【作者】 国华；

【导师】 梁华平； 吕凤林；

【作者基本信息】 第三军医大学 ， 外科学， 2002， 硕士

【摘要】 目的：过度炎症反应不仅在经久难愈的慢性病中严重影响着人类的生活质量，而其过度、失控性的全身炎症反应所导致的广泛而严重的组织结构和生理功能的损害则直接危害着患者的生命，因此，除了原发病的治疗以外，抗炎已成为临床治疗的基本策略。基于NF-κB是控制炎性因子基因表达的瓶颈，靶向NF-κB的抗炎治疗成为国内外众多学者研究的热点，圈套策略以其特异性及高效性有着其他策略无可比拟的优势，成为一种新型的基因治疗方案。本实验设计合成靶向NF-κB的哑铃形圈套ODNs，并验证其抗炎作用。材料与方法：(1)以阳离子脂质体为载体，将荧光标记的圈套ODNs (2.0μg/ml)转染入纯化的大鼠pMφ细胞,分别在30min、1h，3h，6h，9h，12h，18h，24h用荧光显微镜及激光共聚焦显微镜观察荧光标记的圈套ODNs在细胞内的动态变化。(2)用免疫组化法测大鼠pMφ细胞内NF-κBp65在LPS刺激前及刺激后1h的变化。(3)用ELISA法及RT-PCR法测TNFα、IL-6、IL-10在LPS刺激前及刺激后1、2、6、12、18、24h蛋白及mRNA水平的变化。(4)以阳离子脂质体为载体，将圈套ODNs转染大鼠pMφ细胞6h后用LPS(10μg/ml)刺激，以阳离子脂质体、无关ODNs及单纯LPS刺激组作对照，用ELISA法及RT-PCR法测圈套ODNs的不同浓度(2、4、8μg/ml)及不同时间(转染后8、12、18h)对TNFα、IL-6、IL-10在蛋白及mRNA水平的影响。结果：(1)圈套寡核苷酸在pMφ细胞中的分布：荧光显微镜下转染30min细胞内便有荧光标记的圈套ODNs出现，随着时间的延长，细胞内的荧光越来越强，转染6～12h细胞内的荧光最强。激光共聚焦显微镜显<WP=9>示：转染30min细胞内荧光标记的圈套ODNs主要分布在胞浆内，随着时间的延长，细胞内尤其胞核内的荧光越来越强，转染6～12h细胞内的荧光最强。(2)LPS刺激前大鼠pMφ细胞NF-κBp65大多数集中于胞浆，细胞呈"空泡"现象，LPS刺激后NF-κBp65大多数集中于胞核，细胞呈"实心"现象，且NF-κBp65的表达较刺激前明显增加。(3)LPS刺激后TNFα、IL-6、IL-10的表达较刺激前显著增高，且分别以2～6h、12～18h和4～12h时相最明显。(4)LPS刺激后TNFα、IL-6、IL-10mRNA的表达较刺激前显著增高，且分别以2h、12h和6h时相最明显。(5)在蛋白水平及mRNA水平，圈套ODNs 4μg/ml、8μg/ml组可显著降低pMφ表达TNFα、IL-6，且具有剂量依赖性，而只有8μg/ml剂量组对IL-10的抑制有统计学上的差别，且抑制效果随着时间的延长而明显。阳离子脂质体组、无关ODNs组与LPS刺激组相比，无显著性差异。结论：(1)以阳离子脂质体为载体的哑铃形圈套ODNs可转染入大鼠pMφ细胞并分布于胞浆及胞核(2)LPS可引起NF-κB的激活及TNFα、IL-6、IL-10在mRNA及蛋白水平的显著升高(3)哑铃形圈套ODNs可明显降低TNFα、IL-6在mRNA及蛋白水平的表达，而对IL-10的抑制作用较弱。更多还原

【Abstract】 Objective Over-inflammatory response severely influence the life quality of patients suffered from chronic diseases, and extensive and severe insult on the tissue and the physical function induced by over-inflammatory response imperil patients’ lives. Therefore, besides treatment for primary diseases, anti-inflammation is the basic strategy in clinic. Considering nuclear factor-κB (NF-κB) is the key point to control gene expression of inflammatory factors, decoy-ODNs in dumbbell shape with the nuclear sequence similar to NF-κB cis-elements were synthetized in this study. To measure the anti-inflammatory action of decoy-ODNs targeting NF-κB, decoy-ODNs wrapped up by cationic liposomes were transfected into pMφ cells from rats. Then the effects of decoy-ODNs on expression of inflammatory mediators (TNFαand IL-6) and anti-inflammatory mediators (IL-10) in protein and gene levels were observed with enzyme-linked immunoadsorbent assay (ELISA) and reverse transcription polymerase chain reaction (RT-PCR).Materials and Methods 1. Using cationic liposomes as carriers, decoy-ODNs (0.2 μg/ml) labeled with fluorescein were transfected into the purified pMφ cells from rats, and the dynamic intracellular changes of decoy-ODNs were tested at 30 minutes, 1, 3, 6, 9, 12, 18 and 24 hours after transfection by fluorescent microscope and confocal laser scanning microscope. 2. The changes of NF-κB p65 in pMφ cells were measured at 1 hour before and after LPS stimulation, respectively, with immunohistochemistry method. 3.<WP=6>The changes of TNFα, IL-6 and IL-10 in protein and gene levels were measured before LPS stimulation and at 1, 2, 4, 6, 12, 18 and 24 hours after LPS stimulation. 4. With carriers of cationic liposomes, decoy-ODNs were transfected into pM cells from rats. The pMφ transfected with ODNs of unrelated sequence or stimulated by LPS only to be as control. Then the inhibiting effects of the decoy-ODNs with different concentrations on protein and gene of TNFα, IL-6 and IL-10 were tested at the different intervals after stimulation of LPS (10 ug/ml).Results 1. Decoy-ODNs labeled with fluorescein could be observed in cells under the fluorescent microscope at 30 minutes after transfection. With time passing by, the intracellular fluorescent light became brighter and brighter, reached to the brightest at 6-12 hours after transfection. The confocal laser scanning microscope showed decoy-ODNs was distributed mainly in plasma at 30 minutes after transfection. With time passing by, the intracellular especially the nuclear fluorescent light became brighter and brighter, and it also reached the brightest at 6-12 hours after transfection. 2. Before stimulation of LPS, NF-κBp65 mainly existed in nucleus, and cells showed "vacuole" phenomenon. After stimulation, NF-κBp65 mainly existed in nucleus and cells showed "solid" phenomenon. 3. After stimulation of LPS, the expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2-6, 12-18 and 4-12 hours after stimulation, respectively. 4. The gene expressions of TNFα, IL-6 and IL-10 were higher significantly compared with that before stimulation (P < 0.01), especially at 2, 12 and 6 hours after stimulation, respectively. 5. Decoy-ODNs could decrease the expression of TNFα and IL-6 by dose-dependent fashion. There existed statistical inhibiting significance in IL-10 expression and gene expression when only 0.8μg/ml decoy-ODNs was used. And the inhibiting effect increased with time passing by.<WP=7>Conclusions 1. With carriers of cationic liposomes, Decoy-ODNs in dumbbell shape can be transfected into the pMφ cells from rats and distributed in plasma and nucleus. 2. LPS can activate NF-κB and significantly increased the expressions of TNFα, IL-6 and IL-10 in protein and gene levels. 3. Decoy-ODNs in dumbbell shape can significantly decrease the expressions of TNFαand IL-6 in protein and gene levels, but have weaker inhibiting effect on IL-10.更多还原