【作者】 范俊明；

【导师】 杜继曾； 陈学群；

【作者基本信息】 浙江大学 ， 神经生物学， 2008， 博士

【摘要】 生命早期所处环境的不良因素对生后个体正常生理功能发育产生重要影响,已经引起医学界的广泛重视。流行病学调查、临床实践和实验研究发现,母体妊娠和胎儿期内外环境的不利因素对子代各种生理功能发育产生严重影响,甚至导致多种疾病或增高疾病易感性。HPA轴是由下丘脑-垂体-肾上腺所组成的神经内分泌网络,参与机体各种生理功能和对应激反应的调节,在神经内分泌、免疫、自主神经功能以及行为反应等方面具有重要作用。妊娠母体应激影响子代HPA轴功能,HPA轴功能的异常是生后个体生理功能异常和诱发疾病的基础。妊娠低氧或缺氧是临床常见的病理生理现象,诸如呼吸窒息、哮喘、梗塞、心血管系统疾病、贫血、高血压、脐带梗塞、胎盘体积过小、子宫血流量降低、吸烟嗜酒以及在高海拔低氧环境居住,都会导致母体氧供应不足,造成组织器官氧供应量降低。由于妊娠母体所处社会环境、家庭和经济状况种种危机,都可以形成心理应激或诱发焦虑、抑郁和精神分裂,进而影响妊娠母体生理功能,妊娠母体生理性或病理性低氧和心理应激对胎儿和子代的生理功能发育产生明显影响,因此研究妊娠缺氧或低氧并复合心理应激对子代不同发育期HPA轴功能和行为学研究具有重要的理论意义和临床应用价值。本研究建立了一种妊娠母体应激模型,即将妊娠母鼠自受孕之日起（EO）,至开始分娩（E21）的整个妊娠期,全时程（21天）给予间歇性低氧（Hypo,10.8%O₂）、束缚（Res）以及低氧+束缚（Hypo+Res）复合应激,每天4小时,观察生后子代处于青春前期、后期和成年期HPA轴功能和类焦虑样行为反应。首先研究了其对新生大鼠出生体重和生后子代体重增重的影响。其次,研究了其对青春前期（生后35天,P35）和青春后期（P63）子代HPA轴基础水平和应激水平的影响,并比较了妊娠母体应激和非应激对不同发育阶段的子代HPA轴水平变化的差异。最后,研究了其对成年子代（P120）HPA轴基础水平和行为反应的影响。实验研究采用免疫组织化学法（IHC）检测下丘脑室旁核（PVN）促肾上腺皮质激素释放激素（CRH）、CRH受体Ⅰ型（CRHR1）以及CRH受体Ⅱ型（CRHR2）蛋白水平的变化;采用激光共聚焦免疫荧光法检测下丘脑PVN区CRH/CRHR1、尿皮素Ⅰ（UCNⅠ）/CRHR1、CRH/CRHR2以及尿皮素Ⅲ（UCNⅢ）/CRHR2的共表达,检测了脑干蓝斑（LC）区CRH/CRHR1的共表达;原位杂交（ISH）和RT-PCR检测腺垂体CRHR1 mRNA和CRHR2 mRNA的表达水平;十字迷宫（EPM）检测成年期子代类焦虑样行为的发生;高效液相色谱法（HPLC）检测脑干LC组织去甲肾上腺素（NE）和多巴胺（DA）的水平;放射免疫分析法（RIA）测定血浆促肾上腺皮质激素（ACTH）的水平;荧光法测定血浆皮质酮（CORT）的水平。实验结果:1.妊娠期间歇性低氧、束缚及其复合应激对新生大鼠出生体重和生后体重增重的影响妊娠期间歇性低氧、束缚及其复合应激对新生动物出生率及性别比无显著影响,但复合应激显著降低新生大鼠出生体重。妊娠母体复合应激导致生后早期（生后1周,P7）雄性子代体重增长显著降低,这种变化一直持续到出生后第3周（P21）,此后渐至对照组水平,雌性4周后才至对照组水平。2.妊娠期间歇性低氧、束缚及其复合应激对青春前期（生后35天）和青春后期（生后63天）子代大鼠HPA轴基础水平的影响2.1青春前期妊娠期间歇性低氧、束缚及其复合应激对青春前期子代雄性大鼠下丘脑PVN区CRH蛋白基础水平无显著影响。但低氧组和束缚组CRHR1和CRHR2蛋白基础水平,垂体CRHR1 mRNA基础水平均显著低于对照组。低氧组和复合应激组垂体CRHR2 mRNA基础水平显著降低,束缚组无明显变化。复合应激组血浆CORT基础水平显著降低,而低氧组和束缚组无显著变化。2.2青春后期妊娠期间歇性低氧、束缚及其复合应激后,青春后期子代雄性大鼠下丘脑PVN区CRH和CRHR2蛋白基础水平无显著变化。复合应激组CRHR1蛋白基础水平显著降低。低氧组垂体CRHR1 mRNA的基础水平显著降低。低氧组和束缚组垂体CRHR2 mRNA基础水平均显著降低,而复合应激组显著增加,且显著高于低氧组和束缚组。复合应激组血浆ACTH基础水平显著高于对照组和低氧组,而低氧组和束缚组无明显变化。低氧、束缚及复合应激组血浆CORT的基础水平显著升高。3.妊娠期间歇性低氧、束缚及其复合应激对青春前期（生后35天）和青春后期（生后63天）子代大鼠HPA轴应激水平的影响3.1青春前期妊娠期间歇性低氧、束缚及其复合应激没有改变青春前期子代雄性大鼠下丘脑PVN区CRH蛋白应激水平。束缚组子代下丘脑PVN区CRHR1蛋白应激水平显著增加,复合应激组子代CRHR1蛋白应激水平显著降低,低氧组子代CRHR2蛋白应激水平显著增加,而束缚组和复合应激组子代无显著变化。妊娠母体应激后,子代垂体CRHR1 mRNA的应激水平显著降低。低氧组子代垂体CRHR2 mRNA应激水平显著增加。子代相应应激后,各处理组血浆CORT水平显著增加。3.2青春后期妊娠期间歇性低氧、束缚及其复合应激后,青春后期子代雄性大鼠下丘脑PVN区CRH蛋白应激水平显著降低。子代下丘脑PVN区CRHR1蛋白应激水平和对照组相比无显著差异。复合应激组子代下丘脑PVN区CRHR2蛋白应激水平显著增加,而低氧组和束缚组无变化。妊娠母体复合应激组子代垂体CRHR1 mRNA应激水平显著降低,而低氧组无显著变化。低氧组子代垂体CRHR2 mRNA应激水平显著高于对照组,束缚组和复合应激组子代与对照组相比无显著变化。子代相应应激后,血浆ACTH和CORT水平均显著升高。4.妊娠期应激和非应激对青春前期（生后35天）和青春后期（生后63天）子代大鼠HPA轴应激水平的比较4.1青春前期子代给予相应的应激后,妊娠期间歇性束缚组和复合应激组子代下丘脑PVN区CRH蛋白水平显著低于非应激对照组（None-PS）中相应的束缚组和复合应激组。束缚组子代CRHR1蛋白水平显著升高,而低氧组子代显著低于None-PS中相应的应激组。低氧组子代垂体CRHR1 mRNA应激水平显著低于None-PS组中的低氧组,而复合组子代显著高于None-PS组中的复合组。束缚组子代垂体CRHR2 mRNA应激水平显著高于None-PS组中的束缚组。4.2青春后期妊娠期复合应激后,复合应激组青春后期子代雄性大鼠下丘脑PVN区CRH蛋白应激水平显著低于None-PS组中相应复合应激组。子代下丘脑PVN区CRHR1蛋白应激水平显著低于None-PS组中相应应激组。复合应激组子代下丘脑PVN区CRHR2蛋白应激水平显著增加,而低氧组和束缚组无变化。妊娠母体低氧组子代垂体CRHR1 mRNA应激水平显著低于None-PS组中相应低氧组。复合应激组子代垂体CRHR2 mRNA应激水平显著高于None-PS组中复合应激组。相应应激后,复合应激组子代血浆ACTH水平显著高于None-PS处理中复合应激组。5.妊娠期间歇性低氧、束缚及其复合应激提高成年期子代大鼠HPA轴基础水平和诱导类焦虑样行为妊娠母体应激后,子代成年雄性大鼠HPA轴活性显著增加,下丘脑PVN区CRH和CRHR1蛋白表达水平显著升高,而CRHR2显著降低。激光共聚焦免疫荧光检测显示,复合应激组子代下丘脑PVN区存在CRH/CRHR1、UCNⅠ/CRHR1、CRH/CRHR2以及UCNⅢ/CRHR2高度共表达,脑干LC区中存在CRH/CRHR1高度共表达。子代腺垂体CRHR1 mRNA和CRHR2 mRNA、血浆ACTH和CORT水平以及肾上腺重量（%BW）均显著增加。子代脑干LC组织NE和DA水平显著增加,复合应激组显著高于低氧组和束缚组。应激组雄性子代出现类焦虑样行为,复合应激组更加明显。结论:1.妊娠期间歇性低氧+束缚复合应激导致新生大鼠出生体重和生后早期子代大鼠体重增长降低。2.妊娠期间歇性低氧、束缚及其复合应激降低青春前期但升高青春后期子代HPA轴基础水平。3.妊娠期间歇性低氧、束缚及其复合应激提高了成年子代雄性大鼠HPA轴活性,提高脑干LC组织NE和DA水平,诱导产生类焦虑样行为,复合应激作用更显著。此作用可能与PVN区CRH/CRHR1、UCNⅠ/CRHR1、CRH/CRHR2和UCNⅢ/CRHR2以及脑干LC区中CRH/CRHR1共存和激活,并分别驱动HPA轴和交感神经系统相关。提示CRH神经元可能通过其CRHR1和CRHR2两型受体,共同整合调节自身合成和分泌。更多还原

【Abstract】 The early life adverse environments pose a profound effect on physiological function of fetus and offspring. There are epidemiological investigations and clinic researchs suggest maternal stress has an effect on the endocrine function and behavioral response of offspring, induces disorders or susceptibility to diseases. HPA axis is a nerve-endocrine network which consists of hypothalamic, pituitary and adrenal, involved in the regulation of a varity of physiological function and stress response and plays an important role in neuroendocrine, immune, autonomic nerve and behavioral response. Pregnancy stresses influences the physiological function of HPA axis in offspring, dysfunction of HPA axis is associated with the abnormal physiological function of offspring.Pregnant maternal hypoxia stress is a common clinical pathological and physiological phenomenon. For example, apnea, asthma, infarct, cardiovascular disease, anemia, hypertension, umbilical occlusion, little placental, uterus blood flow decrease, smoking, drinking, and live in the high altitude that oxygen supply is deficient for pregnant animal and oxygen concentration decrease in organ and tissue, which will have an impact on the fetus and offspring, and eventually induced pathological symptom and abnormal behavioral response in offspring.In this study, we established a model that the pregnant rats were given simulated intermittent normobaric hypoxia （5 km, 10.8% O₂） （Hypo）, restraint （Res） and combination of both （Hypo+Res） throughout the pregnant period, 4 hours per day. We examined the change of the body weight gain in postnatal early rats, basal level of HPA axis and the level of response to stress in early （P35） and late puberty （P65） male rats; and anxiety-like behavior of adult （P120） male rats. The protein of CRH, CRHR1 and CRHR2 in the hypothalamic PVN were detected by immunohistochemistry （IHC）. The colocalization of CRH family in PVN and locus ceruleus （LC） of brain stem was studied by confocal immunofluroscence. CRHR1 mRNA and CRHR2 mRNA in pituitary were measured by in situ hybridization （ISH） and reverse-transcript polymerase chain reaction （RT-PCR）. The elevated-plus maze （EPM） was applied to test the anxiety-like behavior and the high performance liquid chromatography （HPLC） was applied to measure noradrenaline （NE） and dopamine （DA） in LC of brain stem, and levels of plasma ACTH and CORT were measured by radioimmunoassay （RIA） and fluorimetric method respectively. Results:1. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the birth weight and body weight gain of rats offspring.The birth weight was significantly decreased in rat offspring after pregnancy intermittent Hypo, Res and Hypo+Res. There was no change on the maternal body weight gain, gestation length, litter size or proportion of male and females. The body weight gain of male rat offspring was significantly decreased in postnatal 1-2 w and female was inl-3w after pregnancy intermittent Hypo, Res and Hypo+Res. After that it returned to the control. The body weight of the male was higher than that of the female.2. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on basal levels of HPA axis in early and late puberty rats.2.1 Early puberty （P35）There was no change of the basal levels of CRH peptides in PVN of early puberty male rats after pregnancy intermittent Hypo, Res and Hypo+Res. Compared with control or Hypo+Res group, basal levels of CRHR1 and CRHR2 peptides were significantly decreased in Hypo and Res groups. The basal levels of CRHR1 mRNA in pituitary were significantly decreased among all stress groups, CRHR2 mRNA was significantly decreased in Hypo and Hypo+Res groups, but was no change in Res group. Compared with control, CORT basal level in Hypo+Res group was significantly decreased, but was no change in Hypo or Res group.2.2 Late puberty （P65）There was no change of the basal levels of CRH or CRHR1 peptides in PVN of late puberty male rats after pregnancy intermittent Hypo, Res and Hypo+Res. Compared with control or Hypo group, the basal levels of CRHR1 or CRHR2 peptides were markedly decreased in Hypo+Res. Compared with control, Res or Hypo+Res group, the basal levels of CRHR1 mRNA in pituitary were significantly decreased in Hypo, CRHR2 mRNA was significantly decreased in Hypo or Res, but a significant increase in Hypo+Res （vs. control）. ACTH basal levels were significantly increased in Hypo+Res （vs. control or Hypo group）, CORT basal levels were significantly increased among Hypo, Res or Hypo+Res group compared with controls.3. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the HPA axis activity after stress in early and late puberty rats.3.1 Early puberty There was no change of the levels of CRH peptides in PVN of early puberty male rats offspring after exposed to the corresponding stress （vs. control）. The levels of CRH peptides in PVN of Res and Hypo+Res groups were both markedly decreased （vs. corresponding group in None-PS）. CRHR1 peptides levels in Res group were significantly increased （vs. control or corresponding group in None-PS）. CRHR1 peptides levels in Hypo+Res group were significant decreased （vs. control）. CRHR2 peptides levels in Hypo group were significantly increased （vs. control）, but there was change for Res or Hypo+Res group.CRHR1 mRNA in pituitary was significantly decreased in Hypo, Res and Hypo+Res group （vs. control）. CRHR1 mRNA was significantly decreased in Hypo group, but increased in Hypo+Res group （vs. corresponding group in None-PS）. CRHR2 mRNA were significantly increased in Hypo group （vs. control） and Res group （vs. corresponding group in None-PS）. Plasma CORT levels were significantly increased after stress exposure （vs. controls）.3.2 Late pubertyPregnancy intermittent Hypo, Res or Hypo+Res exposure significantly decreased the levels of CRH peptides in PVN of late puberty male rats. CRH peptides levels in Hypo+Res group were markedly decreased （vs.corresponding group in None-PS）. Although there was no change in Hypo, Res or Hypo+Res group （vs.control）, CRHR1 peptides levels were significantly increased （vs. corresponding group in None-PS）. CRHR2 peptides levels in Hypo+Res group were significantly increased （vs. control）, but there was no change in Hypo or Res group.CRHR1 mRNA in pituitary was significantly decreased in Hypo+Res group and a trend for Res group （vs. control）. CRHR2 mRNA was significantly increased in Hypo group （vs. control or corresponding group in None-PS） and Hypo+Res group （vs. corresponding group in None-PS）. Plasma ACTH levels were greatly increased in Hypo and Res group （vs. control） and Hypo+Res group （vs. control or corresponding group in None-PS）. Plasma CORT levels were significantly increased （vs. control）.4. Comparison of HPA axis reactivity of early and late puberty rat offspring in pregnancy stress and none-pregnacy stress （none PS）4.1 Early puberty （P35）CRH peptides in PVN of Res and Hypo+Res groups were significantly decreased （vs. the corresponding group None-PS）. CRHR1 peptides in Res group were significantly increased, and decreased in Hypo group （vs. the corresponding group in None-PS）. CRHR1 mRNA in pituitary were significantly decreased in Hypo, and increased in Hypo+Res group （vs. the corresponding group in None-PS）. CRHR2 mRNA in Hypo group were significantly increased （vs. control）, and increased in Res group （vs. the corresponding group in None-PS）.4.2 Late puberty （P65）CRH peptides in PVN of late puberty male rats were significantly decreased after pregnancy intermittent Hypo+Res stress （vs. the corresponding group in None-PS）. CRHR1 peptides were significantly decrease in all groups （vs. None-PS）; and CRHR2 peptides in Hypo+Res group were enhanced （vs. the corresponding group in None-PS）, but was no change in Hypo or Res group. CRHR1 mRNA in pituitary were significantly decreased in Hypo group （vs. None-PS）, and CRHR2 mRNA in Hypo+Res group were significant increased （vs. the corresponding group in None-PS）. Plasma ACTH levels in Hypo+Res group were markedly increased （vs. the corresponding group in None-PS）.5. Effects of pregnancy intermittent Hypo, Res and Hypo+Res on the anxiety-like behavior in adult male rats （P120）.Pregnancy intermittent Hypo, Res or Hypo+Res exposure affects the HPA axis and behavior in adult male rats. CRH and CRHR1 protein in PVN of the hypothalamus significantly increased, but CRHR2 significantly decreased. By confocal immunofluroscence, CRH and CRHR1, UCN I and CRHR2, CRH and CRHR2, UCN III and CRHR2 in PVN of the hypothalamus, and CRH and CRHR1 in LC were colocalized in Hypo+Res group. Pituitary CRHR1 and CRHR2 mRNA were markedly enhanced in PS （pregnancy stress） offspring. Plasma ACTH, CORT levels and adrenal weight were significantly increased （vs.control）. Also, the norepinephrine and dopamine levels significantly increased in the locus coeruleus, the highest level was in Hypo+Res offspring. Pregnancy intermittent Hypo, Res or Hypo+Res significantly increased anxiety-like behavior of adult male rats, the percentage of open arm entries in the elevated-plus maze test markedly decreased in rats offspring from all stressed groups.Conclusion:1. Pregnancy intermittent Hypo, Res or combination of both decreases the birth weight and body weight gain in postnatal early life.2. Pregnancy intermittent Hypo, Res or combination of both decrease HPA axis basal activity in early puberty, but increase in late puberty rat offspring. 3. Pregnancy intermittent Hypo, Res or combination of both alter the CRFR1 in the PVN as well as the drive on the cascade of responses of the HPA axis, resulting in an increase of anxiety-like behavior in adult male rat offspring. The colocalization of CRH and CRHR1, UCN I and CRHR2, CRH and CRHR2, UCN III and CRHR2 in PVN of the hypothalamus, and CRH and CRHR1 in LC in Hypo+Res group are involved in the proceeding, and it maybe drive from HPA axis and NE-sympathetic nerve system. The results indicate that CRH regulates its synthesis and release through integration of CRHR1 and CRHR2 in offspring after pregnancy intermittent Hypo, Res or combination of both.更多还原