Oligofectamine介导转录因子SP1诱骗性寡核苷酸转染猪内皮细胞系条件的优化研究  

OPTIMIZING CONDITION FOR OLIGOFECTAMINE-MEDIATED SP1 DECOY OLIGODEOXYNUCLEOTIDES TRANSFECTION INTO SV-40-PED CELLS

作　　者：黄亚冰[1] 徐婧[1] 陈松[1] 谢林[1] 王璐[1] 尹注增[1] 左利群[1] 陈实[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院器官移植研究院,武汉430030

出　　处：《中国修复重建外科杂志》2007年第8期877-881,共5页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家高技术研究发展计划(863)资助项目(2003AA205009);高等学校博士学科点专项基金资助项目(20040487077)~~

摘　　要：目的确定SP1诱骗性寡核苷酸(Decoy Oligodeoxynucleotides,ODNs)在Oligofectamine介导下转染猪血管内皮细胞系SV-40-PED的最佳转染条件和效果。方法以SV-40-PED为转染对象,在6孔培养板中,接种SV-40-PED细胞2×105/孔,将4μlOligofectamine和不同浓度的SP1ODNs(2.5、5.0、7.5、10.0和12.5μl)分别溶于100μl无血清、无抗生素的DMEM培养基,室温放置20min后转染细胞,SP1ODNs最终浓度分别为50、100、150、200和250nmol/L、转染26h检测不同浓度转染情况,确定最佳SP1ODNs与Oligofectamine的比率、转染时间;流式细胞仪检测细胞内的相对荧光强度及摄取率评价转染效率;荧光显微镜观察细胞内荧光分布;测定细胞上清乳酸脱氢酶(lactate dehydrogenase,LDH)含量,观察细胞损伤情况。结果SV-40-PED在Oligofectamine的介导下可以摄取SP1ODNs。SP1ODNs终浓度为50、100、150nmol/L组细胞形态无明显变化,终浓度为200、250nmol/L组细胞收缩、变圆后逐渐恢复;SP1ODNs终浓度为250nmol/L时,转染26h时细胞形态也无明显变化,48、72h时有细胞漂浮现象。荧光下观察,转染成功各组荧光物质分布于细胞核内,其中SP1ODNs终浓度为200、250nmol/L组可见清晰核仁,浓度越高,荧光强度越强,细胞内荧光物质主要聚集于细胞核。SP1ODNs终浓度为250nmol/L时,转染72h组LDH浓度为137.12±3.92U/L,显著高于26h的49.61±17.13U/L和48h的120.26±8.42U/L,均有统计学意义(P<0.01);当转染时间为26h时,各浓度组上清液LDH值差异无统计学意义(P>0.05)。结论SP1ODNs终浓度为250nmol/L、转染26h时可以为SV-40-PED转染提供良好效果。Objective To determine the optimizing parameters in transfecting the SV-40-PED cells mediated by oligofectamine. Methods With a change of Decoy oligodeoxynucleotides （ODNs）/oligofectamine in ratio and the transfection time, the uptake rate and the mean fluorescence intensity of SP10DNs in the SV-40-PED cells were measured by flow cytometry to evaluate the transfection efficiencies. 4 μl oligofectamine with different concentrations of ODNs（2.5,5.0,7.5,10. 0 and 12.5 μl） were put into 100 μl of DMEM without serum and antibiotics, the （SV-40- PED） cells were transfected after 20 min at room temperature, the final concentration of SP1 decay ODNs were 50,100, 150,200 and 250 nmol/L. Transfection efficiency was detected at 26 h after transfection. The intracellular distribution of SP10DNs was determined with a fluorescence microscope. The lactate dehydrogenase （LDH） activity in the supernatant was measured to assess the cytotoxicity. Results The uptake of SP10DNs into the SV-40-PED cells was significantly improved by oligofectamine. The cell appearance did not change much in the groups of 50, 100 and 150 nmol/L. In the groups of 200 and 250 nmol/L, the cell reverted after being shrinked and altered to round. At 26 h after the transfection, there was no marked change in the cell form at the concentration of 250 nmol/L. There was floatation at 48 and 72 h after the transfection. Under the fluorescence microscope, we observed fluorescent materials distributed in the cell nucleus in the successfully-transferred groups. We could see the nucleoli clearly in the groups of 200 nmol/L and 250 nmol/L. There was a stronger fluorescence intensity with a higher concentration and the fluorescent materials gathered at the cell nucleus. At the final concentration of 250 nmol/L, the LDH level was 137. 12±3. 92 U/L in the 72-h group, which was significantly higher those that in the 26-h group（49.61±17.13 U/L）and the 48-h group（120.26 ±8.42 U/L）（P〈0.01）. At 26 h aher the transfeetion, there were no sta

关 键 词：SP1诱骗性寡核苷酸 转录因子 转染 猪内皮细胞系 

分 类 号：Q78[生物学—分子生物学]