Clara细胞在胎肺发育中和肺损伤后的表达及其意义

发布时间：2018-02-25 15:20

本文关键词： Clara细胞胎肺 Clara细胞分泌蛋白溶菌酶转化生长因子-β 降钙素基因相关肽正己烷　出处：《暨南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的观察胎肺发育中Clara细胞的发生、发育，探讨Clara细胞对胎肺发育过程的作用意义。构建正己烷静式吸入染毒致SD大鼠和昆明小鼠器官损伤的模型，为开展正己烷吸入致肺损伤的研究提供实验方法。观察经正己烷染毒损伤的实验大鼠和小鼠肺组织中Clara细胞的变化情况，并检测Clara细胞分泌蛋白(CCSP)及其相关细胞因子表达特征，探讨正己烷致肺损伤中Clara细胞的功能作用。方法 “昆明小鼠正己烷慢性吸入静式染毒模型”的建立：24只昆明小鼠随机分为4组，每组6只，，分为对照组和3个染毒组(4w、8w和12w组)，初始染毒浓度17.6g／m~3，在特制染毒箱中8h／d进行静式吸入染毒。各染毒组鼠均于染毒结束后0.5 h内取血，及时送气相色谱质谱仪检测全血正己烷浓度。“SD大鼠正己烷急性吸入静式染毒模型”按本研究小组以往的实验条件重复建立，也进行必要的检测予以验证。 16～30周胎儿肺组织共12例，取自暨南大学第一附属医院妇产科。染毒实验的昆明小鼠和SD大鼠处死后，新鲜组织及时取材。胎儿及动物的新鲜肺组织，4％多聚甲醛固定，常规石蜡包埋、切片，供HE染色、VG胶原纤维染色、免疫组化及显微镜观察。用免疫组化S-P法，检测胎肺和正己烷致损伤的实验鼠肺中CCSP蛋白、TGF-β因子、降钙素基因相关肽(CGRP)和溶菌酶(Lysozyme)的表达。结果 1．急性染毒的SD大鼠和慢性染毒的4w、8w和12w组昆明小鼠，经检测血液正己烷平均浓度均明显高于正常对照组，达到染毒要求。慢性染毒的4w、8w和12w组昆明小鼠，经显微镜观察结果证实，肺、肝、肾等正己烷主要代谢器官的结构都出现明显病理学变化。随着染毒时间的延长，病理损伤呈逐渐加重趋势，昆明小鼠正己烷慢性损伤模型构建已达到目的。 2．免疫组化检测胎肺结果：20w胎肺最先出现CCSP阳性细胞，位于细支气管上皮，此后数量逐渐增多，至30w时CCSP阳性细胞数量接近成体肺水平。在16w胎肺内已检出CGRP阳性细胞，主要定位于气道上皮内，阳性细胞数量在25w时最多，至30w时数量有所下降，揭示Clara细胞与肺神经内分泌细胞(PNECs)在发生和发育过程中存在密切联系。 3．检测正己烷损伤的肺组织结果：CCSP蛋白的表达结果显示正己烷染毒后，肺内Clara细胞严重受损，甚至脱落，且随着染毒时间的延长，Clara细胞数量明显减少。急性和慢性损伤的肺组织内，随染毒时间的延长，肺巨噬细胞数目逐渐增加；TGF-β表达区域扩大和强度增加；细支气管内CGRP阳性表达的神经内分泌细胞(PNECs)逐渐增多，急性损伤的肺内还出现较多NEB(神经上皮小体)。VG染色显示随染毒时间延长，损伤的肺组织内胶原纤维逐渐增多，肺纤维化病变严重。结论 1．“SD大鼠正己烷急性吸入静式染毒模型”和“昆明小鼠正己烷慢性吸入静式染毒模型”可以导致肺、肝和肾等正己烷主要代谢器官的进行性损伤，操作简便并有重复性，具有一定的科学依据和可行性。可以作为研究正己烷毒性损伤机制的实验模型。 2．Clara细胞在胎肺发育中经历逐渐成熟的过程，发育成熟的Clara细胞不但合成和分泌CCSP蛋白，还分泌肺表面活性物质的特异性SP蛋白，CCSP蛋白和SP蛋白对于确保胎肺的成熟发育和呼吸功能的建立具有极其重要意义。实验还揭示Clara细胞与肺神经内分泌细胞(PNECs)在肺发生发育过程中存在密切联系。 3．正己烷中毒致肺损伤过程中，Clara细胞是正己烷毒性作用的靶细胞。Clara细胞广泛参与肺损伤后的炎症细胞浸润、炎症因子抑制和抗纤维化等病理生理过程并起调节作用，因此认为Clara细胞是肺内气道上皮中具重要防御作用的细胞类型。
[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.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321

【参考文献】