聚乳酸纳米粒介导decoy片段调控大鼠脑微血管内皮细胞组织因子表达的体外研究  

Regulation of tissue factor expression in brain microvascular endothelial cells by PLA nanoparticles coating NF-κB decoy oligonucleotides

作　　者：胡豫[1] 王华芳[1] 孙望强[2] 谢长生[2] 魏文宁[1] 郑金娥[1] 姚军霞[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,武汉430022 [2]华中科技大学材料技术与工程学院

出　　处：《中华血液学杂志》2005年第9期534-538,共5页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(30170392)

摘　　要：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 a new strategy, of polylactic acid （PLA） nanoparticles delivery system coating nuclear factor-kappaB （NF-κB） decoy oligonucleotides （ODNs） for inhibiting TF expression in cultured brain microvascular endothelial cells（BMECs）. Methods PLA nanoparticles coating FITC-labeled NF-κB decoy ODNs were formulated by nano-deposition method and the characteristics of nanopartlcles were detected. BMECs were isolated and cultured in vitro. The cellular uptake and intracellular localization of nanoparticles in BMECs was detected by flow eytometry and confocal microscopy. Changes in the expressions of TF and nuclear protein P65 were examined by RT-PCR and Western blot in NF-κB decoy ODNs transfected BMECs by LPS stimulation. Results The decoy-nanoparticles obtained were uniform spherical particles with an effective diameter of 162.1 nm and a polydispersity index of 0.118. NF-κB decoy ODNs encapsulated in nanoparticles could be released in a controlled manner in phosphate-buffered saline for up to 28 days. It was observed that the cellular uptake of nanoparticles were increased with the time of incubation and the concentration of nanoparticles in the medium. Nanoparticles localized mainly in the BMECs cytoplasm. LPS-induced upregulation of TF transcription was inhibited by NF-κB decoy ODNs transfection but not by missense ODNs transfection. Furthermore, changes in the transcription level of TF were paralleled by a reduction of capacity of P65 in nuclear extract of NF-κB decoy ODNs transfected cells. Conclusions These findings offer a potential therapeutic strategy in the control of TF expression in BMECs in vitro.

关 键 词：聚乳酸纳米粒 decoy寡核苷酸片段 核冈子κB 脑微血管内皮细胞 组织因子 血管内皮细胞组织因子 DECOY寡核苷酸 大鼠脑 体外研究 片段 

分 类 号：R743.3[医药卫生—神经病学与精神病学]