發(fā)布時間：2018-06-12 08:10

  本文選題：哮喘 + mCLCA3　； 參考：《華中科技大學(xué)》2012年碩士論文

【摘要】：研究背景 支氣管哮喘是一種復(fù)雜的可遺傳的氣道慢性炎癥性疾病，出現(xiàn)反復(fù)發(fā)作的喘息，胸悶，氣急，咳嗽，哮鳴音等非特異性癥狀及體征，常伴有可逆性氣道阻塞及氣道高反應(yīng)性[1]，而這種特征性的可逆性氣道阻塞及氣道高反應(yīng)性被認(rèn)為是由支氣管粘膜慢性炎癥所引起的[2,3]，這些炎癥以不同程度氣道上皮粘液高分泌，杯狀細胞化生及纖維化為特征[4]。因此氣道上皮在氣道防御機制中起重要作用，有證據(jù)表明氣道上皮功能紊亂是慢性哮喘病人的根本所在[5]。 CLCA是鈣激活性氯離子通道蛋白家族，mCLCA3（別名gob-5）是其成員之一[6,7]，目前已有15種CLCA成員在6種哺乳動物中被發(fā)現(xiàn)。其中四種為人源hCLCA1、hCLCA2、hCLCA3及hCLCA4（別名為hCaCC2），六種為鼠源mCLCA1、mCLCA2、mCLCA3（別名為gob-5）、mCLCA4、mCLCA5及mCLCA6，兩種為牛源性成員bCLCA1（別名為CaCC）及bCLCA2（別名為Lu-ECAM-1），一種豬源性成員pCLCA1，一種馬源性成員eCLCA1，以及一種大鼠同源性成員[8,9]。 CLCA基因表達與氣道疾病之間的聯(lián)系非常有趣，因為這些疾病都與氣道粘液過度分泌有關(guān)，并且CLCA家族中至少一個成員是選擇性表達于粘液細胞的[10-12]。在最早的研究中，人們用CLCA蛋白基因轉(zhuǎn)染人胚腎293細胞引起鈣激活性氯離子電流，發(fā)現(xiàn)了它的類蛋白通道功能，并將其視為通道蛋白，但后經(jīng)證實CLCA蛋白是可溶性的分泌分子[8,13]，是否具有鈣激活性氯離子通道功能卻尚未被證實。這些家族成員具有相似的特征，蛋白結(jié)構(gòu)分析表明大多數(shù)CLCA成員初級翻譯產(chǎn)物蛋白分子量一致，均為大小為100KD含有氨基末端信號肽的蛋白質(zhì)，這一初級蛋白糖基化形成大小為130KD的糖蛋白，然后斷裂成兩個大小分別為90KD和40KD的亞基。另有研究表明在距離C-端240個氨基酸處有一個水解酶位點[14]，120KD的糖蛋白被水解為N-端85KD及C-端35KD，兩個片段均含有多個N-linked糖基化位點。CLCA蛋白結(jié)構(gòu)的相似性是其功能及表達部位具有高度同源性的結(jié)構(gòu)基礎(chǔ)。 mCLCA3最早發(fā)現(xiàn)并克隆自小鼠腸道組織，mCLCA3及其人類同源的hCLCA1蛋白已經(jīng)被鑒定為是與分泌腺功能紊亂性疾病具有臨床相關(guān)性的分子，包括哮喘及囊性纖維化等。它存在于胃腸道、呼吸系統(tǒng)及子宮杯狀細胞，大量表達于正常小鼠的胃、小腸、結(jié)腸及子宮，僅少量表達于氣道組織[15]；有研究表明mCLCA3在哮喘小鼠肺組織中呈現(xiàn)高表達，我們前期的研究也證實了這一點，并發(fā)現(xiàn)其表達水平與過敏原的刺激呈現(xiàn)時間依賴性[16,17]。mCLCA3在正常小鼠肺組織幾乎無表達，說明mCLCA3并不參與正常小鼠肺生理，其表達與哮喘性疾病具有緊密相關(guān)性。Atsushi Nakanishi的研究中，經(jīng)過敏原刺激后，小鼠胃、小腸及結(jié)腸中mCLCA3的表達量與正常小鼠處于同一水平，但在肺組織中卻有顯著增高，說明mCLCA3選擇性表達于致敏小鼠的氣道杯狀細胞[18]。該實驗還將mCLCA3高表達及低表達質(zhì)粒包裝成腺病毒，通過氣管內(nèi)給藥的方式轉(zhuǎn)染致敏小鼠，結(jié)果氣道粘液表達相應(yīng)增高及降低，表明mCLCA3是氣道粘液表達的重要調(diào)節(jié)因子。mCLCA3高表達質(zhì)粒轉(zhuǎn)染粘液腺樣細胞NCI-292，致粘液蛋白Muc5ac表達量增高[18,19]，進一步證實了上述結(jié)論，即mCLCA3高表達不論在體內(nèi)還是體外實驗均與粘液高分泌及杯狀細胞化生有關(guān)。 本實驗旨在探討小鼠模型中，mCLCA3在氣道粘液高分泌、氣道炎癥及趨化因子合成中的地位。為此我們將mCLCA3高表達質(zhì)粒及轉(zhuǎn)染試劑通過滴鼻的方式轉(zhuǎn)染正常小鼠，觀察氣道炎癥的變化，檢測Muc5ac、炎性介質(zhì)CCL2、CCL5、CCL11及細胞因子IL13、IL-4、IL-5、IFN-γ的表達水平。明確mCLCA3與粘液蛋白Muc5ac、趨化因子及Th1/Th2細胞因子表達之間的關(guān)系，以助進一步研究mCLCA3上調(diào)粘液表達的信號轉(zhuǎn)導(dǎo)通路，為治療哮喘提供新的思路。 目的：研究哮喘相關(guān)基因mCLCA3對粘液蛋白Muc5ac、趨化因子及Th1/Th2細胞因子表達的影響及其在小鼠氣道炎癥中的作用。方法：實驗共分為三組：①正常小鼠+陰性對照質(zhì)粒組，，②正常小鼠+mCLCA3高表達質(zhì)粒組，③哮喘小鼠。通過滴鼻的方式將轉(zhuǎn)染試劑和質(zhì)粒配制成的轉(zhuǎn)染緩沖液滴入各組小鼠鼻腔內(nèi)，隨呼吸進入氣道。用Real-time PCR的方法檢測各組小鼠肺組織內(nèi)mCLCA3、Muc5ac，趨化因子CCL2、CCL5、CCL11及細胞因子IL13、IL-4、IL-5、IFN-γ在mRNA水平的表達變化。用Western-blotting和免疫組織化學(xué)方法檢測mCLCA3在蛋白水平的表達量及表達部位。對各組小鼠肺泡灌洗液中總炎性細胞進行計數(shù)，觀察肺組織炎癥變化；小鼠肺組織行HE染色對氣道進行炎癥分?jǐn)?shù)評分了解氣道炎癥情況。 結(jié)果：免疫組織化學(xué)表明mCLCA3僅表達于小鼠氣道上皮細胞。Real-time PCR表明mCLCA3mRNA在哮喘組及正常+高表達質(zhì)粒組均明顯高于正常對照組，P＜0.05；而哮喘組與正常+高表達質(zhì)粒組相比較，P＞0.05，mRNA水平的表達無統(tǒng)計學(xué)差異。mCLCA3Western-blotting結(jié)果表明：①mCLCA3在蛋白水平的變化趨勢與mRNA水平相同；②條帶分布：正常對照組小鼠肺組織幾乎無mCLCA3蛋白表達，哮喘組及正常+高表達質(zhì)粒組在75KD、90~100KD及125~130KD均有顯著表達；③選取90~100KD條帶做灰度分析：哮喘組及正常+高表達質(zhì)粒組均較正常對照組顯著升高，P㩳0.05；正常+高表達質(zhì)粒組與哮喘組相比較，P＜0.05，表明正常+高表達質(zhì)粒組在蛋白水平的表達量較哮喘組低。Muc5ac和IL-13的Real-time PCR結(jié)果表明：哮喘組及正常+高表達質(zhì)粒組均較正常對照組明顯升高，P＜0.05；哮喘組與正常+高表達質(zhì)粒組相比較，P＞0.05，二者表達量無顯著差異。趨化因子CCL-2、CCL-5、CCL-11及Th2細胞因子IL-4、IL-5的Real-timePCR結(jié)果均表明,哮喘組較正常對照組明顯升高，P＜0.05；正常+高表達質(zhì)粒組較正常對照組有升高，但P＞0.05，未達統(tǒng)計學(xué)差異標(biāo)準(zhǔn)；Th1細胞因子IFN-γ的mRNA表達量在哮喘組或正常+高表達質(zhì)粒組均無顯著升高。相關(guān)性分析表明：肺組織中mCLCA3mRNA與粘液蛋白Muc5ac mRNA（r=0.759，P=0.000）及IL13mRNA（r=0.776，P=0.000）的表達成總體正相關(guān)，在0.01水平上具有顯著相關(guān)性；IL13mRNA與Muc5ac mRNA成總體正相關(guān)（r=0.895，P=0.000），在0.01水平上具有顯著相關(guān)性。BALF中總炎性細胞計數(shù)及肺組織切片HE染色氣道炎癥評分結(jié)果均表明：哮喘組及正常+高表達質(zhì)粒組均較正常對照組明顯增高，P＜0.05；哮喘組與正常+高表達質(zhì)粒組相比較，P＞0.05，差異無統(tǒng)計學(xué)意義。結(jié)論：哮喘相關(guān)基因mCLCA3上調(diào)Muc5ac的表達致小鼠氣道粘液高分泌。外源性mCLCA3引起肺組織及氣道炎癥，但并不直接參與氣道上皮細胞趨化因子的分泌。這一發(fā)現(xiàn)為指導(dǎo)哮喘的治療提供了新的線索。
[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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R562.25

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相關(guān)期刊論文 前2條

1 何麗;張惠蘭;趙建平;甄國華;張波;;哮喘小鼠氣道上皮高表達mCLCA3及其與趨化因子和Th1/Th2細胞因子的關(guān)系[J];華中科技大學(xué)學(xué)報(醫(yī)學(xué)版);2010年05期

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本文編號：2008991