STAT6圈套ODN对哮喘小鼠脾淋巴细胞IL-13表达影响的实验研究

发布时间：2018-07-03 17:48

本文选题：支气管哮喘 + 白介素-13　；参考：《泸州医学院》2012年硕士论文

【摘要】：目的：支气管哮喘是由多种细胞（如嗜酸性粒细胞，肥大细胞，T细胞，中性粒细胞和气道上皮细胞等）和细胞组分参与的气道慢性炎症性疾病，反复发作性喘息，可逆性气道狭窄和气道重塑是其主要特征。依赖于信号转导和转录激活因子6（STAT6）持续活化与过度表达，使T细胞向Th2优势分化和Th2分泌IL-4，IL-5，，IL-13等炎性介质，是哮喘主要的病理生理基础。本研究通过STAT6圈套寡核苷酸（ODN）和哮喘小鼠脾淋巴细胞的共同培养来观察STAT6圈套寡核苷酸（ODN）对哮喘小鼠脾淋巴细胞IL-13表达的影响。方法：（1）实验分组：将48只BALB/c雌性小鼠分成两组：Ⅰ组，正常小鼠8只；Ⅱ组，哮喘小鼠40只；其中Ⅱ组哮喘小鼠脾淋巴细胞分成5组：空白对照组（A组）、哮喘OVA组（B组）、哮喘脂质体组（C组）哮喘圈套ODN组（D组）、哮喘无序ODN组（E组）。哮喘组用卵白蛋白(OVA)和氢氧化铝建立哮喘模型，分别于第0、7、14天腹腔注射OVA致敏，第21天至28天雾化吸入5%OVA激发，每天一次，每次30分钟。正常小鼠组以生理盐水代替OVA。（2）末次激发24小时后，处死小鼠收集肺泡灌洗液分别行白细胞，淋巴细胞和嗜酸性粒细胞的分类计数；HE染色肺组织切片观察气道炎症变化。取出哮喘小鼠的脾脏，分离脾淋巴细胞，进行体外培养并由阳离子脂质体2000转染剂携带合成的全链硫代修饰的STAT6寡核苷酸（ODN）转染细胞，再用OVA激发淋巴细胞使其激发活化。24小时后离心收集培养的脾淋巴细胞上清液并采用酶联免疫吸附（ELISA）法测定其中的IL-13含量，离心后沉淀的细胞抽提总RNA逆转录转为cDNA，建立实时荧光PCR反应条件进行PCR扩增，分析IL-13mRNA的相对含量。实验数据采用均值±标准差((?)±s)表示,用SPSS17.0统计软件进行统计学分析。结果：（1）Ⅱ组与Ⅰ组比较，支气管肺泡灌洗液中白细胞，淋巴细胞，嗜酸性粒细胞均增高，有统计学意义(P0.01)；（2）病理切片HE染色显示：Ⅰ组支气管上皮完整，杯状细胞少见，基底层及平滑肌层较薄，血管及气道周围见极少量炎症细胞，支气管管腔内无大量分泌物；Ⅱ组气管和血管周围及肺泡隔内见大量嗜酸性粒细胞浸润，气道上皮细胞增生肥大，上皮不完整，上皮下基底层明显增厚，气管内分泌物潴留，支气管平滑肌增生（3）脾淋巴细胞IL-13mRNA的表达水平：空白组（A组）、哮喘OVA组(B组)、哮喘脂质体组（C组）哮喘圈套ODN组（D组）、哮喘无序ODN组（E组）的淋巴细胞IL-13mRNA的相对表达量分别为：1.97253±0.17883、4.54200±0.51474、4.22287±0.29981、2.09736±0.20819、4.37192±0.31691。哮喘ODN组(D组)和空白组（A组）低于哮喘OVA组(B组)、哮喘脂质体组（C组），哮喘无序ODN组（E组）(P0.01)；哮喘ODN组(D组)和空白组（A组）间的差别没有统计学意义(P0.05)；哮喘脂质体组（C组）哮喘无序ODN组（E组）和哮喘OVA组(B组)之间的差别没有统计学意义(P0.05)；4.脾淋巴细胞上清液中IL-13的含量：空白组（A组）、哮喘OVA组(B组)、哮喘脂质体组（C组）、哮喘ODN组(D组)、哮喘无序ODN组（E组）IL-13含量分别为：1.51093±0.10415、2.97970±0.12980、2.88808±0.08958、1.57130±0.10443、2.93010±0.10128。哮喘ODN组(D组)和空白组（A组）低于哮喘OVA组(B组)、哮喘脂质体组（C组）和哮喘无序ODN组（E组）(P0.01)；哮喘ODN组(D组)和空白组（A组）间的差别没有统计学意义(P0.05)；哮喘脂质体组（C组）哮喘无序ODN组（E组）和哮喘OVA组(B组)之间的差别没有统计学意义(P0.05)。结论：用荧光显微镜观察脂质体2000转染FITC标记的STAT6圈套ODN能顺利进入哮喘小鼠的脾淋巴细胞；STAT6圈套ODN能特异性抑制哮喘小鼠脾淋巴细胞中IL-13ｍRNA的转录和IL-13的分泌；脂质体和无序ODN对哮喘小鼠脾淋巴细胞表达IL-13的影响没有差异性，说明STAT6圈套ODN对哮喘小鼠脾淋巴细胞IL-13的表达影响具有特异性而非脂质体和核酸的作用。
[Abstract]:Objective: bronchial asthma is an airway chronic inflammatory disease involving multiple cells, such as eosinophils, mast cells, T cells, neutrophils and airway epithelial cells, and cell components. Repeated paroxysmal wheezing, reversible airway stenosis and airway remodeling are its main characteristics. It depends on signal transduction and transcription activator 6. STAT6) continuous activation and overexpression, the differentiation of T cells to Th2 and Th2 secretion of IL-4, IL-5, IL-13 and other inflammatory mediators, which are the main pathophysiological basis of asthma. This study observed STAT6 trap oligonucleotides (ODN) on the splenic lymphocyte IL in asthmatic mice by co culture of STAT6 trap oligonucleotides (ODN) and asthmatic mice splenic lymphocytes The effect of -13 expression. Methods: (1) experimental group: 48 female mice were divided into two groups: group I, 8 normal mice, group II and 40 asthmatic mice, of which group II asthma mice spleen lymphocytes were divided into 5 groups: blank control group (group A), asthma OVA group (B group), asthma liposome group (C group) ODN group (D group), asthma disorder ODN, ODN disorder ODN Group (group E). Asthma group was established with OVA and aluminum hydroxide to establish asthma model, OVA sensitized by intraperitoneal injection on day 0,7,14, and 5%OVA stimulated by inhalation from twenty-first days to 28 days, once a day, 30 minutes each time. Normal mice were treated with saline instead of OVA. (2) for 24 hours, and the mice were sacrificed to collect bronchoalveolar lavage fluid. The taxonomy of cells, lymphocytes and eosinophils were counted; HE stained lung tissue sections were used to observe the changes in airway inflammation. The spleen of the asthmatic mice was taken out and splenic lymphocytes were separated. The cells were cultured in vitro and carried by the cationic liposome 2000 transfection agent to transfect the whole chain thiosulfate modified STAT6 oligodeoxynucleotides (ODN). Then OVA was used to stimulate the cells. The lymphocyte supernatant was collected and cultured after activation of.24 hours, and the content of IL-13 was measured by enzyme linked immunosorbent assay (ELISA). After centrifugation, the precipitated cells extracted total RNA reverse transcriptase to cDNA, and established the real-time fluorescent PCR reaction conditions for PCR amplification and analyzed the relative content of IL-13mRNA. Experimental data collection was collected. By means of mean mean standard deviation ((?) + s), statistical analysis was performed with SPSS17.0 statistical software. Results: (1) compared with group I, the white blood cells, lymphocytes and eosinophils in the bronchoalveolar lavage fluid were all higher, and were statistically significant (P0.01). (2) pathological section HE staining showed that the bronchial epithelium in group I was intact and the goblet cells were rare. The basal layer and smooth muscle layer were thin, and there were very small amount of inflammatory cells around the vessel and the airway. There was no large secretion in the bronchial tube. In group II, a large number of eosinophilic granulocytes were found in the trachea and the perivascular and alveolar septum, the epithelial cells were hyperplastic, the epithelium was not complete, the subepithelial basement layer was thickened, the secretory retention of the trachea and the branch in the trachea. The expression level of IL-13mRNA in the splenic lymphocyte of tracheal smooth muscle hyperplasia (3): blank group (group A), asthma OVA group (group B), asthma liposome group (group C) asthma trap ODN group (group D), and the relative expression of IL-13mRNA in lymphocyte of asthma disorder ODN group (E group): 1.97253 + 0.17883,4.54200 + 0.51474,4.22287 + + + + 7192 + 0.31691. asthma group ODN (group D) and blank group (group A) were lower than group OVA of asthma (group B), asthma liposome group (C group), asthma disorder ODN group (E group) (P0.01), and there was no statistical difference between asthmatic ODN group (D group) and blank group; the difference between asthma disorder group and asthma group There was no statistical significance (P0.05); 4. the content of IL-13 in the supernatant of spleen lymphocyte: blank group (group A), asthma OVA group (B group), asthma liposome group (C group), asthma ODN group (D group), and asthmatic ODN group (E group) IL-13 content respectively: 1.51093 + + + + + + + + + + asthmatic asthma Group (group D) and blank group (group A) were lower than group OVA of asthma (group B), asthma liposome group (C group) and asthma disorder ODN group (E group) (P0.01), and there was no significant difference between asthmatic group ODN group (D group) and blank group (A group), and there was no significant difference between asthma disorder group and asthma group (Group). 0.05) conclusion: it is concluded that the STAT6 trap ODN labeled by liposome 2000 transfected with FITC can successfully enter the spleen lymphocytes of the asthmatic mice, and STAT6 trap ODN can specifically inhibit the transcription of IL-13mRNA and the secretion of IL-13 in the spleen lymphocytes of the asthmatic mice, and the expression of IL-13 in the spleen lymphocytes of the asthmatic mice by liposomes and unordered ODN The effect of STAT6 decoy ODN on the expression of IL-13 in spleen lymphocytes of asthmatic mice is specific, rather than that of liposomes and nucleic acids.
【学位授予单位】：泸州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R562.25

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