一种改良大鼠肝脏Kupffer细胞分离方法

目的观察改良大鼠肝脏Kupffer细胞(KCs)分离方法获取KCs的效果。方法参照Akira提供的方法进行以下改进:①前灌注液在体灌注,Ⅳ型胶原酶离体灌注消化;②Percoll分离液不连续密度梯度离心;③台盼蓝染色检测分离细胞的活度;④选择性贴壁法纯化获取的细胞;⑤吞墨实验、DAB染色及CD163细胞免疫荧光法鉴定所分选细胞。结果肝脏KCs的获得量为(3±1.5)×105/g鼠肝,细胞活度>92%;光镜下细胞呈圆形,培养24 h后呈梭形或多角形;具有较强的吞噬能力,DAB染色呈"煎蛋"样,荧光显微镜下>99%为KCs。结论改良的大鼠肝脏KCs分离方法较Akira法能够获取更高纯度的KCs,简捷经济,值得推广。     

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。     

Kupffer细胞在细菌感染性疾病中的作用

Kupffer细胞(Kupffer cells,KCs)是体内最大的巨噬细胞群,他们参与了肝脏多种疾病的发生发展.体外原代培养是研究KCs生物学功能的重要手段,获得较多数量、较高纯度和活性的KCs是研究其作用机制的首要条件.许多吞噬颗粒和可溶性物质都可以和KCs细胞膜上的受体结合进而激活KCs,其中最重要的是脂多糖(lipopolysaccharide,Kupffer细胞(Kupffer cells,KCs)是体内最大的巨噬细胞群,他们参与了肝脏多种疾病的发生发展.体外原代培养是研究KCs生物学功能的重要手段,获得较多数量、较高纯度和活性的KCs是研究其作用机制的首要条件.许多吞噬颗粒和可溶性物质都可以和KCs细胞膜上的受体结合进而激活KCs,其中最重要的是脂多糖(lipopolysaccharide,LPS).LPS经过Toll样受体4(Toll-like receptor4,TLR4)信号途径直接激活K Cs,导致一系列炎症因子产生增多.高浓度的LPS还可以直接进入细胞内,导致Caspase11途径的激活,促进白介素-1β(interleukin 1 beta,IL-1β)的成熟和释放.KCs在脓毒症、内毒素耐受以及急性胰腺炎中扮演了重要角色,本文将对其在上述疾病中的作用及相关分子机制做一综述.     

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.     

胰岛β细胞的分离纯化与原代培养技术

胰岛β细胞是糖尿病研究的中心环节之一。目前体外研究胰岛β细胞的模型系统有两类，一是从胰岛β细胞瘤组织克隆产生的β细胞株，二是从活体胰岛组织分离纯化获取胰岛β细胞。国内利用细胞株作为研究模型者较多，而β细胞的体外原代培养尚未见研究报道。     

大鼠肝贮脂细胞Kupffer细胞的分离,培养和鉴定

贮脂细胞是目前肝纤维化研究的热点，本文参考Ｆｒｉｅｄｍａｎ等的不连续密度梯度心法并作改良，建立了一种经济，简便，可靠的分离大鼠肝贮脂细胞，Ｋｕｆｆｅｒ细胞的方法，贮脂细胞得率的１０＾７／大鼠，纯度在９０％以上，Ｋｕｐｆｆｅｒ细胞纯度约６０％，其培养上清基本适合进一步研究用。本方法的建立，为进一步研究肝贮脂细胞与肝纤维化的关系，尤其是细胞水平及分子生物学水平的研究奠定了基础。     

原代培养大鼠肝细胞分离方法比较研究

目的:探索原代培养大鼠肝细胞的最佳分离方法。方法:对比观察不同的肝细胞分离技术的培养效果。培养前,以4％台盼蓝染色判定肝细胞活性,培养48h后,观察肝细胞贴壁及生长状况。结果:①灌流消化法优于剪切消化法;②在多种灌流消化法中,经门静脉灌流消化法优于经胆总管灌流消化法及经腹主动脉灌流消化法;③在门静脉灌流的基础上,0.1％胶原酶Ⅰ37℃热消化10～15分钟,效果最好,而0.25％胰蛋白酶消化10～12分钟次之,用含EDTA的D-Hank液直接灌流分离法效果极差。结论:原代培养肝细胞的得率及活性与不同分离法包括灌流途径、灌流液、消化时间等有关。     

大鼠冠状动脉平滑肌细胞的原代培养及鉴定

目的探讨大鼠冠状动脉平滑肌细胞的原代培养方法,为冠状动脉疾病的研究提供理想的细胞模型。方法无菌条件下分离大鼠冠状动脉,采用酶消化法分离培养平滑肌细胞,进行形态学观察及台盼蓝染色测定细胞活力,采用免疫荧光染色技术对平滑肌α肌动蛋白进行鉴定。结果形态学、免疫荧光染色鉴定表明培养的细胞为血管平滑肌细胞,细胞存活率达97%。原代培养14d左右即可进行传代,可以传6代以上,且细胞形态及生长状态良好。结论酶消化法分离大鼠冠状动脉平滑肌细胞,方法简单、高效,细胞纯度高、且活性好、生长迅速。     

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.     

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.     

Transferrin receptor on rat Kupffer cells in primary culture

Kupffer cells may play a role in the turnover of iron in acute viral hepatitis. The transferrin receptor of rat Kupffer cells in primary culture was therefore investigated in this study. Daily specific bindings on 125I-diferric transferrin (Tf) to rat Kupffer cells in primary culture from day 3 to day 6 of culture were 1.64 +/- 0.08%, 4.16 +/- 0.05%, 4.34 +/- 0.07% and 2.63 +/- 0.07%, respectively. The specificity of the Tf binding sites was examined by competition studies showing that galactose (30 mmol x l-1) and ovalbumin (90 mumol x l-1) did not compete for the binding sites, but human lactoferrin (50 mumol x l-1) competed for the binding sites by about 30%. The affinity and capacity of Tf receptor on rat Kupffer cells in 5-day culture were analyzed according to the method of Scatchard. A single class of 125I-diferric Tf binding sites with an affinity constant of 1.65 x 10(7) l x l-1) and a capacity of 6.86 x 10(6) sites/cell was found. After zymosan (500 micrograms/ml) preincubation for 30 min, the binding capacity increased about 1.7-fold, and this increase depended upon the increase of the affinity of Tf receptor. These data suggest that Kupffer cells in the activated state accelerate the removal of elevated serum iron.     

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.     

Release of lysosomal enzymes is not correlated with superoxide and prostaglandin production by stimulated rat Kupffer cells in primary culture

A substantial production of prostaglandin E2 (PGE2) was induced in primary cultures of rat Kupffer cells by zymosan, calcium ionophore A23187, phorbol ester and arachidonic acid, whereas contact with latex particles, glucan or immunocomplexes led to a minor PGE2 release only. Superoxide generation, on the other hand, was observed after administration of zymosan, glucan and the phorbol ester but not after treatment with the calcium ionophore, arachidonate, latex particles or immunocomplexes. Lysosomal enzymes like beta-glucuronidase and N-acetyl-beta-D-glucosaminidase were detected in the medium of rat Kupffer cells in primary culture after contact with zymosan or calcium ionophore A23187. Other particulate matter, e.g., latex particles, glucan and immunocomplexes, lipopolysaccharides or soluble agents such as phorbol ester, arachidonic acid and gamma-interferon did not provoke the release of lysosomal enzymes. The activities of beta-glucuronidase and N-acetyl-beta-D-glucosaminidase found following prolonged exposure to zymosan or to A23187 were accompanied by the appearance of typical cytosolic enzymes like lactate dehydrogenase and glucose-6-phosphate dehydrogenase in similar proportions and with the same time course. The release of lysosomal enzymes seen after administration of zymosan or calcium ionophore is thought to be the result of unspecific leakage rather than a specific response of elicited Kupffer cells.     

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.     

Kupffer cells and their function

The intention of this review is to stress new information regarding the quite versatile functions of Kupffer cells. Although their main function is phagocytosis and defence of the liver against bacteria, endotoxaemia and viral infections, they also fulfil other important roles. They will phagocytose and partially degrade bacterial antigens before handing them on to the hepatocytes for excretion into the bile. They handle LDL lipoproteins, whilst the HDL proceed directly into the hepatocytes. They produce lymphokine mediators that direct protein synthesis by the hepatocytes. Also they normally produce prostaglandins that are cyto-protective for the hepatocytes. Conversely, if they are required to attack infected hepatocytes or cancer cells, then they switch to the production of leukotrienes. Thus they function as specialised macrophages, and it is not surprising that other "activated macrophages" have to be recruited into the liver to support them in inflammatory reactions.     

大鼠胰星状细胞的分离与培养

目的建立大鼠胰星状细胞(pancreatic stellate cells,PSCs)的分离培养方法.方法大鼠胰腺组织经胶原酶和链霉蛋白酶E消化后,用Nycodenz不连续密度梯度离心法分离PSC,在328 nm紫外光激发下观察细胞的自发荧光现象,并以免疫组化技术检测结蛋白(desmin)、神经胶质原纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和α-平滑肌动蛋白(α-smooth muscle actin,α-SMA)的表达,同时观察培养细胞的形态和生长特性.结果新鲜分离的大鼠PSCs产率、活力和纯度分别约为2.5×106/g胰腺、95%和90%.培养的PSCs可自发活化,表达α-SMA,细胞由静止型转化为肌成纤维样细胞表型.原代培养10天后细胞纯度＞95%,传代培养后细胞纯度可达99%以上.结论利用Nycodenz密度梯度离心方法可成功分离大鼠PSCs,其细胞产率、活力和纯度均可满足体外研究需要.     

大鼠胰星状细胞的分离与培养

目的　建立大鼠胰星状细胞 (pancreatic stellate cells,PSCs)的分离培养方法。方法　大鼠胰腺组织经胶原酶和链霉蛋白酶 E消化后 ,用 Nycodenz不连续密度梯度离心法分离 PSC,在 32 8nm紫外光激发下观察细胞的自发荧光现象 ,并以免疫组化技术检测结蛋白 (desmin)、神经胶质原纤维酸性蛋白 (glial fibrillary acidic protein,GFAP)和α-平滑肌动蛋白 (α- sm ooth m uscle actin,α- SMA )的表达 ,同时观察培养细胞的形态和生长特性。结果　新鲜分离的大鼠 PSCs产率、活力和纯度分别约为 2 .5 ×10 6 /g胰腺、95 %和 90 %。培养的 PSCs可自发活化 ,表达 α- SMA,细胞由静止型转化为肌成纤维样细胞表型。原代培养 10天后细胞纯度 >95 % ,传代培养后细胞纯度可达 99%以上。结论　利用 Nycodenz密度梯度离心方法可成功分离大鼠 PSCs,其细胞产率、活力和纯度均可满足体外研究需要。