本文關(guān)鍵詞：茅臺(tái)酒對(duì)二乙基亞硝胺引發(fā)小鼠HCC發(fā)生的影響及分子機(jī)制　出處：《貴陽(yáng)醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 茅臺(tái)酒 乙醇 肝癌形成 氧化應(yīng)激 基因表達(dá) 凋亡

【摘要】：目的觀察長(zhǎng)期適量飲用茅臺(tái)酒對(duì)DEN引發(fā)小鼠HCC發(fā)生的影響及可能的機(jī)制。方法192只C57BL/6J雄性小鼠隨機(jī)分為對(duì)照組(C)、茅臺(tái)酒組(M)、乙醇組(E)、茅臺(tái)酒+DEN組(MD)、乙醇+DEN組(ED)及DEN組(D),未作任何處理的小鼠即為對(duì)照。茅臺(tái)酒組和乙醇組小鼠分別灌胃給予53%,5ml/kg的茅臺(tái)酒和乙醇,5天/周直至35周；DEN組小鼠首先于實(shí)驗(yàn)第一周腹腔注射一次100mg/kg DEN,第二周再次腹腔注射50mg/kg DEN,以后正常飼養(yǎng)；茅臺(tái)酒+DEN組及乙醇+DEN組小鼠除接受DEN處理外,分別灌胃給予53%,5ml/kg的茅臺(tái)酒和乙醇,5天/周直至35周；密切監(jiān)測(cè)小鼠體重等一般情況。分別于實(shí)驗(yàn)3周末、13周末及35周末戊巴比妥鈉麻醉后處死小鼠,收集血液及肝組織樣本。測(cè)定各組小鼠肝臟指數(shù)及血清丙氨酸轉(zhuǎn)氨酶(alanine transaminase, ALT)、天冬氨酸轉(zhuǎn)氨酶(aspartate transaminase, AST);檢測(cè)肝組織勻漿丙二醛(Malondialdehyde,MDA)的活性,并進(jìn)行肝組織病理學(xué)檢查(HE、Masson和網(wǎng)狀纖維染色及透射電鏡)；采用免疫組織化學(xué)法分析肝組織磷脂酰肌醇聚糖(Glypican-3,GPC3)的表達(dá)。采用實(shí)時(shí)熒光定量RT-PCR、免疫組織化學(xué)法和Western-blotting分析小鼠肝組織金屬硫蛋白-1/2(MT-1/2)、核因子E2延長(zhǎng)因子2(Nrf2)、谷氨酰半胱氨酸連接酶催化亞單位(GCLC)和調(diào)節(jié)亞單位(GCLM)的表達(dá)。運(yùn)用定制的SABioscience PCR芯片、實(shí)時(shí)熒光定量RT-PCR、免疫組織化學(xué)法和Western-blotting分析實(shí)驗(yàn)鼠肝組織中與乙醇反應(yīng)、肝纖維化、凋亡、腫瘤抑制及肝臟保護(hù)等相關(guān)指標(biāo)的表達(dá)；采用POD法和磷脂結(jié)合蛋白V/碘化丙啶(AnnexinV/PI)流式細(xì)胞分析法分別檢測(cè)肝組織細(xì)胞凋亡指數(shù)及早期凋亡率。結(jié)果實(shí)驗(yàn)35周后,與其它組比較,ED組小鼠體重顯著降低(P0.05),肉眼及鏡下可見(jiàn)明顯的肝臟病理改變甚至HCC發(fā)生(50%),具有顯著增加的肝臟指數(shù)(P0.05)和肝纖維化程度(P0.05)及GPC3陽(yáng)性表達(dá)(P0.05)；而MD組小鼠未見(jiàn)明顯的生長(zhǎng)影響和肉眼可見(jiàn)的肝臟改變,鏡下可見(jiàn)部分肝細(xì)胞氣球樣變性、水腫、脂肪變性、局灶性壞死和輕.中度纖維化發(fā)生,除1例樣本具有輕度的GPC3陽(yáng)性表達(dá)外,其余無(wú)GPC3陽(yáng)性表達(dá)。無(wú)論3周末、13周末或35周末,M組總是具有與對(duì)照組相似的ALT、AST血清水平(P0.05)；3周末時(shí),與C組比較,E組、D組、MD組和ED組顯示了增加的ALT、AST血清水平(P0.001),ED組ALT、AST血清水平顯著高于MD組和/或D組(P0.05)；13周末及35周末時(shí),ED組仍然具有比其它所有組別顯著增高的ALT、AST血清水平(P0.05),其余各處理組與對(duì)照組ALT水平相似。在觀察的3個(gè)時(shí)間點(diǎn),除M、MD組外,其余各處理組均呈現(xiàn)比C組顯著增加的肝組織MDA水平(p0.05),尤其ED組(p0.01),但無(wú)遞增趨勢(shì)可見(jiàn)。與其它組比較,MD組具有顯著增加的MT-1/2mRNA水平(P0.001)和MTs蛋白水平(P0.05)；ED組也顯示比C組顯著增加的MT-1mRNA表達(dá)(P0.05),但具有同C組相似或更低的MTs蛋白表達(dá)水平。與C組比較,MD組顯示輕度增加的Nrf2或GCLC mRNA和蛋白表達(dá)(P0.05),但ED組總是顯示與之相似的Nrf2或GCLC表達(dá)水平；無(wú)論MD或ED組均未見(jiàn)增加的GCLM mRNA或蛋白表達(dá)(P0.05)。PCR芯片結(jié)果顯示,單純的乙醇喂養(yǎng)比等劑量的茅臺(tái)酒引起顯著增加的肝臟基因mRNA表達(dá)改變(P0.05)；與ED組比較,MD組顯著上調(diào)TAp63、p21、caspase9、Wt1等基因的mRNA表達(dá)(P0.05)；與MD組比較,ED組上調(diào)Atm、Fzd7、NQO1、Irs1、 Lefl、Tgfbr2、CK8、CK18基因的mRNA表達(dá)(P0.05)；與C組比較,MD組輕度上調(diào)Pten、Rbl、p53等基因的mRNA表達(dá)(P0.05),而ED組顯示與C組相似或減少的mRNA表達(dá)水平；蛋白水平檢測(cè)也提示MD組較其它組別顯著上調(diào)TAp63、p21及paspase9的表達(dá)水平(P0.05).POD原位細(xì)胞凋亡檢測(cè)顯示對(duì)照組少量肝組織細(xì)胞凋亡,單純的茅臺(tái)酒或茅臺(tái)酒+DEN處理顯示明顯增加的肝組織細(xì)胞凋亡(P0.05),乙醇+DEN顯示個(gè)別、少量的肝組織細(xì)胞凋亡,與單純的DEN處理組肝組織細(xì)胞凋亡程度相似(P0.05)；單純的茅臺(tái)酒或茅臺(tái)酒+DEN處理顯示了比對(duì)照組或DEN組增加的早期凋亡率,但差異無(wú)統(tǒng)計(jì)學(xué)意義,卻具有比乙醇+DEN處理顯著增加的早期凋亡率(P0.05)。結(jié)論與飲用等劑量乙醇比較,5 ml/kg/d飲用茅臺(tái)酒35周后,DEN引發(fā)小鼠具有減輕的肝損傷,未見(jiàn)明顯的HCC癌前病變。茅臺(tái)酒能夠顯著上調(diào)DEN引發(fā)小鼠肝組織抗氧化應(yīng)激指標(biāo)MTs、Nrf2及其靶基因GCLC表達(dá)水平,以及上調(diào)腫瘤抑制相關(guān)指標(biāo)TAp63、caspase9及p21的表達(dá)水平,這些可能是茅臺(tái)酒減少酒精與DEN協(xié)同引起肝損傷的重要機(jī)制之一。為進(jìn)一步探討茅臺(tái)酒與HCC發(fā)生的相關(guān)性提供了實(shí)驗(yàn)依據(jù)。
[Abstract]:Objective To observe the effect of long-term moderate drinking wine Moutai HCC the initiation of DEN mice and its possible mechanism. Methods 192 male C57BL/6J mice were randomly divided into control group (C), Moutai Liquor Group (M), alcohol group (E), Moutai liquor group +DEN (MD), ethanol (ED) and DEN +DEN group group (D), without any treatment of the mice as control. Moutai liquor group and ethanol group mice were lavaged with 53% 5ml/kg, Moutai liquor and ethanol, 5 days / week until 35 weeks; DEN group were first in the first week of the experiment with a single intraperitoneal injection of 100mg/kg DEN, again for second weeks by intraperitoneal injection 50mg/kg DEN, after the normal feeding; Moutai liquor group +DEN and group +DEN mice received DEN ethanol treatment, were orally given 5ml/kg 53%, Moutai liquor and ethanol, 5 days / week until 35 weeks; close monitoring of body weight of mice in general. At the 3 week, 13 and 35 weeks at the end of the weekend pentobarbital were small Rat, collect blood and liver tissue samples. The mice liver index and serum alanine transaminase (alanine, transaminase, ALT), aspartate aminotransferase (aspartate, transaminase, AST); detection of liver homogenate malondialdehyde (Malondialdehyde, MDA) activity and liver histopathological examination (HE, Masson and reticular fiber staining and transmission electron microscopy); immunohistochemistry analysis of liver tissue glypican (Glypican-3, GPC3). The expression by real-time quantitative RT-PCR, immunohistochemistry and Western-blotting analysis of mouse liver metallothionein -1/2 (MT-1/2), nuclear factor E2 elongation factor 2 (Nrf2), glutamate cysteine ligase the catalytic subunit (GCLC) and regulatory subunit (GCLM) expression. Using SABioscience PCR chip customized, real-time fluorescence quantitative RT-PCR, immunohistochemistry and Western-blotting Analysis of experimental rat liver tissue and ethanol reaction, hepatic fibrosis, apoptosis, expression of tumor suppressor index and liver protection etc.; using POD and annexin V/ and propidium iodide (AnnexinV/PI) flow cytometry apoptosis of liver index and early apoptosis rate were detected by analysis. Results after 35 weeks, and compared to other groups, the weight of mice in ED group decreased significantly (P0.05), the macroscopic and microscopic pathological changes were visible even HCC (50%), with a significant increase in the liver index (P0.05) and liver fibrosis (P0.05) and GPC3 expression (P0.05); MD group mice had no obvious effects on the growth of and visible changes in the liver, fatty degeneration microscopically part ballooning degeneration of liver cells, edema, focal necrosis and mild moderate fibrosis. In 1 cases, the positive expression of GPC3 sample is mild, while the positive expression of GPC3. 鏃犺3鍛ㄦ湯,13鍛ㄦ湯鎴，

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