IN VITRO METHOD OF ACCELERATING LYMPHOCYTE SENESCENCE

加速淋巴细胞衰老的体外方法

• 批准号: 2500997
• 负责人: Robert Thomas Woodland
• 金额: $ 7.75万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2500997
• 关键词: B lymphocyte; CD40 molecule; aging; genetically modified animals; immunosenescence; laboratory mouse; nuclear factor kappa beta; tissue /cell culture

We propose to test the hypothesis that long term culture of primary B lymphocytes accelerates senescence in these cells making them a useful and accessible model to study signal transduction defects in aging lymphocytes. Studies to examine the biology of primary lymphocytes from aged murine donors is severely compromised by the expense of animal aging and by the difficulty of finding disease-free donors whose lymphocytes have not been stimulated by exposure to environmental antigens. An in vitro model that would allow an acceleration of lymphocyte senescence could be a powerful tool, making aging studies more accessible to interested researchers, and reducing the variation in results among workers drawing from significantly different animal colonies. We have found that normal primary B cells expressing a human bcl-2 transgene survive unstimulated for prolonged periods in culture. B cells from these long term cultures are stimulated to proliferate with LPS and anti-CD38 as vigorously as freshly isolated input cells. However, in striking contrast cultured B cells become increasingly refractory to anti-Ig and CD4OL driven proliferation. We hypothesize that deficiencies in Ig and/or especially CD40 stimulatory pathways could be the basis for the hyporesponsive phenotype of aged B cells to thymus dependent antigens and that cultured B cells develop these deficiencies in an accelerated fashion. We propose three experimental aims to validate this new experimental model: (l) To compare proliferation of cultured B cells to aged B cells using 4 polyclonal activators; anti-Ig, CD4OL, CD38, and LPS; (2) To quantitate by flow cytometry the expression of ligand receptors on unactivated cells, and the induction of costimulators and activation molecules on young, aged and cultured B cells after stimulation with selected polyclonal activators; (3) To assess NF-kappa B activation induced by all four activators in cultured and aged B lymphocytes.

我们建议检验以下假设：原代 B 的长期培养 淋巴细胞加速这些细胞的衰老，使它们成为有用的细胞 和可用于研究衰老信号转导缺陷的模型 淋巴细胞。检查原代淋巴细胞生物学的研究 老年小鼠捐赠者因动物费用而受到严重损害 老龄化以及难以找到无病捐献者 淋巴细胞未因暴露于环境而受到刺激 抗原。体外模型可以加速 淋巴细胞衰老可能是进行衰老研究的有力工具 感兴趣的研究人员更容易接触到，并减少变异 工人从显着不同的动物中提取的结果 殖民地。我们发现正常的原代 B 细胞表达人类 bcl-2 转基因可以在培养物中长时间不受刺激地存活。 来自这些长期培养物的 B 细胞被刺激增殖 使用 LPS 和抗 CD38 与新鲜分离的输入细胞一样有力。 然而，与此形成鲜明对比的是，培养的 B 细胞变得越来越 对抗 Ig 和 CD4OL 驱动的增殖具有抵抗力。我们假设 Ig 和/或尤其是 CD40 刺激途径的缺陷 可能是衰老 B 细胞反应低下的基础 胸腺依赖性抗原，培养的 B 细胞会产生这些抗原 缺陷以加速的方式出现。我们提出了三个实验 旨在验证这个新的实验模型：（l）比较 使用 4 种多克隆细胞将培养的 B 细胞增殖为老化的 B 细胞 活化剂；抗 Ig、CD4OL、CD38 和 LPS； (2) 流量定量 细胞计数未激活细胞上配体受体的表达，以及 对年轻人和老年人共刺激剂和激活分子的诱导 并用选定的多克隆刺激后培养 B 细胞 活化剂； (3) 评估所有四种物质诱导的 NF-kappa B 激活 培养和老化的 B 淋巴细胞中的激活剂。