Functional dissection and medical application of the 4 modules in the chondrocyte-derived growth factor, ecogenin/CTGF.

1.生态生长素/结缔组织生长因子各模块特异性抗体的制备及检测体系的建立：首先，由短杆菌生产的S菌茎独立制备各模块，定位抗结缔组织生长因子抗体的表位。结果，在我们建立的ELISA体系中，至少获得了一种针对IGFBP、VWC、TSP1或CT每个模块的抗体，其中最敏感的一个已经用于后续的研究。此外，还建立了其他定量检测CTGF亚片段的酶联免疫吸附试验体系。研究模块化结构在细胞生长和分化中的作用：利用每个纯化的模块，我们检测软骨细胞HCS-2/8细胞中的信号转导通路是否被激活。结果，一些不同的通路被特定的模块激活。由于对其他细胞类型的研究结果不同，CTGF被认为可以不同地使用多种信号通路。值得注意的是，几种模块特异性抗体对HCS-2/8的蛋白多糖合成有相当的促进作用，而这些抗体则抑制了细胞的增殖。这些发现表明抗体以及CTGF衍生物在治疗学中的用途。接下来，我们评估了这些模块对细胞黏附的影响，发现了所有模块对细胞黏附的促进作用。在四种治疗方法中，CT效果最好。最后，评价了这些模块的过表达对细胞周期的影响。建议采用双模块TSP-CT和单层螺旋CT使细胞周期停滞于M期。因此，在考虑治疗应用时，应特别注意CT模块。动物模型实验治疗：在应用模块化突变体之前，将野生型CTGF应用于实验性大鼠软骨缺损模型，并对其效果进行评估。然后发现CTGF可以再生受损的关节软骨。这一方法将被应用于模块突变体的评估。

1. Establishment of the production and detection system with specific antibodies of each module of ecogenin/CTGF : Firstly, each module was independently prepared by a Brevibacillus production s stem, and the epitopes of anti-CTGF antibodies were located. As a result at least one antibody against each module, IGFBP, VWC, TSP1 or CT, was obtained Among the ELISA systems we established, the most sensitive one is already in use for subsequent research. Additionally, the other ELISA systems to quantify CTGF subfragments were also established.2. Investigation of the roles of modular structure in cell growth and differentiation : Utilizing each purified module, we examined if the signal transduction cascades in chondrocytic HCS-2/8 cells were activated. As a result, a few different pathways were activated by specific modules. Since different results were obtained with other cell types, CTGF is supposed to use multiple signaling pathways differentially. Of note, proteoglycan synthesis by HCS-2/8 was rather enhanced by a few module-specific antibodies, whereas proliferation was inhibited by those antibodies. These findings suggest the utility of the antibodies in therapeutics as well as the CTGF derivatives. Next, we evaluated the effects of the modules on cell adhesion and found the promoting effects of all of the modules. Among the four, CT was the most effective. Finally, effects of overexpression of the modules on cell cycle was evaluated. Then, dimodular TSP-CT and single CT were suggested to arrest the cell cycle at M-phase. As such, particular care should be taken for CT module in considering therapeutic application.3. Experimental therapeutics with animal models : Prior to the application of modular mutants, wild type CTGF was applied to the experimental rat cartilage defect models and the effects were evaluated. Then CTGF was found to regenerate damaged articular cartilage. This methodology is going to be applied to the evaluation of modular mutants.