大鼠原代肝细胞培养方法的建立

目的：探索混合胶原凝胶培养肝细胞的方法,观察培养鼠肝细胞的功能与形态特征,用于评价中药十八反的作用机理。方法：两步法分离鼠肝细胞,与鼠尾胶原溶液混合接种于培养板,观察培养鼠肝细胞的形态学特征和生化指标。采用RT-PCR技术。检测药物对P450亚酶CYP3A1表达的影响,进一步确证该体系的可靠性。结果：双层胶原培养体系可观察到典型的肝细胞形态特征,肝细胞功能检测显示肝细胞合成分泌的尿素、白蛋白,而乳酸脱氢酶漏出量较少。药物对P450亚酶CYP3A1表达的影响呈良好的剂量依赖性,同时双层胶原具有保持肝细胞活性的优点。可作为原代肝细胞培养的条件。结论：混合胶原凝胶培养能保留体内的细胞功能和活性,特别是保留药物代谢酶的活性。     

小鼠Kupffer细胞分离及细胞培养

目的 采用在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离Kupffer细胞（Kupffer cells,KCs）,探讨其在分离小鼠KCs的应用及其对KCs生物活性的影响.方法 根据原位灌注和梯度离心方法不同随机分为4组：无胶原酶原位灌注＋3层梯度离心组（A）、无胶原酶原位灌注＋双层梯度离心组（B）、胶原酶原位灌注＋3层梯度离心组（C）和胶原酶原位灌注＋双层梯度离心组（D）.采用F4/80（BM8）免疫染色及吞墨实验判断细胞纯度和功能、台盼蓝拒染实验判断细胞的活力,探讨不同方法KCs分离的效果及细胞活性.结果 刚分离的KCs细胞近似圆形,接种l h后收获细胞纯度较高,但细胞得率相对较低.培养4 h后KCs得率相对较高,培养28 d仍能存活.免疫荧光可显示分离的为KCs,台盼蓝染色显示各组细胞的活力均在90 %左右,在体胶原酶灌注和双层梯度离心可以增加KCs的得率,双层梯度离心法可以增加分离KCs的纯度.结论 在体胶原酶灌注对提高KCs得率较为重要,在体胶原酶灌注、不连续密度梯度离心、选择性贴壁3步法分离小鼠KCs的的方法简便、高效、稳定,培养的KCs具有良好的细胞生物学性状.     

Kupffer细胞起源及其免疫学功能的研究进展

Kupffer细胞(Kupffer cells,KCs)主要存在肝血窦中,是机体单核巨噬细胞系统的重要组成部分。传统观点认为肝脏的KCs来是由单核巨噬细胞系统分化而来,但是新近研究发现具有分化潜能的造血干细胞能被诱导分化为具有KCs功能的巨噬细胞。KCs通过模式识别受体(pattern recognition receptors,PRR)识别外源和内源的危险信号,激活后分化为M1型和M2型。在体内,硅化合物、脂质体封装混合物以及氯化钆类可以特异性消除KCs和(或)阻断KCs的功能。KCs的基本功能包括:吞噬清除病原;抗原递呈,启动适应性免疫;促进肝细胞再生等。     

乙型肝炎病毒假病毒体外感染树鼩原代肝细胞模型的建立

目的：建立乙型肝炎病毒假病毒（HBVpp）体外感染树鼩原代肝细胞（PTH）模型。方法：通过肝脏原位两步灌注法分离PTH并对其冻存方法进行优化,通过共转染293T细胞生产基于慢病毒包装系统的HBVpp并考察其感染的种属和组织特异性。结果：通过肝脏原位两步灌注法分离了PTH,并优化了PTH冻存液的配方;包装的HBVpp具有与HBV真病毒类似的感染种属和组织特异性。结论：该模型的建立对深化HBV进入肝细胞机制的研究具有重要意义。     

敲除Usp13促进棕榈酸诱导的小鼠肝实质细胞凋亡*

目的: 研究去泛素化酶Usp13对棕榈酸诱导的肝实质细胞凋亡的影响,并对其机制进行初步探究。方法: 两步胶原酶灌注法分离小鼠原代肝实质细胞并采用棕榈酸处理;甘油三酯酶法及油红O染色检测肝实质细胞中脂质的累积;MTS和LDH试剂盒检测细胞活力;流式细胞术检测细胞凋亡;qPCR检测凋亡相关基因的表达以及免疫印迹法检测Caspase-3的活化。结果: 敲除Usp13基因并不影响棕榈酸诱导下肝实质细胞的甘油三酯累积,但导致细胞损伤加重,凋亡增加。机制研究显示,敲除Usp13促使肝实质细胞中抗凋亡基因Bcl-2、Bcl-xl表达下调,促凋亡基因Bid、p53表达上调,以及Caspase-3活化增强。结论: 初步揭示了Usp13调控棕榈酸诱导下肝实质细胞凋亡的机制,为深入研究Usp13调控非酒精性脂肪性肝炎的发生、发展奠定了基础。     

原代培养大鼠前列腺细胞建立前列腺增生筛药模型

目的建立原代培养大鼠前列腺上皮细胞体外筛药模型。方法无菌状态下取雄性SD大鼠腹侧叶前列腺,称量后,剪成1 mm3小块,经Ⅱ型胶原酶消化1 h后,过滤、离心获取前列腺细胞,接种于24孔板培养并对细胞进行形态学和免疫组织化学鉴定。获取的大鼠前列腺上皮细胞分别接种于96孔板和24孔板培养12 d后给予系列剂量(0.1～1000μmol/L)的他莫昔芬和阳性药物爱普列特处理72 h,利用CCK-8法检测前列腺上皮细胞存活率并计算药物IC50值,并进一步通过Giemsa染色后观察细胞生长及形态变化。结果细胞鉴定结果显示,获取的细胞具典型上皮细胞形态特征,细胞角蛋白和前列腺特异性抗原表达阳性,提示所获细胞为前列腺上皮细胞。爱普列特与前列腺上皮细胞孵育72 h后,CCK-8法检测结果显示能够明显抑制前列腺上皮细胞生长,其IC50值为42.7μM,进一步镜下观察结果显示,爱普列特未明显改变大鼠前列腺上皮细胞形态,但能导致存活细胞数减少。他莫昔芬则对大鼠前列腺上皮细胞生长无明显抑制作用,镜下观察前列腺上皮细胞数量和形态未见明显改变。结论利用原代培养SD大鼠前列腺上皮细胞可以成功建立前列腺增生体外筛选模型。     

草鱼肝细胞的分离与原代培养

目的以草鱼(Ctenopharyngodon idellus)肝细胞为实验对象,在不同条件下进行原代培养,以探讨适合草鱼肝细胞生长的最佳条件及培养方法,用于饲料营养与非营养物质对草鱼肝细胞代谢、损伤作用机制的研究。方法采用温胰蛋白酶消化法和红细胞裂解液分离、纯化肝细胞,MTT法测定细胞增殖率,并测定不同时期培养液上清液中LDH、Alb和BUN的含量,分析肝细胞生长状况。结果采用0.25%浓度的温胰蛋白酶消化法,消化20min,分步收集肝细胞,经台盼蓝染色检测和血球计数板计数,活细胞数≥99%。结论在含10%胎牛血清、10μg/mL胰岛素的M199培养基中,以接种浓度1.7×106cell/mL左右为宜,置于27℃、4.5%CO2浓度的恒温培养箱中,可成功培养草鱼原代肝细胞。     

肝细胞生长因子对四氯化碳损伤原代培养大鼠肝细胞的保护作用

本工作采用无血清原代培养大鼠肝细胞法,观察了重组人肝细胞生长因子（ｒ－ｈＨＧＦ）对四氯化碳（ＣＣｌ４）致大鼠肝细胞损伤的保护作用。结果表明,ｒ－ｈＨＧＦ对ＣＣｌ４染毒肝细胞有明显的保护作用。ｒ－ｈＨＧＦ保护组较ＣＣｌ４染毒组细胞存活率显著升高,细胞内丙氨酸转氨酶、钾离子漏出明显降低。结果提示,ｒ－ｈＨＧＦ可减轻ＣＣｌ４对肝细胞膜的损伤,提高细胞膜的结构完整性     

茶多酚对NASH 大鼠肝脏组织VEGF 及氧化应激的影响

目的:茶多酚对NASH大鼠肝脏组织VEGF及氧化应激的影响。方法:雄性SD大鼠30只,随机分为3组,正常对照组、模型组、茶多酚治疗组。正常组普通饲料喂养,模型组喂高脂饮食,茶多酚治疗组在高脂饮食12周后茶多酚(150mg(/kg.d)灌胃治疗,16周末处死各组大鼠,留取肝脏组织,观察各组大鼠肝组织病理改变,测定其肝脏丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性以及血管内皮生长因子(VEGF)、Ⅰ、Ⅲ型胶原的表达。结果:模型组大鼠肝组织中SOD活性降低而MDA含量以及VEGF、Ⅰ、Ⅲ型胶原表达均明显高于正常组。茶多酚治疗可减轻肝纤维化程度,显著升高肝组织中SOD活性、降低MDA含量以及VEGF、Ⅰ、Ⅲ型胶原表达水平。结论:茶多酚可通过抑制肝纤维化组织VEGF表达,降低肝组织氧化应激水平而发挥抗肝纤维化作用。     

Isolation and characterization of rat primary lung cells

Summary Lung cell culture may be useful as anin vitro alternative to study the susceptibility of the lung to various toxic agents. Lungs from female Wistar rats were enzymatically digested by recirculating perfusion through the pulmonary artery with a sequence of solutions containing deoxyribonuclease, chymopapain, pronase, collagenase, and elastase. Lung tissue was microdissected and resuspended and the cells obtained were washed by centrifugation. By this isolation method, 2×10⁸ cells per rat lung were obtained with an average viability of 97%. Lung cells cultured in medium containing antibiotics and serum maintained a viability of >70% for 5 d. Rat primary lung cells were exposed to various toxic agents and their viability was assessed by formazan production capacity after 18 h of incubation. Compared to rat and mouse hepatocyte cultures (EC⁵⁰=5.8 mM), rat primary lung cells were much more susceptible to hydrogen peroxide (EC₅₀=0.6 mM). All cell types were equally sensitive to the more potent toxicanttert-butylhydroperoxide (EC₅₀=0.1 mM). Paraquat was more toxic to lung cells (EC₅₀=0.03 mM) than to rat (EC₅₀=2.8 mM) and mouse (EC₅₀=0.2 mM) hepatocytes. In contrast, rat lung cells were less sensitive to sodium nitroprusside (EC₅₀=2.6 mM) compared to rat (EC₅₀=0.2 mM) and mouse (EC₅₀=0.03 mM) hepatocytes. Nitrofurantoin and menadione (at EC₅₀=0.04 mM and 0.006 mM, respectively) were more toxic to rat lung and liver cells than to murine hepatocytes (EC₅₀=0.2 mM and 0.04 mM, respectively). Our findings demonstrate the applicability of this rat primary lung cell culture for studying the effects of lung toxicants. Parts of the study had been presented orally at the meeting of the German Society of Toxicology and Pharmacology in Mainz (FRG), March 15–17, 1994.     

Isolation of rat Kupffer cells: a combined methodology for highly purified primary cultures

We report a four-step procedure that optimizes the methodology for isolation of highly purified rat Kupffer cells (KC). We combined the previously reported techniques of enzymatic tissue treatment, density gradient centrifugation, centrifugal elutriation and selective adherence. ED-2 immunophenotyping and non-specific esterase histochemistry were used for cell identification. This combination resulted in a satisfactorily high yield of 80-100 x 10(6)KCs per liver, over 95% positive for ED-2 and 98% viable cells. Cultures of isolated KCs were functionally intact and exhibited a concentration and time-dependent LPS-induced TNF-alpha and nitric oxide production.     

Methylenetetrahydrofolate reductase gene polymorphisms in patients with nonalcoholic steatohepatitis (NASH)

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of abnormal hepatic steatosis in the absence of a history of alcohol use. Nonalcoholic steatohepatitis (NASH) is the progressive form of NAFLD. Hyperhomocysteinemia causes steatosis, and the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms result in hyperhomocysteinemia. To examine whether the C677T and A1298C polymorphisms of the MTHFR gene were associated with NASH, we analysed the allele and genotype distribution of the MTHFR C677T and A1298C polymorphisms in 57 well-diagnosed NASH patients, 324 healthy controls in a case-control study of Turkish subjects of Caucasian origin. The diagnosis of the NASH patients was based on liver biopsy. The method used in the analysis of genotypes was PCR-RFLP. The MTHFR A1298C polymorphism was significantly associated with NASH (chi(2) = 8.439; p = 0.015) in the total NASH patients compared with healthy controls. The MTHFR 1298C allele (odds ratio (OR) = 2.480; 95%CI = 1.286-4.782; chi(2) = 7.703; df = 1; p = 0.006) was significantly associated with NASH in the total NASH patients. The MTHFR C677C/A1298C compound genotype (OR = 2.218; 95%CI = 1.003-4.906; chi(2) = 3.998; df = 1; p = 0.046) in men patients was also significantly associated with NASH. Likewise the MTHFR C1298C genotype was significantly associated with NASH in women patients with NASH (OR = 2.979; 95%CI = 1.027-8.641; chi(2) = 4.343; df = 1; p = 0.037). In conclusion, the MTHFR 1298C allele in all NASH patients, C1298C genotype, C677C/C1298C compound genotype in women NASH patients and C677C/A1298C compound genotype in men NASH patients were genetic risk factors for NASH.     

Oxidative products from alcohol metabolism differentially modulate pro-inflammatory cytokine expression in Kupffer cells and hepatocytes

Pro-inflammatory cytokines play a vital role in the pathogenesis of alcoholic steatohepatitis. The present study was to determine the role of alcohol-induced oxidative stress in modulating cytokine production. A rat model of alcohol consumption was used to determine alcohol-induced hepatic cytokine expression. Chronic alcohol exposure caused lipid accumulation, oxidative stress, and inflammation in the livers of Wistar rats. The role of oxidative stress in regulating cell type-specific cytokine production was further dissected in vitro. Lipopolysaccharide (LPS) dose-dependently upregulated TNF-α, MIP-1α, MCP-1, and CINC-1 in Kupffer cells-SV40, whereas TNF-α dose-dependently induced CINC-1, IP-10, and MIP-2 expression in H4IIEC3 hepatoma cells. An additive effect on cytokine production was observed in both Kupffer cells-SV40 and hepatocytes when combined hydrogen peroxide with LPS or TNF-α, respectively, which was associated with NF-κB activation and histone H3 hyper-acetylation. Unexpectedly, an inhibitory effect of 4-hydroxynonenal on cytokine production was revealed in LPS-treated Kupffer cells-SV40. Mechanistic study showed that 4-hydroxynonenal significantly enhanced mRNA degradation of TNF-α, MCP-1, and MIP-1α, and decreased the protein levels of MCP-1 in LPS-stimulated Kupffer cells-SV40 through reducing the phosphorylation of mRNA binding proteins. This study suggests that Kupffer cells and hepatocytes express distinct pro-inflammatory cytokines/chemokines in response to alcohol intoxication, and oxidative products (4-hydroxynonenal) differentially modulate pro-inflammatory cytokine/chemokine production via NF-κB signaling, histone acetylation, and mRNA stability.     

Stimulation of liver functions in hierarchical co-culture of bone marrow cells and hepatocytes

A hierarchial co-culture, in which rat hepatocytes and non-parenchymal liver cells (NPLCs) were separated by a collagen layer and which was designed to mimic the in vivo microenvironment, was carried out with the aim of developing a module for bio-artificial liver support. Compared with a monolayer co-culture and hepatocytes cultured alone in a monolayer, higher urea synthesis activity was maintained for 6 d in the hierarchical co-culture. When a rat hepatoma cell line H4-II-E-C3, which retains the induction of tyrosine aminotransferase (TAT), was co-cultured in a monolayer with NPLCs, dose-dependent stimulation of TAT induction was observed. In a hierarchical co-culture, NPLCs further stimulated TAT induction in H4-II-E-C3 cells. Since peritoneal macrophages could stimulate TAT induction in hepatocytes in both monolayer and hierarchical co-cultures, bone marrow cells, which can proliferate and differentiate into macrophages in vitro, were investigated as a possible substitute for NPLCs. Bone marrow cells isolated from rat femurs were cultivated in the presence of IL-3 and macrophage colony-stimulating factor (M-CSF), and co-cultured with hepatocytes. Urea synthesis and TAT induction of hepatocytes were stimulated in the co-culture. The co-culture of bone marrow and H4-II-E-C3 cells, both of which have proliferation ability in vitro, was also shown to be effective in stimulating liver functions. The hierarchical configuration, in which two cell types can communicate with the soluble factor(s) through a collagen layer, was found to be more effective than a monolayer in long-term co-culture.     

Tripartite motif 16 ameliorates nonalcoholic steatohepatitis by promoting the degradation of phospho-TAK1

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New scoring system combining the FIB-4 index and cytokeratin-18 fragments for predicting steatohepatitis and liver fibrosis in patients with nonalcoholic fatty liver disease

Purpose: To establish a new scoring system as a noninvasive tool for predicting steatohepatitis and liver fibrosis in patients with nonalcoholic fatty liver disease (NAFLD).

Methods: A total of 170 patients histologically diagnosed with nonalcoholic steatohepatitis (NASH) (n?=?130) or nonalcoholic fatty liver (NAFL) (n?=?40) were enrolled. We analyzed receiver operating characteristic (ROC) curves and performed multivariate analysis to predict steatohepatitis and liver fibrosis.

Results: Multivariate analysis showed that cytokeratin-18 fragment (CK18-F) levels (≥278?U/L) (odds ratio [OR], 4.46; 95% confidence interval [CI], 1.42–14.00; p?=?0.010) and the FIB-4 index (≥1.46) (OR, 4.54; 95% CI, 1.93–29.50; p?=?0.004) were independently associated with prediction of NASH. We then established a new scoring system (named the FIC-22 score) for predicting NASH using CK18-F levels and FIB-4 index. The areas under the ROC curve (AUROCs) of the FIC-22 score and NAFIC score were 0.82 (95% CI, 0.75–0.89) and 0.71 (95% CI, 0.62–0.78) (p?=?0.044). Additionally, the AUROC of the FIC-22 score for predicting the presence of fibrosis (F?≥?1) was 0.78 (95% CI, 0.70–0.85).

Conclusions: In patients with NAFLD, the FIC-22 score had high predictive accuracy not only for steatohepatitis but also for the presence of liver fibrosis.