發(fā)布時(shí)間：2018-06-04 01:05

  本文選題：Wistar大鼠 + 聯(lián)苯雙酯��； 參考：《河北醫(yī)科大學(xué)》2014年碩士論文

【摘要】：目的：肝纖維化是各種慢性肝病向肝硬化發(fā)展的共同病理階段，過度和異常細(xì)胞外基質(zhì)沉積是其組織學(xué)特征，表現(xiàn)為肝竇毛細(xì)血管化、肝小葉內(nèi)及匯管區(qū)纖維組織增生。與肝硬化不同，肝纖維化階段可逆轉(zhuǎn)，因此肝纖維化發(fā)生發(fā)展機(jī)制的研究是目前肝病研究的熱點(diǎn)之一。 脂肪型脂肪酸結(jié)合蛋白(adipocyte fatty acid binding protein,A-FABP,FABP4)是脂肪酸結(jié)合蛋白家族9個(gè)成員中最具特征的一員，F(xiàn)ABP4不僅在脂肪細(xì)胞和巨噬細(xì)胞表達(dá)，還表達(dá)于全身多種臟器微血管內(nèi)皮細(xì)胞。研究發(fā)現(xiàn)巨噬細(xì)胞和脂肪細(xì)胞中FABP4的缺失可降低炎癥性轉(zhuǎn)錄因子信號(hào)和多種促炎反應(yīng)。這種炎癥-代謝反應(yīng)促進(jìn)脂肪炎癥和胰島素抵抗，同時(shí)也促進(jìn)動(dòng)脈粥樣硬化和心血管疾病的發(fā)生。 我們前期的研究結(jié)果發(fā)現(xiàn)：正常大鼠和人的多種組織的內(nèi)皮細(xì)胞和巨噬細(xì)胞表達(dá)FABP4，但肝臟枯否氏細(xì)胞，小血管和肝竇內(nèi)皮細(xì)胞均未見其表達(dá)。人乙肝纖維化和肝硬化組織部分小血管內(nèi)皮細(xì)胞表達(dá)FABP4，肝纖維化程度增加其表達(dá)增加。這種表達(dá)形式提示FABP4在肝損傷過程中發(fā)揮作用。 四氯化碳(Carbon tetrachloride,CCl4)誘導(dǎo)的肝纖維化大鼠模型是經(jīng)典的化學(xué)性肝損傷后的肝纖維化模型，聯(lián)苯雙酯是公認(rèn)治療肝纖維化藥物，常作為評(píng)價(jià)肝纖維化藥物療效的陽性對(duì)照藥。本實(shí)驗(yàn)用免疫組化和Western blot法檢測(cè)CCl4性肝纖維化和聯(lián)苯雙酯治療后大鼠肝組織中FABP4的表達(dá)和變化，關(guān)注肝組織損傷及修復(fù)過程中FABP4，特別是內(nèi)皮細(xì)胞中的FABP4與纖維化消長(zhǎng)的關(guān)系，試圖為揭示或闡明肝纖維化的發(fā)生發(fā)展和轉(zhuǎn)歸提供新視角。 方法： 1大鼠CCl4肝纖維化模型和聯(lián)苯雙酯干預(yù)模型的建立 30只Wistar大鼠隨機(jī)分為3組，正常對(duì)照組(Control組)、四氯化碳模型組(CCl4組)及聯(lián)苯雙酯干預(yù)組(干預(yù)組)，每組10只，雌雄各半。CCl4組和干預(yù)組大鼠皮下給予40%四氯化碳花生油溶液(首次劑量0.5ml/100g，以后0.3ml/100g)；Control組皮下給予等量花生油溶液。同時(shí)干預(yù)組給予2mg/kg的聯(lián)苯雙酯灌胃，其他兩組大鼠羧甲基纖維素鈉灌胃，連續(xù)6周。三組停止給予CCl4或花生油后繼續(xù)灌胃2周。CCl4組和干預(yù)組在給予CCl4次日飲用乙醇水、Control組飲用常規(guī)凈化水各8周。 2血天門冬氨酸氨基轉(zhuǎn)移酶(Aspartate aminotransferase,AST),丙氨酸氨基轉(zhuǎn)移酶(Alanine aminotransferase,ALT)測(cè)定 腹主動(dòng)脈取血并常規(guī)分離血清，血清于-80℃保存用于血AST、ALT的測(cè)定。 3肝臟形態(tài)學(xué)觀察 放血處死動(dòng)物，觀察肝臟、脾臟，稱重，計(jì)算肝臟、脾臟系數(shù)。肝組織常規(guī)石蠟包埋、切片、HE染色。觀察肝臟組織學(xué)改變。 4免疫組織化學(xué)檢測(cè)FABP4、ED1和α-SMA的表達(dá) 免疫組織化學(xué)檢測(cè)石蠟切片中FABP4(PV二步法)，ED1和α-SMA(α-smooth muscle actin)(SP三步法)在各組肝組織的表達(dá)，免疫熒光雙標(biāo)(一抗非共孵育法)檢測(cè)FABP4和ED1共表達(dá)。 5FABP4、ED1和α-SMA表達(dá)的定量分析 采用OLYMPUS BX63顯微成像系統(tǒng)，每張切片照相后使用Image-ProPlus6.0軟件對(duì)圖片進(jìn)行FABP4定量分析。ED1和α-SMA采用陽性細(xì)胞計(jì)數(shù)法：每張切片計(jì)數(shù)高倍鏡下(400倍)10個(gè)視野肝小葉內(nèi)有核陽性細(xì)胞數(shù)。 6Western blot法進(jìn)行FABP4和α-SMA半定量分析 結(jié)果： 1成功建立大鼠CCl4肝纖維化模型和聯(lián)苯雙酯干預(yù)組模型 (1)肝臟形態(tài)學(xué)改變：與Control組比較，CCl4組肝臟色黃，質(zhì)地變硬，表面粗糙呈顆粒狀。干預(yù)組大鼠肝臟色粉紅，表面沒有明顯顆粒狀物質(zhì)，質(zhì)地較CCl4組軟。CCl4組肝臟系數(shù)(3.74±0.73)較Control組(2.54±0.26)高(P0.01)；干預(yù)組肝臟系數(shù)(3.00±0.28)較CCl4組明顯下降(P0.05),但仍高于Control組(P0.05)。脾臟系數(shù)三組間未見差異。 (2)組織學(xué)改變：CCl4組正常肝小葉結(jié)構(gòu)破壞，肝細(xì)胞水樣和脂肪變性、灶狀壞死及炎細(xì)胞浸潤(rùn)，增生的纖維組織從匯管區(qū)呈星芒狀伸向肝小葉。干預(yù)組肝小葉結(jié)構(gòu)基本清晰，肝細(xì)胞的變性、壞死及炎細(xì)胞浸潤(rùn)較CCl4組明顯減輕，匯管區(qū)可見少量纖維增生。 (3)血清AST、ALT：CCl4組血清AST和ALT水平較Control組顯著升高[AST:394±160(CCl4組),189±99(Control組),P0.05;ALT:335±252(CCl4組),73±55(Control組),P0.01]。干預(yù)組AST和ALT較CCl4組顯著下降(干預(yù)組AST:183±71,P0.01;ALT:66±31,P0.05)，與Control組相比無顯著差異。 2大鼠肝組織FABP4的表達(dá)變化 免疫組化結(jié)果，Control組肝組織幾乎未見FABP4的表達(dá)；CCl4組肝小葉內(nèi)壞死區(qū)和纖維間隔內(nèi)可見單個(gè)散在分布的FABP4陽性細(xì)胞，部分小血管和肝竇內(nèi)皮細(xì)胞表達(dá)FABP4；干預(yù)組FABP4的表達(dá)方式與CCl4組相似。FABP4(綠色)和ED1(紅色)的免疫熒光雙標(biāo)顯示：?jiǎn)蝹�(gè)散在分布的FABP4陽性細(xì)胞為枯否氏細(xì)胞(黃色熒光)。定量分析結(jié)果：CCl4組肝組織FABP4積分光密度(Integra optical density,IOD)(70367.99,81339.00)比Control組(285.63,302.00)有意義升高(P0.05)；干預(yù)組IOD值(6123.50,8609.00)則明顯低于CCl4組(P0.05)，但仍高于Control組(P0.05)。WesternBlot分析證實(shí)CCl4組肝組織FABP4的表達(dá)明顯高于Control組；聯(lián)苯雙酯干預(yù)后FABP4的表達(dá)量有意義下降,但仍高于Control組(P均0.05)。 3大鼠肝臟組織ED1和α-SMA的表達(dá)變化 Control組ED1陽性細(xì)胞位于肝竇和匯管區(qū)。CCl4組肝竇、匯管區(qū)陽性細(xì)胞增多、壞死區(qū)可見陽性細(xì)胞。干預(yù)組表達(dá)形式與CCl4組基本相同。細(xì)胞計(jì)數(shù)結(jié)果：CCl4組陽性細(xì)胞數(shù)(47.00,27.00)明顯高于Control組(7.00,7.00)(P0.05)；聯(lián)苯雙酯干預(yù)(23.00,14.00)可明顯降低CCl4引起的ED1陽性細(xì)胞數(shù)(P0.05)，但仍高于Control組(P0.05)。CCl4組的α-SMA表達(dá)于肝竇、纖維間隔、壞死灶及小血管平滑肌。干預(yù)組α-SMA表達(dá)形式與CCl4組基本相同。細(xì)胞計(jì)數(shù)結(jié)果：與Control組(0.00,0.00)相比，CCl4組α-SMA陽性細(xì)胞有意義增多(25.50,49.00)(P0.05)；聯(lián)苯雙酯干預(yù)(1.00,3.00)能明顯降低CCl4引起的α-SMA陽性表達(dá)(P0.05)，但仍高于Control組(P0.05)。Western Blot分析證實(shí)，CCl4組肝組織α-SMA的表達(dá)明顯高于Control組，與CCl4組相比，聯(lián)苯雙酯干預(yù)后α-SMA的表達(dá)量有意義下降，但仍高于Control組(P均0.05)。 結(jié)論： 1與正常肝組織比較，肝纖維化組織中FABP4呈現(xiàn)出陽性表達(dá)；陽性表達(dá)的細(xì)胞為枯否氏細(xì)胞和小血管內(nèi)皮細(xì)胞。這種與生理狀態(tài)下完全不同的表達(dá)方式和表達(dá)量的變化提示FABP4作為功能蛋白在肝炎癥性損傷和修復(fù)過程中扮演了重要角色。 2聯(lián)苯雙酯干預(yù)明顯降低CCl4誘發(fā)的FABP4表達(dá)和ED1陽性細(xì)胞數(shù)，，同時(shí)α-SMA表達(dá)有意義降低，提示聯(lián)苯雙酯抑制炎癥和纖維化的作用可能至少部分是通過抑制枯否氏細(xì)胞中FABP4的表達(dá)來完成。 3肝小血管和肝竇內(nèi)皮細(xì)胞中FABP4在肝纖維化組織的高表達(dá)與肝損傷和修復(fù)之間的關(guān)系及機(jī)制需要進(jìn)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 偽 - SMA in Immunohistochemical Detection

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

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

Image - ProPlus6 . 0 software was used to quantitatively analyze the images of the images after each slice was photographed . ED1 and 偽 - 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 偽 - 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 鹵 0.73 ) was higher than that in the control group ( 2.54 鹵 0.26 ) ( P0.01 ) .
The liver coefficient of the intervention group ( 3.00 鹵 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 鹵 252 ( CCl _ 4 group ) , 73 鹵 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 鹵 71 , P0.01 ; ALT : 66 鹵 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 偽 - 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 ) , 偽 - SMA positive cells increased significantly ( 25.50 , 49.00 ) compared with control group ( 0.00 , 0.00 ) ( P0.05 ) .
The expression of 偽 - SMA was significantly higher than that in control group ( P < 0.05 ) , but the expression of 偽 - 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 偽 - 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 .
【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R575

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