LIPOPOLYSACCHARIDE ENDOTOXIN RESPONSE GENE IN CELLS

细胞内脂多糖内毒素反应基因

• 批准号: 2004497
• 负责人: PETER M WONG
• 金额: $ 26.1万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2004497
• 关键词: G protein; Retroviridae; bacterial genetics; cell line; endotoxins; fluorescent in situ hybridization; genetic mapping; host organism interaction; immune response genes; laboratory mouse; lipopolysaccharides; macrophage

DESCRIPTION (Adapted from applicant's abstract): The interaction of the outer membrane components of bacteria with host cells in one form or another has been investigated extensively over the past decades. The lipopolysaccharide endotoxin (LPS) of Gram-negative bacteria is an example of an essential element of the outer membrane of these organisms that as amphipathic molecule has the property of binding to and stimulating various types of mammalian cells. The results of this interaction are multiple pathophysiological, pharmacological and immunological responses by the host. Over the preceding years, our long term objective has been to gain a better understanding of how LPS endotoxin affects cells of the host. The knowledge that host responses are under genetic control is key to this understanding. Consequently, we have pursued the goal of isolating the LPS gene by the use of the C3H/HeJ mouse strain whose cells possess a specific defect for LPS. By employing a differential functional screening approach, we have isolated a cDNA from splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/OuJ responder mice, and its expression in splenic B cells of C3H/HeJ non-responder mice resulted in polyclonal B cell activation in response to LPS stimulation. In this proposal, our specific aims, which are a logical consequence of this finding, are as follows: 1) to complete the confirmatory analysis that expression of the cDNA we isolated can functionally reconstitute a macrophage cell line derived from a C3H/HeH mouse, 2) to refine the mapping analysis indicating that the gene is located on mouse chromosome four, 3) to define the nature of the Lpsd gene we isolated from C3H/HeH mice, 4) to investigate its genomic organization and to verify two single-base substitutions found in C3H/HeJ cDNA, 5) to introduce the Lpsn gene into various primary cells of the C3H/HeJ non-responder mouse and measure their responsiveness after exposure to LPS, 6) to introduce the Lpsn gene into C3H/HeJ non-responder mice using a retrovirus vector and measure the key parameters of endotoxin responsiveness in these mice including B cell mitogenesis, macrophage activation and cytokine elaboration, and resistance to endotoxemia or lethal shock, and 7) to assess the role of Lpsn in resistance to Gram negative bacterial infection.

描述（改编自申请人摘要）： 细菌的外膜成分与一种或另一种形式的宿主细胞 在过去的几十年里被广泛研究。 的 革兰氏阴性菌的脂多糖内毒素（LPS）是一个例子 这些生物体外膜的一种基本元素， 两亲分子具有结合并刺激各种细胞的性质。 哺乳动物细胞的类型。 这种相互作用的结果是多重的 宿主的病理生理学、药理学和免疫学反应。 在过去的几年里，我们的长期目标是获得一个更好的 了解LPS内毒素如何影响宿主细胞。 知识 宿主的反应受遗传控制是理解这一点的关键。 因此，我们追求的目标是通过使用 的C3 H/HeJ小鼠品系，其细胞具有对LPS的特异性缺陷。 通过采用差异功能筛选方法，我们分离出了 C3 H/OuJ应答小鼠脾B细胞的cDNA，及其在 C3 H/OuJ应答小鼠脾B细胞及其在脾B细胞中的表达 C3 H/HeJ无应答小鼠的细胞导致多克隆B细胞活化 对LPS刺激的反应。 在这项建议中，我们的具体目标， 是这一发现的逻辑结果，如下：1）完成 通过对所分离cDNA表达的验证性分析， 功能性地重建衍生自C3 H/HeH的巨噬细胞细胞系 小鼠，2）改进指示基因定位的作图分析 3）为了确定Lpsd基因的性质，我们 分离自C3 H/HeH小鼠，4）研究其基因组组织， 为了验证在C3 H/HeJ cDNA中发现的两个单碱基取代，5）至 将Lpsn基因导入C3 H/HeJ的各种原代细胞， 非应答小鼠并测量它们在暴露于LPS后的应答性， 6)为了将Lpsn基因引入C3 H/HeJ无应答小鼠， 逆转录病毒载体，并测量内毒素反应性的关键参数 包括B细胞有丝分裂、巨噬细胞活化和 细胞因子的产生和对内毒素血症或致死性休克的抵抗，以及7） 探讨脂蛋白酶N在革兰氏阴性杆菌耐药中的作用 感染