MALLORY BODIES INDUCED BY ALCOHOL AND DRUGS

长期目标：确定马洛里体（MB）的发病机制 酒精性肝病的病因有哪些？ 假设是 MBs在肝细胞中形成不溶性蛋白复合物 这是对细胞应激的热休克样反应的结果。 这 一种假说认为，MB的形成是翻译后的结果， 引起细胞角蛋白（CK）构象变化的事件 蛋白 推测导致这种情况的热休克相关蛋白质 变化包括泛素、转氨酶和热休克蛋白（hsp） 70. 已知这些蛋白质会改变蛋白质构象， 在经历细胞应激的细胞中的热休克蛋白反应。 到 为实现这一目标，具体目标是：1）建立 MB形成的实验模型，以研究其 使用喂食灰黄霉素或DDC的小鼠的发病机制。 诱导后 喂养、热休克或热休克反应诱导剂4个月时的MB 例如高剂量酒精或砷将被应用于体内 体外预处理小鼠肝脏和对照。 随后小鼠喂食 gris或DDC，然后停药一个月，让MB 消失，将在体外激发或在体外诱导MB 阵 2)分子生物学技术将使用 实验模型：1）使用原代组织培养和体内肝脏 用于转氨酶、细胞角蛋白mRNA表达的切片制备物 49和55，泛素和热休克蛋白70。3)合成率和周转率 在CK 49和55中使用（35）S标记的甲硫氨酸掺入的CK蛋白 将通过凝胶电泳分离和放射自显影进行研究。 4)CK 49和55的相对合成速率将在 用电子显微镜放射自显影法研究中间纤维和微束 用（3）H-甲硫氨酸作细胞骨架的薄切片。第五章） 细胞角蛋白和MB的磷酸化将使用（32）P 通过2D电泳放射自显影检测掺入。6)MB 利用高压傅立叶变换测定蛋白质构象 红内光谱学 因此，通过研究热休克蛋白的作用 在MB的形成过程中，我们希望能证明翻译后改变 在细胞角蛋白蛋白构象导致MB的形成， 不溶且抵抗细胞角蛋白正常转换。 到 进一步证实了热休克样反应与微生物群的关系 我们将研究MB消失的恢复，看看热休克是否 蛋白质表达和其他翻译后改变也恢复 在肝细胞的组织培养物中恢复到正常。

Long term objectives: To determine the pathogenesis of Mallory body (MB) formation in the liver of alcoholic liver disease. The hypothesis to be tested is that MBs form as an insoluble protein complex in liver cells as the result of a heat shock-like response to cellular stress. This hypothesis suggests that MBs form as a result of post translational events which cause conformational changes in the cytokeratins (CK) protein. The heat shock associated proteins postulated to cause this change include ubiquitin, transglutaminase and heat shock protein (hsp) 70. These proteins are known to alter protein conformation as a result of the hsp response in cells which have undergone cellular stress. To meet this goal, the following specific aims are: 1) establish experimental models for MB formation in order to investigate their pathogenesis using mice fed griseofulvin or DDC. After the induction of MBs at 4 months of feeding, heat shock or heat shock response inducers such as high dose alcohol or arsenic will be applied in vivo and in in vitro to pretreated mouse livers and controls. Subsequently mice fed gris or DDC, then withdrawn from the drugs for a month to allow MBs to disappear, will be challenged in vitro or in vitro to induce MB formation. 2) Molecular biology technology will be employed using the experimental models in 1) using primary tissue culture and in vivo liver slice preparations for mRNA expression for transglutaminase, cytokeratins 49 and 55, ubiquitin and hsp 70. 3) Rate of synthesis and turn over of CK proteins using (35)S labeled methionine incorporation in CK 49 and 55 will be studied by gel electrophoresis separation, and autoradiography. 4)Relative rates of synthesis of CK 49 and 55 will be studied in intermediate filaments and MBs using electron microscopy autoradiography of thin sections of cytoskeletons using (3)H-methionine. 5) Phosphorylation of the cytokeratins and MBs will be studied using (32)P incorporation detected by 2D electrophoresis autoradiography. 6) MB protein conformation determination using high pressure fourier transform intrared spectroscopy. Thus, by studying the role of heat shock proteins during MB formation we hope to show that post translational alteration in cytokeratin protein conformation causes MBs to form and become insoluble and resistant to normal turnover of cytokeratin proteins. To further substantiate the relationship of heat shock-like response and MBs we will study the recovery where MBs disappear to see if the heat shock proteins expression and other post translational alterations also revert to normal in tissue cultures of hepatocytes.