低温缺氧条件下脂质体介导的寡核苷酸对人脐静脉内皮细胞-304的转染

Transfection of human umbilical vein endothelial cell line ECV-304 with liposome-oligonucleotide complexes under hypothermic and anoxic conditions

目的研究在Euro-Collins溶液(ECs)中,低温(4 ℃)缺氧条件下体内阳离子脂质体转染剂(Invivoliposome)介导NF-κB诱捕物寡核苷酸(NF-κB decoy ODN)在人脐静脉内皮细胞株ECV-304中的转染效率。方法(1) 将ECV-304在含10%胎牛血清的RPMI 1640培养液中以37 ℃、5% CO2的条件培养,待生长至单层融合后,进行试验。(2) 使用前配制脂质体-ODN复合物,脂质体与ODN 的+/-电荷比率为2。(3) 应用荧光显微镜和流式细胞仪,观察在ECs中、4 ℃条件下,ODN浓度分别为0.50、0.75、1.00、1.25 μmol/L时,缺氧保存2、4、6、8 h后ECV-304的平均荧光强度(MFI)和ODN的胞内分布;同时设立裸ODN转染为对照组,观察对ECV-304的转染效率。结果在ECs中、4 ℃缺氧保存条件下,随着保存时间的延长和脂质体-ODN浓度的升高,ECV-304的MFI增强,保存6 h后MFI基本达到了最大强度,并且大部分ODN均位于细胞核内;而ODN的细胞摄取率无差异。但与裸转染相比,MFI和细胞摄取率均有显著差异性。结论在ECs中和低温缺氧条件下,脂质体可以高效地将NF-κB decoy ODN转染到ECV-304细胞核,为供体器官在基因调控水平的保护研究提供了实验依据。

Objective To investigate the transfection efficiency of nuclear factor (NF)-κB decoy oligodeoxynucleotides (ODN) mediated by in vivo liposome in human umbilical vascular endothelial cell line ECV-304 under hypothermic and anoxic conditions. Methods ECV-304 cells were cultured in RPMI 1640 culture medium containing 10% fetal bovine serum at 37 ℃ in the presence of 5% CO2. Liposome-ODN complexes were prepared just before use and added to the cells with a liposome-ODN charge ratio of 2∶1. ECV-304 cell monolayers were transfected with liposome-ODN complexes containing 0.50, 0.75, 1.00 and 1.25 μmol/L ODN respectively in Euro-Collins solution (ECs) at 4℃ and then stored for 2, 4, 6 and 8 hours respectively under anoxic condition. The ODN without liposome was transfected into ECV-304 cells under identical conditions as the control. The distribution of fluorescein isothiocyanate (FITC)-labeled ODN in ECV-304 cells was observed by fluorescence microscope, and the transfection efficiency and mean fluorescence intensity (MFI) were evaluated by flow cytometry. Results MFI was enhanced as the storage time extended and ODN concentration increased, reaching the peak level at 6 h (P<0.05). After a 6-hour storage, most of the ODN was found to locate in the cell nuclei, and the transfection efficiency did not vary significantly between the groups. Compared with the control group, however, the differences in transfection efficiency and MFI were significant. Conclusion ODN can be highly efficiently transfected into ECV-304 cells by in vivo liposome in ECs under hypothermic and anoxic conditions, which provides an experimental basis for further study of the donor organ preservation at the level of genetic regulation.