α-SMA阳性的HSC消失.结论:腹腔注射CCl_48 wk后大鼠肝功能明显损伤,形成肝硬化,KCs激活和HSCs活化相关,停止注射CCl_4 4 wk后大鼠肝纤维化发生自发逆转.     

内毒素诱导肝损伤库普弗细胞活化的分子机制

肠源性内毒素是肝损伤发病机制中的一个重要因子.然而,内毒素发挥效应的确切分子机制至今尚未完全阐明.近年的研究显示炎性细胞介质在肝细胞损害中具有重要作用.在肝内,库普弗细胞是炎性细胞介质的重要来源.在内毒素的作用下,库普弗细胞的内毒素信号转导系统表达增加,进而引起库普弗细胞活化、合成和释放一系列的炎症介质,引起肝脏的损害.     

肝细胞性肝癌中库普弗细胞的分布及意义

往认为,库普弗细胞不存在于肝细胞性肝癌(HCC)组织中;现在认为,库 抗－CD68(鼠抗－CD68,D8kO公司)工普弗细胞也存在于高分化肝癌及早期肝 作浓度为二:50,用高压蒸气祛(121℃、癌[l]。本实验应用组织芯片技术(专利号:LZ大气压10＿)进行抗原修复,DABZL99241752、ZL99241767．8)研究了HCC 显色。     

内毒素诱导肝损伤库普弗细胞活化的分子机制

肠源性内毒素是肝损伤发病机制中的一个重要因子。然而,内毒素发挥效应的确切分子机制至今尚未完全阐明。近年的研究显示炎性细胞介质在肝细胞损害中具有重要作用。在肝内,库普弗细胞是炎性细胞介质的重要来源。在内毒素的作用下,库普弗细胞的内毒素信号转导系统表达增加,进而引起库普弗细胞活化、合成和释放一系列的炎症介质,引起肝脏的损害。     

一种改进的大鼠肝星状细胞分离方法

目的 改进大鼠肝星状细胞的分离方法,提高肝星状细胞分离的纯度和活力。方法 SD大鼠在静脉注射1 ml含二氯亚甲基二磷酸盐的脂质体3 d后,用含100 U/ml肝素的D-Hanks液灌注肝脏10～15 min,改用含0.05％胶原酶溶液的D-Hanks液灌注25～30 min,将肝脏细胞悬液置于0.025％胶原酶、0.005％DNase Ⅰ的溶液中振荡消化30 min,细胞悬液过200目筛网,50×g离心2 min去除肝细胞,300×g离心10 min得到肝脏非实质细胞沉淀,将细胞悬浮于Nycodenz使终浓度为11.5％,1400×g离心17 min,吸取Nycodenz上层的细胞即为肝星状细胞、台盼蓝拒染实验鉴定细胞活力,自发荧光、Desmin免疫细胞化学染色鉴定细胞纯度,内源性过氧化物酶染色检测库普弗细胞。结果 肝星状细胞得率每只大鼠约3×10~7个,细胞活力在95％以上,初分离的肝星状细胞在328 nm激发光下自发蓝绿色荧光,Desmin免疫细胞化学染色鉴定纯度达到90％,未检测到内源性过氧化物酶阳性细胞。结论 建立了一种无库普弗细胞混杂的大鼠肝星状细胞分离方法,可以用于原代肝易状细胞的生物学行为的研究。     

RNA干扰供体大鼠库普弗细胞B7分子表达对受体大鼠淋巴细胞激活的影响

目的:观察RNA干扰供体Lewis大鼠库普弗细胞(KC)B7分子表达对受体BN大鼠淋巴细胞增殖和生成IL-2的影响.方法:分离培养供体Lewis大鼠KC, 设计大鼠B7分子的干扰片段, 构建并鉴定含B7干扰片段的RNA干扰载体Psilencer 3.1H1-Neo-B7, 将RNA干扰载体转染供体大鼠的KC, 转染后采用RT-PCR方法检测KC上B7分子表达的变化. 将转染后的KC分为3组, 对照组(A); 空载体组(B); RNA干扰B7表达组(C). 分离培养受体BN大鼠的淋巴细胞, 将以上各组细胞分别与BN大鼠的淋巴细胞进行共培养, 采用MTT法检测各组淋巴细胞的增殖情况. 采用ELISA方法检测各组培养上清中IL-2的含量.结果: 分离培养的供体Lewis大鼠KC得率为5×107, 活率大于98%. 构建的RNA干扰载体经酶切和测序鉴定正确. RNA干扰KC后其B7的表达降低了22%(P<0.01). 将干扰B7表达的KC与BN大鼠的淋巴细胞进行共培养, 与对照组相比, 受体BN大鼠的淋巴细胞增殖降低了49%(P<0.01), 细胞培养上清中IL-2的分泌量下降了67%(P<0.01).结论:RNA干扰供体Lewis大鼠KC B7分子的表达可明显抑制受体BN大鼠淋巴细胞的增殖和IL-2的产生.     

鼠肝Kupffer细胞的分离、培养和鉴定

目的:Kupffer细胞是固定于肝脏的吞噬细胞,Kupffer细胞的分离、培养对肝脏疾病发生机制中的有关细胞和分子生物学的研究具有重要意义。方法:用链霉蛋白酶和胶原酶原位灌流,Nycodenz密度梯度离心分离大鼠Ku-pffer细胞,再经贴壁培养,并应用免疫组织化学、吞噬功能试验、电镜等方法进行鉴定。结果:本法能成功地获得高纯度的Kapffer细胞,Kapffer细胞得率为3～5×10~6/肝,贴壁后呈典型的星形及多角形,免疫组化染色示溶菌酶阳性、胞浆内见吞噬的印度墨汁及乳胶珠颗粒,电镜观察细胞表面有发达的伪足、微绒毛,胞浆内含大量溶酶体及吞噬的乳胶珠颗粒。结论:本实验所用的Kupffer细胞分离培养方法简单易行、可靠、细胞纯度高,可用于进一步研究Ku-pffer细胞的生物学功能。     

一种简便实用的大鼠Kupffer细胞的分离与鉴定方法

目的：研究一种简便实用的大鼠Kupffer细胞（KCs）的分离与鉴定方法.方法：原位两步灌流法对大鼠肝脏进行冲洗消化;利用Percoll液进行不连续密度梯度离心分离KCs;;选择性贴壁纯化KCs;台盼蓝染色法鉴定细胞存活率;吞噬实验鉴定细胞功能;ED1单克隆抗体免疫荧光细胞化学鉴定KCs;显微镜下观察KCs形态变化.结果：获取的KCs数量为（2.41±0.32）×107/只,其中活细胞数量占（92.3±2.12）％;吞噬实验显示（95.2±2.58）％的细胞内存在碳素颗粒;免疫荧光化学检测证明KCs纯度为（96.3±1.46）％;在显微镜下观察KCs形态,36h细胞形态变得不规则,3d后呈星形或多角形,体外培养可以存活7～10d.结论：此种KCs分离方法操作相对简便,获取的细胞数量、活性功能、纯度等方面均能达到进一步的实验要求,值得推广.     

Long-term alcohol exposure changes sensitivity of rat Kupffer cells to lipopolysaccharide

BACKGROUND: Chronic ethanol treatment enhances Kupffer cell sensitivity to lipopolysaccharide (LPS). In this model, CD14 in Kupffer cells was increased significantly 4 weeks after ethanol. Moreover, it was shown that prostaglandin E2 produced by activated Kupffer cells participated in the mechanism of ethanol-induced fatty liver. This study was designed to elucidate the temporal effect of chronic ethanol exposure on Kupffer cell sensitization to LPS. METHODS: Rats were given ethanol every 24 hr intragastrically for up to 12 weeks, and Kupffer cells were isolated 24 hr after the final ethanol administration and cultured in RPMI 1640 with 10% fetal bovine serum. After addition of LPS to Kupffer cells, intracellular calcium ([Ca2+]i) was measured. RESULTS: CD14 in Kupffer cells was increased approximately 2-fold, and then it decreased and returned to control levels. The LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha by Kupffer cells were also increased approximately 3-fold over control values, but they also returned to control levels. Triglyceride content increased with the duration of chronic ethanol treatment. At 8 weeks, prostaglandin E2 produced by Kupffer cells increased approximately 3-fold over control values and triglycerides by approximately 4-fold before gradually decreasing to basal levels. After 12 weeks of ethanol exposure, LPS-induced increases in [Ca2+]i and tumor necrosis factor-alpha production were only approximately 50% as high as peak levels at 4 weeks. Liver triglyceride content at 12 weeks was reduced significantly compared with values at 8 weeks. CONCLUSIONS: Kupffer cells at the early stage of chronic ethanol exposure exhibited sensitization to LPS, but this sensitivity was blunted later. This correlated with triglyceride accumulation in the liver. These data indicate that long-term alcohol exposure changes the sensitivity of rat Kupffer cells to LPS but that the magnitude of the effect is time dependent.     

Isolation and primary culture of rat Kupffer cells

The aim of this study was to describe a reproducible method for the isolation, purification and primary culture of rat Kupffer cells. Kupffer cells were isolated following sequential pronase/collagenase digestion of the liver and enrichment of a non-parenchymal cell fraction by a single-density gradient centrifugation step using 30% metrizamide. Kupffer cells were isolated and further purified from this cell fraction by centrifugal elutriation. Kupffer cells were isolated at 1017 g at 48–110 mL/min. All Kupffer cell fractions exhibited phagocytosis of 3 μm latex beads. Kupffer cell fractions isolated at 48 and 60 mL/min were predominantly ED2 negative while later fractions (80–110 mL/min) were ED2 positive. Kupffer cells were adherent in culture after 2 h. This method for Kupffer cell isolation resulted in a yield of 80–120 times 10⁶ Kupffer cells per liver.     

脂联素对大鼠原代Kupffer细胞白细胞介素10表达的影响

目的本研究观察外源性脂联素对体外培养的大鼠原代Kupffer细胞白细胞介素（IL）-10表达的调节作用,以阐明脂联素的抗炎作用机制。方法通过两步胶原酶灌流、密度梯度离心和2 h选择性贴壁方法分离纯化大鼠肝脏Kupffer细胞。酶联免疫吸附试验方法检测脂联素作用下原代Kupffer细胞上清液中IL-10的表达水平。荧光定量PCR法检测脂联素作用下原代Kupffer细胞IL-10 mRNA表达。结果 0.5、1.0、1.5μg/ml脂联素处理Kupffer细胞4、8、12、16 h,与对照组相比,同一浓度的脂联素随着培养时间的延长,Kupffer细胞IL-10分泌逐渐增加;而同一作用时间下,随着脂联素浓度的增加,Kupffer细胞IL-10分泌量并没有逐渐增加。1.0和1.5μg/ml脂联素作用Kupffer细胞16 h,IL-10基因表达量与对照组相比显著增加,各处理组间无明显差异。结论脂联素可上调大鼠原代Kupffer细胞IL-10的表达,这可能是其抗炎作用机制之一。     

Relationship between cellular shape and receptor-mediated endocytosis: an ultrastructural and morphometric study in rat Kupffer cells

ABSTRACT— The relationship between cellular shape (i.e., size, volume, presence of microvilli, pseudopodia, flat or round shape) and receptor-mediated endocytotic activities (i.e., binding and internalization) was investigated using intact liver as well as freshly isolated Kupffer cells and Kupffer cells in culture. The morphological features of Kupffer cells were reconstructed by three-dimentional analysis from in situ experiments and by densitometric analysis of cells in suspension and in culture. By morphometry at the ultrastructural level, different cellular shapes were compared with the respective capacities for binding and internalization of glycoproteins with terminal galactosyl residues. The number of asialoglycoprotein-gold particles bound to the cell surface or internalized into endosomes was calculated. Our data show that differences in cellular shape, mainly related to the reduction of projection and microvilli and to the roundness of cell surface, accompany modulation of galactose-specific receptors in rat Kupffer cells, thus supporting the hypothesis that cell morphology is affected by endocytic activities. In fact, the progressive reduction in microvilli projections and cellular roundness is paralleled by the progressive decrement of both binding and uptake capacity from in situ, freshly isolated and cultured Kupffer cells.     

雷抑素对大鼠移植肝Kupffer细胞的影响

目的探讨肝移植术前应用雷抑素对大鼠肝Ｋｕｐｆｅｒ细胞的影响方法以ＳＤ大鼠为供受体建立原位肝移植模型．受体移植术前３ｄ连续口服１％羧甲基纤维素１ｍｌ／ｄ（对照组）或雷抑素１０ｍｇ／ｋｇ·ｄ（用药组）．分别于术后１，２，３，２４ｈ采血并取肝组织，检测血清ＴＮＦ，ＡＬＴ及肝ＭＤＡ水平，观察肝超微结构及大鼠１周存活率变化．结果对照组移植术后３ｈ血清ＴＮＦ（５３ｋＵ／Ｌ±０４１ｋＵ／Ｌ），肝ＭＤＡ（４８４６ｎｍｏｌ／ｇ±２３６ｎｍｏｌ／ｇ）显著增加，ＴＮＦ表达呈强阳性；而且药组ＴＮＦ（０９ｋＵ／Ｌ±０１１ｋＵ／Ｌ）肝ＭＤＡ（３６１８ｎｍｏｌ／ｇ±１５４ｎｍｏｌ／ｇ）无明显变化，ＴＮＦ表达阴性，两者相差显著Ｐ＜００１）．电镜检查，对照组肝Ｋｕｐｆｅｒ细胞呈活化表现，而用药组肝Ｋｕｐｆｅｒ细胞呈非活化状态．对照及用药组术后１周存活率分别为０％和６０％．结论术前应用雷抑素可抑制移植肝ＴＮＦ和Ｏ２的产生，抑制Ｋｕｐｆｆｅｒ细胞活化，以减轻肝冷缺血再灌注损伤．