人肺对流感病毒固有免疫应答机制研究

发布时间：2018-05-12 05:05

本文选题：人肺精确薄片模型 + 移植培养　；参考：《苏州大学》2011年博士论文

【摘要】：目的通过实验探索人肺体外移植培养的条件,构建人肺体外移植培养液的配置方案,优化人肺标本的处理和保存,稳定切片后细胞,规范模型设备的使用和精确切片厚度的选择,从而建立标准化流程,创建一个稳定的、强大的、高产量、高重复性和实用性的多细胞共生长的人肺器官体外培养模型。方法选取与俄克拉荷马大学合作的五家医院中接受肺癌手术的志愿者的按University of Oklahoma Institutional Review Board批准的标准程序切除的正常肺叶组织,以及交通事故死亡遗体捐赠者的肺器官。参照改良的琼脂糖充填肺组织的方法,以含1.5%低熔低胶琼脂糖培养液,经充膨、钻取、切片及培养等步骤建立人肺精确薄片体外移植模型,并将人肺薄片进行长时间的体外培养,选取不同时间点对其生长状态进行严密的显微镜下组织形态的监测。以乳酸脱氢酶释放毒性实验评价该模型建立体系对细胞的毒性。结果建立的人肺精确薄片模型体系稳定,无明显的细胞毒性损伤,易重复、高产量。按此模型流程制备的人肺精确薄片经长时间(14天)的体外培养,整体结构完整,三维立体结构清楚,支气管清晰可见,肺泡细胞随培养时间活力无改变,共同维持了肺正常的结构和功能。结论按标准流程建立的人肺精确薄片体外移植模型体系稳定,人肺薄片组织能经受长时间体外培养,结构清晰、细胞稳定,是一个可重复使用替代人肺器官功能的体外培养模型。第二部分流感病毒对移植培养人肺薄片的影响目的利用新建的人肺精确薄片体外移植培养模型,侦测暴露于流感病毒的人肺薄片里是否支持流感病毒的感染和病毒复制。方法按照以低熔低胶琼脂糖充填肺组织建立人肺精确薄片体外移植培养模型的标准流程获取人肺薄片,分别暴露于6x106 PFU/ml流感病毒A/PR/8/34 (H1N1)(PR8)和A/Oklahoma/309/06 (H3N2)(OK/06),经不同时间的体外培养后收获肺切片,以半定量RT-PCR和共聚焦显微镜侦测人肺薄片对流感病毒暴露的反应。结果暴露于流感病毒PR8和OK/06体外培养24h后的人肺薄片免疫结果显示,在激光共聚焦显微镜下,暴露于流感病毒的人肺切片,可见在肺组织的多种细胞中存在红色的荧光素信号,提示抗-PR8-NP和抗-OK/06-N P的存在,而在无病毒颗粒的稀释液组仅看到染成兰色的细胞核,说明人肺薄片可被暴露的流感病毒感染。另外,半定量RT-PCR结果显示,流感病毒PR8和OK/06 NS1mRNA的表达在流感病毒接触后8h有显著的高峰生成,说明体外培养的人肺薄片支持流感病毒的病毒复制。结论暴露于流感病毒OK/06和PR8的人肺薄片,支持流感病毒的感染和病毒复制。第三部分人肺多源细胞对流感病毒感染的反应目的利用可被流感病毒A/PR/8/34(H1N1) (PR8)感染的人肺薄片体外移植培养的模型,侦测人肺内流感病毒感染诱导的细胞因子反应及人肺多细胞成分在参与人肺对流感病毒的固有免疫反应中的作用。方法按照新建的人肺薄片体外移植培养模型标准流程获取人肺组织切片,暴露于流感病毒PR8的刺激下,分别以RNase protection assay (RPA)方法在转录水平检测流感病毒刺激后人肺薄片内炎症因子mRNA的表达,以ELISA检测流感病毒刺激后的炎症因子的蛋白释放,侦测人肺切片对流感病毒的固有免疫应答,并以共聚焦显微镜免疫荧光方法侦测流感病毒诱导人肺炎症因子表达的多细胞来源。结果流感病毒刺激后的人肺中流感病毒诱导产生了多种类的炎症因子,在转录水平发现MIP-1α和IP-10 mRNA存在高表达,并在翻译水平证实了流感病毒刺激引起诱导生成炎症因子蛋白释放增加。共聚焦显微镜免疫结果表明,由流感病毒诱导生成的多种细胞因子反应是来源于人肺上皮细胞、肺泡巨噬细胞和间质细胞的多种细胞公共参与的固有免疫反应。结论流感病毒引起人肺内激活多种固有细胞产生炎症因子反应,诱导生成的MIP-1α和IP-10等多种炎症因子参与了流感病毒感染后启动的人肺固有免疫应答。第四部分CSE抑制流感病毒触发的人肺固有免疫目的进一步明确流感病毒A/PR/8/34(H1N1) (PR8)感染体外移植培养人肺薄片后人肺多种固有细胞参与诱导细胞因子和抗病毒因子生成的固有免疫应答。评价香烟烟雾提取物(Cigarette Smoke Extract,CSE)在人肺器官培养模型上对流感病毒引起的细胞因子的影响并探讨其可能的作用机制。方法采用标准流程获得的香烟烟雾提取物(CSE)处理人肺薄片组织,以ELISA方法检测流感病毒PR8诱导的多种炎症因子蛋白的表达及CSE对炎症因子蛋白表达的影响,明确CSE是否对流感病毒诱导的人肺固有免疫存在抑制作用;进而以RT-PCR检测CSE处理或联合抗氧化剂处理对流感病毒感染后诱导的炎症因子及PRRs mRNA表达的影响;再以Western blot检测CSE处理或联合抗氧化剂对流感病毒刺激后人肺RIG-I蛋白表达的影响。另外,以LDH释放毒性实验测定CSE对人肺切片的细胞毒性。结果在基因转录和翻译水平发现CSE减弱流感病毒触发的人肺细胞因子反应以及RIG-I mRNA和蛋白的表达,适宜浓度CSE处理是安全的。同时发现,CSE对RIG-I及其它PRRs信号通路的抑制作用可被抗氧化剂保护。结论CSE减弱流感病毒触发人肺炎症因子的反应,可能主要是通过减弱由RIG-I为主的PRRs信号通路介导的人肺对流感病毒触发的固有免疫反应机制所致。
[Abstract]:Objective to explore the conditions of human lung transplantation in vitro, to construct a configuration scheme for human lung transplantation culture, to optimize the treatment and preservation of human lung specimens, to stabilize the cells after slicing, to standardize the use of model equipment and to select the precise slice thickness, and to establish a standardized process to create a stable, powerful, high output and high level. A reproducible and practical multicellular growth model of human lung organ in vitro.
Methods the normal lobectomy tissues were removed by standard procedures approved by University of Oklahoma Institutional Review Board in five hospitals with the cooperation with the University of Oklahoma, and the lung organs of the donors who died from the dead body of the traffic accident were selected, and the improved method of filling the lung with the improved agarose was used. With 1.5% low melt low gel agarose culture medium, the model of human lung precise thin slice in vitro transplantation was established through filling, drilling, slicing and culture. The human lung slices were cultured for a long time in vitro. The tissue morphology of the human lung slices was monitored under the microscope at different time points. The toxicity test of lactate dehydrogenase was evaluated. The model establishes the toxicity of the system to the cell.
Results the established human lung thin slice model system was stable, no obvious cytotoxic damage, easy repetition and high yield. The human lung thin slices prepared by this model were cultured in vitro for a long time (14 days), the whole structure was complete, the three-dimensional structure was clear, the bronchus was clear and visible, and the alveolar cells were not changed with the culture time. It maintains the normal structure and function of the lung.
Conclusion the model system of human lung precise thin slice in vitro is stable, and the tissue of human lung slice can be cultured in vitro, the structure is clear and the cell is stable. It is an in vitro culture model that can be reused to replace the function of human lung organ.
The second part is the influence of influenza virus on transplanted human lung slices.
Objective to detect the infection and replication of influenza virus in human lung slices exposed to influenza virus by using a new model of human lung precise thin film in vitro transplantation.
Methods human lung slices were obtained according to the standard flow of human lung accurate thin slice culture model with low melt and low gel agarose filling lung tissue. The lung slices were exposed to 6x106 PFU/ml influenza virus A/PR/8/34 (H1N1) (PR8) and A/Oklahoma/309/06 (H3N2) (OK/06) respectively. The lung slices were harvested after different time in vitro culture, and half quantitative RT-PCR was used. Confocal microscopy was used to detect the response of human lung slices to influenza virus exposure.
Results the results of human lung slice immunization exposed to influenza virus PR8 and OK/06 in vitro showed that the human lung slices exposed to influenza virus under confocal laser scanning microscope showed red fluorescein signals in various cells of the lung tissue, suggesting the presence of anti -PR8-NP and anti -OK/06-N P, and the dilution of the virus free particles. The liquid group saw only the infected blue nuclei, indicating that the human lung slices could be exposed to influenza virus infection. In addition, the semi quantitative RT-PCR results showed that the expression of influenza virus PR8 and OK/06 NS1mRNA had a significant peak generation after influenza virus contact, indicating that human lung slices in vitro supported the replication of influenza virus.
Conclusion human lung slices exposed to influenza virus OK/06 and PR8 support influenza virus infection and virus replication.
The third part is the response of human lung multisource cells to influenza virus infection.
Objective to detect the cytokine response induced by human lung influenza virus infection and the role of human lung multicellular components in human lung's inherent immune response to influenza virus, using a model of human lung slices infected by influenza virus A/PR/8/34 (H1N1) (PR8) infection in vitro.
Methods human lung tissue sections were obtained according to the standard flow model of the new human lung slice in vitro transplantation culture model. Under the stimulation of influenza virus PR8, RNase protection assay (RPA) method was used to detect the expression of inflammatory factor mRNA in human lung slices after influenza virus stimulation, and ELISA was used to detect the inflammation after influenza virus stimulation. The protein release of the disease factor detects the inherent immune response of the human lung slices to influenza viruses and detects the multicellular sources of influenza virus induced human pneumonia factor expression by the confocal microscopy immunofluorescence.
Results influenza virus induced influenza virus induced many kinds of inflammatory factors in human lung. The high expression of MIP-1 alpha and IP-10 mRNA was found at the transcriptional level, and at the level of translation, it was proved that influenza virus stimulation induced the increase of induced production of inflammatory factor protein. The immunological results of confocal microscopy showed that the virus was induced by influenza virus. A variety of cytokine responses are derived from the innate immune responses of various cells of human lung epithelial cells, alveolar macrophages and interstitial cells.
Conclusion influenza virus induces inflammatory factors in human lung activation in human lung. Many inflammatory factors, such as MIP-1 alpha and IP-10, are induced to participate in the natural immune response of human lungs initiated by influenza virus infection.
The fourth part of CSE inhibits the innate immunity of human lung triggered by influenza virus.
Objective to further clarify the inherent immune response of human lung lamellae after influenza virus A/PR/8/34 (H1N1) (PR8) infection in human lung slices to induce the generation of cytokine and antiviral factor, and to evaluate the effect of cigarette smoke extract (Cigarette Smoke Extract, CSE) on influenza virus in human lung organ culture model. The influence of cytokines and its possible mechanism.
Methods the cigarette smoke extract (CSE) obtained by standard process was used to treat the tissue of human lung slices. The expression of various inflammatory factor proteins induced by influenza virus PR8 and the effect of CSE on the expression of inflammatory factor protein were detected by ELISA method. It was clear whether CSE inhibited the inherent immunity of human lung induced by influenza virus; then RT-PCR was detected. The effects of CSE treatment or combined anti oxidant treatment on the expression of inflammatory factors and PRRs mRNA induced by influenza virus infection were measured, and Western blot was used to detect the effect of CSE treatment or combined antioxidants on the expression of human lung RIG-I protein after influenza virus stimulation. In addition, the cytotoxicity of CSE to human lung slices was tested by LDH release toxicity.
Results CSE attenuated the human lung cytokine response and the expression of RIG-I mRNA and protein at the gene transcription and translation levels. The appropriate concentration of CSE treatment was safe. Meanwhile, the inhibitory effect of CSE on RIG-I and other PRRs signaling pathways could be protected by antioxidants.
Conclusion CSE attenuates the response of influenza virus triggered pneumonia factor, which may be mainly caused by the inherent immune response mechanism of human lung to influenza virus mediated by the RIG-I based PRRs signaling pathway.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392

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