本文選題：血瘀 + 肝纖維化　； 參考：《中國人民解放軍醫(yī)學(xué)院》2014年博士論文

【摘要】：背景及目的：肝硬化是一種嚴(yán)重危害人類健康的疾病，由不可逆性肝纖維化發(fā)展而來，預(yù)后差。中醫(yī)從血瘀病機出發(fā)常用活血化瘀藥物治療肝纖維化、肝硬化，臨床療效顯著。中醫(yī)血瘀與西醫(yī)淤血微循環(huán)障礙有病理學(xué)交叉之處，近年來研究發(fā)現(xiàn)肝臟微循環(huán)障礙為肝纖維化主要發(fā)病機制和病理基礎(chǔ)之一，但基于中醫(yī)血瘀的肝纖維化形成機制研究還不夠清晰，，因此我們擬建立一種能夠去除其他致病因素干擾，獨立體現(xiàn)血瘀致肝纖維化這一病理過程的動物模型，嘗試觀察血瘀致肝纖維化的形成特點，分析可能的發(fā)病機理，單純探索血瘀與肝纖維化之間的關(guān)系，尋找活血化瘀中藥防治肝纖維化作用的關(guān)鍵靶點，為防治肝纖維化、肝硬化中藥新藥物篩選提供實驗室依據(jù)及參考。 方法：（1）采用經(jīng)肝后段下腔靜脈部分結(jié)扎術(shù)（partial ligation of thesuprahepatic inferior vena cava，pIVCL）制作慢性瘀血性肝纖維化小鼠模型，部分小鼠無特殊處理措施常規(guī)喂養(yǎng)，部分小鼠給予抗凝劑華法林口服，部分采用組織旁路因子抑制劑(Tissue factor pathway inhibitor,TFPI)轉(zhuǎn)基因小鼠制作模型。于術(shù)后6周處死小鼠。（2）記錄小鼠體重及肝脾重量，門靜脈壓力等一般情況，收集血清、肝臟或細(xì)胞樣本，分別進(jìn)行肝功能檢測、組織學(xué)檢查（蘇木精-伊紅染色、天狼猩紅染色），羥脯氨酸含量檢測、免疫熒光染色、RT-PCR、Western Blot、ELISA以及脫氧膽酸溶解性分析實驗等方法檢測，觀察評估血瘀致肝纖維化后，肝纖維化相關(guān)指標(biāo)平滑肌肌動蛋白（Smoothmuscle acetin α，α-SMA）、膠原、纖維連接蛋白、纖維蛋白、羥脯氨酸等變化，行磁共振掃描評估肝硬變程度及其與門靜脈壓力、肝內(nèi)羥脯氨酸含量關(guān)系分析。（3）以人或小鼠肝星形細(xì)胞（Hepatic stellate cell，HSC）為目標(biāo)細(xì)胞進(jìn)行體外實驗，評估細(xì)胞受到周期性機械拉伸、細(xì)胞周圍纖維蛋白含量變化、應(yīng)用β1整合素受體或肌動蛋白抑制劑對HSC分泌纖維連接蛋白及溶解性的影響。 結(jié)果：①本研究采用肝后段下腔靜脈部分結(jié)扎術(shù)（pIVCL）成功建立了瘀血性肝纖維化小鼠實驗?zāi)Ｐ�，描述了模型小鼠�?體重量比、脾/體重量比、門靜脈壓力、肝功等一般情況。②體內(nèi)試驗：pIVCL手術(shù)組6周時膠原沉積、肝內(nèi)羥脯氨酸含量、α-SMA較假手術(shù)組均顯著增加；術(shù)后口服抗血凝劑華法林6周或采用TFPI轉(zhuǎn)基因小鼠進(jìn)行pIVCL術(shù)后，膠原沉積、肝內(nèi)羥脯氨酸含量、α-SMA表達(dá)較野生型小鼠pIVCL術(shù)后增加幅度顯著減少； pIVCL手術(shù)后小鼠肝臟MRE提示肝硬度增加，與門靜脈壓力升高及肝內(nèi)羥脯氨酸含量增加成正比。③體外實驗：HSC受到周期性機械拉伸、予纖維蛋白刺激，則纖維連接蛋白（Fibronectin，F(xiàn)N）產(chǎn)量增加，不可溶性FN增加；給予β1整合素受體或肌動蛋白抑制劑刺激則使HSC分泌FN減少，不可溶性FN含量減少；在纖維蛋白膠內(nèi)孵育HSC，MRE檢測結(jié)果示纖維蛋白膠硬度增加。 結(jié)論：①通過瘀血性肝纖維化小鼠模型的建立，證實血瘀可以作為獨立致病因素導(dǎo)致肝纖維化產(chǎn)生；②該小鼠模型搭建了傳統(tǒng)中醫(yī)血瘀與現(xiàn)代西醫(yī)淤血之間聯(lián)系橋梁，為中醫(yī)血瘀在肝纖維化中作用的認(rèn)識找到現(xiàn)代生物學(xué)依據(jù)提供了實驗室數(shù)據(jù)；③一些抗凝處理措施抑制肝纖維化形成的結(jié)果，反證了血瘀可以導(dǎo)致肝纖維化形成這一結(jié)論，并為活血化瘀類中藥抗肝纖維化作用病理基礎(chǔ)研究找到了關(guān)鍵靶點，為下一步篩選抗肝纖維化、肝硬化新藥物提供了實驗室參考。
[Abstract]:Background and objective: liver cirrhosis is a serious disease which endangers human health. It is developed from irreversible liver fibrosis, and the prognosis is poor. Traditional Chinese medicine is used to treat liver fibrosis and cirrhosis with significant clinical effect. It is found that the liver microcirculation disorder is one of the main pathogenesis and pathological basis of liver fibrosis, but the mechanism of the formation of liver fibrosis based on blood stasis of traditional Chinese medicine is not clear. Therefore, we intend to establish an animal model which can remove the interference of other pathogenic factors and reflect the pathological process of liver fibrosis independently, and try to observe it. The characteristics of the formation of liver fibrosis caused by blood stasis, analysis of possible pathogenesis, simple exploration of the relationship between blood stasis and liver fibrosis, looking for the key targets for the prevention and treatment of liver fibrosis by activating blood and removing stasis, providing laboratory basis and reference for the prevention and control of liver fibrosis and the screening of new drugs of traditional Chinese medicine.
Methods: (1) a mouse model of chronic stagnant liver fibrosis was made by partial ligation of the posterior inferior vena cava (partial ligation of thesuprahepatic inferior vena cava, pIVCL). Some mice were fed without special treatment, and some mice were given the anticoagulant to warfarin, and some of the tissue bypass factor inhibitors (Tissu) were used. E factor pathway inhibitor, TFPI) transgenic mice were made in mice. 6 weeks after the operation, mice were killed. (2) the weight of the mice and the liver and spleen weight, the pressure of the portal vein were recorded, the serum, liver or cell samples were collected, the liver function test, histology examination (hematoxylin eosin staining, Sirius scarlet stain), and hydroxyproline content examination Test, immunofluorescence staining, RT-PCR, Western Blot, ELISA and deoxycholic acid dissolution test and other methods, observe and evaluate the changes of smooth muscle actin (Smoothmuscle acetin alpha, alpha -SMA), collagen, fibrin protein, hydroxyproline and other changes of hepatic fibrosis related indexes after blood stasis induced liver fibrosis. Analysis of the relationship between the degree of liver cirrhosis and the pressure of portal vein and the content of hydroxyproline in the liver. (3) in vitro experiments were conducted on human or mouse Hepatic stellate cell (HSC) for target cells, and the cells were subjected to periodic mechanical tension, the changes of fibrin content around the cells, and the application of beta 1 integrin receptor or actin inhibitor Effects of HSC on fibronectin secretion and solubility.
Results: (1) a mouse model of blood stasis induced liver fibrosis was successfully established by partial ligation of the posterior inferior vena cava (pIVCL). The liver / body weight ratio, spleen / weight ratio, portal pressure and liver function were described. (2) in vivo test: collagen deposition, intrahepatic hydroxyproline content and alpha -S in the pIVCL operation group at 6 weeks. MA was significantly higher than that in the sham operation group; after 6 weeks of oral antiblood coagulant and TFPI transgenic mice, the collagen deposition, the content of hydroxyproline in the liver and the increase in the expression of alpha -SMA were significantly lower than that of the wild type mice after pIVCL; the MRE of the liver of mice after pIVCL operation showed the increase of the liver hardness and the pressure of the portal vein. High and intrahepatic hydroxyproline content increased in proportion. (3) in vitro experiment: HSC was subjected to periodic mechanical tension and stimulated by fibrin protein, the yield of fibronectin (Fibronectin, FN) increased, and the insoluble FN increased; the secretion of HSC by beta 1 integrin receptor or actin inhibitor resulted in the decrease of HSC secretion and the decrease of the insoluble FN content. HSC was incubated in fibrin glue, and the hardness of fibrin glue was increased by MRE.
Conclusion: (1) through the establishment of a mouse model of blood stasis of liver fibrosis, it is proved that blood stasis can lead to liver fibrosis as an independent pathogenic factor. 2. The model set up a bridge between traditional Chinese medicine blood stasis and modern western medicine, which provides a modern biological basis for the understanding of blood stasis in liver fibrosis. Some measures of anticoagulant treatment inhibit the formation of liver fibrosis and prove that blood stasis can lead to the formation of liver fibrosis, and the key target is found for the basic research on the pathological basis of liver fibrosis, which provides a laboratory reference for the next screening of liver fibrosis and new drugs for liver cirrhosis. Exam.

【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R575.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

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2 張寧;肖小河;周雙男;郭玉明;羅生強;宮Z

本文編號：1890868