mCLCA3在小鼠气道粘液高分泌及气道炎症中作用的研究

发布时间：2018-06-12 08:10

本文选题：哮喘 + mCLCA3　；参考：《华中科技大学》2012年硕士论文

【摘要】：研究背景支气管哮喘是一种复杂的可遗传的气道慢性炎症性疾病，出现反复发作的喘息，胸闷，气急，咳嗽，哮鸣音等非特异性症状及体征，常伴有可逆性气道阻塞及气道高反应性[1]，而这种特征性的可逆性气道阻塞及气道高反应性被认为是由支气管粘膜慢性炎症所引起的[2,3]，这些炎症以不同程度气道上皮粘液高分泌，杯状细胞化生及纤维化为特征[4]。因此气道上皮在气道防御机制中起重要作用，有证据表明气道上皮功能紊乱是慢性哮喘病人的根本所在[5]。 CLCA是钙激活性氯离子通道蛋白家族，mCLCA3（别名gob-5）是其成员之一[6,7]，目前已有15种CLCA成员在6种哺乳动物中被发现。其中四种为人源hCLCA1、hCLCA2、hCLCA3及hCLCA4（别名为hCaCC2），六种为鼠源mCLCA1、mCLCA2、mCLCA3（别名为gob-5）、mCLCA4、mCLCA5及mCLCA6，两种为牛源性成员bCLCA1（别名为CaCC）及bCLCA2（别名为Lu-ECAM-1），一种猪源性成员pCLCA1，一种马源性成员eCLCA1，以及一种大鼠同源性成员[8,9]。 CLCA基因表达与气道疾病之间的联系非常有趣，因为这些疾病都与气道粘液过度分泌有关，并且CLCA家族中至少一个成员是选择性表达于粘液细胞的[10-12]。在最早的研究中，人们用CLCA蛋白基因转染人胚肾293细胞引起钙激活性氯离子电流，发现了它的类蛋白通道功能，并将其视为通道蛋白，但后经证实CLCA蛋白是可溶性的分泌分子[8,13]，是否具有钙激活性氯离子通道功能却尚未被证实。这些家族成员具有相似的特征，蛋白结构分析表明大多数CLCA成员初级翻译产物蛋白分子量一致，均为大小为100KD含有氨基末端信号肽的蛋白质，这一初级蛋白糖基化形成大小为130KD的糖蛋白，然后断裂成两个大小分别为90KD和40KD的亚基。另有研究表明在距离C-端240个氨基酸处有一个水解酶位点[14]，120KD的糖蛋白被水解为N-端85KD及C-端35KD，两个片段均含有多个N-linked糖基化位点。CLCA蛋白结构的相似性是其功能及表达部位具有高度同源性的结构基础。 mCLCA3最早发现并克隆自小鼠肠道组织，mCLCA3及其人类同源的hCLCA1蛋白已经被鉴定为是与分泌腺功能紊乱性疾病具有临床相关性的分子，包括哮喘及囊性纤维化等。它存在于胃肠道、呼吸系统及子宫杯状细胞，大量表达于正常小鼠的胃、小肠、结肠及子宫，仅少量表达于气道组织[15]；有研究表明mCLCA3在哮喘小鼠肺组织中呈现高表达，我们前期的研究也证实了这一点，并发现其表达水平与过敏原的刺激呈现时间依赖性[16,17]。mCLCA3在正常小鼠肺组织几乎无表达，说明mCLCA3并不参与正常小鼠肺生理，其表达与哮喘性疾病具有紧密相关性。Atsushi Nakanishi的研究中，经过敏原刺激后，小鼠胃、小肠及结肠中mCLCA3的表达量与正常小鼠处于同一水平，但在肺组织中却有显著增高，说明mCLCA3选择性表达于致敏小鼠的气道杯状细胞[18]。该实验还将mCLCA3高表达及低表达质粒包装成腺病毒，通过气管内给药的方式转染致敏小鼠，结果气道粘液表达相应增高及降低，表明mCLCA3是气道粘液表达的重要调节因子。mCLCA3高表达质粒转染粘液腺样细胞NCI-292，致粘液蛋白Muc5ac表达量增高[18,19]，进一步证实了上述结论，即mCLCA3高表达不论在体内还是体外实验均与粘液高分泌及杯状细胞化生有关。本实验旨在探讨小鼠模型中，mCLCA3在气道粘液高分泌、气道炎症及趋化因子合成中的地位。为此我们将mCLCA3高表达质粒及转染试剂通过滴鼻的方式转染正常小鼠，观察气道炎症的变化，检测Muc5ac、炎性介质CCL2、CCL5、CCL11及细胞因子IL13、IL-4、IL-5、IFN-γ的表达水平。明确mCLCA3与粘液蛋白Muc5ac、趋化因子及Th1/Th2细胞因子表达之间的关系，以助进一步研究mCLCA3上调粘液表达的信号转导通路，为治疗哮喘提供新的思路。目的：研究哮喘相关基因mCLCA3对粘液蛋白Muc5ac、趋化因子及Th1/Th2细胞因子表达的影响及其在小鼠气道炎症中的作用。方法：实验共分为三组：①正常小鼠+阴性对照质粒组，，②正常小鼠+mCLCA3高表达质粒组，③哮喘小鼠。通过滴鼻的方式将转染试剂和质粒配制成的转染缓冲液滴入各组小鼠鼻腔内，随呼吸进入气道。用Real-time PCR的方法检测各组小鼠肺组织内mCLCA3、Muc5ac，趋化因子CCL2、CCL5、CCL11及细胞因子IL13、IL-4、IL-5、IFN-γ在mRNA水平的表达变化。用Western-blotting和免疫组织化学方法检测mCLCA3在蛋白水平的表达量及表达部位。对各组小鼠肺泡灌洗液中总炎性细胞进行计数，观察肺组织炎症变化；小鼠肺组织行HE染色对气道进行炎症分数评分了解气道炎症情况。结果：免疫组织化学表明mCLCA3仅表达于小鼠气道上皮细胞。Real-time PCR表明mCLCA3mRNA在哮喘组及正常+高表达质粒组均明显高于正常对照组，P＜0.05；而哮喘组与正常+高表达质粒组相比较，P＞0.05，mRNA水平的表达无统计学差异。mCLCA3Western-blotting结果表明：①mCLCA3在蛋白水平的变化趋势与mRNA水平相同；②条带分布：正常对照组小鼠肺组织几乎无mCLCA3蛋白表达，哮喘组及正常+高表达质粒组在75KD、90~100KD及125~130KD均有显著表达；③选取90~100KD条带做灰度分析：哮喘组及正常+高表达质粒组均较正常对照组显著升高，P㩳0.05；正常+高表达质粒组与哮喘组相比较，P＜0.05，表明正常+高表达质粒组在蛋白水平的表达量较哮喘组低。Muc5ac和IL-13的Real-time PCR结果表明：哮喘组及正常+高表达质粒组均较正常对照组明显升高，P＜0.05；哮喘组与正常+高表达质粒组相比较，P＞0.05，二者表达量无显著差异。趋化因子CCL-2、CCL-5、CCL-11及Th2细胞因子IL-4、IL-5的Real-timePCR结果均表明,哮喘组较正常对照组明显升高，P＜0.05；正常+高表达质粒组较正常对照组有升高，但P＞0.05，未达统计学差异标准；Th1细胞因子IFN-γ的mRNA表达量在哮喘组或正常+高表达质粒组均无显著升高。相关性分析表明：肺组织中mCLCA3mRNA与粘液蛋白Muc5ac mRNA（r=0.759，P=0.000）及IL13mRNA（r=0.776，P=0.000）的表达成总体正相关，在0.01水平上具有显著相关性；IL13mRNA与Muc5ac mRNA成总体正相关（r=0.895，P=0.000），在0.01水平上具有显著相关性。BALF中总炎性细胞计数及肺组织切片HE染色气道炎症评分结果均表明：哮喘组及正常+高表达质粒组均较正常对照组明显增高，P＜0.05；哮喘组与正常+高表达质粒组相比较，P＞0.05，差异无统计学意义。结论：哮喘相关基因mCLCA3上调Muc5ac的表达致小鼠气道粘液高分泌。外源性mCLCA3引起肺组织及气道炎症，但并不直接参与气道上皮细胞趋化因子的分泌。这一发现为指导哮喘的治疗提供了新的线索。
[Abstract]:Research background
Bronchial asthma is a complicated, hereditary, chronic airway inflammatory disease with recurrent episodes of wheezing, chest tightness, breath, cough, wheezing, and other nonspecific symptoms and signs, often accompanied by reversible airway obstruction and airway hyperresponsiveness, and this characteristic reversible airway obstruction and airway hyperresponsiveness are considered to be supported by a branch of [1]. [2,3] caused by chronic inflammation of the trachea mucous membrane, the inflammation is characterized by hypersecretion of mucous mucus in the airway epithelium, goblet cell metaplasia and fibrosis, and therefore the airway epithelium plays an important role in the airway defense mechanism. There is evidence that airway epithelial dysfunction is the fundamental [5]. of chronic asthma patients.
CLCA is a calcium activated chloride channel protein family, mCLCA3 (alias gob-5) is one of its members, [6,7]. Currently, 15 kinds of CLCA members have been found in 6 mammals. Four of them are human hCLCA1, hCLCA2, hCLCA3 and hCLCA4 (alias is hCaCC2), six are mCLCA1, mCLCA2, two species, and two species BCLCA1 (alias CaCC) and bCLCA2 (alias Lu-ECAM-1), a porcine source member pCLCA1, a horse source member eCLCA1, and a rat homologous member of the [8,9]. CLCA gene expression associated with airway disease is very interesting, because these diseases are associated with the excessive secretion of airway mucus, and the CLCA family. At least one member is a [10-12]. that is selectively expressed in mucous cells in the earliest study. People use the CLCA gene to transfect human embryonic kidney 293 cells to calcium activated chlorine ion current, and discover its protein like channel function and consider it as a channel protein, but it is confirmed that CLCA protein is a soluble secretory molecule [8,13], or whether it is a soluble secretory molecule. The functions of calcium activated chloride channels have not been confirmed. These family members have similar characteristics. Protein structure analysis shows that most of CLCA members are identical in the molecular weight of primary translation products, all of which are 100KD containing amino terminal signal peptides, and this primary protein is glycosylated to form a sugar egg of 130KD size. White, then fractured into two subunits of 90KD and 40KD, respectively. Another study showed that there was a hydrolase site [14] at the 240 amino acids at the C- end, and the 120KD glycoprotein was hydrolyzed to N- terminal 85KD and C- end 35KD, and the two fragments all contained multiple N-linked glycosylation sites, the similarity of the.CLCA egg white structure was its function and expression site. A structural basis with high homology.
MCLCA3 was first discovered and cloned from the mouse intestinal tissue. MCLCA3 and its human homologous hCLCA1 protein have been identified as a molecule that is clinically relevant to the secretory disorder of the disease, including asthma and cystic fibrosis. It exists in the gastrointestinal tract, the respiratory system and the goblet cells of the subuterine uterus, which are expressed in the stomach of normal mice. The small intestine, colon and uterus were only a small amount of expression in the airway tissue [15]; some studies showed that mCLCA3 was highly expressed in the lung tissue of the asthmatic mice. Our previous study also confirmed this, and found that the expression level of the allergen and the irritation of the allergen presented time dependent [16,17].mCLCA3 in the normal mice lung tissue almost no expression, indicating that mCLCA3 The expression of mCLCA3 in the stomach, small intestine and colon of mice was at the same level as normal mice, but in the lung tissue, the expression of mCLCA3 in the lung was significantly higher, indicating that mCLCA3 was selectively expressed in the sensitized mice. Airway goblet cell [18]. also packed mCLCA3 high expression and low expression plasmid into adenovirus and transfected sensitized mice through intratracheal administration. The results showed that the expression of airway mucus increased and decreased, indicating that mCLCA3 was an important regulator of airway mucus expression and.MCLCA3 high expression plasmid transfected to mucous adenoid cells NCI-292. The expression of liquid protein Muc5ac is increased by [18,19], which further confirms the conclusion that high expression of mCLCA3 is related to mucus hypersecretion and goblet cell metaplasia in both in vivo and in vitro.
The purpose of this study was to explore the role of mCLCA3 in airway mucus hypersecretion, airway inflammation and chemokine synthesis in the mouse model. To this end, we transfected mCLCA3 high expression plasmids and transfection agents into normal mice through nasal drops to observe the changes in airway inflammation, detect Muc5ac, CCL5, CCL11 and cytokines IL13, IL-4, I. L-5, IFN- gamma expression level. Clarify the relationship between mCLCA3 and mucin Muc5ac, chemokines and the expression of Th1/Th2 cytokine, in order to further study the signal transduction pathway of mCLCA3 to increase the expression of mucus, and to provide a new way of thinking for the treatment of asthma.
Objective: To study the effect of asthma related gene mCLCA3 on the expression of mucin Muc5ac, chemokine and Th1/Th2 cytokine and its role in airway inflammation in mice. Methods: the experiment was divided into three groups: (1) normal mice + negative control plasmids, (2) normal mice, +mCLCA3 high expression plasmid group, (3) asthmatic mice. The transfection buffer solution prepared by transfection reagents and plasmids dropped into the nasal cavity and entered the airway with respiration. The expression of mCLCA3, Muc5ac, chemokine CCL2, CCL5, CCL11 and cytokine IL13, IL-4, IL-5, IFN- gamma in the mRNA level were detected by Real-time PCR. The expression and expression of mCLCA3 at the protein level were detected by the method of learning. The total inflammatory cells in the alveolar lavage fluid in each group were counted and the inflammatory changes in the lung tissue were observed. The lung tissues of mice were stained with HE to evaluate the airway inflammation.
Results: immunohistochemistry showed that the expression of mCLCA3 only in mouse airway epithelial cells.Real-time PCR showed that mCLCA3mRNA in the asthma group and the normal + high expression plasmid group were significantly higher than that of the normal control group, P < 0.05, while the asthma group was compared with the normal + high expression plasmid group, P > 0.05, and the expression of mRNA level was not statistically different.MCLCA3Western-b. The results of lotting showed that: (1) the change trend of mCLCA3 in protein level was the same as that of mRNA; (2) the distribution of band distribution: there was almost no mCLCA3 protein expression in the lung tissue of normal control mice, and the expression of 75KD, 90~100KD and 125~130KD in the asthma group and the normal + high expression plasmid group were significantly expressed. (3) the 90~100KD strip was selected as the gray level analysis: asthma group and The normal + high expression plasmid group was significantly higher than the normal control group, P? 0.05, and the normal + high expression plasmid group was compared with the asthma group, P < 0.05. The results showed that the expression of normal + high expression plasmid group in protein level was lower than that of.Muc5ac and IL-13 in the asthma group. The results showed that the asthma group and the normal + high expression plasmid group were all compared with the normal control group. The group was significantly higher, P < 0.05; the asthma group was compared with the normal + high expression plasmid group, P > 0.05, and there was no significant difference in the expression of two. The Real-timePCR results of chemokine CCL-2, CCL-5, CCL-11 and Th2 cytokine IL-4 and IL-5 were all significantly higher in the asthma group than in the normal control group, P < 0.05, and the normal + high expression plasmid group was more than the normal control group. Increase, but P > 0.05, no statistical difference standard, the mRNA expression of Th1 cytokine IFN- gamma was not significantly increased in the asthmatic group or normal + high expression plasmid group. The correlation analysis showed that the expression of mCLCA3mRNA and mucin Muc5ac mRNA (r=0.759, P=0.000) and IL13mRNA (r=0.776, P=0.000) were positively correlated in the lung tissue, and in 0.01 There was a significant correlation on the level of IL13mRNA and Muc5ac mRNA (r=0.895, P=0.000). There was a significant correlation between the total inflammatory cells count in.BALF and the HE staining airway inflammation score in the lung tissue section at the 0.01 level, all showed that the asthma group and the normal + high expression plasmid group were all significantly higher than the normal control group, P < 0.05 The asthma group was compared with the normal + high expression plasmid group, P > 0.05, the difference was not statistically significant. Conclusion: the asthma related gene mCLCA3 up-regulated Muc5ac induced the hypersecretion of airway mucus in mice. Exogenous mCLCA3 causes lung tissue and airway inflammation, but does not directly participate in the secretion of chemokines in the airway epithelial cells. This discovery is the guidance of this discovery. The treatment of asthma provides a new clue.
【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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