SENESCENE AND PREB CELL DEVELOPMENT

描述(改编自调查者摘要)：B淋巴细胞生成 由于老化过程而急剧下降。在小鼠身上，衰老 导致前B细胞数量减少，前B细胞功能改变，以及 产生一种与此截然不同的抗体库 在幼年成年老鼠身上可见。研究人员假设前B细胞 改变了RAG-1和RAG-2的表达，以及终末 脱氧核苷酸转移酶(TDT)，并因此改变 Mu重链VH曲目的生成。他进一步提出了假设 VH曲目的更新换代和多样化有助于 减少前B细胞受体复合体的形成或功能障碍 (MU/lambda-5/V-Pre-B)和随后未能招募新的Pre-B细胞进入 有丝分裂。这将导致前B细胞的低产量和 老年小鼠的改变的特异性谱系。为了测试这些 假设，他在具体目标1中提出了评估对 前B细胞受体蛋白的转录和蛋白表达， Lambda-5和V-Pre-B转化为前B细胞(CD43+CD25-B220+)，新形成 前B细胞(CD43+CD25+B220+)和晚期前B细胞(CD43-CD25+B220+) 与老年人隔离(24个月)VS Young(3个月)BALB/c小鼠的荧光检测 激活了细胞分选。他还将评估这些蛋白质的能力 通过以下途径组装成表面膜相关的前B细胞受体 利用替代轻链和前B细胞受体复合体特异性 前B细胞受体组装的单抗染色及分析 以及A-MuLV前B细胞系幼鼠和老年鼠的翻转。这个 老年前B细胞受体表达对功能的影响 RAG-1和RAG-2的表达；TDT；蛋白酪氨酸激酶Blk； 将评估^X5本身的表达。在具体目标2中， 前B细胞受体依赖的MU重链谱系的选择 将在新生前B细胞(CD43+CD25+B220+)中进行评估，而不是以后 (选择后)老年小鼠与年轻小鼠的前B细胞(CD43-CD25+B220+) 特别强调CDR3的多样性。在具体目标3中， 衰老小鼠骨髓微环境在决定衰老过程中的重要性 观察到的B系细胞发育的变化将使用 体内过继移植模型和体外基质细胞培养 系统。此应用程序中的实验将提供一种细胞和 了解B淋巴细胞亚群时间丢失的分子基础 在衰老过程中VH谱系的生成发生了变化。

DESCRIPTION (Adapted from the Investigator's abstract): B lymphopoiesis declines dramatically due to the aging process. In the mouse, senescence results in decreased pre-B-cell numbers, altered pro-B-cell function, and generation of an antibody repertoire which is critically different from that seen in young adult mice. The investigator hypothesizes that pro-B-cells from aged mice have altered expression of Rag-1, and Rag-2, and terminal deoxynucleotidyl transferase (TdT) enzymes, and, consequently, altered generation of the mu heavy chain VH repertoire. He further hypothesizes that altered generation and diversification of the VH repertoire contributes to diminished formation or dysfunction of the pre-B-cell receptor complex (mu/lambda-5/V-pre-B) and subsequent failure to recruit new pre-B-cells into mitosis. This would result in both low production of pre-B-cells and an altered specificity repertoire in aged mice. In order to test these hypotheses, he proposes in Specific Aim 1 to assess the regulation of transcription and protein expression of the pre-B-cell receptor proteins mu, lambda-5, and V-pre-B into pro-B-cells (CD43+CD25-B220+), newly formed pre-B-cells (CD43+CD25+B220+), and late stage pre-B-cells (CD43-CD25+B220+) isolated from aged (24 mo.) vs. young (3 mo.) BALB/c mice via fluorescence activated cell sorting. He will also assess the capacity of these proteins to assemble into surface membrane associated pre-B-cell receptors by utilizing surrogate light chain and pre-B-cell receptor complex specific monoclonal antibody staining and analysis of pre-B-cell receptor assembly and turn-over in A-MuLV pre-B-cell lines from young vs. aged mice. The capacity of aged pre-B-cell receptor expression to functionally affect the expression of Rag-1 and Rag-2; TdT; the protein tyrosine kinase Blk; and the expression of ^X5 itself will be assessed. In Specific Aim 2, the pre-B-cell receptor dependent selection of the mu heavy chain repertoire will be evaluated in nascent pre-B-cells (CD43+CD25+B220+) vs. later (post-selection) pre-B-cells (CD43- CD25+B220+) from aged vs. young mice with particular emphasis on diversity in CDR3. In Specific Aim 3, the importance of the bone marrow microenvironment of aged mice in dictating the changes observed in B lineage cell development will be investigated using both in vivo adoptive transfer models and in vitro stroma cell culture systems. The experiments in this application will provide a cellular and molecular basis for understanding the temporal loss of B lymphopoiesis and altered generation of VH repertoire during senescence.