INTESTINAL CELL DIFFERENTIATION IN VITRO

我们的长期目标是阐明调节 肠粘膜功能，特别强调上皮细胞 增殖和分化，以及生长因子对它们的调节 和荷尔蒙。 拟议的研究将利用一种新的体外模型， 以人类肠上皮细胞为中心 条件性永生化的温度敏感性SV 40大肿瘤 抗原（tsFHI细胞系）。 这些细胞可以在没有 通过在32 ℃下培养，在增殖状态下的明显限制，和 通过向更高温度的转变， 使癌基因失活。 利用这些细胞，我们将研究 最近发现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剂（CKIs），以及其他 在肠细胞中， 分化过程。 我们的具体目标是：1）直接获得 CKI p21/Waf-1/Cip 1在不可逆损失中的关键作用的证据 标志着tsFHI细胞开始增殖的可能性 2）将表达变化与复杂的 其他已知CKI与tsFHI细胞生长和分化的形成; 和3）鉴定、克隆和表征其他细胞组分 其表达在tsFHI细胞被转染时特异性改变， 诱导分化。 这些研究中使用的方法 将包括免疫沉淀、蛋白质印迹和北方印迹， 用野生型或缺失的cDNA转染，mRNA差异显示 PCR、cDNA克隆、测序和克隆基因的功能分析。 建议的研究应提供重要资料，以进一步 了解各种肠道生理活动， 疾病状态，特别是营养和致癌作用。

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.