论文部分内容阅读
目的探讨由聚乳酸纳米粒包裹的核因子(NF)-κΒdecoy寡核苷酸片段对体外培养的大鼠脑微血管内皮细胞组织因子表达活性的调控功能。方法以聚乳酸为材料,纳米沉积法制备载荧光标记NF-κΒdecoy-纳米粒混悬液,检测其物理表征、包封率和体外释放情况。共聚焦显微镜和流式细胞术观察和检测培养的脑微血管内皮细胞摄取decoy-纳米粒的效率和细胞内分布,运用逆转录-聚合酶链反应(RT-PCR)及Western blot技术分别比较摄取纳米粒的细胞在脂多糖(LPS)刺激下TFmRNA和P65表达的变化。结果制备的decoy-纳米粒平均粒径为162.1nm,多分散指数为0.118,体外释放实验显示经28d其总释放率达92.3%,流式细胞术检测到细胞对decoy-纳米粒摄取随浓度和作用时间的增加而增加,被摄取的decoy-纳米粒位于细胞胞浆内,RT-PCR结果提示经纳米粒包载的NF-κΒdecoy具有生物活性,可以显著抑制LPS作用下脑微血管内皮细胞的TF表达水平,Western blot结果显示核提取物中P65的表达显著降低。结论聚乳酸纳米粒能将NF-κΒdecoy寡核苷酸片段递送至细胞内,且能保持生物活性,该方法为脑血栓病基因治疗提供了依据。
OBJECTIVE: To investigate the regulatory effect of NF-κBdecoy oligonucleotide fragments encapsulated with poly (D, L-lactic acid) nanoparticles on the tissue factor expression activity of cultured rat brain microvascular endothelial cells in vitro. Methods Polylactic acid (PLA) was used as a material to prepare fluorescent-labeled NF-κBdecoy-nanoparticle suspension by nano-deposition method. The physical characterization, entrapment efficiency and release in vitro were measured. Confidence microscope and flow cytometry were used to observe and detect the efficiency and intracellular distribution of decoy-NPs uptake in cultured brain microvascular endothelial cells. The uptake of nano-particles by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot Changes of TFmRNA and P65 expression in granulosa cells stimulated with lipopolysaccharide (LPS). Results The average particle size of the prepared decoy nanoparticles was 162.1 nm with a polydispersity index of 0.118. The in vitro release assay showed that the total release rate of decoy nanoparticles reached 92.3% after 28 days. The uptake of decoy nanoparticles by flow cytometry The effect of NF-κBdecoy on the activity of NF-κBdecoy was significantly inhibited by LPS-treated NF-κBdecoy Western blot showed that the expression of P65 in nuclear extracts was significantly decreased. CONCLUSION: Polylactic acid nanoparticles can deliver NF-κBdecoy oligonucleotide fragments into cells and maintain biological activity. This method provides the basis for gene therapy of cerebral thrombosis.