Control of synovitis using gene therapy.

在临床应用中，需要我们探索更安全的基因治疗载体。所以我们选择了相对安全的非病毒载体。首先，通过三种阳离子聚合物将LacZ基因转导至软骨细胞样细胞系（HCS2/8），并通过X-gal染色和ONPG检测该基因的表达情况。聚酰胺胺（PAMAM）树突状物在3种病毒中最有效，显示出非病毒载体的有效性。将该阳离子聚合物和基于ebv的质粒载体注射到大鼠关节内，可以提高体内转导效果。然后在体内观察电穿孔的效果。将含有标记基因的ebv质粒载体注射到大鼠膝关节内，并通过一对特殊电极施加不同电压的电脉冲。基因表达量在150V时达到最高水平，且明显优于阳离子聚合物。我们对凋亡信号调控激酶(ASK) -1基因进行了研究。该基因在细胞中的表达有望诱导细胞凋亡。将含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染色法中，60 h后约50%的细胞被诱导凋亡。在这项研究中，我们发现与其他非病毒载体相比，电穿孔在体内将基因转导到滑膜组织中是有用的，并表明ASK-1可能通过转导到滑膜而对滑膜炎有效。

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.