雷帕霉素与大鼠肝纤维化演变的相关研究

的肝纤维化动物模型大鼠应用雷帕霉素后,检测大鼠肝组织切片中转移生长因子（TGF-β1）表达,探讨雷帕霉素与肝纤维化病理演变的相关性。方法作者将实验大鼠随机分为3组：①正常对照组大鼠（A组）;②肝纤维模型组大鼠（B组）;③肝纤维模型灌注雷帕霉素组（C组）。C组大鼠按2mg/kg体重胃内灌注雷帕霉素,每天1次,持续2周。3组大鼠同期处死,取肝组织进行形态学及组织学研究分析。结果 3组大鼠肝组织切片经HE及免疫组织化等染色后,图片经Image-pro plus 6.0图像分析软件整理。A、B、C 3组大鼠肝组织切片中TGF-β1表达呈显著性差异,具有统计学意义（P〈0.01）。结论转化生长因子表达在肝纤维化的发生发展中具有重要的正相关性作用,实验中B组大鼠肝组织内TGF-β1高表达,加重大鼠肝组织的纤维进程,本实验显示雷帕霉素具有调控降低肝组织内TGF-β1的表达,缓解模型大鼠肝脏纤维变性的进程。     

Targeting Endothelial Erk1/2-Akt Axis as a Regeneration Strategy to Bypass Fibrosis during Chronic Liver Injury in Mice

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SWE评估非酒精性脂肪肝病大鼠肝纤维化程度

目的 探讨剪切波弹性成像(SWE)技术评估非酒精性脂肪肝病(NAFLD)大鼠肝纤维化程度的应用价值。 方法 SD大鼠88只,随机分为对照组(40只)和实验组(44只),对照组给予普通饲喂,实验组通过不同饮食建立不同纤维化分期的NAFLD大鼠模型,分别于第 1、2、3、8、12周末行大鼠肝脏SWE检查及病理分析。 结果 所有大鼠肝脏Emean与肝纤维化程度呈显著正相关(r=0.82,P＜0.001)。不同纤维化分期的Emean组间差异有统计学意义(F=25.5,P＜0.05);除了F1和F2组之间Emean没有显着差异(P＞0.05),其余任意两组之间的肝 Emean 比较差异均有统计学意义(均P＜0.05)。SWE诊断肝纤维化≥F1、≥F2、≥F3、＝F4的截值分别为6.3、7.4、8.6、9.7kPa,曲线下面积(AUC)分别为0.87、0.89、0.93、0.98,敏感性和特异性分别为83.3%、93.3%、100%、100%和85.5％、83.1%、91.3%、92.7%。 结论 SWE技术可有效评估NAFLD的肝纤维化程度。     

Targeting AngII/AT1R signaling pathway by perindopril inhibits ongoing liver fibrosis in rat

The renin–angiotensin system (RAS) has a substantial role in liver fibrosis, cirrhosis, and portal hypertension. Hence, targeting RAS through angiotensin‐converting enzyme (ACE) inhibitors can mend hepatic fibrosis; the current study was designed to examine the potential fibrosis inhibition activity of perindopril using a rat model of liver fibrosis induced by thioacetamide (TAA). Four groups of rats were used throughout this study, Group I (control group); rats received the vehicle. TAA was used for inducing liver fibrosis in rats by intraperitoneal injection of 200‐mg/kg body weight twice a week for 6 weeks. Group II served as (TAA group). Rats of Groups III and IV were given perindopril at doses of 2 and 8 mg/kg 2 weeks after TAA administration and continued concomitantly with TAA till the end of the experiment. Injection of TAA resulted in a significant increase in aminotransferases' activities and bilirubin with a significant decrease in serum albumin and total protein and a significant decrease in hepatic content of GSH and SOD. Additionally, TAA injection raised the hepatic content of TGF‐β1, α‐SMA, TNF‐α, and level of MDA. Histological and immunohistochemical data presented marked fibrosis in liver sections of TAA‐administrated rats with increased collagen deposition, elevated METAVIR scoring, and increased expression of α‐SMA, caspase‐3, and AT1R. Oral dosing of perindopril for 4 weeks concomitant with TAA could mend the altered parameters near to normal values and abolished the ongoing fibrosis extension. In conclusion, these results demonstrated that perindopril, as ACE inhibitor, could grant a superior remedial nominee in preventing liver fibrosis progression through targeting angiotensin II formation.     

sEVs from tonsil-derived mesenchymal stromal cells alleviate activation of hepatic stellate cells and liver fibrosis through miR-486-5p

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Transplanted fibroblast cell sheets promote migration of hepatic progenitor cells in the incised host liver in allogeneic rat model

Cell sheet engineering has been noted as a new and valuable approach in the tissue‐engineering field. The objective of this study was to explore a procedure to induce hepatic progenitor cells and biliary duct structures in the liver. Sprague–Dawley rat dermal fibroblast (DF) sheets were transplanted into the incised surface of the liver of F344 nude rats. In the control group, an incision was made without transplantation of the DF sheets. Bile duct (BD)‐like structures and immature hepatocyte‐like cells were observed in the DF sheet transplant sites. These BD‐like structures were cytokeratin‐8‐positive, while the hepatocyte‐like cells were both OV‐6‐positive and α‐fetoprotein‐positive as well. The proliferation and differentiation of liver progenitor cells were not influenced by hepatectomy. We also transplanted DF sheets transfected with a plasmid encoding the enhanced yellow fluorescent protein target to mitochondria (pEYFP–Mito) by electroporation, and found that the new structures were pEYFP–Mito‐negative. We observed new BD‐like structures and immature hepatocytes after transplantation of DF sheets onto incised liver surfaces, and clarified that the origin of these BD‐like structures and hepatocyte‐like cells was the recipient liver. The present study described an aspect of the hepatic differentiation process induced at the site of liver injury. Copyright © 2013 John Wiley & Sons, Ltd.     

剪切波弹性成像评估大鼠非酒精性脂肪肝病肝纤维化程度

目的探讨剪切波弹性成像(SWE)技术评估大鼠非酒精性脂肪肝病(NAFLD)肝纤维化程度的应用价值。方法将88只SD大鼠随机分为对照组(40只)和实验组(48只),对照组给予普通饲喂,实验组通过饮食调配建立不同肝纤维化分期的NAFLD模型,分别于第1、2、3、8、12周末随机抽取对照组大鼠8只,与预定喂养结束的实验组大鼠一起行SWE检查获取肝脏杨氏模量平均值(Emean),并处死行病理检查;绘制受试者工作特征(ROC)曲线分析Emean预测不同肝纤维化程度的诊断效能。结果肝纤维化F0期、F1期、F2期、F3期、F4期分别有52只、6只、6只、18只、6只,考虑建模成功,其对应的Emean分别为(5.46±0.37)kPa、(6.28±0.33)kPa、(6.42±0.41)kPa、(9.01±0.95)kPa及(15.88±2.49)kPa,不同肝纤维化分期Emean比较差异有统计学意义(F=25.5,P<0.05);除F1期与F2期比较差异无统计学意义外,其余任意两两比较差异均有统计学意义(均P<0.05)。所有大鼠肝脏Emean与肝纤维化程度呈显著正相关(r=0.82,P<0.001)。SWE诊断肝纤维化≥F1、≥F2、≥F3、F4期的截断值分别为6.3 kPa、7.4 kPa、8.6 kPa、9.7 kPa,曲线下面积分别为0.87、0.89、0.93、0.98,敏感性和特异性分别为83.3%、93.3%、100%、100%和85.5%、83.1%、91.3%、92.7%。结论SWE技术可有效评估大鼠NAFLD肝纤维化程度。     

Liver extracellular matrix promotes BM‐MSCs hepatic differentiation and reversal of liver fibrosis through activation of integrin pathway

In cell‐based therapies for liver injuries, the clinical outcomes are closely related to the surrounding microenvironment of the transplanted bone marrow mesenchymal stem cells (BM‐MSCs). However, whether liver‐specific ECM (L‐ECM), as one of major microenvironment signals, could regulate the therapeutic effect of BM‐MSCs through changing their biological characteristics is unclear. This study aimed to investigate the hepatogenicity and underlying mechanism of L‐ECM as well as its potential regulative role in the MSC‐based liver recovery. L‐ECM was prepared by homogenization of decellularized whole porcine liver. After three‐dimensional culture with or without the presence of L‐ECM, BM‐MSCs expressed hepatocyte‐specific genes and proteins in an L‐ECM concentration‐dependent manner. Further analysis showed that L‐ECM could activate specific types of integrins (ITGs) as well as their downstream signalling pathways. When the cell/ECM interaction was enhanced by incorporating BM‐MSCs with Mn²⁺, ITGs were activated and the hepatogenic capacity of L‐ECM was improved. The regeneration of rat livers from either acute or chronic fibrosis could also be accelerated after transplantation of Mn²⁺‐treated BM‐MSCs. L‐ECM therefore promotes hepatic differentiation of BM‐MSCs via the ITG pathway and plays a therapeutically beneficial role for stem cell‐based liver regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

骨髓基质细胞体外分化移植对局灶脑缺血大鼠分子表达及行为的影响

目的观察大鼠骨髓基质细胞分化为内皮细胞后静脉移植治疗大鼠永久大脑中动脉闭塞模型(MCAO)后缺血脑血管内皮细胞生长因子(VEGF)及其受体FLK-1的表达以及动物行为学改变。方法利用400 ng/ml重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)在体外诱导骨髓基质细胞分化为内皮细胞。线栓法制作永久SD大鼠MCAO模型(n=45),24 h后细胞移植组(n=15)舌静脉注射3×106分化后的内皮细胞,对照组1(n=15)注射等量PBS,对照组2(n=15)血管闭塞后不予任何处理。在细胞移植前及移植后1、3、5、7、14 d分别采用姿势反射实验、肢体不对称应用实验和角落实验观察大鼠动物行为学评分的变化,并通过免疫组化染色方法观察缺血脑组织血管内皮细胞生长因子(VEGF)及其受体FLK-1的表达。结果细胞移植组动物行为学评分优于对照组1、2,在移植后第7天和第14天较两个对照组有显著性差异(P<0.05);各组缺血侧脑组织缺血周边和皮层VEGF、FLK-1阳性细胞较非缺血侧增多(P<0.05),其中以细胞移植组最明显(P<0.05);VEGF主要表达在神经元、神经胶质细胞和部分内皮细胞,FLK-1主要表达在内皮细胞和部分神经元、神经胶质细胞;细胞移植组缺血侧毛细血管增生较对照组多(P<0.05)。结论骨髓基质细胞体外诱导分化后的内皮细胞静脉移植可以改善大鼠局灶脑缺血的神经功能;内皮细胞移植后可促进缺血脑组织VEGF、FLK-1的表达;动物神经功能的改善机制可能与VEGF、FLK-1的表达增加,内皮细胞及毛细血管增殖有关。     

Abnormal chemokine-induced responses of immature and mature hematopoietic cells from motheaten mice implicate the protein tyrosine phosphatase SHP-1 in chemokine responses.

Chemokines regulate a number of biological processes, including trafficking of diverse leukocytes and proliferation of myeloid progenitor cells. SHP-1 (Src homology 2 domain tyrosine phosphatase 1), a phosphotyrosine phosphatase, is considered an important regulator of signaling for a number of cytokine receptors. Since specific tyrosine phosphorylation of proteins is important for biological activities induced by chemokines, we examined the role of SHP-1 in functions of chemokines using viable motheaten (me(v)/me(v)) mice that were deficient in SHP-1. Chemotactic responses to stromal call-derived factor 1 (SDF-1), a CXC chemokine, were enhanced with bone marrow myeloid progenitor cells as well as macrophages, T cells, and B cells from me(v)/me(v) versus wild-type (+/+) mice. SDF-1-dependent actin polymerization and activation of mitogen-activated protein kinases were also greater in me(v)/me(v) versus +/+ cells. In contrast, immature subsets of me(v)/me(v) bone marrow myeloid progenitors were resistant to effects of a number of chemokines that suppressed proliferation of +/+ progenitors. These altered chemokine responses did not appear to be due to enhanced expression of CXCR4 or lack of chemokine receptor expression. However, expression of some chemokine receptors (CCR1, CCR2, CCR3, and CXCR2) was significantly enhanced in me(v)/me(v) T cells. Our results implicate SHP-1 involvement in a number of different chemokine-induced biological activities.     

原发性肝癌患者血清sTRAIL、MMP-3水平及其与肝纤维化指标的相关性

目的分析原发性肝癌患者血清可溶性肿瘤细胞坏死因子相关凋亡诱导配体(sTRAIL)、基质金属蛋白酶-3(MMP-3)水平,以及其与肝纤维化指标的相关性。方法 52例原发性肝癌患者纳入原发性肝癌组,63例良性肿瘤患者纳入良性肿瘤组,39例健康体检者纳入对照组,比较各组血清sTRAIL、MMP-3及肝纤维化指标水平,并分析血清sTRAIL、MMP-3及肝纤维化指标[透明质酸(HA)、层黏蛋白(LN)、Ⅲ型前胶原肽(PCⅢ)、Ⅳ型胶原(Ⅳ-C)]的相关性,绘制受试者工作特征(ROC)曲线分析血清sTRAIL、MMP-3水平对原发性肝癌的诊断价值。结果原发性肝癌组血清sTRAIL、MMP-3水平高于良性肿瘤组及对照组,良性肿瘤组血清sTRAIL、MMP-3水平高于对照组,原发性肝癌组HA、LN、PCⅢ、Ⅳ-C水平高于良性肿瘤组及对照组,差异均有统计学意义(P<0.05);良性肿瘤组与对照组的HA、LN、PCⅢ、Ⅳ-C水平比较,差异无统计学意义(P>0.05)。TNM分期Ⅰ期、Ⅱ期、Ⅲ期患者血清sTRAIL、MMP-3、HA、LN、PCⅢ、Ⅳ-C水平依次升高,两两比较,差异有统计学意义(P<0.05)。ROC曲线分析结果显示,sTRAIL、MMP-3、sTRAIL+MMP-3的ROC曲线下面积分别为0.686、0.730、0.806。相关分析显示,原发性肝癌患者血清sTRAIL、MMP-3与HA、LN、PCⅢ、Ⅳ-C之间均呈正相关(r=0.501~0.651,P<0.05)。结论血清sTRAIL、MMP-3可能参与原发性肝癌的发生、发展,且与肝纤维化指标呈正相关,对原发性肝癌的早期诊断有重要价值。     

慢性乙型肝炎肝组织中星状细胞与肝纤维化关系的研究

目的研究肝星状细胞(HSC)与肝纤维化和肝硬化的关系.方法检测60例慢性乙型肝炎患者肝功能及乙型肝炎e抗原(HBeAg),所有患者均进行了肝组织活检,其中S1 10例, S2 24例, S3 12例,S4 14例;采用免疫组织化学法,通过应用特殊的单克隆抗体(McAb)检测慢性乙型肝炎患者肝组织石蜡切片中α-平滑肌肌动蛋白(α-SMA)阳性细胞(即HSC).结果苦味酸天狼猩红染色法胶原面积的百分比与肝纤维化分期有关(r=0.885,P＜0.01);HSC与慢性乙型肝炎患者肝组织的纤维化分期呈正相关(r=0.761,P＜0.01);HSC与慢性乙型肝炎患者肝组织中苦味酸天狼猩红染色法胶原面积的百分比正相关(r=0.854,P＜0.01).结论 HSC与慢性乙型肝炎肝纤维化和肝硬化呈正相关.     

Expression of platelet‐bound stromal cell‐derived factor‐1 in patients with non‐valvular atrial fibrillation and ischemic heart disease

Summary. Aims: Blood cell infiltration and inflammation are involved in atrial remodelling during atrial fibrillation (AF) although the exact mechanisms of inflammatory cell recruitment remain poorly understood. Platelet‐bound stromal cell‐derived factor‐1 (SDF‐1) is increased in cases of ischemic myocardium and regulates recruitment of CXCR4⁺ cells on the vascular wall. Whether platelet‐bound SDF‐1 expression is differentially influenced by non‐valvular paroxysmal or permanent atrial fibrillation (AF) in patients with stable angina pectoris (SAP) or acute coronary syndrome (ACS) has not been reported so far. Methods and results: A total of 1291 consecutive patients with coronary artery disease (CAD) undergoing coronary angiography were recruited. Among the patients with SAP, platelet‐bound‐SDF‐1 is increased in patients with paroxysmal AF compared with SR or to persistent/permanent AF (P < 0.05 for both). Platelet‐bound SDF‐1 correlated with plasma SDF‐1 (r = 0.488, P = 0.013) in patients with AF and ACS, which was more pronounced among patients with persistent AF (r = 0.842, P = 0.009). Plasma SDF‐1 was increased in persistent/permanent AF compared with SR. Patients with ACS presented with enhanced platelet‐bound‐SDF‐1 compared with SAP. Interestingly, among patients with ACS, patients with paroxysmal or persistent/permanent AF presented with an impaired platelet‐bound SDF‐1 expression compared with patients with SR. Conclusions: Differential expression of platelet‐bound and plasma SDF‐1 was observed in patients with AF compared with SR which may be involved in progenitor cell mobilization and inflammatory cell recruitment in patients with AF and ischemic heart disease. Further in vivo studies are required to elucidate the role of SDF‐1 in atrial remodeling and the atrial fibrillation course.     

犬肝纤维化MR弥散加权成像及VEGF表达与病理对照研究

目的：分析磁共振弥散加权成像参数ADC值、eADC值及VEGF表达水平在中华田园犬肝纤维化进程中的变化规律;并与肝脏组织常规病理结构改变进行对照分析,探讨肝纤维化的诊断方法。方法：采用腹腔注射50%四氯化碳（CCL4）油溶液配合高脂饮食,建立犬肝纤维化的动物模型,定期对其行MR常规扫描、DWI及肝穿刺,对肝脏组织进行常规病理学染色及VEGF免疫组织化学染色,根据病理分期分组,分析各组ADC值、eADC值及VEGF表达水平的变化规律及与肝纤维化的相关性。结果：各期肝纤维化模型：S0 （n=12）、S1 （n=9）、S2 （n=9）、S3 （n=10）、S4 （n=10）;纤维化程度加重,ADC值减低、eADC值增高,差异均有统计学意义（P〈0.05）。ADC值及eADC值与肝纤维化程度有很好的相关性;随肝纤维化程度加重,VEGF表达水平明显增高。结论：四氯化碳油溶液腹腔注射配合高脂饮食可以成功建立不同分期犬肝纤维化模型。ADC值及eADC值能够定量分析不同程度的肝纤维化,是诊断不同程度纤维化较有意义的评价指标。VEGF可能在慢性肝病所致肝纤维化过程中起重要作用。     

SDF-1基因沉默对脑缺血后强制性运动疗法调控海马齿状回神经发生过程的影响

目的探讨脑缺血后强制性运动疗法对海马齿状回的神经干细胞的影响。方法应用免疫组化并结合行为学方法,探讨强制性运动疗法对脑缺血后海马齿状回神经干细胞及SDF-1基因沉默对该过程的作用。结果脑缺血后强制性运动疗法能够促进大鼠学习记忆功能的恢复,增加大鼠海马齿状回新生神经干细胞的数量,而SDF-1基因沉默后会逆转强制性运动疗法该作用。结论SDF-1／CXCR4在脑缺血后强制性运动疗法调控海马齿状回神经发生的过程中起到了重要的作用。     

赖氨大黄酸对胆汁淤积性肝纤维化大鼠Smad2、Smad3表达的影响

目的探讨赖氨大黄酸(RHL)对胆汁淤积性肝纤维化模型大鼠Smad2、Smad3表达的影响。方法采用胆总管结扎(BDL)的手术方法建立大鼠胆汁淤积性肝纤维化模型,将大鼠随机分为5组:对照组、模型组、赖氨酸组、低剂量RHL组[35mg/(kg·d)]、高剂量RHL组[70mg/(kg·d)]。应用实时荧光定量PCR的方法检测大鼠肝脏组织中Smad2、Smad3mRNA的相对表达量。Western blot的方法检测Smad2、Smad3蛋白时相对表达量的变化。结果与模型组大鼠比较,经RHL治疗后胆汁淤积性肝纤维化大鼠肝脏组织中Smad2、Smad3mRNA相对表达量降低,差异有统计学意义(P0.05);与模型组大鼠相比,经RHL治疗后胆汁淤积性肝纤维化大鼠肝脏组织中Smad2、Smad3蛋白的相对表达量降低,差异有统计学意义(P0.05)。结论 RHL能够抑制肝纤维化的发展,可能是通过抑制Smad2、Smad3mRNA和蛋白的表达,阻断TGF-β1/Smads信号传导通路实现的。     

应用FibroTouch技术评价2型糖尿病肾病患者肝脏脂肪变性和纤维化程度及二者相关性

目的 利用FibroTouch技术,评价2型糖尿病肾病（DN）患者肝脏脂肪变性和肝脏纤维化的程度及二者的相关性。方法 选取2型糖尿病肾病患者90例。根据肾小球滤过率（eGFR）将其分为三组,eGFR≥90ml?min-1(1.73m2)-1为A组(30例),60ml?min-1?(1.73m2)-1≤eGFR＜90ml?min-1?(1.73m2)-1为B组（30例）,eGFR＜60 ml?min-1?(1.73m2)-1为C组（30例）,同时选取30名健康志愿者为对照组。应用FibroTouch技术获得肝脏脂肪衰减值（db/m）和肝脏硬度值（Kpa）。结果 各组肝脏脂肪衰减值和肝脏硬度值比较,A、B、C组均明显高于对照组（P均＜0.05）,B、C组均高于A组（P均＜0.05）,C组高于B组（P＜0.05）;肝脏脂肪衰减值和肝脏硬度值呈正相关关系（r=0.733,P＜0.01）。结论 应用FibroTouch技术可以准确、无创地评价DN患者的肝脏脂肪变性和纤维化程度,具有重要的临床意义。     

Genesis of hepatic fibrosis and its biochemical markers

Liver fibrosis is characterized by an abnormal hepatic accumulation of extracellular matrix (ECM) that results from both increased deposition and reduced degradation of collagen fibres. Fibrotic liver injury results in activation of the hepatic stellate cell (HSC). Surrogate markers are gradually being substituted for biomarkers that reflect the complex balance between synthesis and degradation of the extracellular matrix. Once the hepatic stellate cell is activated, the preceding matrix changes and recurrent injurious stimuli will perpetuate the activated state. The ECM directs cellular differentiation, migration, proliferation and fibrogenic activation or deactivation. The metabolism of the extracellular matrix is closely regulated by matrix metalloproteinases (MMP) and their specific tissue inhibitors (TIMP). Although liver biopsy combined with connective tissue stains has been a mainstay of diagnosis, there is a need for less invasive methods. These diagnostic markers should be considered in combination with liver function tests, ultrasonography and clinical manifestations.     

脑缺血后强制性运动疗法对内源性神经干细胞及基质细胞衍生因子-1水平的影响

目的目前强制性运动疗法广泛地应用于康复领域,并被证实能够有效地促进肢体功能恢复,但其确切的机制尚不清楚。方法应用免疫组化及ELISA方法并结合行为学探讨了强制性运动疗法对脑缺血后内源性神经干细胞及间质细胞源性因子-1表达的影响。结果发现脑缺血后强制性运动疗法能够促进患侧肢体运动功能的恢复,增加大鼠侧脑室下区新生神经干细胞的数量,并能提高脑组织基质细胞衍生因子-1蛋白的表达水平。结论强制性运动疗法可能通过影响脑缺血后的神经再生过程从而达到增强中枢神经系统修复能力的作用。