摘要
目的 研究核因子(NF)κB结合位点诱捕物寡核苷酸(NF-κB decoy ODN)对肾小管上皮细胞的NF-κB活性及下游炎症因子表达的影响。方法 (1)鱼精蛋白-脂质体法转染NF-κB decoyODN:将NF-κB decoy ODN与鱼精蛋白、脂质体混合,转染至TNF-α刺激培养的大鼠肾小管上皮细胞内,测定转染率。(2)凝胶电泳迟滞分析测定转染 NF-κB decoy ODN后细胞NF-κB活性。(3)RT-PCR方法检测转染NF-κB decoy ODN后细胞 ET-1、iNOS、ICAM-1、VCAM-l以及MCP-1 mRNA表达水平。结果 当+/-比例为4时,鱼精蛋白-脂质体法转染率高达93.20％;转染NF-κB decoy ODN可抑制TNF-α激活的NF-κB活性,从而进一步抑制ET-1、iNOS、ICAM-1、VCAM-I和MCP-1的mRNA表达。结论(1)鱼精蛋白-脂质体法是一种不受血清影响的高效率转染方法。(2)NF-κB decoy ODN在体外通过抑制NF-κB活性,进而影响肾小管上皮细胞炎症因子的表达。
Objective To evaluate the effect of NF-KB decoy oligodeoxynucleotides (ODN) strategy mediated by protamine liposome on NRK-52E renal tubular epithelial cells in vitro. Methods Lipsome (1 : 1 DOTAP/cholesterol) and ODNs complexes were prepared at + /-charge ratio of 4 and then transfected into TNF-αstimulated, complete cultured (contained 20% fetal bovine serum) NRK-52E cells. The transfection efficiency was observed by fluorescence microscope and evaluated by flow cytometry. NF-κB DNA binding abilities were analyzed by electrophoretic mobility shift assay (EMSA) . Genes expression of endothelin-1 (ET-1), iNOS, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecular-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were assessed by reverse transcription polymerase chain reaction (RT-PCR). Results Mediated by protamine liposome produced high transfection efficiency compared to naked transfection (transcription efficiency was 93. 20% and 39. 0%, respectively) . NF-κB DNA binding abilities activated by TNF-α were abolished by NF-κB decoy ODN, instead of scrambled ODN. TNF-α-induced gene expression of ET-1, iNOS, ICAM-1, VCAM-1 and MCP-1 was significantly inhibited by NF-κB decoy ODN (all P < 0. 01) . Conclusion Protamine liposome mediated ODN transfection method is highly efficient in vitro and not affected by serum. Transfection of NF-KB decoy ODN results in attenuation of NF-κB activities, leading to the inhibition of inflammatory genes expression in TNF-α activated renal tubular epithelial cells.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2003年第4期244-249,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金(39800070)
