本文選題：肝細(xì)胞 + 凝膠包埋培養(yǎng)　； 參考：《浙江大學(xué)》2010年碩士論文

【摘要】：隨著人民生活水平的提高,過量飲酒引起的肝臟損傷日益增多,已經(jīng)成為影響公眾健康的問題。盡管對酒精性脂肪肝(簡稱酒精肝)的損傷機(jī)理,包括脂肪肝誘因和形成過程等,已有較為全面的認(rèn)識,然而,目前尚無有效控制和治療酒精肝的方法。其原因在于,缺少合適的酒精肝模型,因此難以篩選和找到有效的防治手段。目前酒精肝模型以動物為主,建立模型一般需要八周時間,高成本和低效率限制了其應(yīng)用。而體外酒精肝模型則可以在短時間(1-2天)內(nèi)建立,具有成本低、適合快速篩選治療藥物的優(yōu)勢。但是,傳統(tǒng)的體外酒精肝模型采用單層貼壁肝細(xì)胞培養(yǎng)方式,并以高濃度的乙醇進(jìn)行短時間刺激,該方法大大偏離了體內(nèi)乙醇慢性肝損傷的情況,不能模擬體內(nèi)酒精肝的發(fā)生過程。本文針對傳統(tǒng)體外模型的缺點(diǎn),采用能長期維持肝功能的凝膠包埋培養(yǎng)肝細(xì)胞,通過條件優(yōu)化和體內(nèi)外比較驗(yàn)證該模型的可靠性,并用于篩選抗酒精肝的中藥有效成分,為尋找有效的酒精肝治療藥物奠定基礎(chǔ)。 論文分為三部分。第一部分為酒精肝模型的建立,考察了不同細(xì)胞培養(yǎng)方式、培養(yǎng)基和乙醇濃度對模型的影響,并采用針形電極在線檢測了有無ParafilmM封口膜密封的培養(yǎng)皿中乙醇揮發(fā)速率差異。結(jié)果發(fā)現(xiàn),肝細(xì)胞在Parafilm M封口膜密封的培養(yǎng)皿內(nèi)以Williams' medium E培養(yǎng)基和凝膠包埋培養(yǎng)的方式并經(jīng)過48h的200 mM乙醇作用,可以反映乙醇導(dǎo)致的體內(nèi)肝細(xì)胞損傷。 第二部分對該模型進(jìn)行了詳細(xì)評價,分析了包括脂質(zhì)積累、過氧化壓力和CYP 2E1誘導(dǎo)等各項(xiàng)指標(biāo)。研究表明,經(jīng)過乙醇處理后,體外模型的損傷指標(biāo)呈現(xiàn)與體內(nèi)酒精肝一致的變化趨勢,說明該模型可以表征體內(nèi)酒精肝的情況。然后,使用針形電極對肝灌流中的乙醇濃度變化(表征體內(nèi)肝臟中乙醇濃度)進(jìn)行了檢測,初步嘗試關(guān)聯(lián)體內(nèi)外模型的乙醇濃度,進(jìn)一步驗(yàn)證了體外模型的可靠性。 第三部分采用已建立的模型進(jìn)行保肝藥物篩選,找到了一種有效抗酒精肝的中藥有效成分——納米水飛薊賓。 總之,本文建立的體外酒精肝模型,可用于抗酒精肝藥物篩選,尤其在利用我國豐富中藥資源發(fā)掘有效保肝藥物方面,具有廣闊的應(yīng)用前景。
[Abstract]:With the improvement of people's living standard, liver injury caused by excessive drinking has become a public health problem. Although there has been a comprehensive understanding of the mechanism of alcoholic fatty liver injury, including the inducement and formation process of fatty liver, there is no effective method to control and treat alcoholic liver. The reason is that it is difficult to screen and find effective prevention and treatment because of the lack of suitable alcoholic liver model. At present, the alcoholic liver model is mainly animal. It usually takes eight weeks to establish the model, and its application is limited by its high cost and low efficiency. The model of alcoholic liver in vitro can be established in a short time of 1-2 days. It has the advantages of low cost and suitable for rapid screening of drugs. However, the traditional model of alcoholic liver in vitro was cultured by monolayer adherent hepatocytes and stimulated with high concentration of ethanol for a short time. This method deviates greatly from the situation of chronic liver injury caused by ethanol in vivo. It is not possible to simulate the development of alcoholic liver in vivo. In view of the shortcomings of the traditional model in vitro, the hepatocytes were cultured with gel entrapment which could maintain liver function for a long time. The reliability of the model was verified by optimizing the conditions and comparing in vivo and in vitro, and it was used to screen the effective components of traditional Chinese medicine for anti-alcoholic liver. In order to find effective alcohol liver treatment drugs to lay the foundation. The paper is divided into three parts. The first part was the establishment of alcoholic liver model. The effects of different cell culture methods, culture medium and ethanol concentration on the model were investigated. The ethanol volatilization rate in the culture dish with or without ParafilmM sealing membrane was detected online by needle electrode. The results showed that the hepatocytes were cultured in Parafilm's medium E medium and gel in the culture dish sealed by Parafilm M sealing membrane and treated with 200mm ethanol for 48 h, which could reflect the damage of hepatocytes induced by ethanol in vivo. In the second part, the model was evaluated in detail, including lipid accumulation, peroxidation pressure and CYP 2E1 induction. The results showed that the damage index of the model showed the same change trend as that of the alcoholic liver in vivo after ethanol treatment, which indicated that the model could represent the alcoholic liver in vivo. Then, the change of ethanol concentration in liver perfusion was detected by needle electrode, which was used to characterize the concentration of ethanol in vivo and in vivo. The reliability of the model in vitro was further verified by correlating the ethanol concentration of the model in vivo and in vitro. In the third part, the established model was used to screen hepatoprotective drugs, and an effective Chinese medicine component, silibine-nanometer silybin, was found. In conclusion, the in vitro alcoholic liver model established in this paper can be used for screening anti-alcoholic liver drugs, especially in exploiting effective hepatoprotective drugs by using rich Chinese medicine resources in China, which has a broad application prospect.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R575.5;R-332

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 Osama El-Assal;Won-HoKim;SvetlanaRadaeva;;IL-6-deficient Mice Are Susceptible to Ethanol-induced Hepatic Steatosis:IL-6 Protects against Ethanol-induced Oxidative Stress and Mitochondrial Permeability Transition in the Liver[J];Cellular & Molecular Immunology;2004年03期

2 付康;;非酒精性脂肪肝發(fā)病機(jī)制的研究進(jìn)展[J];河南醫(yī)學(xué)研究;2007年04期

3 劉軍;潘珩;;脂肪肝發(fā)病機(jī)制研究進(jìn)展[J];臨床和實(shí)驗(yàn)醫(yī)學(xué)雜志;2006年04期

4 方志紅;崔劍巍;曹健美;彭景華;馮琴;胡義揚(yáng);;Lieber-DeCarli酒精性肝損傷模型的復(fù)制[J];中西醫(yī)結(jié)合學(xué)報(bào);2006年06期

5 陸倫根;酒精性肝病的治療[J];現(xiàn)代醫(yī)藥衛(wèi)生;2002年07期

6 閻彩鳳;向紅丁;平凡;陳偉;;不同類型非酒精性脂肪肝與糖脂代謝的相關(guān)分析[J];中國慢性病預(yù)防與控制;2006年05期

7 李龍輝;李龍江;湯為學(xué);左國慶;廖于;;甘利欣作用前后體外酒精性脂肪肝細(xì)胞模型甘油三酯水平變化及其可能作用機(jī)制[J];重慶醫(yī)科大學(xué)學(xué)報(bào);2010年02期

8 ;Evaluation of diffusion in gel entrapment cell culture within hollow fibers[J];World Journal of Gastroenterology;2005年11期

9 ;CYP2E1-dependent hepatotoxicity and oxidative damage after ethanol administration in human primary hepatocytes[J];World Journal of Gastroenterology;2005年29期

10 Myeong Soo Lee;Myung-Sunny Kim;Soo Young Park;Chang-Won Kang;;Effects of betaine on etnanol-stimulated secretion of IGF-I and IGFBP-1 in rat primary hepatocytes: involvement of p42/44 MAPK activation[J];World Journal of Gastroenterology;2006年11期

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