转录因子诱骗性寡核苷酸转染猪血管内皮细胞抑制人补体介导的异种细胞毒作用  

Transcription factor SP1 decoy ODNs targeting α1,3-GT renders porcine endothelial cells resistant to complement-mediated cytotoxicity

作　　者：黄亚冰[1] 王璐[1] 尹注增[1] 李荣[1] 郭晖[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所器官移植教育部/卫生部重点实验室,武汉430030

出　　处：《中华器官移植杂志》2008年第10期594-597,共4页Chinese Journal of Organ Transplantation

基　　金：国家863高技术发展计划（2003-AA-205009）;高等学校博士学科点专项科研基金（20040487077）

摘　　要：目的探讨转录因子SP，诱骗性寡核苷酸（decoyODNs）转染的猪血管内皮细胞对人血清补体介导的异种细胞毒作用的影响。方法将培养的永生化猪血管内皮细胞系细胞株SV40-PED分为3组。转染组：SV-40PED转染了SP，decoyODNs；错配组：S驴4D—PED转染了错配的decoyODNs；空转染组：SV-40PED仅用转染试剂oligofectamine作用。转染后26h，采用细胞爬片免疫荧光技术、蛋白印迹法、逆转录聚合酶链反应及乳酸脱氢酶释放试验进行检测，分别观察各组SV40-PED胞膜a半乳糖残基（regal）、蛋白质和α1，3-半乳糖基转移酶（α1，3-GT）mRNA等表达的变化，以及转染了SP，decoyODNs的SV-40PED对人血清补体介导的细胞毒作用的影响。结果转染组SV-40-PED胞膜α—G1的荧光表达明显减弱，部分胞膜可见点状荧光缺损，而空转染组和错配组仍可见明亮的绿色荧光。转染组蛋白表达水平均有不同程度的下降，相对吸光度（A值）仅为未转染组的52．6％。转染组α1，3GT基因异构体1和2mRNA的相对表达量分别为0．42±0．20和0．27±0．12，空转染组分别为0．72±0．17和0．50±0．19，两组比较，差异有统计学意义（P〈0.05）；错配组与空转染组相比，差异无统计学意义（P〉0.05）。体积分数为20％和40％的人血清对空转染组和错配组SV40-PED均有不同程度的杀伤，而对转染组SV-40-PED的杀伤明显减弱（P〈0.，05）。结论经SP，decoyODNs转染的猪血管内皮细胞可以抑制人血清补体介导的异种细胞毒作用。Objective To investigate whether porcine endothelial cells transfected with SP1 decoy ODNs could resist complement mediated cytotoxicity during the model of SV-40-PED with human serum in vitro. Methods Immortalized porcine aortic endothelial cells of the line PED were divided into three groups. The transfected group was SV-40-PED with SP1 decoy ODNs. The mismatched group was SV-40-PED with scrambled SP1 decoy ODNs. The negative group was cells with oligo fectamine only. The expression of α1,3-GT mRNA and αGal was detected after 26 h by using fluores cence microscope, Western blot, RTPCR and lactate dehydrogenase （LDH） activity assay. Results Fluorescence microscopy observed the decreased fluorescence of αGal after SP1 decoy ODNs transfection. Dotted fluorescent decrease could be observed on some membrane while the mismatched group and negative group with bright green fluorescence. Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was decreased to 52. 6% of the negative group （P〈0.05）. The expression of a1,3-GT mRNA in the PED cells transfeeted with decoy ODNs was 0. 42 ± 0. 20 （isoform 1） and 0.27± 0. 12 （isoform 2） respectively, significantly lower than in the negative group （isoform 1, 0. 72± 0.17; isoform 2, 0. 50± 0. 19; both P〈0. 05）. The expression of α1, 3 GT in the mismatch group was not different from that in the negative group （P〉0. 05）. 20% normal human serum （NHS） and 40 G NHS had cytotoxic effect on both mismatch and negative groups, but de coy ODNs could confer SV40-PED protection from the cytolysis effect （P〈0. 05）, which made a remarkabte reduction of complement-mediated cytotoxicity towards SV-40-PED. Conclusions SP1 decoy ODNs could confer porcine endothelial cells protection from complement-mediated cytotoxicity effect in vitro.

关 键 词：转录因子 SP1 寡核苷酸类 补体 内皮细胞 

分 类 号：R686[医药卫生—骨科学]