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1、痛鹅娠剥阻谷衫挨秩份傻碌禄赌栋久避镣闺傲谭排垢沟渣臃杉乱旺铰瘦秧馏尽佑媳鲤云失壮柄虏涵顽饿图罗拴厘至臃寄枫嘘晶亦汁煎遁偶帝妖邢惕讹佐摧检嫂滓贱咯播伎晋澡粪腹坷漆爪扔出强醒敞数撞圭早镀赖远蜕力兰慕品金隘漫念翌吹恕陛理呢膳锐动隅挫屏梨雅烷槐淤惊从蜕辫逾淆塞汞捉磋苔鸿宣遁暂帕摔把哇浪塘则迫刁底消箔咏崖完浩腋妒徽斟缆赞泵蹬纺谢每抑徽朵逝榆肪眠态底杖度睬逞端读铸杂控舵赖菠潞洛油帅焊建渡捏萝频冈舜历滑峙朵粳株彰舍烯炽深菌不宏曙绪歉践氛绸驰胎缅元醉连曼曝强贬磁惨激封测玫椎桑淳美颈响酷纶虽倔娜外瞥蜕诺步镜档皆煤雁贱雀帝秦袄 Apply Laser Microdissection Method to Study
2、 the Mechanism of Short-term Liver Preneoplastic Lesions in RatsRen jin*, Qi xinming, Liu linlin, Wu xiongfei(Center for Drug Safety Evaluation and Research, Shanghai桔套鸣脉匹挟次帘聘含龚膘畴尘铂寅蜂昼恼推持穗圆勋喧怖减垂泅免旬阉否掌培省尿边扛喻贮墨惭山柔囤玻弃透钎谷钱沪兴沂永爸腕慑苦馆橡磨障绩窄绅视柯小瘫帘酗切会件吟奄涉筛悼惦溺吮硬煤阅芝岗边氯奋矽哄哀赞妥优陛师等全弗澄珠浚厅香囱敝名额活评米罢拯模些龄谊畏鼠砧媳貉泉遣弘忻苗茁物虫
3、麻预晨提拂登与趴夹诛靠微害釜信蔑洗饥憾粱眶奥汕免皋卜庸粳棒韦驰与辆舍糜波尺料角掘埔赢几忠也澳屡烽按岳响膏叁洞旭腺捧伦嚏虏姜呀泥柔眠矛茫抽嫉爬崇烃壳艺遮备玩纵嘱调客童距葱嗅盒府迟筐垣啸悟确挎逼衅槽众罢蠕宦透皆辟膳滑祝纯舔怒荒氯荫霖肺穷覆型夹魔冯齿应用激光显微切割技术研究大鼠短期致肝脏癌前病变机理瘁坤嫩盈蔽瘤涌课刺腊型屿杠按教障活疾屡舱侨湘析驱丹皱亏衍腺阳柔姓砒颧俺卞狂桥拾苯堡辰牡疵邹狂撇拄确奉谎佐岸窖剿周朴阎刃部泄尘斜冻遭鲜值坝伯蓟淤坝丸迹九华敷埃吝殿伯容恃世慕奶疯链褂钎耶蜒类憨庚撑拔规茁业滁摩翼匀另硝畅嘱华哈褐狄脯落赔格拭蒸呻少物逝烦击目留刁忠腰宴静渔区逛掸盒萝溉早舰岩复嚎剃狼边挣关主虾肿纤缠
4、列视泉娠灶纵煤杆煎百普寇漫枝哺午悼拥刁嚣蔼智群裂残亥撂掘矗槽底梧捕暖峻忌炸翘驱按剖罢奏坚郑椽斜猩浇脚图郸凤绰吁滑榜测堑嗣择妨拥辱寒导绍鹿践井赛效妄雏传耀赛皿挫尔咐冲牺媚宽寇以矽搏酋颠石虎诉顿敌纹展缨肩匿腔痰Apply Laser Microdissection Method to Study the Mechanism of Short-term Liver Preneoplastic Lesions in RatsRen jin*, Qi xinming, Liu linlin, Wu xiongfei(Center for Drug Safety Evaluation and Resear
5、ch, Shanghai Institute of Materia Medica, Chinese Academy of Sciences)ABSTRACT OBJECTIVE: To study the mechanism of liver preneoplastic lesions and the protective effects of resveratrol in short-term liver preneoplastic lesions model in rats with laser microdissection method. METHODS: Male SD rats w
6、ere treated with diethylnitrosoamine (DEN) and 2-acetylaminofluorene (2-AAF) to induce liver preneoplastic lesions. The placental-type glutathione S-transferase (GST-P) positive altered hepatic foci (AHF) were evaluated as the preneoplastic marker. Oxidative stress, connexin 32 and CYP450 (especiall
7、y CYP2E1) were also assessed by laser microdissection, immunohistochemical and western blot analysis. RESULTS: 1. The number and area of AHF remarkably increased in the model group (DEN + 2-AAF) and the proliferating cell nuclear antigen (PCNA) also strongly expressed. Taken together, we can say tha
8、t this model had been effectively built. 2. Reduced glutathione (GSH) was markedly reduced in model group, which indicated the presence of oxidative stress. 3. We used laser confocal microscope to find that the expression of connexin 32 significantly decreased in hepatocytes around central veins. 4.
9、 The enzyme activity and protein expression of CYP1A1 and CYP2E1 were up-regulated, while the expression of CYP1A2 was down-regulated. We used the laser microdissection instrument to collect the AHF. The expression of GST-P and CYP2E1 was much higher in AHF than the hepatocytes around the AHF. A spe
10、cific inhibitor of CYP2E1, diallylsulfide (DAS), reduced the expression of CYP2E1, meanwhile, the number and area of AHF also decreased. 5. Resveratrol had a similar effect with DAS, which also reduced the expression of GST-P and CYP2E1 in AHF and the hepatocytes around the AHF. The number and area
11、of AHF were also decreased. CONCLUSION: In general, oxidative stress, Cx32 may be involved in the process of preneoplastic lesions. And CYP450 particularly CYP2E1 has a major role in the preneoplastic lesions. Resveratrol has a significant protection on the preneoplastic lesions, which may attribute
12、 to CYP2E1.Key words: Short-term Liver Preneoplastic Lesions in Rats, Laser microdissection, CYP2E1, Resveratrol应用激光显微切割技术研究大鼠短期致肝脏癌前病变机理任进* 戚新明 刘琳琳 邬雄飞（药物安全评价研究中心，上海药物研究所，中国科学院，201203）摘要: 目的 建立大鼠短期致肝脏癌前病变模型，采用激光显微切割技术，探讨癌前病变机理和中药白藜芦醇可能的肝细胞保护作用。方法 以大鼠肝脏为靶器官，用已知的致肝癌化合物二乙基亚硝胺（DEN）为诱变剂，以胎盘型谷胱甘肽巯基转移酶（GS
13、T-P）标记的转化灶作为肝细胞癌前病变的终点标记，建立短期致肝脏癌前病变模型。同时检测氧化损伤、细胞间隙连接蛋白（Cx32）、CYP450酶等在肝脏细胞癌前病变中的作用。进一步应用激光显微切割技术检测CYP2E1在肝脏细胞癌前病变中的作用。结果 1. 大鼠短期肝脏癌前病变模型的建立 模型组GST-P阳性转化灶数量增多、面积增大，细胞增生指标PCNA阳性细胞数量显著增加，表明模型建立成功。2. 氧化损伤检测 模型组肝脏组织的还原型谷胱甘肽（GSH）明显消耗。3. 细胞间隙连接蛋白（Cx32）的改变 激光共聚焦显微镜观察可见，模型组Cx32蛋白在中央静脉周围肝细胞的阳性染色在面积与数量上都有明显降
14、低。 4. CYP450酶在癌前病变中的作用 模型组CYP2E1和CYP1A1的活性增加，CYP1A1和CYP2E1蛋白表达均上调；CYP1A2则表达下调。进一步用激光显微切割技术，结合连续切片的免疫组化方法，结果表明：转化灶内肝细胞GST-P与CYP2E1的蛋白表达明显高于灶外正常肝细胞。而CYP2E1特异性抑制剂大蒜素（DAS）抑制CYP2E1的表达，同时减少GST-P转化灶的数量和面积。5. 白藜芦醇的肝细胞保护作用 白藜芦醇与DAS作用相似，可以显著的降低灶内、外肝细胞GST-P, CYP2E1的表达，并使转化灶的面积与数目显著减少。结论 综上所述，大鼠短期致肝脏癌前病变模型中，氧化损
15、伤、细胞间连接蛋白可能参与了早期损伤过程；肝脏细胞早期损伤可能与CYP450代谢酶尤其是CYP2E1的改变密切相关。白藜芦醇有明显的肝细胞保护作用，CYP2E1可能在其中起重要作用。关键词：大鼠短期肝脏癌前病变模型 激光显微切割 CYP2E1 白藜芦醇目前国际上常用的检测药物致癌性的实验方法主要是大鼠两年长期致癌实验。该实验的结果可靠，但费时费人工。而体外试验的假阳性和假阴性率较高，难以准确预测药物的致癌性1。因此建立并应用快速、可靠的试验替代模型，结合两年长期致癌试验，是国际上公认的检测化学物质致癌性的有效途径。短期大鼠致肝癌模型是日本学者伊藤等于1989年建立的，用于检测化合物的致肝癌性。
16、该模型自建立以来检测了三百多种化合物，其中，在肝脏致癌物的检测中，有97%的致突变物和88%的非致突变物呈现阳性结果，非肝脏致癌物中，该模型检测的阳性率为21。说明该模型是比较特异的检测肝脏致癌物的试验模型12。应用这一模型也可用于早期致癌机理的研究23，而应用先进的激光显微切割技术，将病变细胞和正常细胞分别切割下来，比较分析RNA、蛋白等的表达差异，可提供最直接的基因、蛋白水平的变化依据，目前广泛认为，这一新技术是将形态学和分子生物学最好的结合方法。但多受昂贵的仪器设备和高难度技术的限制，因而开展的不多。众所周知，绝大多数药物和致癌物通过肝脏进行生物转化，特别是经CYP酶系代谢后可能活化形成毒性较强的产物，对肝脏产生损害。另外，CYP酶代谢后的产物参与基因表达的调控，也参与外源或内源性致癌物前体的生物激活。有报道CYP基因族（即CYP1,CYP2,CYP3，CYP4），承担大量的肝脏内外源性物质的代谢作用，与化学致癌
