茅台酒对二乙基亚硝胺引发小鼠HCC发生的影响及分子机制
发布时间：2018-01-18 07:28
本文关键词：茅台酒对二乙基亚硝胺引发小鼠HCC发生的影响及分子机制 出处：《贵阳医学院》2014年博士论文 论文类型：学位论文
更多相关文章： 茅台酒 乙醇 肝癌形成 氧化应激 基因表达 凋亡
【摘要】：目的观察长期适量饮用茅台酒对DEN引发小鼠HCC发生的影响及可能的机制。方法192只C57BL/6J雄性小鼠随机分为对照组(C)、茅台酒组(M)、乙醇组(E)、茅台酒+DEN组(MD)、乙醇+DEN组(ED)及DEN组(D),未作任何处理的小鼠即为对照。茅台酒组和乙醇组小鼠分别灌胃给予53%,5ml/kg的茅台酒和乙醇,5天/周直至35周；DEN组小鼠首先于实验第一周腹腔注射一次100mg/kg DEN,第二周再次腹腔注射50mg/kg DEN,以后正常饲养；茅台酒+DEN组及乙醇+DEN组小鼠除接受DEN处理外,分别灌胃给予53%,5ml/kg的茅台酒和乙醇,5天/周直至35周；密切监测小鼠体重等一般情况。分别于实验3周末、13周末及35周末戊巴比妥钠麻醉后处死小鼠,收集血液及肝组织样本。测定各组小鼠肝脏指数及血清丙氨酸转氨酶(alanine transaminase, ALT)、天冬氨酸转氨酶(aspartate transaminase, AST);检测肝组织匀浆丙二醛(Malondialdehyde,MDA)的活性,并进行肝组织病理学检查(HE、Masson和网状纤维染色及透射电镜)；采用免疫组织化学法分析肝组织磷脂酰肌醇聚糖(Glypican-3,GPC3)的表达。采用实时荧光定量RT-PCR、免疫组织化学法和Western-blotting分析小鼠肝组织金属硫蛋白-1/2(MT-1/2)、核因子E2延长因子2(Nrf2)、谷氨酰半胱氨酸连接酶催化亚单位(GCLC)和调节亚单位(GCLM)的表达。运用定制的SABioscience PCR芯片、实时荧光定量RT-PCR、免疫组织化学法和Western-blotting分析实验鼠肝组织中与乙醇反应、肝纤维化、凋亡、肿瘤抑制及肝脏保护等相关指标的表达；采用POD法和磷脂结合蛋白V/碘化丙啶(AnnexinV/PI)流式细胞分析法分别检测肝组织细胞凋亡指数及早期凋亡率。结果实验35周后,与其它组比较,ED组小鼠体重显著降低(P0.05),肉眼及镜下可见明显的肝脏病理改变甚至HCC发生(50%),具有显著增加的肝脏指数(P0.05)和肝纤维化程度(P0.05)及GPC3阳性表达(P0.05)；而MD组小鼠未见明显的生长影响和肉眼可见的肝脏改变,镜下可见部分肝细胞气球样变性、水肿、脂肪变性、局灶性坏死和轻.中度纤维化发生,除1例样本具有轻度的GPC3阳性表达外,其余无GPC3阳性表达。无论3周末、13周末或35周末,M组总是具有与对照组相似的ALT、AST血清水平(P0.05)；3周末时,与C组比较,E组、D组、MD组和ED组显示了增加的ALT、AST血清水平(P0.001),ED组ALT、AST血清水平显著高于MD组和/或D组(P0.05)；13周末及35周末时,ED组仍然具有比其它所有组别显著增高的ALT、AST血清水平(P0.05),其余各处理组与对照组ALT水平相似。在观察的3个时间点,除M、MD组外,其余各处理组均呈现比C组显著增加的肝组织MDA水平(p0.05),尤其ED组(p0.01),但无递增趋势可见。与其它组比较,MD组具有显著增加的MT-1/2mRNA水平(P0.001)和MTs蛋白水平(P0.05)；ED组也显示比C组显著增加的MT-1mRNA表达(P0.05),但具有同C组相似或更低的MTs蛋白表达水平。与C组比较,MD组显示轻度增加的Nrf2或GCLC mRNA和蛋白表达(P0.05),但ED组总是显示与之相似的Nrf2或GCLC表达水平；无论MD或ED组均未见增加的GCLM mRNA或蛋白表达(P0.05)。PCR芯片结果显示,单纯的乙醇喂养比等剂量的茅台酒引起显著增加的肝脏基因mRNA表达改变(P0.05)；与ED组比较,MD组显著上调TAp63、p21、caspase9、Wt1等基因的mRNA表达(P0.05)；与MD组比较,ED组上调Atm、Fzd7、NQO1、Irs1、 Lefl、Tgfbr2、CK8、CK18基因的mRNA表达(P0.05)；与C组比较,MD组轻度上调Pten、Rbl、p53等基因的mRNA表达(P0.05),而ED组显示与C组相似或减少的mRNA表达水平；蛋白水平检测也提示MD组较其它组别显著上调TAp63、p21及paspase9的表达水平(P0.05).POD原位细胞凋亡检测显示对照组少量肝组织细胞凋亡,单纯的茅台酒或茅台酒+DEN处理显示明显增加的肝组织细胞凋亡(P0.05),乙醇+DEN显示个别、少量的肝组织细胞凋亡,与单纯的DEN处理组肝组织细胞凋亡程度相似(P0.05)；单纯的茅台酒或茅台酒+DEN处理显示了比对照组或DEN组增加的早期凋亡率,但差异无统计学意义,却具有比乙醇+DEN处理显著增加的早期凋亡率(P0.05)。结论与饮用等剂量乙醇比较,5 ml/kg/d饮用茅台酒35周后,DEN引发小鼠具有减轻的肝损伤,未见明显的HCC癌前病变。茅台酒能够显著上调DEN引发小鼠肝组织抗氧化应激指标MTs、Nrf2及其靶基因GCLC表达水平,以及上调肿瘤抑制相关指标TAp63、caspase9及p21的表达水平,这些可能是茅台酒减少酒精与DEN协同引起肝损伤的重要机制之一。为进一步探讨茅台酒与HCC发生的相关性提供了实验依据。
[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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