KLF2调控CXCR1、CXCR2、L-selectin、CD11a等对类中性粒细胞粘附迁移的作用机制
发布时间：2018-11-04 17:45
【摘要】：背景及目的：支气管哮喘是一种慢性气道炎症性疾病，中性粒细胞是支气管哮喘气道炎症的主要效应细胞之一，中性粒细胞在支气管内聚集是严重类型哮喘的共同特征，但支气管内中性粒细胞增多的机制并不清楚。本研究主要从细胞水平去阐述KLF2是否通过调节CXCR1、CXCR2、L-selectin、CD11a、CD11b、CD18的表达来影响类中性粒细胞粘附迁移过程。 研究方法： （1）培养HL-60细胞，DMSO诱导分化为类中性粒细胞，通过Diff染色观察细胞核形态及Realtime-PCR检测中性粒细胞分化标志物CD11b mRNA的表达，确定是否诱导成功 （2）用空载及KLF2-RNAi慢病毒载体转染类中性粒细胞，随机分为空白组、空载组和siRNA干预组。应用荧光显微镜、Western blot法进行转染鉴定。 （3）Realtime PCR检测三组中CXCR1、CXCR2、L-selectin、CD11a、CD11b、CD18mRNA的表达，Western blot检测三组中CXCR1、CXCR2、L-selectin的蛋白表达，流式细胞术检测CD11a、CD11b、CD18平均荧光强度。 （4）荧光显微镜观察三组中标记有calcein-AM的类中性粒细胞对脐静脉内皮细胞粘附情况及应用Transwell实验计数浓度为10ng/ml的IL-8对三组类中性粒细胞的趋化迁移率。 结果： 1类中性粒细胞的鉴定 CD11b为公认的中性粒细胞分化成熟标志之一，HL60细胞在经1.3%DMSO诱导后，通过Realtime-PCR检测其CD11b mRNA表达量并与未经过诱导的HL60细胞相比较，诱导后呈明显增多（P0.01），Diff染色油镜下观察类中性粒细胞核呈现肾型及双叶。 2KLF2的干扰 荧光显微镜观察带有GFP的慢病毒荧光转染效果，计算感染效率达到90%以上；Westernblot检测KLF2蛋白表达，发现干扰组的表达与其他两组比较明显减少（P0.01），而空白组与空载组比较差异无统计学意义，计算其敲除率约为76%。 3mRNA及蛋白的检测 （1）Realtime PCR结果显示：KLF2干扰组与空白组、空载组相比较，CXCR1、CXCR2、CD11a、CD11b、CD18mRNA的表达呈明显增多，而L-selectin mRNA的表达明显减少，差异均有统计学意义（P0.01）；而以上因子空白组与空载组相比较，差异均无统计学意义。 (2) Western blot结果显示：KLF2干扰组与空白组、空载组相比较，CXCR1、CXCR2蛋白的表达明显增多，而L-selectin蛋白的表达明显减少，，差异均有统计学意义（P0.01）；而以上因子空白组与空载组相比较，差异无统计学意义。 （3）流式细胞术检测结果显示干扰组CD11a、CD11b、CD18的平均荧光强度与空白组，空载组相比较明显增多（P0.01）；而空白组与空载组相比较，差异无统计学意义。 4类中性粒细胞的粘附及迁移实验 （1）荧光显微镜观察结果显示：KLF2干扰组镜下的类中性粒细胞粘附数与空白组、空载组相比较呈明显增多（P0.05）；而空白组与空载组相比较，差别无统计学意义。 （2）10ng/ml浓度的IL-8对类中性粒细胞迁移率，KLF2干扰组迁移率与空白组、空载组相比较呈明显增多（P0.01），而空白组与空载组相比较，差异无统计学意义。 结论： （1）KLF2可以通过调控粘附分子CD11a、CD11b、CD18的表达，影响类中性粒细胞的粘附能力，而KLF2对L-selectin的调控可能对类中性粒细胞的粘附能力影响不大。 （2）KLF2可以通过调控CXCR1、CXCR2，增加IL-8趋化下类中性粒细胞趋化迁移能力
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R562.25
本文编号：2310662
[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.
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R562.25
【参考文献】
相关期刊论文 前1条
1 ;Sputum interleukin-17 is increased and associated with airway neutrophilia in patients with severe asthma[J];Chinese Medical Journal;2005年11期
本文编号：2310662
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