【作者】 丁浩；

【导师】 王胜资； 王纾宜；

【作者基本信息】 复旦大学 ， 耳鼻咽喉科， 2008， 博士

【摘要】 第一部分菌群相关的中耳组织病理学和机体免疫状态研究目的:观察机体微生物级别不同的SPF(specific pathogen free,SPF)级大鼠和普通(CV)级大鼠对于咽鼓管粘膜病理以及机体免疫状态的影响。方法:SPF级和CV级SD雄性大鼠各8只,作两种大鼠的口咽菌群培养和鉴定;大鼠咽鼓管光镜,扫描电镜观察;流式细胞仪分析(FCM)检测大鼠外周血T淋巴细胞及CD4+、CD8+T淋巴细胞亚群表达区别:脾脏系数的比较。结果:①CV级大鼠口咽部细菌培养阳性率为100%,并为铜绿假单胞菌、金黄色葡萄球菌等致病菌,SPF级大鼠口咽部细菌培养阳性率为25%,且为常驻菌奇异变形杆菌:②咽鼓管光镜检查发现:两种大鼠细胞形态和组织结构均正常,且相似,但光镜下SPF级大鼠咽鼓管粘膜杯状细胞数目明显减少(P＜0.05),扫描电镜显示两种大鼠纤毛形态均正常;③FCM显示SPF级大鼠外周血CD4+淋巴细胞比例较低(P＜0.05):④脾脏系数差别无统计学意义,但SPF级大鼠脾脏系数相对较小。结论:SPF级大鼠和CV级大鼠在口咽部细菌阳性率和种类上存在差异:两种大鼠在光镜和电镜下形态基本相同,但CV级大鼠杯状细胞相对较多说明曾经中耳感染或慢性低毒性刺激的可能;FCM检查和脾脏系数提示SPF级大鼠微生物刺激程度较低。第二部分中耳组织放射损伤及咽部菌群状态与机体免疫功能相关性的研究目的:观察SPF级大鼠和CV级大鼠在中耳鼻咽部不同剂量照射下早期中耳的组织病理改变及机体免疫状态变化,探讨放射后全身免疫状态的改变对中耳组织损伤的影响。方法:选取SPF级和CV级雄性SD大鼠各15只,每组5只。以6MX直线加速器以10Gy、20Gy、30Gy照射大鼠鼻咽区域,在放射后24—36小时进行取材,并与第一部分研究的未照射的大鼠相比较,观察照射后大鼠的口咽菌群改变情况;大鼠咽鼓管扫描电镜检查;咽鼓管粘膜ICAM-1表达;放射前后大鼠外周血T淋巴细胞及CD4+、CD8+淋巴细胞亚群及T淋巴细胞表面ICAM-1(CD54)表达变化:统计学分析咽鼓管粘膜ICAM-1表达与外周血T淋巴细胞及亚群的关系。结果:①放射后SPF级大鼠口咽细菌阳性率上升,但只能检出奇异变形杆菌,CV级大鼠细菌培养仍呈现种类多样、致病菌多的特点;②SPF级大鼠和CV级大鼠在放射前咽鼓管粘膜ICAM-1均未表达,照射后ICAM-1表达开始上升,SPF级大鼠ICAM-1表达明显偏弱,统计学组间分析无明显差异,CV级大鼠在放射剂量较大时(30Gy)ICAM-1明显表达,统计学组间分析差异明显;③电离辐射在较早时期就能导致咽鼓管超微结构的变化,辐射剂量是形态病理变化的重要因素,辐射剂量相同时,CV级大鼠咽鼓管超微结构损伤程度均比SPF级大鼠严重;④CV级大鼠在辐射后外周血T淋巴细胞总数和CD4+淋巴细胞均明显减少,CD8+淋巴细胞增加,组间有明显差异(P＜0.01或P＜0.05),并与辐射剂量有一定的效应关系,同时CD4+/CD8+比值呈明显下降趋势,组间差异明显(P＜0.01);SPF级大鼠在电离辐射后外周血T淋巴细胞总数、CD4+和CD8+淋巴细胞亚群在电离辐射后无明显下降,CD4+/CD8+比值随剂量虽有下降,但下降趋势比较普通大鼠程度较轻,统计学组间分析无明显差异。⑤等级相关分析提示SPF级大鼠咽鼓管ICAM-1的表达与外周血CD4+、CD8+和CD4+/CD8+比例无明显相关关系。而CV级大鼠咽鼓管ICAM-1的表达与CD4+、CD4+/CD8+呈现负相关,与CD8+有正相关关系。结论:微生物状态不同的动物对于鼻咽放射后中耳组织的损伤及机体免疫紊乱是有差异的,SPF级动物其放射性损伤的程度较低,提示菌群因素在机体放射性损伤中起着协同作用。同时菌群因素在中耳局部免疫和全身免疫关系中起着一定的作用。更多还原

【Abstract】 PARTⅠ:Pathological observation of middle ear and immunity status difference in relation with different micro biotaObjective:To observe the difference of the immunity status and middle ear pathology between SPF rats and CV rats which were in different microbe levels.Methods:SPF and CV male SD rats,each were 8.To confirm oropharyngeal bacteria cultivation and determination;To observe the ultra structure of Eustachian tube mucosa with light microscope and scanning electron microscope;To detect peripheral blood T lymphocyte and its subtypes which were stained with fluorescence-labeled monoclonal antibodies by flow cytometry(FCM);to compare the spleen organ coefficients.Results:The bacteria cultivation ratio of CV rats is 100%and those of SPF rats is only 25%;The microbes differentiated from oropharynx of CV rats are Pseudomonas aerugnosa,Staphylococcus aureus,et al while the bacteria of SPF rats is only Proteus m irabilis.Light microscopy and electronic scanning microscopy show the ultra structure of Eustachian tube are normal and same,but light microscopy demonstrates the goblet cells on the surface of Eustachian tube of SPF rats are lesser than those of CV ones(P＜0.05);FCM showed the CD4+ T lymphocyte ratio of SPF rats are lesser than those of CV ones(P＜0.05);There is no statistical difference of spleen organ coefficients between two kinds of rats,but those of SPF rats are relatively small.Conclusions:There exist differences of oropharyngeal bacteria cultivation and determination between two kinds of rats.The goblet cell of Eustachian tube of CV rats suggests previous middle ear infection or chronic stimuli;FCM and spleen organ coefficients suggest that SPF rats are in the lower status to the stimulus of micro biota in the body. PARTⅡ:Radiation-induced injuries of otitis media and immunity status in relation with different micro biotaObjective:To observe differences of the radiation-induced injuries of Eustachian tube of middle ear and immune system between SPF rats and CV rats which were irradiated with gradient doses at nasopharyngeal region.To study the relationship of pathologically local and general immunity status of radiation-induced otitis media.Methods:SPF and CV male SD rats,each were 15.The nasopharyngeal fields of two kinds of rats were irradiated with 6MX linear accelerator at dose of 10Gy、20Gy、30Gy,5 each group,All were sacrificed 24-36hs after irradiation;To confirm oropharyngeal bacteria cultivation and determination;To observe the ultra-structural changes of Eustachian tube mucosa with scanning electron microscope;To detect the expression of intercellular adhesion molecule-1 of the Eustachian tube mucosa by SP immuno-histochemistry;To detect peripheral blood lymphocytes’ surface markers(CD3+、CD4+、CD8+、CD54+) which were stained with fluorescence-labeled monoclonal antibodies by FCM.Results:The bacteria distinguished from CV rats are pathogen mostly and the one from SPF rats is still Proteus m irabilis uniquely.The scanning electronic microscopy shows under same irradiation dose the pathologically morphological injuries of CV Eustachian tubes are worse than those of SPF rats.ICAM-1 of Eustachian tube epithelium of SPF rats is lower than CV ones after radiation.The CD3+、CD4+ lymphocyte and the ratio of CD4+/CD8+ of CV rats decreased dramatically after radiation and in close relation with radiation doses while The CD8+ lymphocyte increased a bit.The CD3+、CD4+、CD8+ lymphocyte and the ratio of CD4+/CD8+ of SPF rats remain in a stable level.Statistic analysis shows there is correlation between ICAM-1 and T lymphocyte subtype changes after radiation.Conclusions:There exists the difference of radiation-induced injuries of otitis media and immune system in relation with different micro biota status.Micro-flora plays important roles in the radiation-induced normal tissue injuries and in the relationship between local and genenral immunity.更多还原