Intelectin在哮喘气道上皮细胞炎性细胞因子表达中的作用和机制

发布时间：2018-05-25 02:04

本文选题：IL13 + CCL5　；参考：《华中科技大学》2012年硕士论文

【摘要】：研究背景目前哮喘发病率呈现上升趋势，但是哮喘的发病机制尚未完全阐明。哮喘是以气道高反应性、粘液分泌增多和慢性嗜酸性粒细胞炎症为特点。有些报道已证实主要在哮喘患者气道上皮细胞和单核细胞中，一些趋化因子RANTES,MCP-3,MCP-4,eotaxin-1andeotaxin-2等的mRNA表达是增加的。趋化因子是8-10kDa的小分子细胞因子，根据每个蛋白分子一个或两个半胱氨酸残基在N末端的位置可以分为四类，即，CXC,CC,C,和CXXXC。许多CC类趋化因子是由气道上皮细胞产生的。IL13可以诱导上皮细胞CCL11(eotaxin),CCL24(eotaxin-2),andCCL26(eotaxin-3)的表达，且这些因子的表达在哮喘者是增加的。这些CC类趋化因子是嗜酸性粒细胞和嗜碱性细胞表面CCR受体的选择性激动剂。哮喘是以TH2细胞调节为主，分泌的细胞因子与IL4/5/9/13,虽然都与哮喘相关，但是IL13是现在认为最关键的因子。白介素13是由TH2型细胞分泌的分子量为12kDa多效能分子，位于常染色体5q31上。白介素13是富含嗜酸性粒细胞、淋巴细胞和巨噬细胞炎症、粘液上皮化生、组织纤维化和基底重塑强有力的刺激剂。白介素13能够刺激哮喘相关的许多细胞，例如B淋巴细胞、肥大细胞、嗜酸性粒细胞、肺泡上皮细胞、成纤维细胞和气道平滑肌细胞。一些实验也证明，重组白介素13的应用或是肺中白介素13的过表达可以产生哮喘样的气道炎症和气道重塑。许多实验证实白介素13在哮喘中是过表达的，且与哮喘的特征TH2细胞炎症和气道重塑的发病机制相关。因此，我们用IL13刺激气道上皮细胞，模拟哮喘样作用。人intelectin(ITLN)是一种分泌性的糖蛋白，由295个氨基酸和N端寡糖组成，它的基本结构单位是二硫键连接40kDa多肽成的120kDa的三聚体。可分为intelectin-1和2。人intelectin也被称作甘油磷酸肌醇连接的转铁蛋白受体、血管内皮细胞凝集素或网膜素。人ITLN基因位于染色体1q21.3上，含有8个外显子，，且人intelectin的mRNA表达于心脏、小肠、结肠和胸腺。属于凝集素类，能够识别特定的细菌壁组分，而不被其他凝集素所识别。所熟知的是它在小肠的潘氏细胞和分泌或是杯状细胞表达。最近，也观察到在气道上皮细胞分泌细胞也有表达。可以识别存在于细菌、真菌和原虫壁上右旋戊糖和呋喃半乳糖残基，但在哺乳动物在不然，说明intelectin在抗原识别中起重要作用。Intelectin-1在小鼠小肠暴露于线旋毛虫后，表达是上调的，其同种型intelectin-2在小肠上皮细胞也被诱导。Intelectin-1是一个TH2细胞调节的抗菌蛋白，在哮喘患者支气管上皮细胞中intelectin-1的基因表达水平是上调的，与IL13的表达上调相关。第一部分Intelectin-1在IL13诱导的气道上皮细胞株A549和16HBE中炎性细胞因子表达中的作用目的：探讨intelectin-1基因在人肺泡/气道上皮细胞分泌炎性因子中的作用。方法：实验用两种细胞系A549和16HBE。分为转染高表达质粒分组为：（A）正常对照组（B）IL13组（C）转染人ITLN1质粒组；转染干扰质粒分组：（A）正常对照组+空质粒组（B）IL13+空质粒组（C）IL13+Itln1-shRNA质粒组。通过RealtimePCR方法检测intelectin-1，CCL5和IL6mRNA的表达。ELISA方法检测细胞上清液中相应因子的蛋白水平表达。地塞米松干预细胞：①对照组+DMSO②IL13+DMSO③DEX100+IL13，然后通过RealtimePCR检测细胞因子ITLN1,CCL5和IL6mRNA的表达。结果：A549细胞中Intelectin-1，CCL5,IL6mRNA的表达水平，人ITLN1组和IL13组较对照组均明显升高，p0.05。而16HBE细胞Intelectin-1，CCL5,IL6mRNA的表达水平，人ITLN1组较对照组明显增高，P0.05均有统计学意义；IL13组较对照组Intelectin-1，IL6mRNA表达水平增高，P0.05；但是IL13组较对照组CCL5mRNA表达水平有增高趋势，但是未有统计意义。细胞A549和16HBE转染人ITLN1-shRNA质粒后，A549和16HBE细胞系：IL13+人ITLN1shRNA质粒组较IL13组intelectin-1,CCL5和IL6的表达明显下降，P0.05，均有统计学意义。ELISA方法检测蛋白水平：高表达实验，人ITLN质粒组与对照组相比，IL6,CCL5的蛋白表达水平明显升高P0.05。干扰实验组：IL13组较对照组，CCL5和IL6的蛋白表达水平明显升高P0.05；IL13+ITLN1-shRNA组较IL13组，CCL5和IL6的蛋白表达水平明显降低P0.05。在A549细胞，地塞米松+IL13组较IL13组，ITLN1,CCL5和IL6明显减少的，P0.05;在16HBE细胞，地塞米松组较IL13组，ITLN1,CCL5和IL6均有显著下降，并p0.05,均有统计学意义。结论：转入人ITLN1高表达质粒后，成功的促使细胞intelectin-1mRNA的高表达。一同检测的因子CCL5和IL6的mRNA和蛋白表达水平也相应的升高，可能与intelectin-1的表达升高有关。转染人ITLN1干扰质粒后，抑制了intelectin-1mRNA的表达,一同检测的因子CCL5和IL6的mRNA和蛋白表达水平也相应的降低。Intelectin-1的表达可能抑制了CCL5和IL6的表达。糖皮质激素可以抑制A549和16HBE细胞中ITLN1,CCL5和IL6的基因表达。由此得出，Intelectin-1在IL-13诱导的气道上皮细胞中炎性细胞因子CCL5和IL-6的表达中有重要的作用。第二部分ERK信号通路在intelectin-1介导的气道上皮细胞炎性细胞因子CCL5和IL6表达中的作用目的：研究探讨intelectin-1参与气道上皮细胞中炎性细胞因子CCL5和IL-6表达的机制。方法：用IL-13和PD98059分别干预人肺上皮细胞株A549和16HBE细胞。PD98059实验分组(1)对照组，(2)IL-13组，(3)IL-13+PD98059组。然后用RealTimePCR方法检测细胞因子CCL5和IL6的mRNA的表达。转染干扰质粒分组：（A）正常对照组+空质粒组（B）IL13+空质粒组（C）IL13+humanITLN1shRNA质粒组，通过RNA干扰的方法抑制IL-13诱导的intelectin-1表达后，用Western-Blot检测intelectin-1,ERK1/2和P-ERK1/2蛋白水平的变化。结果：用PD98059干预IL13刺激的A549和16HBE细胞后,CCL5和IL6的mRNA表达水平，IL13+PD98059组较IL13组明显降低，P0.05。转染humanITLN1shRNA质粒后，intelectin-1和P-ERK1/2蛋白表达水平，IL-13组较对照组升高，P0.05；IL-13+shRNA组中intelectin-1和P-ERK1/2蛋白表达较IL-13组降低，P0.05。结论：ERK1/2的一种抑制剂PD98059，可以阻滞IL-13诱导的CCL5和IL6在A549和16HBE细胞中的表达。而且IL-13的刺激增加了ERK1/2磷酸化以及intelectin-1蛋白表达，但HumanITLLN1-shRNA质粒转染抑制了IL-13诱导的intelectin-1蛋白表达及ERK1/2磷酸化。这些数据显示了ERK1/2通路可以调节IL-13诱导CCL5和IL6的表达，Intelectin-1基因参与了IL-13诱导ERK1/2在A549和16HBE细胞的激活表达。因此我们可以这样认为，Intelectin-1基因可能通过激活ERK1/2信号通路，参与IL-13诱导的CCL5和IL6的表达。
[Abstract]:Research background
The incidence of asthma is on the rise, but the pathogenesis of asthma has not been fully elucidated. Asthma is characterized by airway hyperresponsiveness, increased mucus secretion and chronic eosinophil inflammation. Some reports have been confirmed mainly in airway epithelial cells and mononuclear cells in asthmatic patients, and some chemokines RANTES, MCP-3, MCP-4, eotax. The expression of mRNA in in-1andeotaxin-2 is increased. Chemokine is a small molecular cytokine of 8-10kDa. According to each protein molecule, one or two cysteine residues can be divided into four types at the N terminal position, that is, CXC, CC, C, and CXXXC. many CC chemokines are produced by.IL13 that can induce epithelial cell CCL11. (eotaxin), the expression of CCL24 (eotaxin-2), andCCL26 (eotaxin-3), and the expression of these factors is increased in the patients with asthma. These CC chemokines are selective agonists of the eosinophil and the surface of the basophil on the surface of the CCR receptor.
Asthma is mainly regulated by TH2 cells, secreted cytokines and IL4/5/9/13, which are all associated with asthma, but IL13 is now considered the most critical factor. Interleukin 13 is a 12kDa multipotent molecule secreted by TH2 cells, located on the autosomal 5q31. Interleukin 13 is rich in eosinophils, lymphocytes and mega - macrophages. Cell inflammation, mucous epithelialization, tissue fibrosis and a strong stimulant for the remodeling of the base. Interleukin 13 stimulates many of the cells associated with asthma, such as B lymphocytes, mast cells, eosinophils, alveolar epithelial cells, fibroblasts and airway smooth muscle cells. Some experiments have also proved that the application of recombinant interleukins 13 is or is The overexpression of interleukin 13 in the lung can produce asthma like airway inflammation and airway remodeling. Many experiments have confirmed that interleukin 13 is overexpressed in asthma and is associated with the pathogenesis of TH2 cell inflammation and airway remodeling in asthma. Therefore, we use IL13 to stimulate airway epithelial cells to simulate asthma like effects.
Human intelectin (ITLN) is a secretory glycoprotein consisting of 295 amino acids and N terminal oligosaccharides. Its basic structural unit is the 120kDa trimer of the two sulfur bonds connected to the 40kDa polypeptide. It can be divided into intelectin-1 and 2. intelectin, also known as the transferrin receptor of the glycerol phosphoric inositol connection, vascular endothelial cell agglutinin or omentum The human ITLN gene is located on the chromosome 1q21.3, containing 8 exons, and the mRNA of human intelectin is expressed in the heart, the small intestine, the colon and the thymus. It is a lectin class that recognizes specific bacterial wall components and is not recognized by other lectin. It is known that it is expressed in the small intestine of pans cells and secreted or goblet cells. It is also observed that the secretory cells in the airway epithelial cells are also expressed. It can be identified in bacteria, fungi and protozoan walls of dextran and furan galactose residues, but in mammals, it is indicated that intelectin plays an important role in antigen recognition and.Intelectin-1 is up to up after exposure to Trichinella spiralis in the small intestine of mice. The type of intelectin-2 in the small intestinal epithelial cells is also induced by.Intelectin-1 as an antiseptic protein regulated by TH2 cells. The expression of intelectin-1 gene expression in bronchial epithelial cells of asthmatic patients is up regulated, which is related to the up regulation of IL13 expression.
Part I the role of Intelectin-1 in the expression of inflammatory cytokines in IL13 induced airway epithelial cell lines A549 and 16HBE.
Objective: To investigate the role of intelectin-1 gene in the secretion of inflammatory factors in human alveolar / airway epithelial cells.
Methods: the transfected high expression plasmids were divided into two types of cell lines A549 and 16HBE.: (A) normal control group (B) IL13 group (C) transfected to human ITLN1 plasmid group, and transfected interference plasmid group: (A) normal control group + empty plasmid group (B) IL13+ empty plasmid group (C) IL13+ replication plasmid group. L6mRNA expression.ELISA method was used to detect the protein level of the corresponding factors in the cell supernatant. Dexamethasone intervened cells: (1) the control group was +DMSO (IL13+DMSO) DEX100+IL13, and then the expression of cytokines ITLN1, CCL5 and IL6mRNA were detected by RealtimePCR.
Results: the expression level of Intelectin-1, CCL5 and IL6mRNA in A549 cells was significantly higher in the ITLN1 group and the IL13 group than in the control group. The expression level of p0.05. and 16HBE cells in Intelectin-1, CCL5, IL6mRNA was significantly higher than that in the control group, and the expression level of the group was higher than that in the control group. 05, but the expression level of CCL5mRNA in the IL13 group was higher than that of the control group, but there was no statistical significance. After the transfection of A549 and 16HBE to the ITLN1-shRNA plasmid, the A549 and 16HBE cell lines: the ITLN1shRNA plasmid group of IL13+ man was lower than the IL13 group intelectin-1, and the expression of the protein water was detected by the statistical significance method. In the high expression test, the expression level of IL6 and CCL5 protein in the human ITLN plasmid group was significantly higher than that in the control group. The protein expression level of CCL5 and IL6 in the IL13 group was significantly higher than that in the control group, and the level of CCL5 and IL6 in the IL13+ITLN1-shRNA group was significantly higher than that in the IL13 group, and the level of CCL5 and IL6 protein was significantly lower than that in the IL13 group. In group IL13 compared with group IL13, ITLN1, CCL5 and IL6 were significantly reduced, P0.05, and in 16HBE cells, dexamethasone group was significantly lower than that in IL13 group, ITLN1, CCL5 and IL6 were significantly decreased, and P0.05, all had statistical significance.
Conclusion: after transferring to the high expression plasmid of human ITLN1, the high expression of intelectin-1mRNA is promoted. The mRNA and protein expression level of factors CCL5 and IL6 are also increased, which may be related to the increase of intelectin-1 expression. After transfection of human ITLN1 interference plasmid, the expression of intelectin-1mRNA and the factors to be detected together can be suppressed. MRNA and protein expression levels of CCL5 and IL6 also correspondingly decrease the expression of.Intelectin-1, which may inhibit the expression of CCL5 and IL6. Glucocorticoid can inhibit the gene expression of ITLN1, CCL5 and IL6 in A549 and 16HBE cells. Thus, Intelectin-1 is in the expression of inflammatory cytokines in the epithelial cells of the airway. Play an important role.
The second part is the role of ERK signaling pathway in intelectin-1 mediated expression of inflammatory cytokines CCL5 and IL6 in airway epithelial cells.
Objective: To investigate the mechanism of intelectin-1 involved in the expression of inflammatory cytokines CCL5 and IL-6 in airway epithelial cells.
Methods: IL-13 and PD98059 were used to group the human lung epithelial cell line A549 and 16HBE cell.PD98059 experiment group (1) control group, (2) IL-13 group and (3) IL-13+PD98059 group. Then RealTimePCR method was used to detect the mRNA expression of the cytokine CCL5 and IL6. After inhibiting the expression of intelectin-1 induced by IL-13 by RNA interference, the changes in the level of intelectin-1, ERK1/2 and P-ERK1/2 protein were detected by Western-Blot in the +humanITLN1shRNA plasmid group.
Results: after IL13 stimulated A549 and 16HBE cells with PD98059, the mRNA expression level of CCL5 and IL6 decreased significantly in IL13+PD98059 group than in IL13 group. P0.05. transfected to humanITLN1shRNA plasmid, intelectin-1 and protein expression level was higher than that of control group. Group -13 decreased, P0.05.
Conclusion: an inhibitor of ERK1/2, PD98059, can block the expression of CCL5 and IL6 in A549 and 16HBE cells induced by IL-13. Moreover, IL-13 stimulation increases ERK1/2 phosphorylation and intelectin-1 protein expression, but HumanITLLN1-shRNA plasmid transfection inhibits the expression of IL-13 induced proteins and phosphorylation. The ERK1/2 pathway can regulate the expression of IL-13 induced CCL5 and IL6, and the Intelectin-1 gene participates in the activation of ERK1/2 in A549 and 16HBE cells induced by IL-13, so we can think that the Intelectin-1 gene may be involved in IL-13 induced and expressed expressions by activating the ERK1/2 signaling pathway.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

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