非酒精性脂肪肝模型小鼠的建立

背景：以往非酒精性脂肪肝模型建立的常用诱导方法均有其局限性,因此,有必要建立高质量的非酒精性脂肪肝模型。 目的：拟利用复合高脂饮食和低浓度四氯化碳诱导小鼠非酒精性脂肪肝模型。 方法：取昆明小鼠随机分为3组：对照组,高脂模型组和高脂模型+四氯化碳组。观察饲养第2,4,6和8周末肝脏形态和病理变化,测定第8周末血清丙氨酸氨基转移酶,天冬氨酸氨基转移酶,胆固醇和三酰甘油,以及肝脏胆固醇和三酰甘油水平。 结果与结论：复合高脂饮食+5%四氯化碳腹腔注射的方式造模,第2周末开始出现炎症细胞浸润,第4周末出现少量脂滴,第6周末出现脂肪病变,第8周末在脂肪病变的基础上,部分小鼠出现了肝纤维化,8周末小鼠肝指数、血清丙氨酸氨基转移酶,天冬氨酸氨基转移酶,胆固醇和三酰甘油,肝脏胆固醇和三酰甘油浓度明显升高(P < 0.05或P < 0.01)。通过8周复合高脂饮食和5%四氯化碳腹腔注射成功建立小鼠非酒精性脂肪肝模型。     

非酒精性脂肪肝高脂饲养大鼠模型的标准化及评价

随着人民生活水平的提高、生活方式的改变,非酒精性肪性肝病(nonalcoholicfat yliverdisease,NAFLD)在我国发病率正呈现逐年增高趋势,但迄今为止非酒精性脂肪肝的发病机制仍不明确。目前非酒精性脂肪肝大鼠模型运用广泛,尤其是最常用的高脂饲养(highfat ydiet,HFD)大鼠模型。为了对该模型有更进一步地了解以及更深层次把握其运用。本文主要通过查阅近10年运用此模型造模的文献,从中收集相关造模数据等资料,得出该模型合理、常用的造模方法。     

长程高脂饲料对雌雄大鼠非酒精性脂肪肝差异的影响及机制的初步探讨

目的 观察SD雌雄大鼠肝脏脂质沉积情况,探讨形成雌雄大鼠脂肪肝差异的可能机制.方法 肝脏组织石蜡切片HE染色,用光镜观察肝脏脂质沉积情况.测定第14个月大鼠肝脏组织甘油三酯水平.测定第14个月大鼠的空腹血糖和空腹胰岛素,计算胰岛素抵抗指数.测定第14个月大鼠肝脏组织匀浆液中线粒体和微粒体的丙二醛和过氧化氢酶的含量,检测大鼠肝脏组织氧化应激和抗氧化应激水平.结果 ①长程高脂喂养14个月后雌雄大鼠高脂组肝脏组织出现弥散性的脂肪滴沉积,但无明显的炎性细胞浸润,未见纤维化形成,也未观察到明显的局部性肝细胞坏死或细胞凋亡等现象;②雄性高脂组大鼠肝脏组织的脂肪泡比雌性大鼠的面积大并且数量多,且雄性高脂组肝脏甘油三酯水平显著高于雌性高脂组(P<0.001);③第14个月,雌雄大鼠空腹血糖水平无显著差异,高脂雄性大鼠空腹胰岛素水平显著高于高脂雌性大鼠(P<0.05).高脂雄性大鼠胰岛素抵抗水平(HOMA-IR)亦显著高于高脂雌性大鼠(P<0.05);④雌性高脂组大鼠肝脏组织中丙二醛水平都低于雄性大鼠高脂组,过氧化氢酶水平都高于雄性高脂组大鼠,有显著性差异(P<0.05).结论 单纯长程高脂饲料喂养SD雌雄大鼠能引起典型的脂肪肝的形成,并且雄性高脂组大鼠脂肪肝的程度比雌性高脂组严重,其机制可能与雌性大鼠的外周胰岛素抵抗水平和肝脏组织的氧化应激水平低于雄性大鼠有关.     

青少年非酒精性脂肪肝多重危险因素的研究

随着生活水平的提高,青少年肥胖或超重呈逐年上升趋势,而伴有非酒精性脂肪肝病(non-alcoholic fatty liver disease,NAFLD)的肥胖青少年也日益增多,它严重威胁到21世纪青少年的健康.本研究对青少年非酒精性脂肪肝的多重危险因素进行探讨.     

非酒精性脂肪肝C57BL/6小鼠模型的建立

 BACKGROUND: The establishment of a safe, reliable and easily repeatable mouse model of nonalcoholic fatty liver disease is the prerequisite for the study of the diagnosis and treatment of the disease.     

非酒精性脂肪肝病模型小鼠的肝脏病理变化及肝细胞获取方法

目的 检测非酒精性脂肪肝病（NAFLD）模型小鼠的肝脏病理变化,并探索一种获得大量高活性、高纯度的NAFLD小鼠原代肝细胞的实验方法。方法 60只小鼠随机分为正常组（正常饲料,CD）和高脂组（高脂饲料,HFD）,分饮食饲养20周。定期检测小鼠的体质量,苏木精-伊红（HE）染色和天狼猩红染色观察肝脏组织病理特点。高脂饮食15周时,原位灌注Ⅳ型胶原酶6～8 mL消化得到混合肝细胞,铺于40%的Percoll分离液上,进行密度梯度离心获得纯化的肝细胞,锥虫蓝染色确定细胞的活性,显微镜观察其形态。结果 随着建模时间的延长,HFD组小鼠的体质量逐渐增加,且增幅显著高于CD组;HFD组小鼠肝脏脂肪变性程度增加,在第20周伴随出现了纤维化;建模15周时,每只NAFLD小鼠可获得5×10⁷～7×10⁷个肝细胞,细胞存活率≥85%,细胞纯度≥95%,细胞呈圆形或椭圆形,其边缘平滑,且聚团少;通过精准控制温度、降低胶原酶浓度、使用低速离心和低速密度梯度离心等优化条件后,获得的细胞数量和质量均有显著提高。结论 成功构建了NAFLD小鼠模型,并揭示了不同疾病阶段肝脏...     

替米沙坦抑制大鼠非酒精性脂肪肝纤维化

目的 探讨替米沙坦(Tel)对大鼠非酒精性脂肪肝纤维化(NAFLF)的抑制作用.方法 60只Wistar大鼠随机均分为6个组,对照组用含胆碱的氨基酸(CSAA)饲料喂养,添加0(CSAA组)或2.5 mg/(kg BW)(CDAA治疗组)的Tel;剩余4组用胆碱缺乏的氨基酸(CDAA)饲料喂养复制非酒精性脂肪肝纤维化模型,分别添加0(CDAA组)、0.5(低)、1.0(中)和2.5 mg/(kg BW)(高浓度组)的Tel,共10周.常规方法检测血清生化指标.免疫组化法观察肝组织α-平滑肌激动蛋白(α-SMA)和转化生长因子-β1(TGF-β1)的表达;实时定量PCR法测定肝组织Ⅰ型前胶原、金属基质蛋白酶(MMPs)及其抑制物(TIMPs)的表达.结果 CDAA组血浆中透明质酸,碱性磷酸酶,γ-谷氨酰转肽酶和总胆红素均高于CDAA添加Tel组(P＜0.05或P＜0.01);肝组织α-SMA和TGFβ1定量分析表明,CDAA组表达明显高于CDAA添加Tel组(均P＜0.01).CDAA组的Ⅰ型前胶原表达明显高于CDAA添加Tel组(P＜0.01),且随着Tel剂量的增加,表达逐渐减少;同时MMP-13表达上升(P＜0.01),而MMP-2及9和TIMP-1及2的表达下降(P＜0.01);Tel控制CDAA喂养大鼠的体质量增加.结论 替米沙坦可通过抑制肝星状细胞的活化而阻止NAFLF的形成,有望成为控制非酒精性脂肪肝病发展的很有前景的药物.     

非酒精性脂肪肝的发病机制研究进展

非酒精性脂肪性肝病(Non-alcoholic fatty liver disease,NAFLD)是一种常见的慢性肝脏疾病,可以发展成肝硬化,肝癌等严重疾病。本文对近年来关于NAFLD的发病机制进行了归纳和分析整理,发现NAFLD与诸多因素有关。在二次打击中,以胰岛素抵抗和氧化应激为中心环节,并在相关因素如脂联素、抵抗素、内脂素、瘦素、肿瘤坏死因子-α、成纤维细胞生长因子21、视黄醇结合蛋白4、载脂蛋白B、微粒体甘油三酯转运蛋白、固醇调节元件结合蛋白-1c、过氧化物酶增殖体受体、肉碱棕榈酰转移酶、肝型脂肪酸结合蛋白、解偶联蛋白、细胞色素P450、线粒体锰超氧化物歧化酶、线粒体膜通透性转换孔、肝铁沉积的作用下共同促进了NAFLD的进展。希望本文能为今后进一步的研究提供理论依据和思路。     

免疫细胞在非酒精性脂肪肝炎中作用的研究进展

非酒精性脂肪肝炎(NASH)是一种代谢性肝病,其在肝细胞脂肪变性基础上进一步诱发炎症反应、变性坏死及肝纤维化.近年来,许多新的研究发现免疫细胞在NASH的发病中发挥重要的作用.在NASH发病过程中参与免疫反应的细胞很多,包括中性粒细胞、巨噬细胞、树突状细胞、自然杀伤(NK)细胞、NKT细胞、调节性T细胞(Treg)和Th17细胞等.     

Canagliflozin Ameliorates Nonalcoholic Fatty Liver Disease by Regulating Lipid Metabolism and Inhibiting Inflammation through Induction of Autophagy

PurposeNonalcoholic fatty liver disease (NAFLD) is closely associated with metabolic diseases, including obesity and diabetes, and has gradually become the most common cause of chronic liver disease. We investigated the effects of sodium glucose cotransporter 2 (SGLT2) inhibitor canagliflozin on NAFLD in high-fat diet (HFD)-induced obese mice and possible underlying mechanisms.Materials and MethodsMale C57BL/6 mice were fed a normal-diet, HFD, or HFD with canagliflozin for 14 weeks. AML-12 hepatocytes were treated with canagliflozin. Expression of related pathways was assessed.ResultsCanagliflozin administration reduced body weight and fat mass, compared with HFD alone. Canagliflozin improved glucose and lipid metabolic disorders. Compared with HFD-fed mice, liver weight, serum alanine transaminase (ALT) levels, and hepatic lipid accumulation were decreased after canagliflozin administration. Additionally, canagliflozin upregulated lipolysis markers (CPT1a, ACOX1, and ACADM), downregulated lipogenesis markers (SREBP-1c and FASN), and suppressed the production of inflammatory cytokines (TNFα, MCP1, IL-1β, and IL-6), consistent with significantly increased LC3 II/I and Atg7 levels in the liver following canagliflozin treatment. In vitro, canagliflozin increased CPT1a, ACOX1, and ACADM expression, decreased SREBP-1c and FASN protein expression, and reduced TNFα, MCP1, IL-1β, and IL-6 mRNA levels in lipid mixture (LM)-induced hepatocytes in a dose-dependent manner. These changes were reversed by 3-MA, an autophagy inhibitor.ConclusionOur findings suggest that canagliflozin ameliorates the pathogenesis of NAFLD by regulating lipid metabolism and inhibiting inflammation, which may be associated with its promotion of autophagy.     

Label-free Assessment of the Nascent State of Rat Non-alcoholic Fatty Liver Disease Using Spontaneous Raman Microscopy

Spontaneous Raman microscopy, which can detect molecular vibrations in cells and tissues, could be a useful tool for the label-free assessment of non-alcoholic fatty liver disease (NAFLD). However, it is unclear whether it can be used to evaluate the nascent state of NAFLD. To address this, we analyzed the Raman spectra of rat liver tissues in the nascent state of NAFLD upon excitation at 532 nm. Raman and histochemical analyses were performed of liver tissues from rats fed a high-fat, high-cholesterol diet (HFHCD). Raman microscopic imaging analysis of formalin-fixed thin tissue slices showed hepatic steatosis, as revealed by the Raman band at 2,854 cm⁻¹, whereas lipid droplets were not detectable by hematoxylin-eosin staining of images until 3 days after feeding a HFHCD. Raman signals of retinol at 1,588 cm⁻¹ emitted from hepatic stellate cells were distributed alongside hepatic cords; the retinol content rapidly decreased after feeding a HFHCD, whereas hepatic lipid content increased inversely. Raman microscopic analysis of the surface of fresh ex vivo livers enabled early detection of lipid accumulation after a 1-day feeding a HFHCD. In conclusion, spontaneous Raman microscopy can be applied to the label-free evaluation of the nascent state of NAFLD liver tissues.     

血清APN及CG水平对非酒精性脂肪肝肝纤维化患者的诊断价值

目的：研究脂联素（APN）与甘胆酸（CG）的水平对非酒精性脂肪肝肝纤维化（NAFLD）诊断。方法：采用ELISA检测NAFLD患者和正常对照组的血清APN水平及放免法检测CG水平,并以NEFLD和正常对照组作比较。结果：NAFLD组APN水平显著低于正常对照组（P〈0.05）,CG水平显著高于正常对照组（P〈0.05）,APN水平与CG水平呈显著负相关（P〈0.05）。结论：NAFLD患者血清APN水平降低,APN的表达在NAFLD患者肝纤维化进程中可能起重要作用。     

Status of Essential Trace Minerals and Oxidative Stress in Viral Hepatitis C Patients with Nonalcoholic Fatty Liver Disease

Background: Nonalcoholic fatty liver disease (NAFLD) may be an important factor leading to altered trace mineral homeostasis, thereby accelerating the progression of hepatitis C virus (HCV) infection. Our aim was to determine whether NAFLD influenced the status of certain essential trace minerals and oxidative stress in chronic HCV-infected patients.Design and Methods: Blood biochemical parameters were determined in a group of 30 healthy, non-obese, non-diabetic participants (CNL group), and hepatitis C patients without NAFLD (HCV group, n = 30) and with NAFLD (HCV-NAFLD group, n = 32).Results: Concentrations of thiobarbituric acid reactive substances (TBARS; a measure of oxidative stress), C-reactive protein (CRP), ferritin, aminotransferases, lipid profiles, and insulin metabolism were markedly abnormal in both patient groups than in CNL subjects. Compared to patients in the HCV group, those with HCV-NAFLD group had lower high-density lipoprotein concentrations, higher low-density lipoprotein and homeostasis model assessment-insulin resistance (HOMA-IR) values, disrupted antioxidant enzyme activities, and elevated TBARS concentrations, as well as decreased plasma concentrations of trace minerals zinc (Zn) and selenium (Se) and increased copper (Cu). The alterations in mineral homeostasis were also linked to TBARS, CRP, ferritin, lipoproteins, and HOMA-IR values in the HCV-NAFLD group.Conclusions: There is a progressive deterioration in the homeostasis of minerals (Zn, Se, and Cu) in HCV-NAFLD patients, which may reflect greater oxidative stress and inflammation. These results suggest that the disturbance in mineral metabolism by NAFLD has an impact on the effectiveness of treatment for chronic HCV infection.     

The Role of Innate Immune Cells in Nonalcoholic Fatty Liver Disease

Nonalcoholic fatty liver disease (NAFLD) is a very common hepatic pathology featuring steatosis and is linked to obesity and related conditions, such as the metabolic syndrome. When hepatic steatosis is accompanied by inflammation, the disorder is defined as nonalcoholic steatohepatitis (NASH), which in turn can progress toward fibrosis development that can ultimately result in cirrhosis. Cells of innate immunity, such as neutrophils or macrophages, are central regulators of NASH-related inflammation. Recent studies utilizing new experimental technologies, such as single-cell RNA sequencing, have revealed substantial heterogeneity within the macrophage populations of the liver, suggesting distinct functions of liver-resident Kupffer cells and recruited monocyte-derived macrophages with regards to regulation of liver inflammation and progression of NASH pathogenesis. Herein, we discuss recent developments concerning the function of innate immune cell subsets in NAFLD and NASH.     

非酒精性脂肪肝患者血清AFP水平及其应用价值

目的 检测非酒精性脂肪肝（NAFLD）患者血清AFP水平,并探讨其应用价值.方法 对97例患者进行评估,患者通过做腹部超声诊断确定为脂肪肝,并分成3个组.无脂肪肝患者作为对照组.所有患者和对照组进行体格检查、肝功能检测、稳态模型法评估胰岛素抵抗指数（HOMA-IR）检测.结果 体质指数、AST、ALT、血糖、HOMA-IR等在NAFLD患者中高于对照组.TG、总胆固醇、LDL-C、HDL-C水平在NAFLD中高于对照组.AFP水平在NAFLD中比对照组显著升高（5.43±2.65 IU/mL vs 2.33±0.99 IU/mL,P＜0.05）.三级NAFLD AFP水平显著高于一级NAFLD （6.69±2.36 IU/mLVS 3.56±1.60 IU/mL,P＜0.05）和二级NAFLD AFP水平（6.69±2.36 IU/mL VS 4.27±1.79 IU/mL,P＜0.05）.另外,二级NAFLD AFP水平显著高于一级NAFLD（P＜0.05）.ALT、AST与其无相关性.结论 NAFLD患者AFP水平高于非脂肪肝患者,AFP水平与脂肪病分级有关,NAFLD分级与血清AFP水平的相关性独立于其它因素.     

血清YKL-40对非酒精性脂肪肝肝纤维化的预测作用

目的探讨非酒精性脂肪肝患者血清中YKL-40水平及在肝纤维化程度评价中的价值。方法2017年7月至2019年7月于我院就诊的NAFLD患者142例,根据是否发生肝纤维化分为纤维化组(n=60)与非纤维化组(n=82),另纳入40例健康体检者作为健康对照组,采用放射免疫分析法测定血清中透明质酸(HA)、III型前胶原(PC III)、IV型胶原(CIV)及层粘连蛋白(LN)水平。采用双夹心抗体ELISA法检测血清YKL-40水平。结果三组研究对象年龄、性别、BMI指数等一般性资料差异无统计学意义(P>0.05);健康对照组、S0~S4期血清YKL-40水平随着肝纤维化分期的不断增加而不断升高(P<0.05)。血清YKL-40水平与NFS、FIB-4、APRI评分均呈显著正相关关系,具有统计学意义(P<0.05)。ROC分析结果表明,当Cutoff值为158 ng/mL时,YKL-40对NAFLD患者肝纤维化诊断价值最高,其AUC、灵敏度、特异性、PPV、NPV等均高于传统肝纤维化指标,差异具有统计学意义(P<0.05)。结论血清YKL-40在非酒精性脂肪肝患者中水平明显升高,对肝纤维化具有良好的预测价值。     

肠道菌群与非酒精性脂肪肝关系的研究进展

近年来非酒精性脂肪性肝病的发病率不断增加,虽然属于慢性肝病,但是患者有发展为肝硬化的风险,为临床治疗带来了很多难题。本文综述非酒精性脂肪性肝病的发病机制,分析肠道菌群与非酒精性脂肪性肝病的因果关系,寻求潜在有效的新型治疗方案。