转染组织因子胞内段siRNA阻抑人脐静脉内皮细胞凋亡的体外实验  

In vitro study on blocking HUVEC from apoptosis by transfecting siRNA targeting cytoplasmic domain of tissue factor

作　　者：黎纬明[1] 韩红[2] 李泉[1] 周浩[1] 刘峥嵘[1] 葛超[1] 邹萍[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院血液病研究所,430022 [2]华中科技大学同济医学院附属梨园医院血液科

出　　处：《中华器官移植杂志》2010年第2期114-117,共4页Chinese Journal of Organ Transplantation

基　　金：国家自然科学基金青年基金（30600570）

摘　　要：目的探讨转染组织因子胞内段小片段干扰RNA（siRNA）对血管内皮细胞凋亡的影响。方法体外设计组织因子胞内段siRNA I和siRNA II，插入质粒DNA后用脂质体将其转入人脐静脉内皮细胞株（HUVEC）中。3组HUVEC分别转染siRNAI质粒（siRNA I组）、siRNA II质粒（siRNAII组）和pcDNA^TM 6．2GW／-miR质粒（对照组）。取各组HUVEC细胞，分别与CD8^＋T淋巴细胞进行混合淋巴细胞反应。用流式细胞仪检测混合淋巴细胞反应中HUVEC的凋亡率，用磁珠法检测混合淋巴细胞反应上清液中活化部分凝血活酶时间（APTT）。结果插入的siRNA经过测序，证实为正确序列。HUVEC转染siRNA后24h及48h，siRNA I组和siRNA II组HUVEC的凋亡率均低于对照组（P〈0．01）。siRNAI组HUVEC的凋亡率低于siRNA II组（P〈0．05）。3组上清液的APTT较对照（RPMI1640培养基）缩短（4±0．46）s（P〈0．05）。siRNA I组和siRNA II组与对照组相比较，APTT的差异无统计学意义（P〉0．05）。结论组织因子胞内段siRNA构建成功。转染该siRNA可在不影响凝血功能的情况下对混合淋巴细胞反应中的内皮细胞起到一定保护作用，减少内皮细胞的凋亡。Objective To investigate the effect of small interfering RNA （siRNA） targeting cytoplasmic domain of tissue factor on apoptosis of vascular endothelial cells. Methods Specific siRNA targeting cytoplasmic domain of tissue factor were designed, and synthetic oligos were inserted into plasmid DNA. The siRNA constructs were transfected into human umbilical vascular endothelial cells （HUVEC） with liposome. The HUVEC were transfected with the constructs encoding siRNA I, siRNA II and pcDNATM6. 2 GW/-miR plasmid separately. The transfected HUVEC were mixed with CD8^＋ T lymphocytes. The apoptotic rate of tranfected HUVEC mixed with lymphocytes was analyzed by flow cytometry. Magnetic beads were used to measure PT of the supernatant in the mixed lymphocytes culture. Results The siRNA constructs were confirmed by DNA sequence analysis. The apoptotic rate of HUVEC transfected with siRNA I and II plasmids was decreased significantly as compared with the empty control group （P〈0. 01）. The apoptosis rate of HUVEC transfected with siRNA I plasmid was lower than that of HUVEC transfected with siRNA II plasmid （P〈0. 05）. APTT of the culture supernatants in the three transfection groups was lower in the control groups （P 〈0. 05）, but there was significant difference among the three transfection groups. Conclusion The siRNA targeting cytoplasmic domain of tissue factor were successfully constructed, siRNA can protect HUVEC, and reduce the apoptotic rate of endothelial cells in mixed lymphocyte reaction without influencing the coagulation function.

关 键 词：凝血致活酶 细胞内 内皮细胞 细胞凋亡 RNA 小分子干扰 

分 类 号：R346[医药卫生—基础医学]