NOVEL OSTEOCLASTOGENIC T CELL FACTOR

新型破骨细胞 T 细胞因子

• 批准号: 6375261
• 负责人: LEONARD RIFAS
• 金额: $ 7.15万
• 依托单位: BARNES-JEWISH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6375261
• 关键词: SDS polyacrylamide gel electrophoresis; T lymphocyte; affinity chromatography; antiantibody; cytokine; human tissue; interleukin 6; leukocyte activation /transformation; molecular cloning; northern blottings; osteoblasts; polymerase chain reaction; protein purification; protein sequence; recombinant proteins; rheumatoid arthritis

Rheumatoid arthritis (RA) is currently considered an autoimmune disease in which a pathologic immune response attacks synovial cells, cartilage and bone resulting in joint destruction and permanent disability. Loss of bone mass in RA is a common clinical problem occurring as both juxta-articular or localized bone loss and generalized osteoporosis leading to the risk of fracture. The juxta-articular bone loss is associated with an increase in blood- derived T cells infiltrating into the synovial cavity. Activated (Act) T cells are believed to mediate most of the tissue destruction, once the inflammation cascade has begun. The Act T cell is a source of cytokines known to regulate bone turnover, such as IL-6 , TNF-alpha and a new member of the TNF receptor family, TRANCE/RANKL/ODF/OPGL, but a causative T cell cytokine is presently unknown. This project is aimed at discovering whether a cytokine produced by activated T cells, that we have recently found regulates the expression of IL-6 in normal human osteoblasts and regulates osteoclast differentiation, is a novel protein. The identification of a novel T cell factor which may be inducing osteopenia in rheumatoid arthritis and perhaps other autoimmune diseases may lead to the development of inhibitory agents which may prove to be of therapeutic efficacy in preventing bone loss in these diseases. Thus our specific Aims are: To purify, characterize and clone the soluble factor(s) produced by activated T-cells that regulates IL-6 production in human osteoblasts and induces osteoclastogenesis; and 2) Prepare recombinant protein and produce antibodies to the T cell factor for future studies of its function. The factor(s) will be isolated using affinity chromatogphy, anion and cation exchange chromatography, gel filtration chromatography and SDS-PAGE. Identification of the proteins as novel will be performed by N-terminal or internal sequencing. Internal sequence synthetic peptides will be synthesized and used for the production of polyclonal antibodies. Cloning will be performed using the rapid amplification of cDNA ends (RACE) technique to obtain a partial human cDNA from adaptor-ligated human T cell cDNA. Primers will be designed and the full length cDNA reading frame will be PCR amplified, primers prepared and ultimately a fusion protein cDNA prepared and ligated into a prokaryotic expression vector in order to produce recombinant protein. Antibodies will be prepared against the protein to characterize its function in vitro. We propose to determine whether the T cell factor induces TRANCE in hOB by probing isolated mRNA with labeled cDNA, generated by RT-PCR methods, and Northern blot analysis.

类风湿性关节炎（RA）是一种自身免疫性疾病，其病理性免疫反应攻击滑膜细胞、软骨和骨，导致关节破坏和永久性残疾。类风湿性关节炎患者的骨量丢失是一个常见的临床问题，可表现为关节旁或局部骨质丢失以及导致骨折风险的全身性骨质疏松症。近关节骨丢失与血液来源的T细胞浸润到滑膜腔中的增加有关。一旦炎症级联反应开始，激活的（Act）T细胞被认为介导了大部分组织破坏。Act T细胞是已知调节骨转换的细胞因子的来源，如IL-6、TNF-α和TNF受体家族的新成员TRANCE/RANKL/ODF/OPGL，但目前尚不清楚致病性T细胞细胞因子。本项目的目的是发现由活化的T细胞产生的细胞因子是否是一种新的蛋白质，我们最近发现该细胞因子调节正常人成骨细胞中IL-6的表达并调节破骨细胞的分化。一种新的T细胞因子，可能会诱导骨质减少类风湿性关节炎，也许其他自身免疫性疾病的鉴定可能会导致抑制剂的发展，这可能被证明是在预防这些疾病的骨丢失的治疗效果。因此，我们的具体目标是：纯化、鉴定和克隆由活化的T细胞产生的调节人成骨细胞中IL-6产生并诱导破骨细胞生成的可溶性因子; 2）制备重组蛋白并制备针对T细胞因子的抗体以用于其功能的进一步研究。将使用亲和层析、阴离子和阳离子交换层析、凝胶过滤层析和SDS-PAGE分离因子。将通过N-末端或内部测序鉴定新蛋白。将合成内部序列合成肽并用于生产多克隆抗体。将使用cDNA末端快速扩增（RACE）技术进行克隆，以从接头连接的人T细胞cDNA中获得部分人cDNA。设计引物，PCR扩增全长cDNA阅读框，制备引物，最终制备融合蛋白cDNA并连接到原核表达载体中，以产生重组蛋白。将针对蛋白质制备抗体以表征其体外功能。我们建议，以确定是否T细胞因子诱导TRANCE在hOB探针分离的mRNA与标记的cDNA，产生的RT-PCR方法，和北方印迹分析。