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目的优化α1(I)型前胶原基因反义寡核苷酸(ASODN)转染人增生性瘢痕成纤维细胞的条件。方法ASODN经FITC标记,以阳离子脂质体Oligofectamine为载体转染体外培养的第2～6代人增生性瘢痕成纤维细胞,转染后24h激光共聚焦显微镜下记录荧光素的细胞内分布;流式细胞术计数荧光细胞百分率、作细胞凋亡分析,以荧光细胞百分率最高者为最佳转染效率,依次筛选细胞接种数量、ASODN浓度和脂质体量。结果(1)脂质体转染ASODN,所用各转染条件未引起成纤维细胞凋亡;转染后细胞浆和细胞核内均有荧光素聚集。(2)接种细胞数量为14.25×104个/孔的荧光细胞百分率为74.0%,接种数量为30.00×104个/孔的为73.3%,接种数量为32.25×104个/孔的为94.3%,接种数量为39.00×104个/孔的为64.2%。(3)转染液中ASODN浓度100nmol/L,荧光细胞百分率为38.1%;150nmol/L时,为62.6%;200nmol/L时49.5%;250nmol/L时35.8%。(4)转染所用的脂质体量2μl时,荧光细胞百分率为77.01%,3μl时85.05%,4μl为71.64%。结论α1(I)型前胶原基因ASODN脂质体转染人增生性瘢痕成纤维细胞的最佳转染条件为接种细胞数量32.25×104个/孔,反义核酸浓度150nmol/L,脂质体3μl/孔。通过优化转染条件可提高转染效率,为下一步设计药物开发研究提供基础。
Objective To optimize the conditions for the transfection of human α1 (I) procollagen gene antisense oligonucleotide (ASODN) into human hypertrophic scar fibroblasts. Methods ASODN was labeled with FITC, and the second to sixth generation hypertrophic scar fibroblasts were transfected with cationic liposome Oligofectamine as vector. The intracellular distribution of fluorescein was recorded under confocal laser scanning microscope 24h. The number of fluorescent cells was counted by cytometry, and the apoptosis rate was calculated. The highest transfection efficiency was determined by the highest percentage of fluorescent cells. The number of cells inoculated, the concentration of ASODN and the amount of liposomes were screened. Results (1) Lipofectamine was transfected into ASODN. The transfection conditions were not induced apoptosis of fibroblasts. Fluorescein was accumulated in both cytoplasm and nucleus after transfection. (2) The percentage of fluorescent cells with 14.25 × 104 cells / well inoculation was 74.0%, the number of inoculation was 30.3 × 104 cells / well was 73.3%, the number of inoculation was 32.3 × 104 cells / well was 94.3% The number was 39.00 × 104 pcs / hole was 64.2%. (3) The concentration of ASODN in the transfection solution was 100 nmol / L, the percentage of fluorescent cells was 38.1%, 62.6% at 150 nmol / L, 49.5% at 200 nmol / L and 35.8% at 250 nmol / L. (4) When the amount of liposome used for transfection was 2μl, the percentage of fluorescent cells was 77.01%, 85.05% when 3μl, and 71.64% when 4μl. Conclusions The optimal transfection conditions of α1 (I) procollagen gene ASODN liposomes transfected human hypertrophic scar fibroblasts are 32.25 × 104 cells per well, antisense oligonucleotide concentration 150 nmol / L, 3 μl / well. Transfection efficiency can be improved by optimizing the transfection conditions, which provides the basis for further research on drug development.