Control of synovitis using gene therapy.

寻找更安全的载体用于临床基因治疗是非常必要的。因此，我们选择了相对安全的非病毒载体。首先，用三种阳离子聚合物将LacZ基因导入软骨细胞样细胞系(HCS2/8)，X-Gal染色和ONPG法检测LacZ基因的表达。其中以聚酰胺胺(PAMAM)树枝状大分子效果最好，显示了非病毒载体的应用价值。将这种阳离子聚合物和基于EBV的质粒载体注射到大鼠关节内，可以提高体内的转导效率。然后，我们采用体内电穿孔的效果。将含有标记基因的EB病毒载体注射到大鼠膝关节内，用一对特殊电极施加不同电压的电脉冲。基因表达在150V时达到最高水平，明显好于阳离子聚合物。我们对细胞凋亡信号调节蛋白激酶(ASK)-1基因的研究很感兴趣。该基因在细胞中的表达有望诱导细胞凋亡。将携带ASK-1基因的腺病毒载体感染单层培养的人类风湿滑膜细胞。在Hoechst 33342染色中，60h后，约50%的细胞被诱导凋亡。在本研究中，我们通过与其他非病毒载体的比较，发现了电穿孔在体内将基因转导到滑膜组织中的有效性，并证明了ASK-1通过转导到滑膜来治疗滑膜炎的可能性。

It is needed for us to explore safer vector for gene therapy in clinical use. So we chose non-virus vectors that are relatively safe. Firstly, LacZ gene was transduced to chondrocytes-like cell line (HCS2/8) by three types of cationic polymer and this gene expression was evaluated by X-gal staining and ONPG assay. Polyamidoamine (PAMAM) dendrimer was most effective in three, and it showed the usefulness of non-virus vector. Injections into rat joints with this cationic polymer and EBV-based plasmid vector were found to improve the transduction efficacy in vivo. Then we employed the efficacy of electroporation in vivo. EBV-based plasmid vector containing marker gene was injected into rat knee joints and different voltages of electric pulses were applied with a pair of special electrodes. The gene expression reached the highest level in 150V, and it was much better than with cationic polymers. We took an interest in apoptosis signal regulating kinase (ASK) -1 gene. The expression of this gene in cells was expected to induce apoptosis. Adenovirus vector containing ASK-1 gene was infected to the human rheumatoid synovious cells placed in monolayer culture. In Hoechst 33342 staining assay, 60 hours later, apoptosis was induced in about 50% of cells. In this study, we found the usefulness of the electroporation for gene transduction to synovious tissue in vivo, compared with other non-virus vectors and showed the possibility that ASK-1 would be effective for synovitis by transducing to synovium.