脂肪型脂肪酸结合蛋白在大鼠肝纤维化组织的表达及意义

发布时间：2018-06-04 01:05

本文选题：Wistar大鼠 + 联苯双酯　；参考：《河北医科大学》2014年硕士论文

【摘要】：目的：肝纤维化是各种慢性肝病向肝硬化发展的共同病理阶段，过度和异常细胞外基质沉积是其组织学特征，表现为肝窦毛细血管化、肝小叶内及汇管区纤维组织增生。与肝硬化不同，肝纤维化阶段可逆转，因此肝纤维化发生发展机制的研究是目前肝病研究的热点之一。脂肪型脂肪酸结合蛋白(adipocyte fatty acid binding protein,A-FABP,FABP4)是脂肪酸结合蛋白家族9个成员中最具特征的一员，FABP4不仅在脂肪细胞和巨噬细胞表达，还表达于全身多种脏器微血管内皮细胞。研究发现巨噬细胞和脂肪细胞中FABP4的缺失可降低炎症性转录因子信号和多种促炎反应。这种炎症-代谢反应促进脂肪炎症和胰岛素抵抗，同时也促进动脉粥样硬化和心血管疾病的发生。我们前期的研究结果发现：正常大鼠和人的多种组织的内皮细胞和巨噬细胞表达FABP4，但肝脏枯否氏细胞，小血管和肝窦内皮细胞均未见其表达。人乙肝纤维化和肝硬化组织部分小血管内皮细胞表达FABP4，肝纤维化程度增加其表达增加。这种表达形式提示FABP4在肝损伤过程中发挥作用。四氯化碳(Carbon tetrachloride,CCl4)诱导的肝纤维化大鼠模型是经典的化学性肝损伤后的肝纤维化模型，联苯双酯是公认治疗肝纤维化药物，常作为评价肝纤维化药物疗效的阳性对照药。本实验用免疫组化和Western blot法检测CCl4性肝纤维化和联苯双酯治疗后大鼠肝组织中FABP4的表达和变化，关注肝组织损伤及修复过程中FABP4，特别是内皮细胞中的FABP4与纤维化消长的关系，试图为揭示或阐明肝纤维化的发生发展和转归提供新视角。方法： 1大鼠CCl4肝纤维化模型和联苯双酯干预模型的建立 30只Wistar大鼠随机分为3组，正常对照组(Control组)、四氯化碳模型组(CCl4组)及联苯双酯干预组(干预组)，每组10只，雌雄各半。CCl4组和干预组大鼠皮下给予40%四氯化碳花生油溶液(首次剂量0.5ml/100g，以后0.3ml/100g)；Control组皮下给予等量花生油溶液。同时干预组给予2mg/kg的联苯双酯灌胃，其他两组大鼠羧甲基纤维素钠灌胃，连续6周。三组停止给予CCl4或花生油后继续灌胃2周。CCl4组和干预组在给予CCl4次日饮用乙醇水、Control组饮用常规净化水各8周。 2血天门冬氨酸氨基转移酶(Aspartate aminotransferase,AST),丙氨酸氨基转移酶(Alanine aminotransferase,ALT)测定腹主动脉取血并常规分离血清，血清于-80℃保存用于血AST、ALT的测定。 3肝脏形态学观察放血处死动物，观察肝脏、脾脏，称重，计算肝脏、脾脏系数。肝组织常规石蜡包埋、切片、HE染色。观察肝脏组织学改变。 4免疫组织化学检测FABP4、ED1和α-SMA的表达免疫组织化学检测石蜡切片中FABP4(PV二步法)，ED1和α-SMA(α-smooth muscle actin)(SP三步法)在各组肝组织的表达，免疫荧光双标(一抗非共孵育法)检测FABP4和ED1共表达。 5FABP4、ED1和α-SMA表达的定量分析采用OLYMPUS BX63显微成像系统，每张切片照相后使用Image-ProPlus6.0软件对图片进行FABP4定量分析。ED1和α-SMA采用阳性细胞计数法：每张切片计数高倍镜下(400倍)10个视野肝小叶内有核阳性细胞数。 6Western blot法进行FABP4和α-SMA半定量分析结果： 1成功建立大鼠CCl4肝纤维化模型和联苯双酯干预组模型 (1)肝脏形态学改变：与Control组比较，CCl4组肝脏色黄，质地变硬，表面粗糙呈颗粒状。干预组大鼠肝脏色粉红，表面没有明显颗粒状物质，质地较CCl4组软。CCl4组肝脏系数(3.74±0.73)较Control组(2.54±0.26)高(P0.01)；干预组肝脏系数(3.00±0.28)较CCl4组明显下降(P0.05),但仍高于Control组(P0.05)。脾脏系数三组间未见差异。 (2)组织学改变：CCl4组正常肝小叶结构破坏，肝细胞水样和脂肪变性、灶状坏死及炎细胞浸润，增生的纤维组织从汇管区呈星芒状伸向肝小叶。干预组肝小叶结构基本清晰，肝细胞的变性、坏死及炎细胞浸润较CCl4组明显减轻，汇管区可见少量纤维增生。 (3)血清AST、ALT：CCl4组血清AST和ALT水平较Control组显著升高[AST:394±160(CCl4组),189±99(Control组),P0.05;ALT:335±252(CCl4组),73±55(Control组),P0.01]。干预组AST和ALT较CCl4组显著下降(干预组AST:183±71,P0.01;ALT:66±31,P0.05)，与Control组相比无显著差异。 2大鼠肝组织FABP4的表达变化免疫组化结果，Control组肝组织几乎未见FABP4的表达；CCl4组肝小叶内坏死区和纤维间隔内可见单个散在分布的FABP4阳性细胞，部分小血管和肝窦内皮细胞表达FABP4；干预组FABP4的表达方式与CCl4组相似。FABP4(绿色)和ED1(红色)的免疫荧光双标显示：单个散在分布的FABP4阳性细胞为枯否氏细胞(黄色荧光)。定量分析结果：CCl4组肝组织FABP4积分光密度(Integra optical density,IOD)(70367.99,81339.00)比Control组(285.63,302.00)有意义升高(P0.05)；干预组IOD值(6123.50,8609.00)则明显低于CCl4组(P0.05)，但仍高于Control组(P0.05)。WesternBlot分析证实CCl4组肝组织FABP4的表达明显高于Control组；联苯双酯干预后FABP4的表达量有意义下降,但仍高于Control组(P均0.05)。 3大鼠肝脏组织ED1和α-SMA的表达变化 Control组ED1阳性细胞位于肝窦和汇管区。CCl4组肝窦、汇管区阳性细胞增多、坏死区可见阳性细胞。干预组表达形式与CCl4组基本相同。细胞计数结果：CCl4组阳性细胞数(47.00,27.00)明显高于Control组(7.00,7.00)(P0.05)；联苯双酯干预(23.00,14.00)可明显降低CCl4引起的ED1阳性细胞数(P0.05)，但仍高于Control组(P0.05)。CCl4组的α-SMA表达于肝窦、纤维间隔、坏死灶及小血管平滑肌。干预组α-SMA表达形式与CCl4组基本相同。细胞计数结果：与Control组(0.00,0.00)相比，CCl4组α-SMA阳性细胞有意义增多(25.50,49.00)(P0.05)；联苯双酯干预(1.00,3.00)能明显降低CCl4引起的α-SMA阳性表达(P0.05)，但仍高于Control组(P0.05)。Western Blot分析证实，CCl4组肝组织α-SMA的表达明显高于Control组，与CCl4组相比，联苯双酯干预后α-SMA的表达量有意义下降，但仍高于Control组(P均0.05)。结论： 1与正常肝组织比较，肝纤维化组织中FABP4呈现出阳性表达；阳性表达的细胞为枯否氏细胞和小血管内皮细胞。这种与生理状态下完全不同的表达方式和表达量的变化提示FABP4作为功能蛋白在肝炎症性损伤和修复过程中扮演了重要角色。 2联苯双酯干预明显降低CCl4诱发的FABP4表达和ED1阳性细胞数，，同时α-SMA表达有意义降低，提示联苯双酯抑制炎症和纤维化的作用可能至少部分是通过抑制枯否氏细胞中FABP4的表达来完成。 3肝小血管和肝窦内皮细胞中FABP4在肝纤维化组织的高表达与肝损伤和修复之间的关系及机制需要进一步深入研究。
[Abstract]:Objective : Hepatic fibrosis is a common pathological stage in the development of chronic liver disease to liver cirrhosis .

Adipose fatty acid binding protein ( A - FABP 501 ) is one of the most important members of 9 members of fatty acid binding protein family .

The results of our previous studies showed that there were no expression in endothelial cells and macrophages of normal rats and human tissues , but no expression was found in the cells , small vessels and hepatic sinusoidal endothelial cells in normal rats and human tissues .

The rat model of liver fibrosis induced by carbon tetrachloride ( CCl _ 4 ) was a classic model of hepatic fibrosis induced by hepatic fibrosis . Bifendate was a positive control drug for the treatment of hepatic fibrosis .

Method :

Establishment of the Model of Hepatic Fibrosis and Biphenyl Diester Intervention in Rats

Thirty Wistar rats were randomly divided into 3 groups , normal control group ( control group ) , carbon tetrachloride model group ( CC4 group ) and bifendate intervention group ( intervention group ) .
Control group was given an equal amount of peanut oil solution subcutaneously . At the same time , 2 mg / kg of bifendate was administered to the intervention group and the other two groups of rats were perfused with sodium carboxymethyl cellulose for 6 weeks .

Aspartate aminotransferase ( AST ) , alanine aminotransferase ( ALT ) assay

Serum was taken from the abdominal aorta and the serum was routinely separated , and the serum was stored at - 80.degree . C . for the determination of AST and ALT .

3 . Morphological observation of liver

Animals were sacrificed and the liver , spleen and weight were observed and the liver and spleen coefficients were calculated . Liver tissues were paraffin - embedded , sectioned and HE stained . Histological changes in liver were observed .

Expression of 4 , ED1 and 伪 - SMA in Immunohistochemical Detection

Immunohistochemical staining was used to detect the co - expression of FP4 ( PV two - step method ) , ED1 and 伪 - SMA ( 伪 - smooth muscle actin ) ( SP three - step method ) in the liver tissues of each group .

Quantitative Analysis of the Expression of 5BP4 , ED1 and 伪 - SMA

Image - ProPlus6 . 0 software was used to quantitatively analyze the images of the images after each slice was photographed . ED1 and 伪 - SMA were counted by positive cells , and the number of nuclear positive cells was counted in 10 visual liver lobules per slice count ( 400 times ) .

Semi - quantitative Analysis of 伪 - SMA by Western blot

Results :

1 successfully established the model of rat liver fibrosis and the model of biester intervention group .

( 1 ) Morphological changes of the liver : Compared with the control group , the liver color yellow , the texture and the rough surface of the CCl 4 group were granular . The liver color of the rats in the intervention group was pink , the surface was not obviously granular , the texture was softer than that of the CC4 group , and the liver coefficient ( 3.74 卤 0.73 ) was higher than that in the control group ( 2.54 卤 0.26 ) ( P0.01 ) .
The liver coefficient of the intervention group ( 3.00 卤 0.28 ) was significantly lower than that in the group ( P0.05 ) , but it was still higher than that in the control group ( P0.05 ) . There was no difference among the three groups .

( 2 ) Histological changes : normal hepatic lobule structure destruction , hepatocyte watery and fatty degeneration , focal necrosis and inflammatory cell infiltration in the CCl _ 4 group . The fibrous tissue of hyperplasia was extended from the manifold area to the hepatic lobule . The structure of the hepatic lobule in the intervention group was basically clear , the degeneration of the hepatocytes , necrosis and inflammatory cell infiltration were significantly reduced compared with that of the CC14 group , and a small amount of fiber hyperplasia was seen in the manifold area .

( 3 ) The AST and ALT levels of serum AST and ALT were significantly higher in the control group than in the control group . The AST and ALT levels decreased significantly in the control group ( control group ) , P < 0.05 ; ALT : 335 卤 252 ( CCl _ 4 group ) , 73 卤 55 ( Control group ) , P0.01 ) . There was no significant difference in AST and ALT in the intervention group compared with the control group ( AST : 183 卤 71 , P0.01 ; ALT : 66 卤 31 , P0.05 ) .

Expression Change of Liver Tissue in Rats in 2 Rats

The results of immunohistochemistry showed that the liver tissues of the control group were almost not expressed .
There was a single scattered distribution in the hepatic lobule necrosis area and the fiber interval in the CC14 group , and the expression of the partial small blood vessels and the endothelial cells of the hepatic sinus was increased .
The results of quantitative analysis showed that the integral optical density ( IOD ) ( 70367.99 , 81339.00 ) increased significantly ( P < 0.05 ) than Control group ( 285.63 , 302.00 ) .
The IOD value ( 6123.50 , 8609 . 00 ) in the intervention group was significantly lower than that of the control group ( P0.05 ) .
Compared with control group ( P < 0.05 ) .

Expression of ED1 and 伪 - SMA in Liver Tissue of Rats

Results : The number of positive cells ( 47.00 , 27.00 ) was significantly higher than that in control group ( 7.00 , 7.00 ) ( P0.05 ) .
Results : Compared with control group ( 0.00 , 0.00 ) , 伪 - SMA positive cells increased significantly ( 25.50 , 49.00 ) compared with control group ( 0.00 , 0.00 ) ( P0.05 ) .
The expression of 伪 - SMA was significantly higher than that in control group ( P < 0.05 ) , but the expression of 伪 - SMA was significantly lower than that of control group ( P < 0.05 ) .

Conclusion :

1 Compared with normal liver tissues , the positive expression was found in the liver fibrosis tissue .
The expression pattern and the expression level of the cells which are completely different from the physiological state suggest that FP4 plays an important role in the treatment of inflammatory injury and repair of the liver .

2 - Biphenyl biester intervention significantly reduced the expression and number of ED1 - positive cells and the expression of 伪 - SMA at the same time , suggesting that the role of bifendate in inhibiting inflammation and fibrosis could be accomplished , at least in part , by inhibition of the expression of s4P4 in cuffer cells .

The relationship and mechanism between liver injury and liver injury and liver injury were further studied in three hepatic microvessels and hepatic sinusoidal endothelial cells .
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R575

【参考文献】