【作者】 张萱；

【导师】 李廷玉；

【作者基本信息】 重庆医科大学 ， 儿科学， 2009， 博士

【摘要】 第一部分孕期维生素A营养对幼儿智力发育的影响目的:纵向分析新生儿出生时脐带血VA水平和其2岁时智力发育的关系,评价孕期VA状态对儿童2岁时智力发育的影响。方法:本研究为前瞻性队列研究,研究对象为2005年3月2日至2005年5月24日在重庆市铜梁县四所综合型医院和保健院(铜梁县医院、铜梁中医院、铜梁妇幼保健院和铜梁县巴川镇人民医院)随机募集的共158对非吸烟健康妇女及其新生儿。分娩时收集母血和脐带血用高效液相色谱法测定血清VA水平。并进行详细的人口学资料收集和相关因素的个人社会情况问卷调查,新生儿进行体格测量。幼儿2岁时利用Gesell发育诊断量表(GDS)对其进行智力测试。Gesell测试结果以运动、适应、语言、个人-社会和以上四个能区的平均发育商(development quotients,DQs)表示。所有数据用SAS8.1软件进行统计分析。结果:共有158对母亲-新生儿纳入试验,获得157对完整的母婴人口学资料和临床数据,2岁时有122名儿童进行了GDS的测试。①母血VA水平明显高于脐血(p<0.0001),但母血和脐血VA水平之间无明显相关关系(p>0.05)。脐血中有60.1%视黄醇水平<0.7μmol/L (维生素A缺乏, VAD),而35.1%视黄醇水平(0.7~1.05)μmol/L (边缘型VA缺乏或可疑亚临床VA缺乏, MVAD/SSVAD)。母亲VAD和MVAD/SSVAD的发生率分别是12.8%和43.9%。②VA的胎盘转运率(脐血VA水平/母血VA水平,VA-PTR)是(0.72±0.33)。当母血VA水平在(0.18-2.56)μmol/L这一较大范围内波动时,脐血VA水平维持在(0.30-1.40)μmol/L这一相对较窄的范围内。③在校正了胎龄、新生儿性别、母亲文化水平、新生儿出生头围和产前被动吸烟(ETS)暴露等混杂因素影响后,VA-PTR分别与运动发育商(?=?7.21; 95% CI, 1.91 to 12.50; p = 0.0082)和平均发育商( ?=? 7.02; 95% CI, 2.14 to 11.90; p = 0.0053)成正比。在同一模型中,脐血VA水平分别与语言发育商( ?=?10.59; 95% CI, 1.26 to 19.92; p = 0.0265)和个人-社会发育商( ?=? 8.06; 95% CI,1.35 to 14.76; p = 0.0191)成明显正相关。④将脐血VA水平按其中位数划分为高VA水平组和低VA水平组后,比较两个组的各能区DQs值差异。结果显示高脐血VA水平组的适应发育商和平均发育商明显高于低脐血VA水平组(p值均<0.05)。⑤在校正了胎龄、性别、母亲文化水平、母亲头围、产前ETS暴露等因素影响后,脐血VA水平与出生头围成显著正相关( ?=?1.20; 95% CI,0.18 to 2.21; p = 0.0218),而VA-PTR和出生体重成显著正相关(?=?0.27; 95% CI, 0.08 to 0.47; p = 0.0064)。结论:①调查地区产妇的VA缺乏状况已是一个中度公共卫生问题,很有必要采取措施改善这种局面。②脐血VA水平显著低于母体水平,母血VA水平与脐血VA水平间无显著相关关系。③胎盘可能在VA的母胎转运中发挥重要的调节作用,这种作用有助于当母体VA水平波动时,胎儿的血清VA水平维持在相对窄的范围内。④孕期充足的维生素A水平,可能对儿童智力发育具有良好影响。从一定程度上反映孕期胎儿VA营养的脐血VA水平或VA-PTR能不同程度的影响幼儿2岁时多个能区的智力发育。出生时的VA水平对幼儿2岁时运动能区发育的影响较对其它能区的影响更大。⑤脐血VA水平和VA-PTR与出生时体重、头围等出生结局存在相关联系。第二部分孕期开始的边缘型维生素A缺乏对大鼠海马突触可塑性信号通路中重要分子表达的影响目的:探讨孕期开始的MVAD对学习记忆功能的损伤是否比出生时开始的VAD更严重;生后开始的VAD与出生时补充VA比较学习记忆能力的差别。孕期开始的MVAD对大鼠海马突触可塑性Ca2+依赖性信号传导通路相关分子的表达的影响。方法:清洁级健康雌性Wistar大鼠32只随机分为4组,饲以相应饲料,3周后与健康雄性Wistar大鼠1:2合笼:(1)正常对照组(Control),母鼠8只给予VA充足饲料(含VA 6500 U/kg),仔鼠饲料同母鼠;(2)边缘型维生素A缺乏组(MVAD组)母鼠8只给予VA部分缺乏饲料(VAD饲料,含VA 400 U/kg),仔鼠饲料同母鼠;(3)VA补充组(VAS组):母鼠8只给予维生素A缺乏组饲料至分娩,仔鼠与饲VA充足饲料母鼠所生仔鼠交换喂养,出生后开始仔鼠连续口服补充适量VA7d(50 IU视黄醇/g.d),同时母鼠饲予VA充足饲料,断乳后幼鼠饲料同母鼠;(4)生后缺乏组(PVAD组):母鼠8只给予维生素A充足饲料至分娩,其仔鼠与饲VAD饲料母鼠所生仔鼠交换喂养,母鼠饲予VAD饲料,断乳后幼鼠饲料同母鼠。生后1d,2w,4w,8w 4个时间点于Control组和MVAD组分别随机选取8只雌鼠处死。生后7周每组随机选取雌鼠各8只仔鼠进行水迷宫测试。生后8w在Control组随机选取未做水迷宫和已做水迷宫的雌鼠各8只处死。用HPLC监测血清VA浓度。定量PCR检测海马组织中RA受体RARα、β和突触可塑性信号通路中NMDA受体亚单位NR1、NR2A及NR2B、CaM激酶Ⅱα、CBP和Arc的表达。结果:①血清VA浓度:交配时VAD饲料喂养的母鼠血清视黄醇水平明显低于VA充足饲料喂养母鼠,未见明显VA缺乏的症状。仔鼠8周时MVAD组血清VA水平明显低于Control组,VAS组与Control组差异无统计学意义;PVAD组血清视黄醇水平介于Control组和VAS组之间。②水迷宫行为学测试:空间探索实验MVAD组潜伏期在测试第3,4,5天明显长于Control、VAS组和PVAD组,VAS组仍与Control组有差异,PVAD组潜伏期介于MVAD组和VAS组之间。定位航行实验MVAD组和VAS组在目标区域活动的时间明显低于Control组,而VAS组并未恢复到正常水平。PVAD组在目标区域活动的时间介于Control组和VAS组之间,PVAD组与Control组之间差异无统计学意义。反向空间探索实验Control组和VAS组仔鼠在平台所在象限的时间比例在训练第3天时明显大于MVAD组和PVAD组,尤其是Control组在训练第2天,这一比例就明显升高。MVAD组和PVAD组之间差异无统计学意义。③水迷宫训练对RA受体和突触可塑性信号通路分子的影响:做水迷宫与未做水迷宫的仔鼠其血清视黄醇水平差异无统计学意义,但水迷宫以后仔鼠海马RARα、NMDAR1(NR1)、NR2A、Arc、CAMKⅡα和CBP mRNA水平比未做水迷宫仔鼠明显升高(p<0.05),而RARβ和NR2B mRNA水平与未做水迷宫仔鼠比较差异无统计学意义(p>0.05)。RARα与突触可塑性信号通路相关分子NR1、NR2A、NR2B、Arc和CBP均有明显正相关关系。④各组海马NMDA受体生后mRNA水平变化:Control组从P1d至P2w,海马NR1mRNA水平明显升高,2w后逐渐下降;从生后1d至生后8w,海马NR2A的表达有逐渐上升趋势,而NR2B的表达则有逐渐下降趋势。Control组NR1和NR2B的mRNA水平在多数时间点要高于MVAD组。在P2w时,Control组NR2A和NR2B的比值明显低于MVAD组(p<0.05)。⑤各组海马Arc、CAMKⅡα和CBP生后mRNA水平变化:生后1d至8w,海马Arc和CAMKⅡαmRNA表达有逐渐上升趋势。生后4w和8w,Control组Arc mRNA水平明显高于MVAD组(p<0.05);生后2w和4w,海马CAMKⅡαmRNA水平在Control组明显高于MVAD组(p<0.05),CBP mRNA水平在各时间点差异无统计学意义。结论:①孕期开始的MVAD对仔鼠7周时空间学习、短时记忆、长时记忆和学习新事物能力均有明显影响,出生时开始的VAD对仔鼠空间学习、短时记忆和学习新事物能力的影响较对长时记忆的影响大,除了学习新事物的能力外,其余方面的损伤均不如MVAD组明显。出生时开始的VA补充不能使仔鼠的空间学习、短时记忆和长时参考记忆能力完全恢复,仅学习新事物能力能恢复至正常水平。②水迷宫训练后可使海马RA受体RARα及突触可塑性信号通路相关分子NMDA受体亚单位NR1、NR2A,Arc、CAMKⅡα和CBP mRNA表达水平升高,RARα可能在学习记忆形成及维持过程中与突触可塑性信号分子的表达变化有密切关系。③孕期开始的MVAD可降低突触可塑性信号通路中重要分子如NMDA受体亚单位NR1、NR2B、Arc和CAMKⅡα等的生后早期mRNA表达水平,可能对仔鼠海马LTP的形成和维持方面均有损害。第三部分孕期开始的边缘型维生素A缺乏通过视黄酸受体影响大鼠海马NMDA受体关键期的发育目的:探讨孕期开始的边缘型维生素A缺乏是否通过RA受体RARα影响关键期NMDA受体亚单位NR1的发育。方法:清洁级健康雌性Wistar大鼠16只,随机分为正常对照组(Control)和MVAD组2组,具体同第二部分。生后1d,2w,4w,8w四个时间点于2组分别随机选取8只雌鼠处死。定量PCR检测仔鼠海马组织生后发育过程中视黄酸受体RARα、RARβ和NR1表达;免疫荧光和激光共聚焦检测脑组织内RARα、RARβ和NR1的荧光强度及时间、空间分布;Western-blotting测生后各时间点海马组织中RARα、RARβ和NR1蛋白水平。结果:①生后各时间点RARα、RARβ及NR1 mRNA水平变化:随生后海马的发育,RARαmRNA水平逐渐下降。MVAD组RARαmRNA在各个时间点的表达明显低于Control组(P<0.05)。RARβmRNA水平在生后没有明显变化趋势。MVAD组在各个时间点表达均很低,在P2w和P8w时水平明显低于Control组。MVAD组NR1 mRNA在各个时间点的表达水平也明显低于Control组(P<0.05)。②生后海马发育过程中RARα、RARβ及NR1蛋白表达及定位变化:大鼠海马锥体细胞和颗粒细胞中RARα蛋白在P1d时大量表达,而P2w,P4w和P8w都较P1d明显下降(P<0.05),P4w和P8w两个时间点间差异无统计学意义。P1d时RARα在神经细胞胞浆和突起大量表达,P2w时主要表达在细胞核内,而P4w时则胞核和胞浆均有表达,P8w时以胞浆表达为主,部分细胞突起也有表达。Western Blotting结果与免疫荧光结果基本一致。免疫荧光检测未在P1d和P4w时的海马组织见到明显的RARβ的表达。Western Blotting结果显示,RARβ在P1d几乎无表达,P2w时表达很弱,以胞核表达为主,生后P4w和P8w较生后早期表达增强,胞核表达强于胞浆。P1d时NR1大量表达在胞浆和海马锥体细胞放射层神经细胞突起,P2w和P4w时表达较P1d有下降(P<0.05)。Western Blotting结果显示,NR1的表达在P1d时表达最强,此后逐渐减弱,P2w、P4w和P8w表达差异无统计学意义。③水迷宫训练对RARα、NR1蛋白表达的影响:未经水迷宫训练的仔鼠8w时RARα表达较弱,主要以胞浆表达为主,而水迷宫训练后其蛋白表达水平明显升高,仍以胞浆表达为主;NR1表达也较未经训练仔鼠明显增强(p<0.05)。④生后海马组织RARα和NR1表达部位的关系:激光共聚焦显示P1d时RARα和NR1在海马大量共定位表达于胞浆。⑤MVAD对RARα、RARβ和NR1表达强度及亚细胞定位的影响:免疫荧光显示,P1d时MVAD组RARα和NR1的蛋白表达均较Control组明显降低,以胞浆表达为主。Western Blotting结果显示,MVAD组与Control组在生后四个时间点RARα的蛋白表达亚细胞定位基本与Control组一致,但表达量较Control组均降低(p <0.05);MVAD组NR1的表达在P1d、P2w和P4w均低于Control组(p <0.05)。Western Blotting结果显示MVAD组与Control组比较RARβ表达差异无统计学意义(p >0.05)。结论:①孕期开始的MVAD主要影响仔鼠生后发育早期海马组织RARα的mRNA及蛋白表达水平,对RARβ的影响不明显;②生后大鼠海马RARα在时间、亚细胞定位上的发育程序性表达可能与其在生后学习记忆能力形成不同阶段所起的作用有关,RARα的胞浆表达可能与其翻译调节作用有关。③孕期开始的MVAD可能通过RA受体RARα影响突触可塑性信号通路中重要分子NMDA受体亚单位NR1在发育关键期的表达,这可能是孕期开始的MVAD影响突触可塑性信号通路,从而导致仔鼠学习记忆功能障碍的机制之一。更多还原

【Abstract】 PartⅠEffects of vitamin A nutrition during pregnancy on children’s neurodevelopment at two years oldOBJECTIVE: To investigate the correlation of cord blood vitamin A level at delivery and the neurodevelopment at two years age, and evaluate the effect of vitamin A nutrition during pregnancy on neurodevelopment of offspring.METHODS: The present research was a prospective cohort study and the subjects were 158 children born to nonsmoking women who gave birth at four big hospitals in Tongliang, Chongqing, China, between March 2, 2005 and May 24, 2005. Maternal blood and umbilical cord blood were collected at delivery to detect the serum retinol concentration by high-performance liquid chromatography (HPLC). A questionnaire about demographic information as well as personal-social information was administered by trained interviewers after delivery. The neonatal birth weight, length and head circumference were also measured. Intellectual development was evaluated by Gesell Development Schedule (GDS) at two years old.RESULTS: A total of 158 maternal–neonate pairs were recruited and complete epidemiological and clinical data were obtained from 157 pairs. At two years of age, 122 children were tested with the GDS.①Serum retinol level of maternal was significantly higher than that of cord (p<0.0001), but without any correlativity between them (p>0.05). The prevalence of vitamin A deficiency (VAD) and marginal VAD (MVAD)/suspected subclinical VAD (SSVAD) in neonates were 60.1% and 35.1%, respectively. The prevalence of maternal VAD and MVAD/SSVAD were 12.8% and 43.9%, respectively.②The vitamin A placental transport rate (VA-PTR) was (0.72±0.33). When the maternal retinol levels varied from 0.18μmol/L to 2.56μmol/L, the neonatal retinol levels keep in the range of (0.30-1.40)μmol/L.③After adjusting for potential confounders, VA-PTR was positively associated with motor area development quotients (DQ) (p=0.0082) and average DQ (p=0.0053). Cord VA level was positively related with language area (p=0.0265) and social area DQ (p=0.0191).④The adaptive area and average DQs in high cord VA group was higher than those in low VA group (p<0.05).⑤After adjusting for potential confounders, cord VA level and VA-PTR were positively associated with birth head circumference and birth weight, respectively.CONCLUSIONS:①It is a moderate public health problem of pregnant VAD in the locality, which needs urgent measures to make it better.②Cord retinol level is significantly lower than maternal retinol level, while there is no correlation between them.③Placenta plays an important role in regulating VA transportation from mothers to fetus, which might be helpful to maintain the neonatal VA levels in a relatively small scope when the maternal VA levels varied in a large range.④Adequate vitamin A nutrition during pregnancy has beneficial influence on children’s neurodevelopment. The effect of vitamin A status on motor development might be more obvious than adaptation, language and personal-social development.⑤There were relationships between birth weight or head circumference and cord retinol level or VA-PTR.PartⅡEffects of marginal vitamin A deficiency beginning from pregnancy on hippocampus synaptic plasticity signaling pathway related genes expression in young ratsOBJECTIVE: To investigate if the effect of MVAD from pregnancy on learning and memory in offspring rats was more serious than that of VAD from born, and the impact of VA supplementation from born. The effects of MVAD from pregnancy on hippocampus synaptic plasticity signaling pathway related genes expression in young ratsMETHODS: Thirty-two female rats were randomly divided into control, MVAD, vitamin A supplementation (VAS) and postnatal vitamin A deficiency (PVAD) groups in this study. In control group the dams and pups were fed with normal diet(VA 6500 IU/kg). In MVAD group the dams and pups were fed with MVAD diet(VA 400 IU/kg). In VAS and PVAD groups, the pups were exchanged at delivery. The offspring in VAS group were given oral vitamin A (50IU/g/d) from postnatal day 1 to day 7. Eight female pups were respectively killed postnatal day 1(P1d), P2w, P4w and P8w in control and MVAD groups. A ten days Morris water maze (MWM) test was conducted in the four groups at P7w. Eight female pups with MWM test or non-MWM test were killed in control group at P8w. We monitored the serum vitamin A concentration by HPLC. The mRNA expression of retinoic acid receptor RARα, RARβ, NMDA receptor subunit NR1, NR2A, NR2B, CAMKⅡα, Arc and CBP in hippocampus were detected by fluorescence quantitative PCR(FQ-PCR).RESULTS:①The dams’serum VA concentration in MVAD was lower than that of control, and they always did not have the manifestation of VAD. The serum VA concentration of MVAD group was lower than that of control group at P8w (P<0.05), and there was no difference between VAS and control groups. While the VA level in PVAD group was between control and VAS.②Morris water maze test: During the spatial acquisition test, rats in MVAD group showed longer escape latency than control, VAS and PVAD groups. The latency in PVAD group was between that in control and VAS groups. In the probe trial test, the percent time in target quadrant of MVAD and VAS group was significantly smaller than that of control group. There was no difference between PVAD and control group. In the spatial reversal test, the percent time in target quadrant of control and VAS group was significantly bigger than that of MVAD and PVAD groups at the third day. There was no difference between MVAD and PVAD groups.③There was no difference of the serum VA level between MWM and non-MWM pups, while the expression of RARα, NR1, NR2A, Arc, CAMKⅡαand CBP mRNA in hippocampus were higher in MWM pups than those in non-MWM pups (p<0.05), and the mRNA levels of RARβand NR2B remained unchanged after MWM test (p>0.05). There were positively correlation between the mRNA level of RARαand NR1, NR2A, NR2B, Arc and CBP.④FQ-PCR showed that there was an increase of the expression of NR1 in hippocampus from P1d to P2w, and then decrease gradually. The expression of NR2A and NR2B showed ascending trend and downtrend, respectively from P1d to P8w. The mRNA levels of NR1 and NR2B of control group was significantly higher in most time point than those in MVAD group. The ratio of NR2A to NR2B at P2w was lower in control than that in MVAD group (p<0.05).⑤The expression of Arc and CAMKⅡαincreased gradually from P1d to P8w. The expression of Arc mRNA in control group was significantly higher than that in MVAD group at P4w and P8w(p<0.05), and CAMKⅡαlevel in control group was higher than that in MVAD group at P2w and P4w(p<0.05), and there was no difference of CBP level between the two groups.CONCLUSIONS:①There are significantly effects of MVAD from embryonic and postnatal VAD on the spatial learning, short-term memory, long-term memory and the ability to learn new things at P7w. The effects are more serious in MVAD group than that in PVAD group. VA supplementation from born could not fully restore these abilities at P7w.②There are increase of some synaptic plasticity signaling pathway related genes after MWM test, and RARαmay have close relationship with there changes in the formation and maintain of learning and memory.③MVAD from pregnancy have influence on the early postnatal mRNA expression of important genes related to synaptic plasticity pathway, such as NR1, NR2B, Arc and CAMKⅡα, and could impaire the formation and maintain of long-term potentiation (LTP) in pups.Part III Marginal vitamin A deficiency from embryonic influences the development of hippocampus N-methyl-D-aspartate receptor subunit at critical period via retinoic acid receptor in ratsOBJECTIVE: To explore if MVAD from embryonic influenced the development of hippocampus N-methyl-D-aspartate receptor subunit NR1 at critical period via RARα.METHODS: Sixteen female rats were randomly divided into control and MVAD groups. The method of animal model was the same as partⅡ. Eight female pups in each group were respectively killed at P1d, P2w, P4w and P8w. We detected the expression and subcellular localization of RARα、RARβand NR1 in postnatal hippocampus by FQ-PCR, immunofluorescence, confocal laser scanning microscope and Western Blotting.RESULTS:①The expression of RARαmRNA gradually declined from P1d to P8w, while there was no significantly changes of RARβpostnatal. MVAD resulted the decreased levels of RARα, RARβand NR1 mRNA (P<0.05).②Hippocampal RARαprotein expression at P2w, P4w and P8w decreased compared with P1d (P<0.05), but there was no difference between expression at P4w and P8w. At P1d, abundant RARαprotein located at cytoplasm and neuritis, and then located at nucleus of neurons at P2w. Later, it shifted to the cytoplasm gradually, and expressed in the cytoplasm and some neuritis at P8w. We obtained similar results from Western blotting. No distinct expression of RARβwas detected by immunofluorescence at P1d and P4w in hippocampus. The western blotting showed that the expression of RARβincreased gradually postnatal, and located mainly in the nucleus. Immunofluorescence and western blotting results showed that the expression of NR1 declined with development and located in the cytoplasm.③MWM test resulted in the increase of RARαand NR1 protein in the cytoplasm.④Confocal laser scanning microscope displayed that a mass of RARαand NR1 co-located in the cytoplasm at P1d in hippocampus.⑤The fluorescence intensity and western blotting results showed that RARαand NR1 in MVAD group was lower than those in control group (p <0.05) and still located in the cytoplasm postnatal. There was no significantly difference of RARβbetween MVAD and control groups (p>0.05).CONCLUSIONS:①MVAD from embryonic could results in the decreased mRNA and protein expression of RARα, but with no obvious effect on those of RARβ.②The time and subcellular location-developmental programmed expression of RARαin postnatal hippocampus in rats may be related to the different roles it played during the formation of learning and memory. The cytoplasm location of RARαmay participated in the translation regulation.③MVAD from embryonic may influence the development of hippocampus NR1 at critical period via RARα, and this could be one of the mechanisms that how could MVAD from embryonic affect the synaptic plasticity signaling pathway, and result in impairment of learning and memory in offspring rats.更多还原