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

比较考察了降解聚氨基胺（PAMAM）树形大分子（superect转染试剂）、线性聚乙烯亚胺（PEI） （ExGen 500）和支链PEI三种阳离子聚合物在软骨细胞基因传递中的作用。将含有大肠杆菌LacZ （pSES.β）的质粒载体以不同的摩尔比与三种阳离子聚合物中的一种结合，所得复合物（polyplex）用于转导人软骨细胞样细胞系HCS-2/8。通过O-Nitrophenyl β-D-Galactopyranoside （ONPG）检测和X-gal染色检测基因表达。ONPG实验显示，2 μg的pse递送率最高。β分别与21 μg的树状分子、1.7 μg的线性PEI或2.0 μg的支链PEI结合。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31.0%、30%和8%）。树状分子介导转导后第3天LacZ表达达到最高水平，第14天逐渐下降，恢复到背景水平。通过台盼蓝染色和相衬显微镜观察观察可能的细胞毒性。每种聚合物在最佳剂量下均未引起细胞毒性和形态变化。阳离子聚合物，特别是降解的树状大分子和线性PEI，将成为一种有用的非病毒载体，用于基因传递到软骨细胞谱系的细胞。

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.