【作者】 吴秀明；

【导师】 胡成平；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 背景：肾上腺髓质嗜铬细胞(AMCC)分泌肾上腺素在支气管哮喘调节支气管收缩与舒张功能中发挥作用。表观遗传学研究提示,胎儿时期母体宫内环境的异常可以印迹成年疾病的发生,AMCC在发育分化中受到母体宫内各个环节影响,其分化的最终结果可能导致肾上腺素分泌和/合成障碍,加重支气管哮喘的发病。目的：探讨妊娠期哮喘对子代肾上腺髓质细胞向交感神经元转变的的机制及对子代支气管哮喘发病的影响。方法：32只妊娠大鼠分为正常对照组(CP)、哮喘组(AP)、NGF组(NP)和anti-NGF组(ANP)建立妊娠哮喘大鼠模型,产下子代,CP、AP、NP和ANP各组子代分别为OCP、OAP、ONP、OANP,观察各组大鼠及其所产子代在生后3天(P3)、出生后7天(P7)、出生后14天(P14)、出生后30天(P30)、出生后60天(P60)及成年期再给予OVA致敏及激发后血清肾上腺素(EPI)、神经生长因子(NGF)、皮质酮水平,肾上腺髓质的结构的改变,肾上腺髓质中NGF、苯乙醇胺N甲基转移酶(PNMT)、Rho-GDP解离抑制因子α(Rho GDIα)和外周蛋白表达的变化。结果：1、各组母代大鼠的肾上腺髓质变化AP组血清EPI水平无明显升高,电镜下AMCC线粒体肿胀、脂质增多、嗜铬颗粒浓度降低、细胞出现突起样改变；NP组血清EPI水平较CP组降低,肾上腺髓质病变较AP组进一步加重,ANP组中上述病变有所改善。2、各组子代发育期肾上腺髓质的变化OAP组及ONP组大鼠从P3到P14,AMCC肿胀、脱失、空泡化,EPl分泌颗粒出现数量减少,可见突起形成,细胞核梭形样变。从P30到P60,AMCC空泡样变性减少,EPI分泌颗粒逐渐增多。从P3至P14,OAP组大鼠血清EPI水平较对照组减少(P<0.05),从P30至P60血清EPI与OCP组大鼠比较无明显差异,而ONP组组大鼠从P3直至P60,血清EPI水平均较对照组降低(P<0.05)。从P3至P7,OAP及ONP组大鼠血清NGF水平较对照组增加(P<0.05),从P14至P60,两组血清NGF水平与OCP组比较无明显差异。在各组子代成长过程中,P3至P60,OAP及ONP组大鼠血清皮质酮水平较OCP组明显增加(P<0.05)。P3至P14,OAP组大鼠PNMT在肾上腺髓质的表达较OCP组是减少的(P<0.05),而ONP组大鼠从P3至P60,PNMT的表达均较OCP组降低(P<0.05)。从P3至P14,OAP及ONP组大鼠肾上腺髓质嗜铬细胞中NGF表达较对照组是增加的(P<0.05)。3、OVA致敏及攻击后子代肾上腺髓质变化各组子代大鼠成年后给予OVA致敏及攻击,ONP组大鼠气道高反应性及气道炎症明显增加,OAP组大鼠AMCC出现明显的空泡样变性,电镜显示线粒体水肿,脂质增多,EPI分泌颗粒减少。ONP组大鼠AMCC神经元表型的改变较OAP组更为明显,EPI分泌颗粒减少明显,甚至消失,EPI分泌及释放减少,OAP与ONP组大鼠血清中EPI降低,ONP组尤为明显,ONP组大鼠肾上腺髓质中PNMT表达明显减少,NGF表达明显增多(P<0.05)。ONP组大鼠血清皮质酮水平较OCP组明显增加(P<0.05)。4、各组子代发育期及OVA致敏及攻击后外周蛋白与Rho GDIa表达OCP组子代在P3时可以观察到较弱的外周蛋白表达,P7时表达逐渐增强,P14时达其峰值,然后减少,从P30到P60保持较低的水平。OAP及ONP组大鼠从P3至P14较OCP组有较强的外周蛋白表达(P<0.05),从P30到P60,OAP组大鼠的外周蛋白表达OCP组大鼠比较无明显差异,而ONP组大鼠明显减少(P<0.05)。成年后,各组子代大鼠给予OVA致敏及攻击,OAP组、ONP组肾上腺髓质中外周蛋白表达水平增强,且ONP组的外周蛋白表达水平高于OAP组(P<0.05),OANP组外周蛋白水平表达较OCP组降低(P<0.05)。正常组的子代大鼠肾上腺髓质中Rho GDIa从P3到P14表达较弱,从P30到P60表达P14表达开始增加。OAP组大鼠出生后,从P3至P14表达较OCP组弱,从P30到P60,Rho GDIa表达与OCP组无明显差异,但ONP组Rho GDIa表达从P3至P60均较OCP组减少。从P3到P14,OANP组Rho GDIa表达与OAP组比较明显增强(P<0.05),但从P30到P60,与OAP组比较无明显差异。成年后,各组子代大鼠给予OVA致敏及攻击,OAP组Rho GDIa表达水平减少,ONP组的表达低于OAP组(P<0.05)。OANP组的Rho GDIa表达水平较OAP组增加。结论：我们从肾上腺髓质发育学角度发现哮喘妊娠宫内高浓度的NGF暴露时,可以启动子代大鼠肾上腺髓质细胞的功能冗余性使其表型和功能发生转化,当其成年后受到OVA攻击促使AMCC肾上腺嗜铬细胞向交感神经元分化,延缓或/和抑制了肾上腺髓质结构和功能的成熟,导致肾上腺素合成受阻,支气管收缩与舒张功能失衡,加重支气管哮喘发病。Rho GDIα和外周蛋白参与其中并发挥作用。更多还原

【Abstract】 Background:Adrenal neuroendocrine played an important role in asthma. Epigenetic studies suggest that abnormality in maternal intrauterine environment during fetal period can imprint occurrence of adult diseases.The activity of the sympathoadrenal system could be altered by early life events. The effects of maternal asthma during pregnancy on the adrenal medulla of offspring play a role in blocked adrenaline synthesis, imbalance between bronchial systolic and diastolic function, aggravating asthma severity.Subject:This study aims to explore the influence of maternal asthma during pregnancy on the development of adrenal medulla development of offspring and the differentiation of adrenal medulla cells (AMCC) to sympathetic neurons in adult offspring asthma.Methods:32SD rats were randomly divided into four groups:Asthmatic pregnancy rats (AP), nerve growth factor (NGF)-treated pregnant rats (NP) and NGF antibody-treated pregnant rats (ANP) were sensitized and challenged with ovalbumin (OVA), NP and ANP were treated with NGF and NGF antibody respectively. Rats were select from every pregnant group offspring at random and were divided into four groups(n=40per group):offspring from control pregnant rats (OCP), offspring from AP (OAP), offspring from NP (ONP), offspring from ANP (OANP).Adrenal glands were obtained for electron microscopic examination. The expression of NGF, phenylethanolamine N-methyltransferase (PNMT), peripherin and Rho GDIa were were analyzed by immunohistochemistry combine with the micro-image analysis. The concentration of epinephrine (EPI), NGF and corticosterone in serum were measured by ELISA.Result:1. Serum epinephrine levels were not significantly increased in AP group, electron microscopy show swelled mitochondria, decreased chromaffin granules, appearance of processes like change on adrenal medulla chromaffin cells. Serum epinephrine levels decreased in AP group. NP rats showed an aggravation of above mentioned pathological changes, while NP group NGF showed improved such changes.2. The adrenal medulla cells of OAP and ONP rats showed edema of cytoplasm and mitochondrial, vacuolar degeneration, deceased EPI secretory granule. chromaffin cells appeared fiber outgrowth and changed into spindle shape with long fusiform nucleus from postnatal day3(P3) to postnatal day14(P14). From postnatal day30(P30) to postnatal day60(P60), vacuolar degeneration showed decreased and the PEI secretory granule become increasing. Serum levels of EPI decreased in OAP rats from P3to P147compared to OCP rats and regained normal level from P30to P60; however, in ONP rats, from P3to P60, serum levels of EPI were lower than those in OCP rats. Serum levels of NGF increased from P3to P7in OAP and ONP rats and regained normal level from P14to P60. Serum levels of corticosterone in OAP and ONP rats increased significantly compared to OCP rats from P3to P60.The expression of PNMT proteins in the OAP and ONP rats adrenal medulla decreased significantly compared to OCP rats from P3to P14and gradually increased in OAP rats from P30to P60, there was a trend towards a lower expression in ONP rats. The expression of NGF increased in OAP and ONP rats adrenal medulla compared to OCP rats from P3to P14and return to normal level from P30to P60.3. After OVA sensitization and challenge, adult OAP and ONP rats showed significant Airway hyperresponsiveness and airway inflammation. The increase of vacuolar degeneration and lipid in adrenal medulla cells were observed in in OAP and ONP rats, Electron microscopy studies showed that AMCC presented signs of lesions:swelling mitochondrion, increased lipid, decreased EPI chromaffin granules in OAP and ONP rats. AMCC showed significant neuronal phenotype, EPI secretory granules even disappeared in ONP rats. ONP rats significantly reduced the expression of PNMT compared to OCP rats and NGF was significantly higher in ONP rats than OCP rats. Serum levels of EPI decreased in ONP rats. Serum levels of corticosterone in ONP rats increased significantly compared to OCP rats.4. The expression of peripherin increased in OAP and ONP rats adrenal medulla cells compared to OCP rats from P3to P14and return to normal level from P30to P60in OAP rats. However, the expression of peripherin in ONP rats increased from P30to P60compared to OCP. The expressions of peripherin in OANP rats increased from P3to P14compared to OAP. After OVA sensitization and challenge, the expression of peripherin increased in adult OAP and ONP rats compared to OCP rats and ONP rats showed more expression than OAP rats.The expression of Rho GDIa in the OAP and ONP rats adrenal medulla reduced significantly compared to OCP rats from P3to P14and gradually increased in OAP rats from P30to P60, there was a trend towards a lower expression in ONP rats from P30to P60. The expression of Rho GDIa in the OANP rats increased significantly compared to OAP rats from P3to P14and showed no difference from P30to P60. After OVA sensitization and challenge, OAP and ONP rats significantly reduced the expression of Rho GDIa in adrenal medulla and there was a trend towards a lower expression in ONP rats.Conclusion:Exposure to high level of NGF in the intrauterine environment may play an important role in the process of neural stem cell growth, migration and the differentiation of AMCC into sympathetic neurons, interfering with the synthesis, storage, release of EPI, even participating in adult bronchial asthma in offspring rats after OVA sensitization and challenge. Peripherin and Rho GDIa may play an important role in the differentiation of AMCC of the offspring from asthmatic pregnant rats.更多还原