CELL CYCLE CHANGES IN THE GALACTOSEMIC LENS

半乳糖晶状体中的细胞周期变化

• 批准号: 7381554
• 负责人: Karen Vanessa Gonzalez
• 金额: $ 11.02万
• 依托单位: UNIVERSITY OF PUERTO RICO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7381554
• 关键词: CELL; CYCLE; CHANGES; GALACTOSEMIC; LENS

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. To better understand the mechanism for human health consequences of exposure to high concentrations of sugars, as in diabetes, changes in the cell cycle progression will be studied. It has been previously shown that one of the most significant changes in diabetic and galactosemic LECs are changes in the rate of mitosis when compared to controls as demonstrated using several techniques. When LECs are exposed to high galactose (40 mM) in the culture medium for 4 days an increase in the rate of mitosis is observed as evidenced by an increase in the number of mitotic figures and in H3 incorporation experiments. By 7 days, the rate of mitosis decreases dramatically when compared to control using the techniques listed above. The PI analyzed the cell cycle progression of bovine LECs using flow cytometry and acridine orange and determined that galactosemic LECs exhibited a high mitotic rate at 7 days of exposure to 40 mM galactose because of the amount and conformation of the DNA of these cells. This finding contradicts the results obtained by counting mitotic figures and H3-incorporation experiments. The hypothesis is that changes in the cell cycle progression of galactosemic LECs are a consequence of the activation of a checkpoint at the G2 to mitosis transition regulated by the cdc2/p34 complex. The purpose of this study is to determine the status of the cdc2/p34 complex in normal and galactosemic (40 mM galactose treated) bovine LECs in culture. This study will establish the parameters to be used in the future to expand research using real-time RT-PCR and human LECs obtain from local sources. Results could be used to develop strategies to control the onset of sugar cataracts in susceptible populations. The specific aims of this proposal are two-fold: Scientific: - To establish primary cultures of bovine lens epithelial cells. - To synchronize primary cultures of bovine LECs using - To determine the levels and activation status ofcdc2/p34 complex in normal and galactosemic LECs. The complexes will be isolated using a peptide-agarose bead complex available commercially specific for the cdc2/p34 complex and Western blot analysis with antibodies specific for the complex and anti-phosphotyrosine antibodies. The activation status of the cdc2/p34 complex will be studied using a kinase activity assay. - To determine the levels of the regulators, wee-1 protein and cdc25, in normal and galactosemic LECs. This will be accomplished through Western blot analysis and commercially available antibodies to wee-t protein and cdc25. Academic: - To increase the number of minority science students doing research at UMET through exposure to the latest techniques in cellular biology and travel opportunities to scientific meetings to present their findings. - To strengthen the research environment at UMET through enhancing student's awareness to continue graduate studies' in biomedical sciences. - To integrate research achievements and environment into the academic experience.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。为了更好地了解暴露于高浓度糖（如糖尿病）对人类健康造成影响的机制，将研究细胞周期进程的变化。先前已经表明，与对照组相比，糖尿病和半乳糖血症性 LEC 最显着的变化之一是有丝分裂率的变化，如使用多种技术所证明的。当 LEC 暴露于培养基中的高半乳糖 (40 mM) 4 天时，观察到有丝分裂率增加，有丝分裂图数量增加和 H3 掺入实验证明了这一点。到 7 天时，与使用上述技术的对照相比，有丝分裂率显着降低。 PI 使用流式细胞术和吖啶橙分析了牛 LEC 的细胞周期进展，并确定半乳糖 LEC 在暴露于 40 mM 半乳糖 7 天时表现出高有丝分裂率，因为这些细胞的 DNA 数量和构象。这一发现与有丝分裂计数和 H3 掺入实验获得的结果相矛盾。 假设半乳糖 LEC 细胞周期进程的变化是由 cdc2/p34 复合物调节的 G2 到有丝分裂过渡的检查点激活的结果。本研究的目的是确定培养的正常牛 LEC 和半乳糖血症（40 mM 半乳糖处理）牛 LEC 中 cdc2/p34 复合物的状态。这项研究将建立未来使用的参数，以利用实时 RT-PCR 和从当地获取的人类 LEC 来扩大研究范围。研究结果可用于制定控制易感人群糖性白内障发病的策略。 该提案的具体目标有两个： 科学： - 建立牛晶状体上皮细胞的原代培养物。 - 使用同步牛 LEC 的原代培养物 - 确定正常和半乳糖 LEC 中 cdc2/p34 复合物的水平和激活状态。将使用市售的针对 cdc2/p34 复合物的肽-琼脂糖珠复合物来分离复合物，并使用针对该复合物的特异性抗体和抗磷酸酪氨酸抗体进行蛋白质印迹分析。将使用激酶活性测定来研究 cdc2/p34 复合物的激活状态。 - 确定正常和半乳糖血症 LEC 中调节因子 wee-1 蛋白和 cdc25 的水平。这将通过蛋白质印迹分析和针对 wee-t 蛋白和 cdc25 的市售抗体来完成。 学术： - 通过接触细胞生物学的最新技术以及参加科学会议展示其发现的机会，增加在 UMET 进行研究的少数族裔理科学生的数量。 - 通过提高学生继续生物医学科学研究生的意识来加强 UMET 的研究环境。 - 将研究成果和环境融入学术经验中。