發(fā)布時(shí)間：2018-11-04 17:45

【摘要】：背景及目的：支氣管哮喘是一種慢性氣道炎癥性疾病，中性粒細(xì)胞是支氣管哮喘氣道炎癥的主要效應(yīng)細(xì)胞之一，中性粒細(xì)胞在支氣管內(nèi)聚集是嚴(yán)重類型哮喘的共同特征，但支氣管內(nèi)中性粒細(xì)胞增多的機(jī)制并不清楚。本研究主要從細(xì)胞水平去闡述KLF2是否通過(guò)調(diào)節(jié)CXCR1、CXCR2、L-selectin、CD11a、CD11b、CD18的表達(dá)來(lái)影響類中性粒細(xì)胞粘附遷移過(guò)程。 研究方法： （1）培養(yǎng)HL-60細(xì)胞，DMSO誘導(dǎo)分化為類中性粒細(xì)胞，通過(guò)Diff染色觀察細(xì)胞核形態(tài)及Realtime-PCR檢測(cè)中性粒細(xì)胞分化標(biāo)志物CD11b mRNA的表達(dá)，確定是否誘導(dǎo)成功 （2）用空載及KLF2-RNAi慢病毒載體轉(zhuǎn)染類中性粒細(xì)胞，隨機(jī)分為空白組、空載組和siRNA干預(yù)組。應(yīng)用熒光顯微鏡、Western blot法進(jìn)行轉(zhuǎn)染鑒定。 （3）Realtime PCR檢測(cè)三組中CXCR1、CXCR2、L-selectin、CD11a、CD11b、CD18mRNA的表達(dá)，Western blot檢測(cè)三組中CXCR1、CXCR2、L-selectin的蛋白表達(dá)，流式細(xì)胞術(shù)檢測(cè)CD11a、CD11b、CD18平均熒光強(qiáng)度。 （4）熒光顯微鏡觀察三組中標(biāo)記有calcein-AM的類中性粒細(xì)胞對(duì)臍靜脈內(nèi)皮細(xì)胞粘附情況及應(yīng)用Transwell實(shí)驗(yàn)計(jì)數(shù)濃度為10ng/ml的IL-8對(duì)三組類中性粒細(xì)胞的趨化遷移率。 結(jié)果： 1類中性粒細(xì)胞的鑒定 CD11b為公認(rèn)的中性粒細(xì)胞分化成熟標(biāo)志之一，HL60細(xì)胞在經(jīng)1.3%DMSO誘導(dǎo)后，通過(guò)Realtime-PCR檢測(cè)其CD11b mRNA表達(dá)量并與未經(jīng)過(guò)誘導(dǎo)的HL60細(xì)胞相比較，誘導(dǎo)后呈明顯增多（P0.01），Diff染色油鏡下觀察類中性粒細(xì)胞核呈現(xiàn)腎型及雙葉。 2KLF2的干擾 熒光顯微鏡觀察帶有GFP的慢病毒熒光轉(zhuǎn)染效果，計(jì)算感染效率達(dá)到90%以上；Westernblot檢測(cè)KLF2蛋白表達(dá)，發(fā)現(xiàn)干擾組的表達(dá)與其他兩組比較明顯減少（P0.01），而空白組與空載組比較差異無(wú)統(tǒng)計(jì)學(xué)意義，計(jì)算其敲除率約為76%。 3mRNA及蛋白的檢測(cè) （1）Realtime PCR結(jié)果顯示：KLF2干擾組與空白組、空載組相比較，CXCR1、CXCR2、CD11a、CD11b、CD18mRNA的表達(dá)呈明顯增多，而L-selectin mRNA的表達(dá)明顯減少，差異均有統(tǒng)計(jì)學(xué)意義（P0.01）；而以上因子空白組與空載組相比較，差異均無(wú)統(tǒng)計(jì)學(xué)意義。 (2) Western blot結(jié)果顯示：KLF2干擾組與空白組、空載組相比較，CXCR1、CXCR2蛋白的表達(dá)明顯增多，而L-selectin蛋白的表達(dá)明顯減少，，差異均有統(tǒng)計(jì)學(xué)意義（P0.01）；而以上因子空白組與空載組相比較，差異無(wú)統(tǒng)計(jì)學(xué)意義。 （3）流式細(xì)胞術(shù)檢測(cè)結(jié)果顯示干擾組CD11a、CD11b、CD18的平均熒光強(qiáng)度與空白組，空載組相比較明顯增多（P0.01）；而空白組與空載組相比較，差異無(wú)統(tǒng)計(jì)學(xué)意義。 4類中性粒細(xì)胞的粘附及遷移實(shí)驗(yàn) （1）熒光顯微鏡觀察結(jié)果顯示：KLF2干擾組鏡下的類中性粒細(xì)胞粘附數(shù)與空白組、空載組相比較呈明顯增多（P0.05）；而空白組與空載組相比較，差別無(wú)統(tǒng)計(jì)學(xué)意義。 （2）10ng/ml濃度的IL-8對(duì)類中性粒細(xì)胞遷移率，KLF2干擾組遷移率與空白組、空載組相比較呈明顯增多（P0.01），而空白組與空載組相比較，差異無(wú)統(tǒng)計(jì)學(xué)意義。 結(jié)論： （1）KLF2可以通過(guò)調(diào)控粘附分子CD11a、CD11b、CD18的表達(dá)，影響類中性粒細(xì)胞的粘附能力，而KLF2對(duì)L-selectin的調(diào)控可能對(duì)類中性粒細(xì)胞的粘附能力影響不大。 （2）KLF2可以通過(guò)調(diào)控CXCR1、CXCR2，增加IL-8趨化下類中性粒細(xì)胞趨化遷移能力
[Abstract]:Background and Objective: Bronchial asthma is a chronic airway inflammatory disease. Neutrophil is one of the main effector cells of bronchial asthma airway inflammation. Neutrophil aggregation is a common feature of severe asthma in bronchial asthma. However, the mechanism of the increase of neutrophils in the bronchi is not clear. In this study, the expression of KLF2 was studied mainly from the cell level to investigate whether KLF2 was used to regulate the adhesion and migration of neutrophils through the expression of CD1, CD2, L-selectin, CD11a, CD11b and CD18. Study Methods: (1) HL-60 cells were cultured, DMSO was induced to differentiate into neutrophils, and cell nucleus morphology and Realtime-PCR were observed by diff staining. The expression and determination of NA Successful (2) transfection of neutrophils with no-load and KLF2-RNAi lentiviral vectors was randomly divided into blank groups, no-load groups, and siRNA intervention group. Fluorescence microscope, Western blo Three groups were detected by Realtime PCR. Three groups were detected by Realtime PCR. The expression of cyclin D1, CD11b and CD18mRNA in three groups was detected by Realtime PCR. Western blot was used to detect the protein expression in three groups, and the expression of cyclin D1, CD2, L-selectin in three groups was detected by flow cytometry. and the average fluorescence intensity of CD18. (4) fluorescence microscopy was used to observe the adhesion of the neutrophils to the umbilical vein endothelial cells in three groups, and the concentration of IL-8 was 10ng/ ml using the Transwell experiment. Three groups chemotaxis of neutrophils Results: The identification of neutrophils was recognized as one of the mature markers of neutrophil differentiation. HL60 cells were induced by 1. 3% DMSO and detected by Realtime-PCR. 60 cells showed a significant increase after induction (P0.01), Di The Lower View of Dff Dyeing Oil Mirror The fluorescence transfection effect of lentivirus with GFP was observed under the interference fluorescence microscope with GFP, and the efficiency of infection was more than 90%, and KLF2 protein was detected by Western blot. It was found that the expression of interfering group was significantly decreased compared with other two groups (P0.01). Difference between blank group and no-load group There was no statistical significance, and its knock-out rate was about 76%. The detection of mRNA and protein (1) Realtime PCR showed that the expression of KLF2 interfering group was significantly higher than that of blank group and no-load group, while the expression of CD1, CD11a, CD11b and CD18mRNA increased significantly, while L-s The expression of electroin mRNA was significantly decreased and the difference was statistically significant. (2) Western blot showed that the expression of KLF2 interfering group was significantly higher than that of blank group and no-load group, but the expression of L-selectin was significantly decreased and the difference was significant. The results of flow cytometry showed that the mean fluorescence intensity of CD11a, CD11b and CD18 of interfering groups were compared with those of no-load group. There was a significant increase in the white group and no-load group (P 0. 01) There was no statistically significant difference between the blank group and the no-load group. The adhesion and migration of neutrophils (1) showed that the KLF2 interferes with the neutrophils in the group. The number of adhesion was significantly higher than that in blank group and no-load group (P0.05). interference group There was no significant difference between mobility and blank group and no-load group (P0.01). Conclusion: (1) KLF2 can be used to control adhesion molecules CD11a, CD11b, CD1. 8 expression affecting the adhesion of neutrophils, whereas KLF2 regulates the regulation of L-selectin.
【學(xué)位授予單位】：南華大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R562.25

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 ;Sputum interleukin-17 is increased and associated with airway neutrophilia in patients with severe asthma[J];Chinese Medical Journal;2005年11期

本文編號(hào)：2310662