INTESTINAL CELL DIFFERENTIATION IN VITRO

我们的长期目标是阐明基本的调控机制 肠粘膜功能，特别是上皮细胞 增殖和分化及其受生长因子的调控 和荷尔蒙。建议的研究将利用一种新的体外模型 以人类肠道上皮细胞为中心 温度敏感型SV40大肿瘤条件永生化 抗原(tsFHI细胞系)。这些单元格可以在没有 在32摄氏度的培养中处于增殖状态的表观极限，以及 通过改变到更高的温度而诱导随意分化 致癌基因被灭活了。利用这些细胞，我们将调查 新近发现的细胞周期的作用和机制 调节剂、细胞周期蛋白依赖性激酶抑制物(CKIs)和其他 以确定肠道细胞中的细胞成分 分化过程。我们的具体目标是：1)直接获得 CKI p21/WAF-1/Cip1在不可逆性丢失中起关键作用的证据 标志着tsFHI细胞的开始的增殖能力 分化；2)将表达和复合体的变化联系起来 其他已知的CKI与tsFHI细胞的生长和分化形成； 3)鉴定、克隆和鉴定其他细胞成分 其表达在tsFHI细胞被 诱导分化的。这些研究中将使用的方法 将包括免疫沉淀，Western和Northern印迹， 用野生型或缺失的cDNAs转染，通过 对克隆基因进行聚合酶链式反应、基因克隆、测序和功能分析。 拟议的研究应提供重要的信息，以进一步 了解各种肠道生理活动和 疾病状态，特别是营养和致癌。

Our long-term objective is to elucidate basic mechanisms regulating intestinal mucosal functions, with particular emphasis on epithelial cell proliferation and differentiation, and their modulation by growth factors and hormones. The proposed research will utilize a new in vitro model we have developed, centered on human intestinal epithelial cells conditionally-immortalized with a temperature-sensitive SV40 large tumor antigen (tsFHI cell lines). These cells can be maintained without apparent limits in a proliferative state by culture at 32 degrees C, and induced to differentiate at will by a shift to a higher temperature at which the oncogene is inactivated. Using these cells, we will investigate the role and mechanism of action of recently discovered cell cycle regulators, the cyclin-dependent kinase inhibitors (CKIs), and other yet to be identified cellular components, in the intestinal cell differentiation process. Our specific aims are: 1) To obtain direct evidence for a key role of the CKI p21/Waf-1/Cip1 in the irreversible loss of proliferative potential that marks the onset of tsFHI cell differentiation; 2) To correlate changes in expression and complex formation of other known CKIs with tsFHI cell growth and differentiation; and 3) To identify, clone, and characterize other cellular components whose expression is specifically altered at the time tsFHI cells are induced to differentiate. The methods that will be used in these studies will include immunoprecipitation, Western and Northern blotting, transfection with wild-type or deleted cDNAs, mRNA differential display by PCR, cDNA cloning, sequencing, and analysis of function of cloned genes. The proposed studies should provide information important to further our understanding of a variety of intestinal physiological activities and disease states, in particular nutrition and carcinogenesis.