【摘要】：目的：黏液纖毛清除功能(MCC)是呼吸系統(tǒng)抵抗環(huán)境中各種病原體、污染物侵襲的第一道防線。纖毛擺動(dòng)作為MCC的動(dòng)力來源,對(duì)維持MCC的正常功能起到了至關(guān)重要的作用,纖毛擺動(dòng)機(jī)制的研究雖已開展多年,但對(duì)其了解仍遠(yuǎn)未完善。臨床發(fā)現(xiàn)慢性鼻竇炎(CRS)可導(dǎo)致患者鼻腔MCC下降,但與此同時(shí),CRS患者下呼吸道MCC存在異常的體征卻少有發(fā)現(xiàn)。這預(yù)示著上下呼吸道纖毛擺動(dòng)功能可能存在著差異�；谝陨媳尘�,本實(shí)驗(yàn)對(duì)各種狀態(tài)下,上下呼吸道上皮纖毛的擺動(dòng)功能開展了比較研究,以期闡明：各種狀態(tài)下,上下呼吸道纖毛擺動(dòng)功能是否存在差異；通過比較研究,為纖毛擺動(dòng)機(jī)制的研究開辟新的途徑。 材料與方法：體外建立"Puff"系統(tǒng),模擬“噴嚏”這一生理過程。觀察“噴嚏”對(duì)小鼠鼻腔上皮CBF的影響。使用抑制劑(Apyrase、Suramin、EGTA、BAPTA-AM和H-89)干預(yù)實(shí)驗(yàn),研究ATP、鈣離子(Ca2+)和蛋白激酶A(PKA)在這一過程中的作用。觀察“噴嚏”后,鼻腔、氣管上皮CBF反應(yīng)性的差異。觀察上下呼吸道上皮細(xì)胞基礎(chǔ)狀態(tài)下CBF的差異,使用fura-2測(cè)定兩種上皮細(xì)胞中的Ca2+濃度。觀察綠膿桿菌分泌液對(duì)鼻腔、氣管上皮細(xì)胞基礎(chǔ)CBF和“噴嚏”后CBF反應(yīng)性的影響,并且,利用細(xì)菌分泌液對(duì)鼻腔、氣管上皮細(xì)胞進(jìn)行預(yù)處理后,觀察活體綠膿桿菌在上下呼吸道上皮表面附著能力的差異 結(jié)果：“噴嚏”后鼻腔上皮CBF與“噴嚏”前CBF的比率為1.78±0.05,n=10,ATP、Ca2+以及PKA抑制劑可明顯抑制這一反應(yīng)。氣管組中,“噴嚏”后CBF的升高比率僅為1.21±0.03,n=10,p0.01。氣管上皮基礎(chǔ)CBF約為鼻腔上皮基礎(chǔ)CBF的兩倍。氣管纖毛細(xì)胞內(nèi)Ca2+濃度為91.06±9.85nM,n=4,鼻腔組中這一數(shù)據(jù)為54.90±2.52 nM,n=4,p0.05。綠膿桿菌分泌物在降低鼻腔上皮細(xì)胞基礎(chǔ)CBF的同時(shí),也能夠抑制“噴嚏”后CBF的升高；氣管上皮CBF對(duì)綠膿桿菌分泌物的侵襲則具有抵抗力。鼻腔上皮細(xì)胞上細(xì)菌的貼敷數(shù)量在實(shí)驗(yàn)組及對(duì)照組中都顯著多于氣管上皮細(xì)胞。 結(jié)論：上下呼吸道纖毛功能存在明顯差異,具體表現(xiàn)為：鼻腔上皮CBF對(duì)“噴嚏”這一機(jī)械刺激的反應(yīng)能力高于氣管上皮；基礎(chǔ)狀態(tài)下,氣管上皮CBF快于鼻腔上皮CBF,且這一差異與細(xì)胞內(nèi)Ca2+濃度有關(guān)；綠膿桿菌分泌物可降低鼻腔上皮基礎(chǔ)CBF,并可抑制CBF對(duì)“噴嚏”刺激的反應(yīng),而氣管上皮則對(duì)綠膿桿菌的侵襲具有抵抗力。
[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 鹵0.05 and PKA inhibitor could significantly inhibit this reaction. In trachea group, the increase rate of CBF after sneezing was only 1.21 鹵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 鹵9.85nMnMnMnMnMnM, and that in nasal cavity group was 54.90 鹵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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R765.21
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本文編號(hào)：2231660