人增生性瘢痕成纤维细胞最佳转染条件:α_1(I)型前胶原基因反义寡核苷酸转染的实验

Optimal transfection of human hypertrophic scar fibroblasts with alpha 1(Ⅰ ) procollagen gene antisense oligodeoxynucleotide

目的:选择α1(I)型前胶原基因反义寡核苷酸转染人增生性瘢痕成纤维细胞的最佳条件。方法:实验于2003-10/2004-06在中山大学附属第一医院外科实验室进行。以阳离子脂质体Oligofectamine转染体外培养的第2～6代人增生性瘢痕成纤维细胞,应用荧光显微镜记录荧光素在细胞内的空间分布,用流式细胞术作荧光细胞计数和观察细胞凋亡,通过不同细胞接种数量、反义寡核苷酸浓度和脂质体量转染条件的比较,逐步获得各转染过程中的最佳条件。结果:①在6孔板上转染反义寡核苷酸,所用各转染条件未引起细胞凋亡。②其最佳转染条件为:接种细胞数量32.25×104/皿,反义核酸浓度150nmol/L,脂质体量3μL/孔;转染后细胞浆和细胞核内均有荧光素聚集。结论:获得α1(I)型前胶原基因反义寡核苷酸转染的人增生性瘢痕成纤维细胞的最佳转染条件,可为下一步试验提供基础。

AIM:To select the optimal transfection condition for human hypertrophic scar fibroblasts transfected with α 1(Ⅰ ) procollagen gene antisense oligodeoxynucl eotide. METHODS:The study was carried out in the Surgical Laboratory of the First Aff iliated Hospital of Sun Yat- sen University from October 2003 to June 2004.The primarily cultured human hypertrophic scar fibroblasts in vitro from passage 2 t o passage 6 were transfected with cationic liposome OligofectamineTM.The intrace llular fluorescence distribution was recorded under the fluorescent microscope, and flow cytometry was employed to determine the fluorescent cell percentages an d observe the cell apoptosis.The optimal transfection condition was picked out s tep by step by comparing the quantity of inoculated cells, level of antisense ol igonucleotide and all doses of OligofectamineTM. RESULTS:① No apoptotic cells were found in antisense oligodeoxynucleotide on the 6- well plate.② The optimal transfection of the antisense oligodeoxynucle otide to the human hypertrophic scar fibroblasts was with the cell count of 32.2 5× 104 per well, concentration of antisense oligodeoxynucleotide 150 nmol/L, an d OligofectamineTM 3 μ L per well respectively.The fluorescence gathered both i n the cytoplasm and nuclei after transfection. CONCLUSION:The optimal condition for the transfection of human hypertrophic s car fibroblasts is selected, i.e., by using α 1(Ⅰ ) procollagen gene antisense oligodeoxynucleotide, which provides a basis for further investigation.