本文選題：人肺精確薄片模型 + 移植培養(yǎng)　； 參考：《蘇州大學(xué)》2011年博士論文

【摘要】：目的通過實驗探索人肺體外移植培養(yǎng)的條件,構(gòu)建人肺體外移植培養(yǎng)液的配置方案,優(yōu)化人肺標本的處理和保存,穩(wěn)定切片后細胞,規(guī)范模型設(shè)備的使用和精確切片厚度的選擇,從而建立標準化流程,創(chuàng)建一個穩(wěn)定的、強大的、高產(chǎn)量、高重復(fù)性和實用性的多細胞共生長的人肺器官體外培養(yǎng)模型。 方法選取與俄克拉荷馬大學(xué)合作的五家醫(yī)院中接受肺癌手術(shù)的志愿者的按University of Oklahoma Institutional Review Board批準的標準程序切除的正常肺葉組織,以及交通事故死亡遺體捐贈者的肺器官。參照改良的瓊脂糖充填肺組織的方法,以含1.5%低熔低膠瓊脂糖培養(yǎng)液,經(jīng)充膨、鉆取、切片及培養(yǎng)等步驟建立人肺精確薄片體外移植模型,并將人肺薄片進行長時間的體外培養(yǎng),選取不同時間點對其生長狀態(tài)進行嚴密的顯微鏡下組織形態(tài)的監(jiān)測。以乳酸脫氫酶釋放毒性實驗評價該模型建立體系對細胞的毒性。 結(jié)果建立的人肺精確薄片模型體系穩(wěn)定,無明顯的細胞毒性損傷,易重復(fù)、高產(chǎn)量。按此模型流程制備的人肺精確薄片經(jīng)長時間(14天)的體外培養(yǎng),整體結(jié)構(gòu)完整,三維立體結(jié)構(gòu)清楚,支氣管清晰可見,肺泡細胞隨培養(yǎng)時間活力無改變,共同維持了肺正常的結(jié)構(gòu)和功能。 結(jié)論按標準流程建立的人肺精確薄片體外移植模型體系穩(wěn)定,人肺薄片組織能經(jīng)受長時間體外培養(yǎng),結(jié)構(gòu)清晰、細胞穩(wěn)定,是一個可重復(fù)使用替代人肺器官功能的體外培養(yǎng)模型。 第二部分流感病毒對移植培養(yǎng)人肺薄片的影響 目的利用新建的人肺精確薄片體外移植培養(yǎng)模型,偵測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復(fù)制。 方法按照以低熔低膠瓊脂糖充填肺組織建立人肺精確薄片體外移植培養(yǎng)模型的標準流程獲取人肺薄片,分別暴露于6x106 PFU/ml流感病毒A/PR/8/34 (H1N1)(PR8)和A/Oklahoma/309/06 (H3N2)(OK/06),經(jīng)不同時間的體外培養(yǎng)后收獲肺切片,以半定量RT-PCR和共聚焦顯微鏡偵測人肺薄片對流感病毒暴露的反應(yīng)。 結(jié)果暴露于流感病毒PR8和OK/06體外培養(yǎng)24h后的人肺薄片免疫結(jié)果顯示,在激光共聚焦顯微鏡下,暴露于流感病毒的人肺切片,可見在肺組織的多種細胞中存在紅色的熒光素信號,提示抗-PR8-NP和抗-OK/06-N P的存在,而在無病毒顆粒的稀釋液組僅看到染成蘭色的細胞核,說明人肺薄片可被暴露的流感病毒感染。另外,半定量RT-PCR結(jié)果顯示,流感病毒PR8和OK/06 NS1mRNA的表達在流感病毒接觸后8h有顯著的高峰生成,說明體外培養(yǎng)的人肺薄片支持流感病毒的病毒復(fù)制。 結(jié)論暴露于流感病毒OK/06和PR8的人肺薄片,支持流感病毒的感染和病毒復(fù)制。 第三部分人肺多源細胞對流感病毒感染的反應(yīng) 目的利用可被流感病毒A/PR/8/34(H1N1) (PR8)感染的人肺薄片體外移植培養(yǎng)的模型,偵測人肺內(nèi)流感病毒感染誘導(dǎo)的細胞因子反應(yīng)及人肺多細胞成分在參與人肺對流感病毒的固有免疫反應(yīng)中的作用。 方法按照新建的人肺薄片體外移植培養(yǎng)模型標準流程獲取人肺組織切片,暴露于流感病毒PR8的刺激下,分別以RNase protection assay (RPA)方法在轉(zhuǎn)錄水平檢測流感病毒刺激后人肺薄片內(nèi)炎癥因子mRNA的表達,以ELISA檢測流感病毒刺激后的炎癥因子的蛋白釋放,偵測人肺切片對流感病毒的固有免疫應(yīng)答,并以共聚焦顯微鏡免疫熒光方法偵測流感病毒誘導(dǎo)人肺炎癥因子表達的多細胞來源。 結(jié)果流感病毒刺激后的人肺中流感病毒誘導(dǎo)產(chǎn)生了多種類的炎癥因子,在轉(zhuǎn)錄水平發(fā)現(xiàn)MIP-1α和IP-10 mRNA存在高表達,并在翻譯水平證實了流感病毒刺激引起誘導(dǎo)生成炎癥因子蛋白釋放增加。共聚焦顯微鏡免疫結(jié)果表明,由流感病毒誘導(dǎo)生成的多種細胞因子反應(yīng)是來源于人肺上皮細胞、肺泡巨噬細胞和間質(zhì)細胞的多種細胞公共參與的固有免疫反應(yīng)。 結(jié)論流感病毒引起人肺內(nèi)激活多種固有細胞產(chǎn)生炎癥因子反應(yīng),誘導(dǎo)生成的MIP-1α和IP-10等多種炎癥因子參與了流感病毒感染后啟動的人肺固有免疫應(yīng)答。 第四部分CSE抑制流感病毒觸發(fā)的人肺固有免疫 目的進一步明確流感病毒A/PR/8/34(H1N1) (PR8)感染體外移植培養(yǎng)人肺薄片后人肺多種固有細胞參與誘導(dǎo)細胞因子和抗病毒因子生成的固有免疫應(yīng)答。評價香煙煙霧提取物(Cigarette Smoke Extract,CSE)在人肺器官培養(yǎng)模型上對流感病毒引起的細胞因子的影響并探討其可能的作用機制。 方法采用標準流程獲得的香煙煙霧提取物(CSE)處理人肺薄片組織,以ELISA方法檢測流感病毒PR8誘導(dǎo)的多種炎癥因子蛋白的表達及CSE對炎癥因子蛋白表達的影響,明確CSE是否對流感病毒誘導(dǎo)的人肺固有免疫存在抑制作用;進而以RT-PCR檢測CSE處理或聯(lián)合抗氧化劑處理對流感病毒感染后誘導(dǎo)的炎癥因子及PRRs mRNA表達的影響;再以Western blot檢測CSE處理或聯(lián)合抗氧化劑對流感病毒刺激后人肺RIG-I蛋白表達的影響。另外,以LDH釋放毒性實驗測定CSE對人肺切片的細胞毒性。 結(jié)果在基因轉(zhuǎn)錄和翻譯水平發(fā)現(xiàn)CSE減弱流感病毒觸發(fā)的人肺細胞因子反應(yīng)以及RIG-I mRNA和蛋白的表達,適宜濃度CSE處理是安全的。同時發(fā)現(xiàn),CSE對RIG-I及其它PRRs信號通路的抑制作用可被抗氧化劑保護。 結(jié)論CSE減弱流感病毒觸發(fā)人肺炎癥因子的反應(yīng),可能主要是通過減弱由RIG-I為主的PRRs信號通路介導(dǎo)的人肺對流感病毒觸發(fā)的固有免疫反應(yīng)機制所致。
[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.

【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2011
【分類號】：R392

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