【作者】 张聪；

【导师】 谢敏；

【作者基本信息】 华中科技大学 ， 内科学（呼吸病学）， 2021， 硕士

【摘要】 目的:探讨支气管哮喘气道平滑肌雌激素受体α和β表达水平及其在气道平滑肌表型转化、增殖和迁移中的作用。方法:我们通过临床标本,动物实验,细胞实验三个维度对上述目的进行探讨。首先我们共纳入了10名在支气管镜下气道粘膜活检且在粘膜切片上有平滑肌层的哮喘患者和3名健康对照,并收集这些临床受试者的基本临床信息,具体包括受试者性别、年龄、BMI(Body mass index,体重指数)、肺功能。然后我们利用免疫组化对临床受试者的支气管粘膜切片进行ERα和ERβ(Estrogen receptorαandβ,雌激素受体α和β)表达以及表型相关蛋白表达检测。之后我们建立OVA(Ovalbumin,卵清蛋白)诱导的C57小鼠哮喘模型,具体分组为对照组(8只雄性对照和8只雌性对照)和哮喘组(8只雄性哮喘和8只雌性哮喘),取小鼠右肺组织进行石蜡切片,同样利用免疫组化对肺组织切片中大气道平滑肌层雌激素受体α和β表达、表型相关蛋白表达、大气道平滑肌层厚度、大气道平滑肌层PCNA(Proliferating Cell Nuclear Antigen,增殖细胞核抗原)表达进行检测,对免疫组化结果均采用Image Pro plu6软件进行平均积分光密度半定量分析。最后我们用大鼠原代气道平滑肌细胞进行了体外细胞实验,,以IL-4联合E2/PPT/DPN(Estrogen/Propyl pyrazole triol/Diarylpropionitrile,雌激素/特异性ERα激动剂/特异性ERβ激动剂)刺激细胞,检测大鼠气道平滑肌细胞表型标志物表达变化,细胞增殖情况,之后亦检测沉默雌激素受体α和β后气道平滑肌细胞表型标志物蛋白表达变化,细胞增殖程度,另外我们还通Transwell细胞侵袭实验探讨ERα和β对PDGF(Platelet-derived growth factor,血小板衍生生长因子)诱导的细胞迁移的影响。结果:1)哮喘患者男性和女性之间气道平滑肌ERα和ERβ表达无明显差异,ERα和ERβ表达呈正相关(r=0.65,p=0.0161)。临床样本中气道平滑肌ERα的表达与分泌型表型标志物osteopontin呈正相关(r=0.61,p=0.0302)。2)哮喘动物模型雌性哮喘小鼠相比对照组气道平滑肌ERβ表达增加(p=0.0492);雌性哮喘小鼠与对照组相比出现大气道平滑肌由收缩型向分泌型转化,PCNA表达增加(p=0.0021);哮喘小鼠大气道平滑肌层均较对照增厚(p<0.0001),且雌性哮喘小鼠增加更明显。3)哮喘动物模型中哮喘小鼠大气道平滑肌ERβ与PCNA表达阳性率呈正相关(r=0.33,p=0.036)。4)体外IL-4刺激能促进大鼠气道平滑肌ERα与ERβ蛋白表达增加(p<0.05)。5)体外E2联合IL-4刺激能促进大鼠气道平滑肌由收缩型向分泌型转化(p<0.001),沉默ERα后能抑制哮喘大鼠气道平滑肌表型转化(p<0.05)。6)体外E2或DPN刺激能促进大鼠气道平滑肌增殖(p<0.05),沉默ERβ后大鼠气道平滑肌增殖减弱(p<0.05)。7)体外沉默ERα、ERβ后能抑制PDGF诱发的大鼠气道平滑肌迁移(p<0.001)。结论:哮喘患者男性和女性之间气道平滑肌ERα和ERβ表达无明显差异。哮喘动物模型雌性小鼠相比对照组气道平滑肌ERβ表达增加。ERα可能参与了哮喘气道平滑肌的表型转化过程。ERβ可能促进哮喘气道平滑肌增殖。ERα和ERβ在PDGF诱导的气道平滑肌迁移中均可能发挥了一定正性调控作用。更多还原

【Abstract】 Objective: To explore the expression levels of estrogen receptor α and β in airway smooth muscle layer in asthmatics and their role in the phenotypic transformation,proliferation and migration of airway smooth muscle cellsMethods: We explore the above-mentioned purpose through three aspects: clinical specimens,animal experiments,and cell experiments.Firstly,we included a total of 10 asthmatic patients and 3 healthy controls who had a smooth muscle layer in airway mucosal section through bronchoscopy.At the same time,we collected basic clinical information of these clinical subjects,including gender,age,BMI,percentage of peripheral blood eosinophils,and lung function test results.Then we used immunohistochemistry to detect the expression of estrogen receptor α and β and Phenotypic marker of airway smooth muscle on the bronchial mucosal sections of clinical subjects and used Image Pro plu6 to measure average integrated optical density of airway smooth muscle layer;secondly we established an OVA-induced asthma model in C57 mice[control group(8 males and 8 females)and asthmatic group(8 males and 8 females)],The right lung tissue of the mouse was taken for paraffin section,We used the same method to detect the expression of estrogen receptor α and β and Phenotypic marker of airway smooth muscle on the lung tissue sections,besides,we measured the thickness of airway smooth muscle layer and PCNA expression positive rate;At last,In the in vitro experiments,the rat primary airway smooth muscle cells were used as the experimental object,Cells were stimulated with IL-4 combined with E2/PPT/DPN to detect changes in the expression of smooth muscle cell phenotype markers,cell proliferation,and then also to detect phenotype markers changes and Cell proliferation after silencing estrogen receptor α and β.In addition,we also used Transwell cell invasion experiments to explore the effects of estrogen receptor α and β on PDGF-induced cell migration.Results: 1)There are no significant difference in airway smooth muscle ERα and ERβ expression between male asthmatics and male controls,male and female asthmatics the expression of ERα and ERβ in the large airway smooth muscle layer of clinical subjects is positively correlated(r=0.65,p=0.0161);2)The expression of ERβ in the RASMCs of female asthmatic mice increased;the RASMCs of female asthmatic mice changed from contractile to synthetic phenotype,and the expression of PCNA also increased;the airway smooth muscle layer of all asthmatic mice was thickened,and female asthmatic mice increased the most;3)The expression of ERβ and PCNA in the large airway smooth muscle layer of asthmatic mice is positively correlated(r=0.33,p=0.036);4)In vitro IL-4 stimulation can promote the increase of ERα and ERβ protein expression in rat airway smooth muscle;5)In vitro Estrogen stimulation can promote the transformation of RASMCs from contractile to synthetic phenotype.Silencing ERα can inhibit the transformation of RASMCs from contractile to synthetic phenotype;6)In vitro E2 and DPN stimulation can promote the proliferation of RASMCs,and the proliferation of RASMCs is weakened after ERβ silence;7)Silencing ERα and ERβ in vitro can inhibit the migration of rat airway smooth muscle cell migration.Conclusions: ERβ can promote the proliferation of asthmatic airway smooth muscle.Both ERa and ERβ play a certain positive regulatory role in the migration of airway smooth muscle induced by PDGF.更多还原