上下呼吸道纤毛功能内在差异性的比较研究

发布时间：2018-09-08 20:50

【摘要】：目的：黏液纤毛清除功能(MCC)是呼吸系统抵抗环境中各种病原体、污染物侵袭的第一道防线。纤毛摆动作为MCC的动力来源,对维持MCC的正常功能起到了至关重要的作用,纤毛摆动机制的研究虽已开展多年,但对其了解仍远未完善。临床发现慢性鼻窦炎(CRS)可导致患者鼻腔MCC下降,但与此同时,CRS患者下呼吸道MCC存在异常的体征却少有发现。这预示着上下呼吸道纤毛摆动功能可能存在着差异。基于以上背景,本实验对各种状态下,上下呼吸道上皮纤毛的摆动功能开展了比较研究,以期阐明：各种状态下,上下呼吸道纤毛摆动功能是否存在差异；通过比较研究,为纤毛摆动机制的研究开辟新的途径。 材料与方法：体外建立"Puff"系统,模拟“喷嚏”这一生理过程。观察“喷嚏”对小鼠鼻腔上皮CBF的影响。使用抑制剂(Apyrase、Suramin、EGTA、BAPTA-AM和H-89)干预实验,研究ATP、钙离子(Ca2+)和蛋白激酶A(PKA)在这一过程中的作用。观察“喷嚏”后,鼻腔、气管上皮CBF反应性的差异。观察上下呼吸道上皮细胞基础状态下CBF的差异,使用fura-2测定两种上皮细胞中的Ca2+浓度。观察绿脓杆菌分泌液对鼻腔、气管上皮细胞基础CBF和“喷嚏”后CBF反应性的影响,并且,利用细菌分泌液对鼻腔、气管上皮细胞进行预处理后,观察活体绿脓杆菌在上下呼吸道上皮表面附着能力的差异 结果：“喷嚏”后鼻腔上皮CBF与“喷嚏”前CBF的比率为1.78±0.05,n=10,ATP、Ca2+以及PKA抑制剂可明显抑制这一反应。气管组中,“喷嚏”后CBF的升高比率仅为1.21±0.03,n=10,p0.01。气管上皮基础CBF约为鼻腔上皮基础CBF的两倍。气管纤毛细胞内Ca2+浓度为91.06±9.85nM,n=4,鼻腔组中这一数据为54.90±2.52 nM,n=4,p0.05。绿脓杆菌分泌物在降低鼻腔上皮细胞基础CBF的同时,也能够抑制“喷嚏”后CBF的升高；气管上皮CBF对绿脓杆菌分泌物的侵袭则具有抵抗力。鼻腔上皮细胞上细菌的贴敷数量在实验组及对照组中都显著多于气管上皮细胞。 结论：上下呼吸道纤毛功能存在明显差异,具体表现为：鼻腔上皮CBF对“喷嚏”这一机械刺激的反应能力高于气管上皮；基础状态下,气管上皮CBF快于鼻腔上皮CBF,且这一差异与细胞内Ca2+浓度有关；绿脓杆菌分泌物可降低鼻腔上皮基础CBF,并可抑制CBF对“喷嚏”刺激的反应,而气管上皮则对绿脓杆菌的侵袭具有抵抗力。
[Abstract]:Objective: mucociliary clearance function (MCC) is the first line of defense against various pathogens and pollutants in the respiratory system. Cilia swing, as the power source of MCC, plays an important role in maintaining the normal function of MCC. Although the mechanism of cilia swing has been studied for many years, its understanding is still far from perfect. It was found that (CRS) of chronic sinusitis could lead to the decrease of MCC in nasal cavity, but at the same time, the signs of abnormal MCC in lower respiratory tract of patients with chronic sinusitis were rarely found. This indicates that there may be differences in ciliated function of upper and lower respiratory tract. Based on the above background, a comparative study was carried out on the swinging function of the upper and lower respiratory tract epithelial cilia under various conditions, in order to clarify whether there were differences in the function of the upper and lower respiratory tract cilia in various states. It provides a new way to study the mechanism of ciliated swinging. Materials and methods: Puff system was established in vitro to simulate the physiological process of sneezing. To observe the effect of sneezing on CBF in mouse nasal epithelium. The role of ATP, calcium ion (Ca2) and protein kinase A (PKA) in this process was studied using inhibitors (Apyrase,Suramin,EGTA,BAPTA-AM and H-89). To observe the difference of CBF reactivity in nasal cavity and tracheal epithelium after sneezing. To observe the difference of CBF in the basic state of upper and lower respiratory tract epithelial cells, and to measure the concentration of Ca2 in the two kinds of epithelial cells by fura-2. To observe the effect of Pseudomonas aeruginosa secretion on the reactivity of basic CBF in nasal cavity, tracheal epithelial cells and CBF after sneezing, and to pretreat nasal and tracheal epithelial cells with bacterial secretion. The difference of adhesion ability of Pseudomonas aeruginosa on the surface of upper and lower respiratory epithelium was observed. The ratio of CBF to CBF before sneezing was 1.78 卤0.05 and PKA inhibitor could significantly inhibit this reaction. In trachea group, the increase rate of CBF after sneezing was only 1.21 卤0.03NM 10p0.01. The basic CBF of trachea epithelium was twice as much as that of CBF of nasal epithelium. The concentration of Ca2 in trachea ciliated cells was 91.06 卤9.85nMnMnMnMnMnM, and that in nasal cavity group was 54.90 卤2.52 nM,n=4,p0.05.. The secretion of Pseudomonas aeruginosa can also inhibit the increase of CBF after sneezing, while the secretion of Pseudomonas aeruginosa can inhibit the invasion of the secretion of Pseudomonas aeruginosa, while the CBF of tracheal epithelium can resist the invasion of the secretion of Pseudomonas aeruginosa. The number of bacteria on nasal epithelial cells was significantly higher in the experimental and control groups than in the tracheal epithelial cells. Conclusion: there are significant differences in ciliated function of upper and lower respiratory tract, which are as follows: the ability of nasal epithelial CBF to respond to "sneezing" mechanical stimulation is higher than that of tracheal epithelium, and the basic state of nasal epithelium is higher than that of trachea epithelium. Tracheal epithelial CBF is faster than nasal epithelium CBF, and this difference is related to intracellular Ca2 concentration. Pseudomonas aeruginosa secretions can reduce basic CBF, of nasal epithelium and inhibit CBF's response to sneezing. The trachea epithelium is resistant to the invasion of Pseudomonas aeruginosa.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R765.21
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本文编号：2231660