EXPRESSION OF PROTEIN KINASE C IN LENS EPITHELIAL CELLS

蛋白激酶C(PKC)亚型在各种糖尿病组织中的表达变化已被证实。将细胞暴露在高浓度糖中作为研究糖尿病的系统，也表明了这一点。利用蛋白质印迹分析，P.I.确定，与正常(低糖)培养的细胞相比，在培养的牛晶状体上皮细胞中，暴露于高浓度(40 MM)半乳糖中的一种主要PKC亚型的相对数量发生了变化。在荧光显微镜下比较正常和半乳糖血症牛晶状体上皮细胞时，也观察到两种主要的PKC亚型PKC-α和PKC-伽马的细胞定位的变化。该项目的目的是确定在培养的晶状体上皮细胞暴露于40 mM半乳糖后，PKC亚型在RNA水平上是否发生变化。这将通过建立牛晶状体上皮细胞的原代培养并使用Northern印迹分析等技术来确定PKC亚型的表达是否发生变化来实现。原位杂交将用于确定PKC转录本的表达模式。表达将使用适当的PKC亚型特异的cDNA探针进行研究。通过该项目获得的结果将为阐明半乳糖性白内障的发生机制提供信息。该项目的另一个目标是增加在该机构进行研究的少数族裔学生的数量。提高学生继续细胞和分子生物学研究生学习的意识也将被评估。

Changes in protein kinase C (PKC) isoform expression has been demonstrated in various diabetic tissues. This has been also shown with cells exposed to high concentrations of sugars as a system to study diabetes. Using Western blot analysis the P.I. determined that in bovine lens epithelial cells in culture exposure to high concentration of galactose (40 mM) resulted in changes in the relative amounts of one of the major PKC isoforms when compared to cells grown in normal (low sugar) media. Changes in the cellular localization of two of the major PKC isoforms, PKC-alpha and PKC-gamma, was also observed when comparing normal and galactosemic bovine lens epithelial cells in culture using epifluorescent microscopy. The aim of this project is to determine if changes in PKC isoforms occur at the RNA level in lens epithelial cells in culture after exposure to 40 mM galactose. This will be accomplished by establishing primary cultures of bovine lens epithelial cells and using techniques such as Northern blot analysis to determine if the expression of the PKC isoforms changes. In situ hybridization will be used to determine the expression pattern of the PKC transcripts. Expression will be studied using the appropriate PKC isoform specific cDNA probes. The findings obtained through this project will provide information for the elucidation of the mechanism of development of galactosemic cataracts. Another aim of this project is to increase the number of minority students performing research at the institution. Enhancing the awareness of our students to continue graduate studies in Cellular and Molecular Biology will also be assessed.