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Control of synovitis using gene therapy.

使用基因疗法控制滑膜炎。

基本信息

批准号：
12557127
负责人：
KUBO Toshikazu
金额：
$ 8.06万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557127/
关键词：
gene therapy non-viral vector synovitis electroporation ASK in vivo アデノウイルスベクター apoptosis

项目摘要

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.

我们需要探索更安全的基因治疗载体应用于临床。所以我们选择了相对安全的非病毒载体。首先，通过三种类型的阳离子聚合物将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测定评估该基因的表达。聚酰胺胺（PAMAM）树枝状聚合物是三种中最有效的，它显示了非病毒载体的有用性。研究发现，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中可以提高体内转导效率。然后我们在体内应用了电穿孔的功效。将含有标记基因的基于EBV的质粒载体注射到大鼠膝关节中，并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，并且比阳离子聚合物好得多。我们对凋亡信号调节激酶（ASK）-1 基因感兴趣。该基因在细胞中的表达预计会诱导细胞凋亡。将含有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染色测定中，60小时后，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至滑膜组织的有用性，并表明ASK-1通过转导至滑膜而有效治疗滑膜炎的可能性。