EXPRESSION OF PROTEIN KINASE C IN LENS EPITHELIAL CELLS

蛋白激酶C （PKC）异构体表达的变化已在各种糖尿病组织中得到证实。这一点在暴露于高浓度糖的细胞中也被证明是研究糖尿病的一个系统。使用Western blot分析，P.I.确定，与在正常（低糖）培养基中生长的细胞相比，在培养的牛晶状体上皮细胞中，暴露于高浓度半乳糖（40 mM）会导致PKC主要亚型之一的相对数量发生变化。在比较培养的正常和半乳糖牛晶状体上皮细胞时，使用荧光显微镜还观察到PKC α和PKC γ两种主要PKC亚型的细胞定位变化。本项目的目的是确定暴露于40 mM半乳糖后，培养的晶状体上皮细胞中PKC异构体的RNA水平是否发生变化。这将通过建立牛晶状体上皮细胞的原代培养和使用诸如Northern blot分析等技术来确定PKC异构体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。将使用合适的PKC异构体特异性cDNA探针研究表达。本研究结果将为阐明泌乳性白内障的发生机制提供信息。该项目的另一个目的是增加在该机构进行研究的少数民族学生的数量。提高学生继续在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.