中文摘要i・・i英文摘要符号说明 材料与方法果"结614•T6—•20•—28综攻读学位期间发表或已录用的主要学术论文 •••.4 4原创性声明…! 丫翎嬲Bcl-2和Bax在DEN诱发大鼠肝癌细胞上的表达研究生：刘洪付专业：人体解剖与组织胚胎学导师：王增贤教授中文摘要目的本实验用二乙基亚硝胺（diethylnitrosamine,DEN）建立诱发性肝癌模型，观察肝 细胞的凋亡规律，在诱癌过程中细胞凋亡相关基因Bcl・2和Bax表达变化情况，以探讨 凋亡与肝癌发生发展的关系，为探索肝癌的发病机制提供实验依据方法将80只大鼠随机分为模型组（实验组）（72只）和对照组（8只），模型组用DEN 制备诱发性肝癌模型剖腹探查并记录两组肝脏的形态.颜色、质地变化及转移情况, 肝及转移瘤组织切片经HE染色后观察肝组织的病理形态学变化特点，hochst33342荧 光染色后计数凋亡细胞并计算细胞凋亡率，原位杂交技术检测Bd・2 mRNA和Bax mRNA的表达情况结果1. 根据诱癌过程中肝脏组织发生的病理学改变，可将诱癌过程分为肝细胞损伤期、肝细胞增生■硬化期和肝细胞癌变期三个时期1）肝细胞损伤期：大鼠肝脏表 面未见明显异常。
光镜下肝小叶结构完整，小叶内可见局灶性坏死伴炎性细胞浸润, 表现为弥漫性肝细胞水肿，部分肝细胞呈嗜酸性变；同时可见纤维组织增生及肝细胞 再生2）肝细胞增生•硬化期：肝脏表面粗糙，出现弥散分布的灰白色近圆形病灶肝脏有硬化，肝细胞水肿进一步加重光镜下出现嗜酸性变和透明细胞增生灶，并可 见肝细胞增生结节，结节内细胞异型性较小3）肝细胞癌变期：肝脏表面布满多个 大小不一的灰白色结节，切面可见出血和坏死镜检为肝细胞肝癌，可见肝癌细胞排 列成索状和团块状，向周围肝组织浸润，可见不同程度的脂肪变性，部分区域有岀血・ 坏死癌周组织细胞水肿、嗜酸变性、脂肪变性，并有肝细胞增生灶、增生结节及非 典型增生结节对照组大鼠肝脏表面光滑，质地均匀光镜观察可见肝细胞以中央静 脉为中心呈放射状排列肝细胞呈多边形，边界明显；胞质嗜酸性；肝细胞核大居中, 以单核为主2. hochst33342荧光染色后观察可见，模型组肝组织内凋亡的细胞核或细胞质内 可见浓染致密的颗粒状或块状荧光，典型的还可见核固缩和新月形改变，并可见凋亡 小体肝细胞损伤期和肝细胞增生•硬化期的肝组织均可见较多固缩和碎裂的细胞核; 肝细胞癌变期的癌细胞核大小不一，核膜不规则，胞浆内出现大量的凋亡小体。
对照 组，肝细胞可见少量的异常的细胞核；对照组细胞凋亡率为6.21%,在肝细胞损伤期、 肝细胞增生•硬化期和肝细胞癌变期细胞凋亡率分别是17.42%、18.89%和51.25%(#=33.62, P<0.05),且细胞凋亡率的变化随诱癌病程发展呈上升趋势mRNA和BaxmRNA均定位于细胞胞核和胞浆内，阳性反应产物为黄色颗粒，阳性细胞呈弥漫性分布Bcl・2 mRNA表达的平均光密度值在对照组为0.16970.0101,在肝细胞损伤期、肝细胞增生•硬化期、肝细胞癌变期Bcl-2 mRNA表达的平均光密度值分别为0.1431 0.0072、0.1261 0.0164、0.10340.0124,并且随着肝癌病变发生发展逐渐降低(F=70.3297, P<0.01),其差别有统计学意义而Bax mRNA表达的平均光 密度值在对照组为0.13830.0093,在肝细胞损伤期、肝细胞增生•硬化期、肝细胞癌 变期分别为0.1523+0.0103^ 0・16390・0133和0.17850.0191,随着诱癌病变的发生发展逐渐增高(F=20・5623, P<0.01),四者之间比较有显著统计学意义结论DEN诱发肝癌的发生发展过程中伴有细胞增殖与凋亡异常，异常表现与细胞凋亡相关基因Bcl-2和Bax表达异常有关。
关键词：肝癌；细胞凋亡；二乙基亚硝胺；Bcl-2； BaxTHE EXPRESSION OF Bcl-2 AND Bax ON DEN INDUCEDLIVER CANCER OF RATPostgraduate： Liu HongfuSpeciality： Human Anatomy and EmbryologySupervisor：Prof Wang ZengxianABSTRACT❶ bjectiveThe liver cancer model was induced with diethylnitrosamine(DEN) to observe the regularity of liver cell apoptosis and expression of apoptosis related gene Bcl-2 and Bax in the induced cancer, and to explore the relationship between apoptosis and the development of liver cancer, accumulating experimental data underlying the mechanism of liver cancer, methodsEighty-eight rats were used, 72 rats as experimental group induced by drinking water containing DEN, 8 rats as control group. After laparotomy, the form, color, texture of the liver and metastiasis in both control and experimental groups were observed and recorded. The metastatic tumor and the liver tissue were sectioned and stained with hematoxylin^eosin staining to demonstrate the characteristic in morphological changes, the hochst33342 fluorescent staining was applied to show and count the rate of apoptotic cell, and the in situ hybridization technique was used to detect the Bcl-2 mRNA and Bax mRNA expression.resultsL According to the pathological change of liver cells, the process of carcinogenesis can be divided into three periods: hepatic toxic lesion, hepatic proliferation/cirrhosis and hepatic carcinogenesis> (1) In hepatic toxic lesion period^ no obvious abnormal was observed on the liver surface. Under the microscopy the hepatic lobule was in its integrity, but with diffuse edema, focal necrosis accompanied with inflammatory cell infiltration^ some cells in the lobule showed eosinophilic degeneration. The hepatic cell regeneration and fibrous tissue proliferation were also demonstrated. (2) In hepatic proliferation/ cirrhosis period, the liver swelled fiirther and its surface became rough with diffusing foci, grey and nearly circular, the liver cirrhosis started. Under the microscopy, the liver cells with less atypia proliferated into nodules, and eosinophilic degeneration and hyaline 3proliferation were observed. (3) In hepatic carcinogenesis period, the liver was covered by grey nodules in warious sizes, and the heomorrhagic spots and necrosis were also seen on the section. Under the microscopy, the liver showed all signs of hepatic cell cancer: cells arranged in cord and lumphish^ infiltrated into surrounding liver tissue, and various degrees of fatty degeneration^ reginal heomorrhage and necrosis were also detected. Peritumoral tissues showed edema, eosinophilic and fatty degeneration, foci of liver cell proliferation^ hyperplastic and atypical hyperplasia nodules.In control group, smooth surface and even texture of the liver were observed. Under the microscopy, the liver cells with distinct boundaries arranged radially to the central vein, showing polygonal shape with a big basiphilic monokaryon within eosinophilic cytoplasm.2. The hochst33342 fluorescent staining in experimental group demonstrated hyperchromatic dense granular or massive fluorescent within the apoptotic cells. In cells with typical pathological changes, the nuclear condensa。