摘要
目的探讨用核因子诱捕(decoy)方法抑制软骨细胞生成一氧化氮(NO)的可行性。方法培养大鼠软骨细胞,人工合成大鼠诱生型一氧化氮合酶(iNOS)基因上能够与核因子(NF)-κB序列识别和结合并带有荧光标记的特异性双链寡脱氧核苷酸(ODN)序列NF-κBdecoyODN,将不同浓度的NF-κBdecoyODN转染入培养的大鼠软骨细胞,通过荧光显微镜观察其转染效率及降解情况,检测其对白细胞介素(IL)-1β诱导的软骨细胞NO生成及iNOSmRNA转录的影响。结果NF-κBdecoyODN能转染入软骨细胞胞核并能维持一定时间,其中4μmol/L以上浓度的NF-κBdecoyODN能够有效抑制IL-1β诱导的软骨细胞iNOSmRNA的转录及NO生成。结论NF-κBdecoyODN能够转染入软骨细胞并抑制IL-1β诱导的软骨细胞iNOSmRNA的转录及NO生成,为抑制NO的生成提供了新的思路和方法。
Objective To investigate the feasibility of Decoy Nuclear factor kappa B oligodeoxyribonucleotide (decoy NF-kappa B ODN) on chondrocytes nitric oxide (NO) production induced by interleukin-1β (IL-1β). Methods Double strand NF-kappa B decoy ODN with fluorescein isothiocyanate(FITC) that could recognize and combine to NF-kappa B consensus binding site of rat iNOS gene was synthased and double strand scambled NF-kappa B decoy ODN without FITC that could not recognize and combine to NF-kappa B consensus binding site was also sythased as control. Rat chondrocytes were isolated and cultured, scrambled NF-kappa B ODN and NF-kappa B decoy ODN were transfected into rat chondrocytes (0.5? 1? 2? 4? 8? 24? 48? 72 hours later, chondrocytes and were observed through fluroscenscopy) to determine the transfection efficacy and the degradation of ODN. Following this, 10 μmol/L scrambled NF-kappa B ODN and different concentrations of NF-kappa B decoy ODN were transfected into chondrocytes before IL-1β induction. NO assay kit and RT-PCR were used to detect the NO production and iNOS mRNA transcription. Result Double strand ODN could be transfected into chondrocyte nuclear and degraded 48 hours later. According to the concentration, concentrations above 4 μmol/L of NF-kappa B decoy ODN could inhibit NO production and iNOS mRNA transcription induced by IL-1β. Scrambled NF-kappa B decoy ODN had no such effect. Conclusion Decoy ODN can be transfected into chondrocytes. Decoy NF-kappa B ODN can inhibit NO production and iNOS mRNA transcription induced by IL-1β through decoy method. It provides a new theory and method to inhibite NO production for osteoarthritis treatment.
出处
《中华风湿病学杂志》
CAS
CSCD
2005年第6期346-348,i003,共4页
Chinese Journal of Rheumatology
