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1、 第三军医大学硕士学位论文NF-B在迷路炎发病中的作用以及PDTC的影响研究姓名：王朝永申请学位级别：硕士专业：耳鼻咽喉科学指导教师：姬长友20040501 第三军医大学硕士学位论文NF-B在迷路炎发病中的作用以及PDTC的影响研究摘 要背景及目的：既往认为内耳缺乏免疫细胞，是一免疫豁免器官，不受全身免疫反应的影响，而近来的研究发现, 内淋巴囊及其周围含有浆细胞和免疫活性细胞，内耳处于免疫系统的监视之下，对内外源性刺激能发生快速反应而引起迷路炎或免疫应答。核转录因子-B（nuclear transcription factor B, NF-B）是一类重要的转录激活因子，在调节炎症反应中起着关键
2、作用，它的过度活化可引起机体的炎性损害。因此我们推测迷路炎的发生可能与局部 NF-B 的过度活化有关。本研究旨在通过检测迷路炎时NF-B的 p65亚基及其下游因子 TNF-、IL-2 mRNA 的变化，探讨 NF-B与迷路炎的关系，期望为炎性损害引起的内耳功能障碍的防治提供一定的理论依据。方法：1、建立迷路炎动物模型：48只健康豚鼠，随机分为 LPS（lipopolysaccharide）0.01、0.1、1mg/ml实验组和对照组，LPS 各实验组分别用 0.01、0.1、1mg/ml的 LPS注入中耳腔，对照组注入等量生理盐水。在注药后 6h、48h、2w 三个时间点，每组各取 4只，做听
3、性脑干反应（auditory brainstem response，ABR ）检测和光镜、电镜形态观察耳蜗病理变化，选择建立模型的合适浓度。2、NF-B p65和 TNF- 、IL-2 mRNA的检测：健康豚鼠 42只，随机分为三组：LPS实验组 ，1mg/ml的 LPS 注入中耳腔；二硫代氨基甲酸吡咯烷（pyrrolidine dithiocarbamate，PDTC ）治疗组，鼓膜注射 1mg/mlLPS前，以 100mg/kg体重 经腹腔注射 PDTC ； 对照组，双耳注入生理盐水。以 6h、48h、2w为观察点，ABR检测后处死动物，分别用免疫组织化学（immunohistochemi
4、stry，IHC）和原位杂交（in-situ hybridization histochemistry ，ISHH ）技术检测耳蜗 NF-B p65和 TNF-、IL-2 mRNA的表达。结果：1、不同浓度LPS对耳蜗形态和功能的影响：整个观察期内，0.01、0.1mg/ml LPS不能诱发迷路炎和听阈改变；1mg/ml LPS 在6h、48h可引起听阈显著升高和迷路炎，2w时基本恢复正常。2、NF-B p65的表达：NF-B p65主要表达于正常豚鼠螺旋韧带 I型纤维细胞的胞浆。LPS（1mg/ml）刺激后 6h，耳蜗多个部位如螺旋韧带（spiral ligament ，SL）型纤维细 胞、
5、根细胞和螺旋神经节细胞（spiral ganglion cell，SGC）等表达增强，6 第三军医大学硕士学位论文以胞核染色为主；48h 的表达强于 6h (P0.05)，而 2w 与对照组无显著性差异（P0.05）。3、TNF- mRNA的表达：正常耳蜗无 TNF- mRNA的表达。LPS刺激后 6h，耳蜗 SGC、螺旋缘呈阳性染色，血管纹（stria vascularis，StV）、蜗螺旋轴静脉（spiralmodiolar vein，SMV）和 SL呈弱阳性，以胞核染色为主；48h表达增强，2w后仅 SGC呈弱阳性染色。4、IL-2 mRNA的表达：正常耳蜗无 IL-2 mRNA的表达，
6、仅在LPS实验组和 PDTC治疗组 6h时间点，螺旋缘和 SGC有表达，而其余各组均未见表达。5、PDTC对模型 动物的影响： PDTC治疗组豚鼠 6h、48h的听力损失和耳蜗炎症程度以及 NF- B p65和 TNF- mRNA的表达明显轻于相应的LPS实验组，而随着病程的延长，2w时这种差距已不明显(P0.05)。结论1、中耳腔内不同浓度的 LPS对豚鼠内耳及听阈的影响不一， 1mg/ml LPS可诱发迷路炎和听力损害。2、LPS 诱发的急性迷路炎可能与耳 蜗局部 NF- B p65 的活化有关，其机制可能是 LPS的刺激引起 NF-B过度活化、进而诱导以 TNF- 为代表的多种细胞因子和
7、炎症介质的大量转录，最终导致了炎性损伤和听力损害。3、全身应用 NF- B 的活化拮抗剂PDTC 可在一定程度上降低迷路炎时 NF-Bp65和 TNF- mRNA在耳蜗的表达，减轻听力损害和迷路炎症。关键词：耳蜗；迷路炎；豚鼠；LPS；PDTC ； NF- B；TNF- ；IL-27 第三军医大学硕士学位论文Relationship betwwen the Role of NF-B and PDTCin LabyrinthitisAbstract Background: The inner ear had been known as an organ exempted from immunes
8、ystem for lack of immunocytes in the past. However, recent researches have shown that thelymphocytes and immunocompetent cells resident around peri-endolymphatic sac and theinner ear comes under the immuno-surveillance of the immune system. So the labyrinthitisor immunological response may be occurr
9、ed rapidly in cochleae after antigenic stimulus.Nuclear transcription factor B（NF-B ）, a sort of important transcription activator, mayplay an important role in inflammatory reaction. The inflammatory lesion would occur ifNF-B was activated excessively. These conclusions indicated that the labyrinth
10、itis inducedby otitis media may be related with the overactivation of NF-B. Our research is to find outthe relationship between labyrinthitis and NF-B through identifying the changes of NF-Bp65 and the messenger ribonucleic acid (mRNA) of tumor necrosis factor- (TNF-) andinterleukin-2 (IL-2) in laby
11、rinthitis. The results may offer some theoretical foundation toprevent or cure inner ear dysfunction caused by inflammatory lesion.Methods1. Establishing animal model of labyrinthitis: Forty-eight pigmented guinea pigs wererandomly allocated into four groups: LPS 0.01mg/ml group, LPS 0.1mg/ml group,
12、 LPS1mg/ml group and control group. In three experimental animals, different concentrations(0.01, 0.1, or 1.0 mg/ml) of LPS were transtympanically injected respectively, while theequal volume of saline was used in control group. And auditory brain stem responses (ABR)were recorded at 6 hours, 48 hou
13、rs and 2 weeks later. Then the detected animals were killed.The morphology changes of cochlea of those guinea pigs were observed through lightmicroscope and electron microscope. Finally, the suitable concentration of LPS inducinglabyrinthitis was selected.2. Identifying NF-B p65, TNF- mRNA and IL-2
14、mRNA in animal model: Forty-twopigmented guinea pigs were randomly allocated into three groups: LPS 1mg/ml groupinjected transtympanically by LPS (1mg/ml). Pyrrolidine dithiocarbamate （PDTC ）3 第三军医大学硕士学位论文treatment group: PDTC was injected intraperitoneally before giving LPS in this group. Control g
15、roup injected by saline. Those animals were killed after detecting ABR at 6 hours,48 hours and 2 weeks later. Then the expressions of NF-B p65, TNF- mRNA and IL-2mRNA in these cochlea were investigated by the technique of immunohistochemistry (IHC)or in-situ hybridization histochemistry (ISHH).Results1. Influence of different LPS concentration on guinea pigs cochlea: The differenteffect on ABR threshold and pathomorphology of cochlea changed with differentconcentration of LPS （0.01 mg/ml, 0.1 mg/ml, 1mg/ml）in these
