NOVEL OSTEOCLASTOGENIC T CELL FACTOR

新型破骨细胞 T 细胞因子

• 批准号: 6171684
• 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 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6171684
• 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）目前被认为是一种自身免疫性疾病，其中病理性免疫反应攻击滑膜细胞、软骨和骨骼，导致关节破坏和永久性残疾。类风湿关节炎骨量丢失是一个常见的临床问题，发生在关节旁或局部骨质流失和全身性骨质疏松症，导致骨折的风险。关节旁骨质流失与血源性T细胞浸润滑膜腔的增加有关。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的组织破坏。Act T细胞是已知的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，但目前尚不清楚引起T细胞的细胞因子。本项目旨在发现一种由活化的T细胞产生的细胞因子是否是一种新的蛋白，我们最近发现它可以调节正常人类成骨细胞中IL-6的表达并调节破骨细胞的分化。一种可能在类风湿关节炎和其他自身免疫性疾病中诱导骨质减少的新型T细胞因子的鉴定可能导致抑制剂的开发，这种抑制剂可能被证明在预防这些疾病中的骨质流失方面具有治疗功效。因此，我们的具体目标是：纯化、表征和克隆由活化的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发生的可溶性因子；2)制备重组蛋白，制备针对T细胞因子的抗体，为进一步研究其功能做准备。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。鉴定新蛋白将通过n端或内部测序进行。内部序列合成肽将被合成并用于生产多克隆抗体。克隆将使用cDNA末端快速扩增（RACE）技术从接头连接的人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，并将其连接到原核表达载体上，产生重组蛋白。将制备针对该蛋白的抗体，以表征其体外功能。我们建议通过RT-PCR方法生成的标记cDNA探测分离的mRNA和Northern blot分析来确定T细胞因子是否诱导hOB的TRANCE。