权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study on gene therapy using non-viral vector for joint disease

非病毒载体治疗关节疾病的基因治疗研究

基本信息

批准号：
11470314
负责人：
KUBO Toshikazu
金额：
$ 9.41万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470314/
关键词：
gene therapy non-viral vector chondrocyte HCS-2 8 joint disease drug delivery system polymer EBV

项目摘要

Usefulness of three types of cationic polymer, i.e., degraded polyamidoamine (PAMAM) dendrimer (SuperFect Transfection Reagent), linear polyethylenimine (PEI) (ExGen 500) and branched PEI in gene delivery into chondrocytes was comparatively examined. A plasmid vector containing the E.coli LacZ (pSES.β) was combined with one of the three cationic polymers at various molar ratios and the resultant complex (polyplex) was used to transduce a human chondrocyte-like cell line, HCS-2/8. Gene expression was evaluated by the O-Nitrophenyl β-D-Galactopyranoside (ONPG) assay and X-gal staining. The ONPG assay showed that the highest delivery rate was achieved when 2 μg of pSES.β was combined with either 21 μg of dendrimer, 1.7 μg of linear PEI, or 2.0 μg of branched PEI.At the same DNA/polymer ratios, the proportions of X-gal-staining cells were also the highest (31, 30, and 8%, respectively). LacZ expression reached the highest level 3days after the dendrimer-mediated transduction, gradually declined and returned to the background level on day 14. Possible cytotoxicity was examined by trypan blue staining and phase contrast microscopic observations. Neither cytotoxicity nor morphological change was induced at the optimal dose of each polymer. The cationic polymers, particularly the degraded dendrimer and linear PEI, would be a useful non-viral vector for gene delivery to the cells in chondrocyte lineage.

三种类型的阳离子聚合物，即，降解的聚酰胺胺（PAMAM）树状聚合物（SuperFect Transfection Reagent）、线性聚乙烯亚胺（PEI）（ExGen 500）和支化PEI在基因递送到软骨细胞中的作用进行了比较研究。将含有大肠杆菌LacZ（pSES.β）的质粒载体与三种阳离子聚合物中的一种以各种摩尔比组合，并将所得复合物（多聚物）用于培养人软骨细胞样细胞系HCS-2/8。通过邻硝基苯基β-D-吡喃半乳糖苷（ONPG）测定和X-gal染色评估基因表达。ONPG检测显示，当2 μg pSES.β与21 μg树枝状聚合物、1.7 μg线性PEI或2.0 μg支化PEI组合时，获得最高的递送率。在相同的DNA/聚合物比例下，X-gal染色细胞的比例也最高（分别为31、30和8%）。LacZ表达在转染后3d达到最高水平，随后逐渐下降，并在第14天恢复到背景水平。通过台盼蓝染色和相差显微镜观察检查可能的细胞毒性。在每种聚合物的最佳剂量下，均未诱导细胞毒性或形态学变化。阳离子聚合物，特别是降解的树枝状聚合物和线性PEI，将是一个有用的非病毒载体的基因传递到软骨细胞系的细胞。