【作者】 张敏；

【导师】 梅元武；

【作者基本信息】 华中科技大学 ， 神经病学， 2006， 博士

【摘要】 第一部分小鼠单纯疱疹病毒性脑炎主要免疫反应特征研究目的：了解单纯疱疹病毒性脑炎（herpes simplex virus encephalitis，HSE）模型小鼠在单纯疱疹病毒Ⅰ型（herpes simplex virus typeⅠ，HSV1）感染后免疫应答在感应阶段，增生、分化阶段以及效应阶段的主要免疫反应特性，为进一步研究HSE免疫机制及探讨有效治疗HSE的免疫干预措施打下基础。方法：Balb／c小鼠颅内直接注入HSV1病毒制造HSE模型，脑组织病理切片HE染色观察病理形态变化，流式细胞术检测小胶质细胞表面CD11b、CD40、MHCⅠ、MHCⅡ抗原表达，RT-PCR检测小鼠脑内细胞因子（IL-2、IL-4、IL-10、TNF-α、iNOS）mRNA表达，SPSS10.0软件t检验处理实验结果。结果：成功制造HSE小鼠模型，计算HSV1病毒半数致死量(LD₅₀)为10^-5／0.1ml，HSE小鼠脑组织片状坏死出血，部分神经细胞变性，间质水肿。HSE小鼠较正常对照小鼠表达CD11b、CD40、MHCⅠ、MHCⅡ抗原明显增加，MHCⅡ抗原增加较MHCⅠ明显，T_H1（IL-2、TNF-α），T_H2型细胞因子（IL-4、IL-10、）mRNA及iNOS mRNA均较正常对照小鼠显著高表达（P＜0.05）。结论：小胶质细胞作为脑内重要的抗原提呈细胞（APC），在HSV1感染后被大量激活、增殖，细胞表面MHC抗原表达增加，发挥抗原提呈作用，CD40表达增加，有助于淋巴细胞通过血脑屏障和活化，并进一步激活小胶质细胞，分泌大量细胞因子，TH1及TH2型细胞因子反应在HSE中均起重要作用，并相互协调和制约，过氧化反应也是HSE的一个重要方面。第二部分单纯疱疹病毒性脑炎免疫炎性机制研究目的：以小胶质细胞系BV2作为小鼠单纯疱疹病毒性脑炎的替代研究对象，了解小胶质细胞激活并分泌细胞因子的刺激信号，及细胞因子分泌的细胞内信号途径。方法：MTT选择合适的细胞上药物稀释浓度，RT-PCR法检查细胞因子（IL-2、IL-4、IL-10、TNF-α、iNOS）mRNA含量。小鼠颅内注射HSV1(10^-3／0.1ml)制造小鼠单纯疱疹病毒性脑炎（HSE）模型，与接种HSV1病毒(10^-2／0.1ml)的小胶质细胞系BV2细胞比较接种病毒前后以及用药前后细胞表面抗原表达及细胞因子mRNA含量改变。Western blot检测HSV1感染前后细胞JNK MAPK表达情况，及RT-PCR法检测ERK MAPK和P38 MAPK的抑制剂作用前后细胞因子表达变化及预后改变。结果：BV2细胞具有和Balb／c小鼠脑组织相似的免疫反应特性。JNK MAPK在病毒感染后激活，ERK MAPK和P38 MAPK的抑制剂PD98059和SB203580均可改善BV2细胞在HSV1感染后的生存率，并可减少TNF mRNA表达；阿司匹林和地塞米松均可减少iNOS mRNA表达。结论：BV2细胞可作为小鼠单纯疱疹病毒性脑炎的替代研究对象。3条MAPK的主要途径（JNK，ERK和P38 MAPK）在HSE的免疫反应中均发挥作用，ERK和P38 MAPK参与HSV1感染后细胞TNF mRNA表达，TNF的表达认为与病毒感染造成细胞死亡有关。阿司匹林和地塞米松可能具有减少组织过氧化损伤的潜在神经保护作用。第三部分临床用药对单纯疱疹病毒性脑炎疗效研究目的：了解几种临床常用药物对HSE预后及细胞因子分泌的影响。方法：小鼠颅内注射HSV1(10^-3／0.1ml)制造小鼠单纯疱疹病毒性脑炎（HSE）模型，BV2细胞直接接种病毒作为细胞模型，根据MTT结果选用合适的细胞药物浓度及了解细胞生存率，RT-PCR法检测小鼠脑内细胞因子（IL-2、IL-4、IL-10、TNF-α、iNOS）mRNA表达，流式细胞术检测小胶质细胞表面CD11b、CD40、MHCⅠ、MHCⅡ抗原表达，比较在病毒感染后和几种药物作用后小鼠脑组织和BV2细胞生存率、免疫抗原表达和细胞因子表达差异。结果：黄芪、干扰素和脑多肽可显著改善BV2细胞和模型小鼠在HSV1感染后的生存率，同时各种细胞因子表达降低(T_H1／T_H型细胞因子)；阿司匹林、菲克维兹和地塞米松不能改善HSV1感染后的生存率，各种细胞因子分泌仍明显偏高，以地塞米松明显；药物作用后脑组织免疫抗原CD11b、CD40、MHCⅠ、MHCⅡ变化各异。结论：HSV1感染的初期，提高机体抗病毒免疫能力是治疗的主要方向，具有免疫抑制的药物如阿司匹林、地塞米松由于不能抑制病毒复制反而造成晚期的细胞因子表达显著增高，免疫损伤更严重，应避免使用；在HSV1感染晚期病毒复制控制的情况下，可适当选用具有免疫抑制作用的药物。不同神经营养剂的免疫调节作用不同。第四部分单纯疱疹病毒性脑炎治疗中的预后评估及用药指导目的：找到可通过检测的细胞因子指导HSE感染后的预后评估及用药方案制定的有效工具。方法：RT-PCR方法检测在多种实验药物及条件下BV2细胞表达细胞因子mRNA含量，MTT检测细胞存活率。将得到的以上数据进行相关及回归分析以得到一元线性回归方程，通过此方程得到BV2细胞的预后估计值。以2nsoftEditor神经网络建模工具软件作为神经网络软件工具，神经网络通过前38组训练集进行训练后，将剩下的23组数据作为盲法测试集，代入已经训练好的神经网络，得到相应的预后估计值。将以上2种方法得到的预后分析结果与实际生存率比较，以计算值与实测值之间误差在15％为标准，15％以内为符合，＞15％为不符。了解2种不同方法对HSE感染预后分析的符合率。结果：对61组数据进行相关性分析，得到相关系数，同时作实际细胞生存率与各细胞因子关系散点图，发现与生存率相关性最好的参数是TNF-α，IL-23，IL-4，而其中以TNF-α和IL-23较优。但它们之间的关系也并非纯粹的线性关系，或多或少存在一定的非线性关系。回归分析得到的一元线性回归方程为SR=0.184+0.043×（IL-2）-0.013×（IL-4）-0.016×（IL-10）-0.022×P19—0.018×TNF-0.19×iNOS。对BV2细胞预后估计值与实际值比较符合率为19％。以61组数据中的38组数据作为神经网络的训练集对神经网络进行训练，将剩下的23组数据作为盲法测试集，代入已经训练好的神经网络，发现其预后估计值与实际值比较符合率为75％，高于回归分析。结论：经过训练的神经网络可较准确预测生存率，可有效分析细胞因子分泌之间的非线性复杂关系，将成为临床指导评估HSE预后并进行用药方案制定的有效工具。传统的回归分析适合于低维变量和变量线性可分的情况，而对于变量之间有相互影响、多维、非线性的情形并不合适，神经网络与回归分析比较，更擅长处理大样本、非线性、高维变量数据。更多还原

【Abstract】 Objective: In order to know the characteristics of the three courses of immune responses in herpes simplex virus encephalitis （HSE） of Balb/c mouse.Methods: Balb/c mouse were made HSE by injecting herpes simplex virus type I（HSV1） and their brain tissues were obtained. Pathologic slices of brain tissues of mice were used to identify their morphologic difference. The surface antigens on microglia such as CD11b, CD40, MHCI and MHCII were measured by flow cytometry and semiquantitative reverse transcription polymerase chain reaction （RT-PCR） were used to detect the mRNA of IL-2, IL-4, IL-10, TNF-α and iNOS in mice’s brains. The results of the experiment were operated by Student Test with SPSS10.0 software.Results: After herpes simplex virus typel （HSV1） infection, the lesions of haemorrhage and necrosis in mice’s brains could be observed by microscope, and the expression levels of surface antigen CD11b CD40, MHCI MHCII and mRNA of IL-2, IL-4, IL-10, TNF-α and iNOS increased.Conclusions: Microglial cells play important roles in HSE as antigen presenting cells: they are in activation and proliferation and they present surface antigens such as MHC and CD40 in favor of microglia presenting virus antigens and lymphocyte permeating blood-brain barrier and microglia reactivating, secreting cytokines. T_H1 type cytokines and T_H2 type cytokines both play important roles in HSE and they modulate and restrict quantities of each other. Peroxidation is an important aspect in HSE.PART II Study of Immune Mechanism in Herpes Simplex VirusEncephalitisObjective: To find the way of endocellular and extracellular signals in microglia activation and its cytokines secretion.Methods: Balb/c mouse were made HSE by injecting herpes simplex virus type I（HSV1） and their brain tissues were obtained. The dilution concentrations of medication for BV2 cells were determined by MTT method. BV2 cells were inoculated with HSV1 and then medicated by several drugs including ERK and P38 MAPK inhibitor. The mRNA of IL-2, IL-4, IL-10, TNF-α and iNOS in mice brains and BV2 cells were measured by semiquantitative reverse transcription polymerase chain reaction （RT-PCR） and their quantities were compared between mice’s brain and BV2 cells. Western blot was used to know the presentation JNK MAPK in BV2 cells before and after HSV1 inoculation. The results of the experiment were operated　by Student Test with SPSS 10.0 software.Results: There are much comparability in the characteristics of mRNA changes of many cytokines and iNOS. P-JNK was increased after HSV1 inoculation. PD98059 （inhibitor of ERK MAPK） and SB203580（inhibitor of P38 MAPK） could improve the survival rate of infected BV2 cells and decreased TNF mRNA expression. Aspirin and dexamethasone could decrease iNOS mRNA expression.Conclusion: BV2 cells could be the substitute of mice in HSE study. 3 routes of MAPK（JNK, ERK and P38 MAPK） are all play roles in immune responses of HSE. ERK and P38 MAPK are concerned with the TNF mRNA expression in HSE and TNF is thought to lead to the death of HSV1 infected BV2 cells. Aspirin and dexamethasone may have the ability of nevous protection for their anti- peroxidation.PART III Study of Curative Effect of Six Medicines in HerpesSimplex Virus EncephalitisObjective: To find out the remedial roles and effects of cytokines secretion of several medicines including astragalus, interferon-β cerebroprotein hydrolysate, deproteinized calf blood extractives, dexamethasone and aspirin in HSE.Methods: Balb/c mouse were made HSE by injecting herpes simplex virus type I（HSV1） and BV2 cells were made the cell model of HSE by inoculating HSV1. The dilution concentrations of medication for BV2 cells were determined by MTT method. Cell’s survival rate were measured by MTT method and cytokine’s mRNA such as IL-2 IL-4 IL-10 TNF-α iNOS were measured by semiquantitative reverse transcription polymerase chain reaction （RT-PCR）. The surface antigens on microglia such as CD11b, CD40, MHCI and MHCII were detected by flow cytometry. The results of cell’s survival rate, mRNA of cytokines and surface antigens of before and after virus infection and medication were compared.Results: Astragalus, interferon-β and cerebroprotein hydrolysate can makedly improve the survival rate of BV2 cells and Balb/c mice infected with HSV1 and can reduce the cytokines expression including both T_H1 and T_H type cytokines; aspirin, deproteinized calf blood extractives and dexamethasone cannot improve the survival rate of BV2 cells and Balb/c mice infected with HSV1 and cannot reduce the cytokines expression including both T_H1 and T_H type cytokines.Conclusion: Improve the ability of immune response is of primary importance in initial stages of HSE and medicines of restraining immune response such as aspirin and dexamethasone, which made cells could not control the reproduction of virus, result in more severe damages and higher expression of cytokines after several days and should not be used in initial stages. Under the circumstances of virus control, these medicines could be chosen to meliorate the damage of immune response. There are different functions on immune regulation for different medicines.PART IV Prognosis Assessment and Medication in Treatment ofHerpes Simplex Virus EncephalitisObjective: To find effective tools to guide the prognosis assessment and medication.Methods: Cell’s survival rate were measured by MTT method and cytokine’s mRNA such as IL-2 IL-4, IL-10 TNF-α iNOS were measured by semiquantitative reverse transcription polymerase chain reaction （RT-PCR）. These data were analyzed by regression analysis and neural networks and the results of the prognosis analyzed by two methods were compared with actual survival rate in order to find the better way of prognosticating survival rates.Results: A simple regression equation was obtained by regression analysis and its according rate to actual survival rate is 19%, while the data of prognosticate survival rate obtained by neural networks had much higher according rates（75%） to actual survival rates.Conclusion: Neural networks can prognose the survival rates exactly after training for it can efficiently analyze complicated nonlinear relation between the quantities of cytokines. Neural networks should be an effective tool in HSE treatment to guide the prognosis assessment and medication.更多还原