COORDINATE REGULATION OF APOPTOSIS AND CELL CYCLE IN RA

RA中细胞凋亡和细胞周期的协调调控

• 批准号: 6374345
• 负责人: Harris R Perlman
• 金额: $ 11.67万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-15 至 2002-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6374345
• 关键词: BCL2 gene /protein; CD95 molecule; apoptosis; cell cycle; cell proliferation; cellular pathology; fibroblasts; gene therapy; human tissue; laboratory rat; membrane permeability; mitochondrial membrane; nonhuman therapy evaluation; rheumatoid arthritis; ribozymes; synovial membrane

The work proposed for this grant focuses on the coordinate regulation of proliferation and apoptosis in rheumatoid arthritis (RA). Analysis of human RA synovial tissues (ST) sections revealed increased rates of synovial fibroblast proliferation and low rates of apoptosis, though the functional significance of reduced apoptosis remains to be elucidated. However, analysis of the rates of in vivo proliferation and apoptosis are limited in human-STs as tissue sections were taken late in disease course. Thus, utilization of animal models is vital for an understanding of the molecular pathways of proliferation and apoptosis in RA. Recently, adenoviral mediated delivery of Fas ligand (Ad-FasL), a known apoptotic inducer ameliorated experimental arthritis, suggesting that enhancing the rate of apoptosis by gene therapy may be a potential effective therapy. A caveat to Ad-FasL therapy is that high levels of Fas ligand is cytotoxic to many tissues of the body, thus development of other genes to be delivered to the RA joint is essential. We demonstrated that the anti-apoptotic protein and cell cycle modulator, Bcl-2 was highly expressed in RA compared with osteoarthritis synovial tissues, particularly in the CD68- negative, fibroblast-like synoviocyte population. In order to determine the importance of endogenous Bcl-2, an adenoviral vector expressing a hammerhead ribozyme to Bcl-2 (Ad-Rbz-Bcl-2) mRNA was employed. Ad-Rbz-Bcl-2 infection resulted in reduced Bcl-2 expression and cell viability in synovial fibroblasts isolated from RA-synovial tissues. In addition, Ad-Rbz-Bcl-2- induced mitochondrial permeability transition, cytochrome c release, activation of caspases 9 and 3, and DNA fragmentation. These data suggest that Bcl-2 is necessary for synovial fibroblast survival. In this proposal we describe studies to delineate the mechanism of the induction of mitochondrial permeabilty transition following Ad-Rbz-Bcl-2 infection. In addition we will investigate whether adenoviral mediated delivery of the Bcl-2 ribozyme is efficient in ameliorating adjuvant- induced arthritis in rats. The expected outcome is the suppression of AIA through the inhibition of fibroblast proliferation and increased apoptosis. This approach could lead to the development of a new therapeutic strategy for gene therapy in patients with rheumatoid arthritis.

这项拨款的工作重点是类风湿性关节炎(RA)增殖和凋亡的协调调节。对人RA滑膜组织(ST)切片的分析显示，滑膜成纤维细胞增殖率增加，凋亡率低，但凋亡减少的功能意义仍有待阐明。然而，由于组织切片取自病程较晚，对人类STS体内增殖率和凋亡率的分析有限。因此，动物模型的使用对于了解类风湿关节炎增殖和凋亡的分子途径至关重要。最近，腺病毒介导的Fas配体(Ad-FasL)作为一种已知的凋亡诱导剂改善了实验性关节炎，提示通过基因治疗提高细胞凋亡率可能是一种潜在的有效治疗方法。对Ad-FasL治疗的一个警告是，高水平的Fas配体对身体的许多组织具有细胞毒性，因此开发其他基因输送到RA关节是必不可少的。我们发现，与骨关节炎滑膜组织相比，抗凋亡蛋白和细胞周期调节因子Bcl2在RA滑膜组织中高表达，尤其是在CD68阴性的成纤维细胞样滑膜细胞群中。为了确定内源性Bc l-2的重要性，采用了表达抗Bc l-2锤头状核酶的腺病毒载体(Ad-RBZ-Bc l-2)。Ad-RBZ-Bcl2感染导致RA滑膜成纤维细胞Bcl2表达降低，细胞存活率降低。此外，Ad-RBZ-Bcl2还可诱导线粒体通透性转变、细胞色素c释放、caspase9和caspase3的激活以及DNA片段化。这些数据表明，Bcl-2对于滑膜成纤维细胞的存活是必需的。在这项建议中，我们描述了在Ad-RBZ-Bcl2感染后诱导线粒体通透性转变的机制的研究。此外，我们还将研究腺病毒介导的Bcl-2核酶是否能有效改善佐剂诱导的大鼠关节炎。预期的结果是通过抑制成纤维细胞增殖和增加细胞凋亡来抑制AIA。这一方法可能导致开发一种新的治疗策略，用于类风湿性关节炎患者的基因治疗。