α_1(I)型前胶原基因反义寡核苷酸转染人瘢痕成纤维细胞的条件优化  

Transfection optimization of α_1(I) procollagen gene antisense oligodeoxynucleotide to human hypertrophic scar fibroblasts

作　　者：袁即山[1] 利天增[1] 祁少海[1] 谢举临[1] 徐盈斌[1] 潘姝[1] 张珑娟[2] 孔庆瑜[3] 

机构地区：[1]中山大学附属第一医院烧伤外科,广州510080 [2]中山大学附属第一医院外科实验室,广州510080 [3]中山大学附属第一医院流式细胞仪室,广州510080

出　　处：《中华实验外科杂志》2005年第8期998-1000,i0005,共4页Chinese Journal of Experimental Surgery

基　　金：广东省重点科研项目(199827817);广东省医学科研联合攻关项目(98002)

摘　　要：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 transfection of α1 （Ⅰ） procollagen gene antisense oligodeoxynucleotide to human hypertrophic scar fibroblasts. Methods Passage 2 to passage 6 fibroblasts grown out of human hypertrophic scars were subcultured in the 6-well plates in different cell counts ranging from 14.25 × 10^4/well to 39.00 × 10^4/well. The sequence- known antisense oligonucleotide was labeled at the 5 ＇-end with FITC when synthesizing and phosphothioating, and the ASODNs were transfected with cationic liposome OligofectamineTM to these ceils. Then the locations of fluorescence in the cells were observed under laser scanning confocal microscopy 24 h after transfection. At the same time, the percentage of ASODN transfected ceils and apoptic ceils were counted by flow cytometry. The factors affecting the transfection were optimized according to the results of flow cytometry. Firstly, the cell counts were optimized with 2μl of Oligofectamine^TM and 200 nmol/L of ASODN;Secondly, the concentration of ASODN was optimized with the optimal cell count and 2 μl of Oligofectamine^TM; Lastly, the best volume of cationic liposome was determined by applying the chosen optimal cell count and ASODN concentration.Results （1） No apoptosis occurred in this experiment. After transfection the ASODNs located both in the cytoplasm and the nuclei. （2） The percentage of fluorescent cells was 74.0% when the inoculated cells was 14.25 × 10^4/well, and 73.3 % to 30.00 × 10^4/well, 94.3 % to 32.25 × 10^4/well, 64.2 % to 39.00 × 10^4/well, respectively. （3） The percentage of fluorescent cells was 38.1% when the transfecting concentration of ASODN was 100 nmol/L, and 62.6 % to 150 nmol/L, 49.5 % to 200 nmol/L, and 35.8 % to 250 nmol/L, respetively. （4）The percentage of fluorescent cells was 77.01% when 2μ1 of liposome was used during transfection, 85.05 % when 3 μl, 71.64 % when 4μ1, respectively. Conclusion Within the 6-well plate, the optimal transfection condistions of ASODN to the human hypertrophic scar fibr

关 键 词：α1(Ⅰ)型前胶原基因 反义寡核苷酸 基因转染 人瘢痕成纤维细胞 细胞培养 

分 类 号：R346[医药卫生—基础医学]