SENESCENE AND PREB CELL DEVELOPMENT

描述（改编自研究者摘要）：B淋巴细胞生成 由于老化过程而急剧下降。 在小鼠中，衰老 导致前B细胞数量减少，前B细胞功能改变， 抗体库的产生，其与 在年轻的成年小鼠中发现。 研究者假设前B细胞 来自老年小鼠的Rag-1和Rag-2的表达改变， 脱氧核苷酸转移酶（TdT），并因此改变 产生mu重链VH库。 他进一步假设 VH库的产生和多样化的改变有助于 减少前B细胞受体复合物的形成或功能障碍 (mu/λ-5/V-前-B）和随后未能将新的前-B-细胞募集到 分裂。 这将导致前B细胞的低产量和 老年小鼠特异性谱改变。 为了测试这些 假设，他在具体目标1中提出，以评估监管 前B细胞受体蛋白μ的转录和蛋白表达， λ-5和V-前B转化为新形成的前B-细胞（CD 43 + CD 25-B220+ 前B细胞（CD 43 + CD 25 +B220+）和晚期前B细胞（CD 43-CD 25 +B220+） 分离自老年人（24个月）vs.年轻（3个月）通过荧光法测定BALB/c小鼠 激活细胞分选。 他还将评估这些蛋白质的能力 组装成表面膜相关的前B细胞受体， 利用替代轻链和前B细胞受体复合物特异性 前B细胞受体组装的单克隆抗体染色和分析 和来自年轻小鼠与老年小鼠的A-MuLV前B细胞系中的转化。 的 老化前B细胞受体表达功能性影响 Rag-1和Rag-2的表达; TdT;蛋白酪氨酸激酶Blk;以及 将评估^X5本身的表达。 具体目标2 mu重链库的前B细胞受体依赖性选择 将在新生前B细胞（CD 43 + CD 25 +B220+）中评价， 来自老年小鼠与年轻小鼠的（选择后）前B细胞（CD 43-CD 25 +B220+） 特别强调CDR 3中的多样性。 具体目标3中， 老年小鼠骨髓微环境在决定衰老过程中的重要性 在B谱系细胞发育中观察到的变化将使用 体内过继转移模型和体外基质细胞培养 系统. 本申请中的实验将提供一种细胞和 理解B淋巴细胞生成暂时丧失的分子基础， 在衰老期间改变VH库的产生。

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.