發(fā)布時(shí)間：2018-02-25 15:20

  本文關(guān)鍵詞： Clara細(xì)胞 胎肺 Clara細(xì)胞分泌蛋白 溶菌酶 轉(zhuǎn)化生長(zhǎng)因子-β 降鈣素基因相關(guān)肽 正己烷　出處：《暨南大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 目的 觀察胎肺發(fā)育中Clara細(xì)胞的發(fā)生、發(fā)育，探討Clara細(xì)胞對(duì)胎肺發(fā)育過程的作用意義。構(gòu)建正己烷靜式吸入染毒致SD大鼠和昆明小鼠器官損傷的模型，為開展正己烷吸入致肺損傷的研究提供實(shí)驗(yàn)方法。觀察經(jīng)正己烷染毒損傷的實(shí)驗(yàn)大鼠和小鼠肺組織中Clara細(xì)胞的變化情況，并檢測(cè)Clara細(xì)胞分泌蛋白(CCSP)及其相關(guān)細(xì)胞因子表達(dá)特征，探討正己烷致肺損傷中Clara細(xì)胞的功能作用。 方法 “昆明小鼠正己烷慢性吸入靜式染毒模型”的建立：24只昆明小鼠隨機(jī)分為4組，每組6只，，分為對(duì)照組和3個(gè)染毒組(4w、8w和12w組)，初始染毒濃度17.6g／m~3，在特制染毒箱中8h／d進(jìn)行靜式吸入染毒。各染毒組鼠均于染毒結(jié)束后0.5 h內(nèi)取血，及時(shí)送氣相色譜質(zhì)譜儀檢測(cè)全血正己烷濃度�！癝D大鼠正己烷急性吸入靜式染毒模型”按本研究小組以往的實(shí)驗(yàn)條件重復(fù)建立，也進(jìn)行必要的檢測(cè)予以驗(yàn)證。 16～30周胎兒肺組織共12例，取自暨南大學(xué)第一附屬醫(yī)院婦產(chǎn)科。染毒實(shí)驗(yàn)的昆明小鼠和SD大鼠處死后，新鮮組織及時(shí)取材。胎兒及動(dòng)物的新鮮肺組織，4％多聚甲醛固定，常規(guī)石蠟包埋、切片，供HE染色、VG膠原纖維染色、免疫組化及顯微鏡觀察。 用免疫組化S-P法，檢測(cè)胎肺和正己烷致?lián)p傷的實(shí)驗(yàn)鼠肺中CCSP蛋白、TGF-β因子、降鈣素基因相關(guān)肽(CGRP)和溶菌酶(Lysozyme)的表達(dá)。 結(jié)果 1．急性染毒的SD大鼠和慢性染毒的4w、8w和12w組昆明小鼠，經(jīng)檢測(cè)血液正己烷平均濃度均明顯高于正常對(duì)照組，達(dá)到染毒要求。慢性染毒的4w、8w和12w組昆明小鼠，經(jīng)顯微鏡觀察結(jié)果證實(shí)，肺、肝、腎等正己烷主要代謝器官的結(jié)構(gòu)都出現(xiàn)明顯病理學(xué)變化。隨著染毒時(shí)間的延長(zhǎng)，病理?yè)p傷呈逐漸加重趨勢(shì)，昆明小鼠正己烷慢性損傷模型構(gòu)建已達(dá)到目的。 2．免疫組化檢測(cè)胎肺結(jié)果：20w胎肺最先出現(xiàn)CCSP陽(yáng)性細(xì)胞，位于細(xì)支氣管上皮，此后數(shù)量逐漸增多，至30w時(shí)CCSP陽(yáng)性細(xì)胞數(shù)量接近成體肺水平。在16w胎肺內(nèi)已檢出CGRP陽(yáng)性細(xì)胞，主要定位于氣道上皮內(nèi)，陽(yáng)性細(xì)胞數(shù)量在25w時(shí)最多，至30w時(shí)數(shù)量有所下降，揭示Clara細(xì)胞與肺神經(jīng)內(nèi)分泌細(xì)胞(PNECs)在發(fā)生和發(fā)育過程中存在密切聯(lián)系。 3．檢測(cè)正己烷損傷的肺組織結(jié)果：CCSP蛋白的表達(dá)結(jié)果顯示正己烷染毒后，肺內(nèi)Clara細(xì)胞嚴(yán)重受損，甚至脫落，且隨著染毒時(shí)間的延長(zhǎng)，Clara細(xì)胞數(shù)量明顯減少。急性和慢性損傷的肺組織內(nèi)，隨染毒時(shí)間的延長(zhǎng)，肺巨噬細(xì)胞數(shù)目逐漸增加；TGF-β表達(dá)區(qū)域擴(kuò)大和強(qiáng)度增加；細(xì)支氣管內(nèi)CGRP陽(yáng)性表達(dá)的神經(jīng)內(nèi)分泌細(xì)胞(PNECs)逐漸增多，急性損傷的肺內(nèi)還出現(xiàn)較多NEB(神經(jīng)上皮小體)。VG染色顯示隨染毒時(shí)間延長(zhǎng)，損傷的肺組織內(nèi)膠原纖維逐漸增多，肺纖維化病變嚴(yán)重。 結(jié)論 1．“SD大鼠正己烷急性吸入靜式染毒模型”和“昆明小鼠正己烷慢性吸入靜式染毒模型”可以導(dǎo)致肺、肝和腎等正己烷主要代謝器官的進(jìn)行性損傷，操作簡(jiǎn)便并有重復(fù)性，具有一定的科學(xué)依據(jù)和可行性�？梢宰鳛檠芯空�己烷毒性損傷機(jī)制的實(shí)驗(yàn)?zāi)Ｐ汀?2．Clara細(xì)胞在胎肺發(fā)育中經(jīng)歷逐漸成熟的過程，發(fā)育成熟的Clara細(xì)胞不但合成和分泌CCSP蛋白，還分泌肺表面活性物質(zhì)的特異性SP蛋白，CCSP蛋白和SP蛋白對(duì)于確保胎肺的成熟發(fā)育和呼吸功能的建立具有極其重要意義。實(shí)驗(yàn)還揭示Clara細(xì)胞與肺神經(jīng)內(nèi)分泌細(xì)胞(PNECs)在肺發(fā)生發(fā)育過程中存在密切聯(lián)系。 3．正己烷中毒致肺損傷過程中，Clara細(xì)胞是正己烷毒性作用的靶細(xì)胞。Clara細(xì)胞廣泛參與肺損傷后的炎癥細(xì)胞浸潤(rùn)、炎癥因子抑制和抗纖維化等病理生理過程并起調(diào)節(jié)作用，因此認(rèn)為Clara細(xì)胞是肺內(nèi)氣道上皮中具重要防御作用的細(xì)胞類型。
[Abstract]:objective
To observe the occurrence of fetal lung Clara cell development in the development of Clara cells on the role during the development of fetal lung. Construction of n-hexane injury organs in SD rats and Kunming mice inhalation model, experimental method for the study of lung injury caused by n-hexane inhalation. To observe the changes of Clara positive cells hexane exposure injury in rats and mice lung tissues, and the detection of Clara cell secretory protein (CCSP) and the expression of related cytokines characteristic, function of the n-hexane induced Clara cell lung injury.
Method
"The establishment of Kunming mice chronic n-hexane inhalation inhalation model: 24 Kunming mice were randomly divided into 4 groups, 6 rats in each group, divided into control group and 3 experimental groups (4W, 8W and 12W group), the initial exposure concentration of 17.6g / m~3, in a special box in 8h / D in static inhalation exposure. All groups were in the blood within 0.5 h after the end of the exposure time, gas chromatography and mass spectrometry detection of whole blood concentration of n-hexane. SD rats acute n-hexane inhalation inhalation model according to the experimental conditions of this study group to repeat the previous establishment, is also necessary to verify the detection.
16~30 weeks of fetal lung tissue in 12 cases, from the Department of Obstetrics and Gynecology, the First Affiliated Hospital of Jinan University. The toxicity test in Kunming mice and SD rats were killed after fresh tissue timely collected. Fresh fetal lung tissue and animal, 4% paraformaldehyde, embedded in paraffin, sliced, for HE staining, VG staining of collagen fibers, immunohistochemistry and microscope.
Immunohistochemical method was used to detect the expression of CCSP protein, TGF- beta factor, calcitonin gene-related peptide (CGRP) and lysozyme (Lysozyme) in the lungs of rats induced by fetal lung and n-hexane injury by S-P.
Result
1. acute exposure of SD rats and chronic exposure of 4W, 8W and 12W group of Kunming mice by detecting blood n-hexane average concentrations were significantly higher than those in normal control group, achieve weight. Chronic exposure of 4W, 8W and 12W group of Kunming mice, the results confirmed that the lung, liver by microscope, structure of the main metabolic organ kidney n-hexane appear obvious pathological changes. With the extension of exposure time, the pathological damage aggravated, Kunming mice chronic n-hexane injury model has achieved the aim.
2. immunohistochemical detection of fetal lung 20W results: fetal lung first appeared CCSP positive cells in the bronchiolar epithelium, then gradually increased in number, and 30W number of CCSP positive cells reached the adult lung. CGRP positive cells were detected in 16W fetal lungs, mainly located in the airway epithelium and the positive cells number in 25W when the number has fallen to 30W, revealed that the Clara cells and pulmonary neuroendocrine cells (PNECs) are closely related in the process of occurrence and development.
Results 3. lung tissue damage detection of n-hexane: results of the expression of CCSP protein showed that n-hexane poisoning, lung Clara cells severely damaged, or even fall off, and with the extension of exposure time, the number of Clara cells decreased significantly. The acute and chronic injury in lung tissue, with the extension of exposure time, the number of lung macrophages increased gradually; the expression of TGF- increased to expand the area and intensity; neuroendocrine cells positive expression of CGRP in the bronchioles (PNECs) gradually increased, acute injury of the lungs also appeared more NEB (NEB).VG staining showed that with prolonged exposure, lung tissue damage in collagen fibers increased, lung fibrosis is serious.
conclusion
1. SD rats of acute n-hexane inhalation inhalation model "and" Kunming mice with chronic n-hexane inhalation inhalation model can cause lung, liver and kidney etc. the main metabolic organ of n-hexane injury, simple operation and repeatability, with scientific basis and feasibility. It can be used as an experimental model study on damage mechanism of n-hexane toxicity.
2.Clara cells undergo the course of maturation in fetal lung development, not only the development of the synthesis and secretion of CCSP protein in mature Clara cells also secrete pulmonary surfactant specific SP protein, CCSP protein and SP protein to ensure the establishment of fetal lung development and respiratory function has very important significance. The experiment revealed that Clara cells and pulmonary neuroendocrine cells (PNECs) are closely related in the developmental process of the lung.
3. n-hexane poisoning induced lung injury in Clara cells is n-hexane toxicity of target cells.Clara cells participate in lung injury after the infiltration of inflammatory cells, inflammatory factors and inhibit fibrosis and other pathophysiological processes and play a regulatory role, so that the Clara cells are important defensive role in lung airway epithelial cell types.

【學(xué)位授予單位】：暨南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R321

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 汪雋瑛,方鳳,劉楓,蔣瑾瑾;哮喘大鼠肺表面活性物質(zhì)結(jié)合蛋白A的變化及其意義[J];第二軍醫(yī)大學(xué)學(xué)報(bào);2004年11期

2 張大勇;黃中新;;Sonic hedgehog信號(hào)通路在肺發(fā)育及肺癌發(fā)生中的意義[J];解剖學(xué)研究;2007年01期

3 李艷,辛建保,汪世灝,張培芳;吸煙對(duì)大鼠肺組織MCP-1和TGF-β表達(dá)的影響[J];中國(guó)組織化學(xué)與細(xì)胞化學(xué)雜志;2004年01期

4 岳少杰,羅自強(qiáng);肺表面活性物質(zhì)結(jié)合蛋白的生理功能[J];國(guó)外醫(yī)學(xué).呼吸系統(tǒng)分冊(cè);1995年02期

5 沈齊英,劉錄;正己烷急性吸入毒性作用的肝臟形態(tài)學(xué)觀察[J];工業(yè)衛(wèi)生與職業(yè)病;2001年03期

6 沈齊英,劉錄;正己烷致大鼠脂質(zhì)過氧化損傷的研究[J];環(huán)境與健康雜志;2001年02期

7 左敏,黃中新,夏潮涌;人胎肺神經(jīng)內(nèi)分泌細(xì)胞的發(fā)育及其作用[J];解剖學(xué)雜志;2000年01期

8 孫少華,楊國(guó)嶸,孫曉瑋,朱任之;巨噬細(xì)胞在人類肺組織纖維化中的作用[J];臨床與實(shí)驗(yàn)病理學(xué)雜志;1999年05期

9 羅自強(qiáng),岳少杰,趙必紅;Clara細(xì)胞分泌蛋白[J];生理科學(xué)進(jìn)展;2003年03期

10 劉永平,管茶香;肺神經(jīng)內(nèi)分泌細(xì)胞與肺損傷修復(fù)[J];國(guó)外醫(yī)學(xué)(生理、病理科學(xué)與臨床分冊(cè));2004年01期

本文編號(hào)：1534079