Liver histology and histomorphometry in hybrid sorubim (Pseudoplatystoma reticulatum × Pseudoplatystoma corruscans) reared on intensive fish farming

This study aimed to characterize the liver histology and histomorphometry in sorubim hybrid of different categories (nursery, growth and grow‐out) reared on fish farming. The categories were defined considering body weight (BW): nursery category (n = 5): BW = 37.06 ± 6.00 g (31.6–45.3 g); growth category (n = 5): BW = 310.40 ± 53.80 g (242.1–376.4 g) and grow‐out category (n = 5): BW = 874.28 ± 27.59 g (846.2–913.1 g). Liver fragments were processed to paraffin inclusion, and sections were stained by haematoxylin and eosin (H&E), PAS (Periodic Acid Schiff) and Perl's staining to histology, histomorphometry and density volumetric of liver structures; glycogen analysis and to detect ferric irons (Fe³⁺) respectively. The hepatosomatic index decreased between the categories (P < 0.01). The percentage of PAS‐positive hepatocytes in the nursery category was higher (P < 0.05) in relation to the growth and grow‐out categories. The hepatocytes from all fish were positive to Perl's staining. The density volumetric of liver structures did not differ among categories except to blood vessels were higher (P < 0.01) in the nursery and growth. The area (μm²) and perimeter (μm) of hepatocytes, and the area (μm²), perimeter (μm) and volume (μm³) of the nuclei from grow‐out fish were lower (P < 0.01) than those from the nursery and growth categories. Changes in morphometric characteristics of hepatocytes may result from metabolic changes associated with body growth surubins; therefore, these morphometric characteristics of liver tissue can be used as functional biomarkers for the assessment of fish health and nutrition status.     

Porcine hepatocyte–Kupffer cell co‐culture as an in vitro model for testing the efficacy of anti‐inflammatory substances

As Kupffer cells are highly involved in the regulation of hepatic inflammatory response, the main goal of this study was to improve and to characterize a hepatocyte–Kupffer cell co‐culture of pig origin for modelling endotoxin‐induced hepatic inflammation and for testing the efficacy of potential anti‐inflammatory substances. This monolayer co‐culture was prepared from primary isolated swine hepatocytes and Kupffer cells in the ratio of 6:1 and 2:1, mimicking different states of liver inflammation. The prepared cell cultures were characterized by immunohistochemical CD‐68 detection. Lipopolysaccharide (LPS) challenge of both co‐cultures resulted in elevated interleukin‐8 (IL‐8) and that of 6:1 co‐cultures in increased IL‐6 production with a higher extent than on hepatocyte monocultures, justifying the key role of Kupffer cells in pro‐inflammatory cytokine production. LPS‐induced IL‐8 production was successfully attenuated by concomitant application of both sodium butyrate and terpinen‐4‐ol on hepatocyte monocultures, but not on co‐cultures, demonstrating the importance of the presence of Kupffer cells in cell cultures as inflammatory models. Based on these initial data, the applied porcine primary hepatocyte–Kupffer cell co‐culture is suggested to be a proper tool for in vitro investigations on liver physiology and hepatic inflammation in pigs and can be used as a useful model mimicking in vivo conditions in veterinary research.     

Protective effect of butylated hydroxylanisole against hydrogen peroxide-induced apoptosis in primary cultured mouse hepatocytes

Butylated hydroxyanisole (BHA) is a synthetic phenolic compound consisting of a mixture of two isomeric organic compounds: 2-tert-butyl-4-hydroxyanisole and 3-tert-butyl-4-hydroxyanisole. We examined the effect of BHA against hydrogen peroxide (H₂O₂)-induced apoptosis in primary cultured mouse hepatocytes. Cell viability was significantly decreased by H₂O₂ in a dose-dependent manner. Additionally, H₂O₂ treatment increased Bax, decreased Bcl-2, and promoted PARP-1 cleavage in a dose-dependent manner. Pretreatment with BHA before exposure to H₂O₂ significantly attenuated the H₂O₂-induced decrease of cell viability. H₂O₂ exposure resulted in an increase of intracellular reactive oxygen species (ROS) generation that was significantly inhibited by pretreatment with BHA or N-acetyl-cysteine (NAC, an ROS scavenger). H₂O₂-induced decrease of cell viability was also attenuated by pretreatment with BHA and NAC. Furthermore, H₂O₂-induced increase of Bax, decrease of Bcl-2, and PARP-1 cleavage was also inhibited by BHA. Taken together, results of this investigation demonstrated that BHA protects primary cultured mouse hepatocytes against H₂O₂-induced apoptosis by inhibiting ROS generation.     

改进原位循环灌流法分离小鼠肝细胞研究

【目的】建立小鼠肝细胞分离原位循环灌流方法,为肝脏细胞蛋白质组研究提供技术技持。【方法】参考Seglen胶原酶两步原位灌流法,对其进行改进,建立原位循环灌流分离小鼠肝细胞方法,对小鼠肝脏进行原位循环灌流,离体消化肝脏组织,差速离心获得纯化小鼠肝细胞;台盼蓝拒染试验检测肝细胞活性,常规血球计数法计算肝细胞得率;利用显微形态观察、HE、PAS及免疫细胞化学等检测方法鉴定所获得的肝细胞纯度;用含体积分数20%FBS的RPMI-1640细胞营养液对原代小鼠肝细胞进行培养。【结果】建立了原位循环灌流分离小鼠肝细胞的方法,采用该方法可从每只小鼠肝脏中成功分选到细胞活性大于90%、纯度在95%以上的(4.2×107)～(6.5×107)个肝细胞。原代分离的小鼠肝细胞培养4h后,大多数细胞可贴壁,12h后贴壁完全,细胞呈伸张状态,该原代肝细胞可持续培养7d以上,且细胞形态状况良好。【结论】成功建立了小鼠肝脏原位循环灌流试验体系,获得了纯度和活性均较高的小鼠肝细胞,分选所得的小鼠肝细胞完全满足构建肝细胞蛋白质组表达谱试验的需要。     

斜带石斑鱼肝细胞分离及原代培养方法的建立

本研究以斜带石斑鱼肝细胞为实验对象,在不同培养条件下进行原代培养,旨在探讨稳定可靠的斜带石斑鱼肝细胞分离及原代培养方法。采用组织块分离法和胰蛋白酶(含EDTA)消化法分离肝细胞,并通过密度梯度离心法分离纯化肝细胞,细胞悬液于DMEM/F-12、M199和L-15培养液中培养;细胞活力及数量采用血球计数板计数,并通过MTT法测定细胞增殖率;同时,测定不同时间培养上清液中乳酸脱氢酶(LDH)活性、白蛋白(ALB)和尿素氮(BUN)的含量,以分析肝细胞生长状态。结果表明,组织块方法不适于斜带石斑鱼肝细胞的培养,未见细胞从组织块中迁出,而胰蛋白酶消化法获得良好稳定的培养效果,细胞产量达到1.6×108个/g肝重,活细胞数达到95%;L-15培养基细胞生长明显优于DMEM/F-12和M199培养基;启动原代培养的48~72 h阶段肝细胞生长代谢旺盛,培养上清液中LDH活性显著降低,ALB和BUN含量显著升高。结果显示,0.25%的胰蛋白酶常温消化法适合斜带石斑鱼肝细胞的分离,斜带石斑鱼肝细胞原代培养的最适培养基为L-15培养基,肝细胞在启动原代培养的48~72 h生长代谢旺盛。     

试验性氟中毒对肉鸭肝细胞凋亡的影响

为研究肉鸭氟中毒与肝细胞凋亡的关系及其形态学改变,选用8日龄的天府肉鸭160只,随机分成4组,分别在常规饲料中添加0、350、450和550mg/kg的氟化钠攻毒,攻毒后每隔8d,每组扑杀5只,采用末端脱氧核苷酸转移酶(TdT)介导的dUTP缺口末端标记法(TUNEL)对试验性氟中毒肝组织标本进行处理和标记,光镜下观测24、40、56日龄肉鸭肝细胞凋亡的百分率和形态学改变。TUNEL检测结果显示,在肉鸭氟中毒模型中,DAB着色的阳性细胞呈凋亡细胞的典型形态特征;肝细胞凋亡的百分率较对照组明显升高。结果表明,肉鸭氟中毒时以肝细胞早期和中期凋亡为主,随着摄氟时间的延长和摄氟剂量的加大,肝细胞由凋亡逐渐发展为坏死。表明过量氟在体内毒性作用和肝细胞凋亡密切相关。     

CPT-I基因荧光定量PCR检测方法的建立

CPT-ImRNA的表达对研究奶牛肝脏脂肪酸氧化代谢具有重要作用,CPT-I在肝脏中脂肪酸从细胞浆转移到线粒体过程中起关键作用。本研究应用双标准曲线方法,建立了CPT-I基因的相对荧光定量方法。结果表明,所建立的SYBRGreenI荧光定量PCR方法检测CPT-I基因具有较好的效果,为肝细胞CPT-I基因定量分析奠定了基础。     

Construction of a recombinant adenovirus carrying IL- 12 -IRES-CK gene and its expression in hepatocyte

AIM: To construct a bicistronic recombinant adenovirus carrying creatine kinase (CK) and human interleukin 12 (hIL-12) gene, and then detect the expression of both genes in vitro and metabolic product of CK in vivo. METHODS: Two PCR products, CK and hIL- 12 genes linked by IRES, were inserted into adenoviral vector. Adenovirus particles carrying CK-IRES-IL- 12 gene was generated through homologous recombination, packaging and propagation. Rabbit hepatocytes were isolated and transfected with adenovirus particles. CK and IL-12 gene expression was confirmed by Western blotting and ELISA, respectively.RESULTS: The expression of CK and human IL-12 in hepatocyte were confirmed in vitro. Metabolic product of CK, phosphocreatine (PCr), could be detected in liver by non-invasive phosphorus-31 magnetic resonance spectroscopy (³¹P MRS) in vivo.CONCLUSION: In vivo detection of ectopic expression of CK gene in liver can be achieved by non-invasive ³¹P MRS through adenovirus transduction. The bicistronic recombinant adenovirus Ad5-hIL-12-IRES-CKb carrying CK and hIL- 12 provides a useful tool for the further study of using a reporter gene expression (CK) dynamically, non-invasively monitor a therapeutic gene expression (IL-12) in liver.     

Induction of apoptotic lesions in liver and lymphoid tissues and modulation of cytokine mRNA expression by acute exposure to deoxynivalenol in piglets

Six 1-month-old piglets were intravenously injected with deoxynivalenol (DON) at the concentration of 1 mg/kg body weight, with three pigs each necropsied at 6 and 24 h post-injection (PI) for investigation of hepatotoxicity and immunotoxicity with special attention to apoptotic changes and cytokine mRNA expression. Histopathological examination of the DON-injected pigs revealed systemic apoptosis of lymphocytes in lymphoid tissues and hepatocytes. Apoptosis of lymphocytes and hepatocytes was confirmed by the TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method and immunohistochemical staining against single-stranded DNA and cleaved caspase-3. The number of TUNEL-positive cells in the thymus and Peyer''s patches of the ileum was increased at 24 h PI compared to 6 h PI, but the peak was at 6 h PI in the liver. The mRNA expression of interleukin (IL)-1β, IL-6, IL-18, and tumor necrosis factor (TNF)-α in the spleen, thymus and mesenteric lymph nodes were determined by semi-quantitative RT-PCR, and elevated expression of IL-1β mRNA at 6 h PI and a decrease of IL-18 mRNA at 24 h PI were observed in the spleen. IL-1β and IL-6 mRNA expressions increased significantly at 6 h PI in the thymus, but TNF-α decreased at 6 h PI in the mesenteric lymph nodes. These results show the apoptosis of hepatocytes suggesting the hepatotoxic potential of DON, in addition to an immunotoxic effect on the modulation of proinflammatory cytokine genes in lymphoid organs with extensive apoptosis of lymphocytes induced by acute exposure to DON in pigs.     

虎杖提取物对CCl4诱导建鲤损伤肝细胞生化指标的影响

为探明虎杖对鱼类损伤肝细胞是否具有修复作用，分离建鲤肝细胞，设6个试验组：I组(空白对照组)；II组(CCl4模型组)；III组(800 μg/mL虎杖提取物对照组)；IV组(造模前200、400、800 μg/mL虎杖提取物处理组)；V组(造模后200、400、800 μg/mL虎杖提取物处理组)；VI组(造模前、后200、400、800 μg/mL虎杖提取物处理组)。各组细胞经处理后继续培养12 h，收集肝细胞培养液，检测上清培养液中丙氨酸转氨酶(GPT)、天冬氨酸转氨酶(GOT)、乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)等酶活性，丙二醛(MDA)含量及肝细胞的存活率。结果表明：III组肝细胞培养液中GOT、GPT、LDH、SOD、MDA值及细胞存活率与I组相比无明显变化(P＞0.05)，说明800 μg/mL虎杖提取物没有细胞毒性，可用于后续试验；与II组相比，IV组中800 μg/mL虎杖提取物处理组的效果优于其他2个浓度组，可以极显著降低培养液中GOT值(P<0.01)，显著降低培养液中GPT、LDH值(P<0.05)，显著提高肝细胞的存活率(P<0.05)，而MDA含量和SOD值差异无统计学意义(P＞0.05)；与II组相比，V组中400 μg/mL虎杖提取物效果优于其他2个浓度组，可以极显著降低培养液中GOT值(P<0.01)，显著降低培养液中LDH值(P<0.05)，显著提高肝细胞的存活率(P<0.05)，但GPT值、MDA含量、SOD的差异无统计学意义(P＞0.05)；与II组相比，VI组中800 μg/mL虎杖提取物效果优于其他2个浓度组，能极显著降低培养液中GOT、GPT、LDH在肝细胞中的释放(P<0.01)，显著降低培养液中MDA含量(P<0.05)，显著提高肝细胞的存活率(P<0.05)。综合以上结果，以VI组中800 μg/mL的虎杖提取物效果最好，能有效抑制CCl4所造成的肝细胞损伤，其机制可能与其抗氧化作用有关。