【作者】 马燕；

【导师】 李娜萍；

【作者基本信息】 华中科技大学 ， 病理和病理生理学， 2006， 硕士

【摘要】 急慢性支气管炎引起气道粘膜上皮细胞损伤,在其后的修复过程中,周围上皮细胞迁移、分裂增生和分化,以重新覆盖受损区域。细胞迁移时细胞形态改变、胞膜突起的定向及中心体的极化,分裂时染色体的捕获及纺锤体的形成和定向,这些细胞活动过程均有赖于微管、微丝、Rho GTP酶及微管相关蛋白之间复杂的相互作用。Rho GTP酶是Ras超家族中小分子量G蛋白的成员之一,是一组分子量约为20～25kd的鸟苷酸结合蛋白,在细胞的信号转导通路中作为信号转换器或分子开关,作用于细胞骨架或其靶蛋白而产生多种生物效应,包括Cdc42、RhoA、Rac等,其中以Cdc42研究最多。微管相关蛋白包括APC(adenomatous polyposis coli)、EB1(end-binding protein)、CLIP-170(cytoplasmic linker protein-170)和CLASP(CLIP-associated protein)等,它们结合在微管正极上,参与微管动力学和功能的调节。糖原合成酶激酶3β(glycogen synthase kinase 3β,GSK3β)在细胞内呈组成性激活,APC和CLASP都是其底物。GSK3β活性改变及其对一些微管相关蛋白的磷酸化修饰,能够影响微管的动力学和功能,进而影响细胞的迁移、分裂等生命活动。目的研究GSK3β及CLIP-170在博莱霉素引起的小鼠气道上皮损伤和修复中的表达变化及其意义,以进一步阐明上皮损伤后修复的分子机制。方法健康昆明种小鼠(雌雄不拘)36只,腹腔注射麻醉后经气管插管一次性缓慢注入BLMA5 5mg/kg,诱导其气道上皮急性损伤。随后分批处死小鼠,获取肺组织和气管,以6只正常小鼠作为对照。用免疫组织化学S-P法检测GSK3β及CLIP-170蛋白在气道上皮细胞中的定位与表达;用western blot技术检测GSK3β、P-GSK3β、Cdc42及CLIP-170在损伤修复过程中表达的动态变化;并用免疫双标和共聚焦显微镜技术检测GSK3β及CLIP-170分别与微管蛋白β-tubulin的共定位关系。结果一、大体观察:正常组小鼠双肺红润,表面光滑,弹性良好。实验组小鼠肺组织出现点状出血、肿胀,后期肺组织体积缩小、硬度增加等不同程度的病理变化。二、HE镜下观察:光镜下正常气道上皮细胞排列整齐,为假复层纤毛柱状上皮或纤毛柱状上皮。实验组小鼠肺组织炎性细胞浸润,气道及肺泡上皮细胞坏死、脱落,纤毛倒伏,继而炎性细胞减少,上皮细胞修复,肺间质成纤维细胞增生,纤维化。三、免疫组化染色:GSK3β在正常对照组气道上皮细胞质内表达水平较高,BLM刺激后4d及7d时GSK3β表达迅速降低(P<0.01),而在随后的14d及28d时又有所回升(P<0.01),但仍低于对照组;CLIP-170在正常对照组细胞质内呈低表达,实验组则随着损伤的进行而表达逐渐升高,两组间差别具有显著性意义(P<0.01)。四、western blot检测: GSK3β在实验组早期损伤阶段水平降低,修复末期回升(P<0.01); P-GSK3β呈相反变化。Cdc42在实验组早期损伤阶段升高,随后渐呈下降趋势(P<0.05);CLIP-170在实验组给予BLM后呈持续升高趋势,与对照组比较,P<0.01。五、免疫荧光及共聚焦显微镜检测:β-tubulin在正常对照组小鼠气道上皮中分布较弥散,BLM刺激后4d、7dβ-tubulin明显沿气道上皮细胞近腔面和侧缘细胞膜下呈线状分布,肺泡上皮细胞质内也出现表达。CLIP-170及GSK3β的分布与β-tubulin比较一致,BLM14d、28d时,CLIP-170、GSK3β和β-tubulin均出现向胞质内侧转移、弥散分布的趋势。结论1. GSK3β的磷酸化与Cdc42调节有关;2. GSK3β和P-GSK3β在气道上皮损伤与修复过程中呈波动性变化并在形态学上与微管共定位,提示其可能参与调节微管相关蛋白在微管正极末端的聚集与结合;3. CLIP-170在气道上皮损伤后表达增强且可能与微管蛋白结合,促使微管向细胞周边聚集。更多还原

【Abstract】 The acute and chronic bronchitis causes the injury of airway mucous epithelia. In afterward repair process, the peripheral epithelial cells move, splits, proliferate and differentiate to overlay the damaged district again. The cell appearance changes when cell move, pustute of cell membrane direct and the central bodies polarize. When splitting chromosome succeeds in catching and the spindle is formated and directed. These cellses activity prcess is depended on the microtubule, microfilament, Rho GTP enzyme and the complicated interaction between the microtubule associated proteins. Rho GTP enzyme is one of the little-molecular-weight G protein members of Ras super family. They are a group of guanosine monophosphate binding protein whose molecular weight is about 20-25kd that serve as message transmitting organs or molecular switch in the message transmitting path of cell and produce all kinds of biological effect through effecting cystoskeleton or its target protein. These proteins include Cdc42, RhoA, Rac etc. Among these Cdc42 is studied more. Microtubule associated protein includes APC(adenomatous polyposis coli), EB1(end- binding protein), CLIP-170(cytoplasmic linker protein-170) and CLASP(CLIP associated protein) etc. They bind with the positive end of microtubule, participating in regulating the function of microtubule dynamics. GSK3 (glycogen synthase kinase 3) shows constituting activation in resting cell, whose substrates are APC and CLASP. The GSK3 activity changes and its phosphorylation on some MAPs can affect the function of microtubule dynamics, then affect the life activity such as the migration and splitting of the cell further.ObjectiveIn order to study the expression and significance of GSK3β、Cdc42 and CLIP-170 in the process of injury and reparation of airway epithelia in mice to further clarify the molecule mechanism of injury and reparation of epithelia.MethodsThirty-six KM mice in good conditions were treated with bleomycin intratracheally (5mg/kg, body weight) after abdominal anesthesia, inducing its acute injury of airway epithelia. After killed in batch, the lung organizes and trachea of mice were obtained with six normal mice as control. S-P immunohistochemical method were respectively used to examine the expression and distribution of GSK3βand CLIP-170 in the airway epithelial cell. Western blot technique were used to examine the expression change of GSK3β、P-GSK3β、Cdc42 and CLIP-170 in the injury and reparation process of airway epithelial cell. By double-label immunofluorescence technique and Confocal Laser Scanning Microscopy (CLSM) we explored the colocational relationship between GSK3β、CLIP-170 andβ-tubulin respectively.Results1. Observation on the wholeThe two lungs of normal mice are bloom, the surface is smooth and glossy and the elasticity is well. The lungs of experimental mice appear different degree pathological change from dotted hemorrhage、swelling to atrophy and hard etc.2. Morphologic changesThe normal respiratory epithelia cells arranged orderly under light microscope. It was visible as pseudostratified ciliated columnar epithelium or ciliated columnar epithelium. The lungs of experimental mice was infiltrated with many inflammated cells. The epithelium cells of airway and pulmonary alveoli necrosis and shed off with cilia lodging. Subsequently the cells of inflammation obviously decrease, then epithelia is more complete, then fibroblast proliferate, and eventually lung tissue fibrosis overall.3. The expression of GSK3βand CLIP-170 by immunohistochemistry stainingThe expression of GSK3βin cytoplasm is higher in the normal control group, then decrease significantly(P<0.01) after stimulated by Bleomycin four and seven days later. However, at subsequent fourteen and tweenty-eight days the expression have a little bit trend to back up again(P<0.01), which was still lower than normal group. The expression of CLIP-170 in cytoplasm is lower in the normal control group, then increase gradually with the development of injury(P<0.01).4. The results of western blotGSK3βdecreases after injury, but return to increase in last phase of reparation, but the P-GSK3βshows the contrary change. Cdc42 goes up after injury, then it appears a descending trend gradually. Inversely CLIP-170 display a continously advancing trend.5.Results of double-label immunofluorescence technique and CLSMβ-tubulin distributes more dispersedly in the epithelia of normal mice than stimulated by BLM. In BLM 4d and 7d groupβ-tubulin which also expressed in the epithelium cells of pulmonary alveoli distributes like a line obviously along under the cell membrane and lateral margin. CLIP-170 and GSK3βdistribute in accordance with theβ-tubulin. The trend of moving to cytoplasm is existed in BLM 14d and 28d in these three protein.Conclusions1、The phosphorylation of GSK3βis Cdc42-depended.2、The phosphorylation and dephosphorylation of GSK3βcan regulate MAPs concentrating and binding with the end of microtubule, so play a vital role in the injury and reparation process of airway epithelia.3、CLIP-170, who expresses more after injury, binds withβ-tubulin and reinforces the stabilization of microtubule. It can make microtubule to grow、lengthen、fasciculate and concentrate on cellular periphery. CLIP-170 also play important role in the injury and reparation process of mice airway epithelia.更多还原