【作者】 李娜；

【导师】 刘烈刚；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2009， 博士

【摘要】 1．目的通过建立维生素A缺乏（vitamin A deficiency，VAD）及边缘性维生素A缺乏（marginally vitamin A deficiency，MVAD）雌性大鼠模型，观察不同程度维生素A缺乏对胚胎生长发育及先天畸形发生的影响，并通过孕早期补充适量维生素A观察干预效果；检测器官形成期胚胎组织5个Hox基因mRNA水平的表达；检测3种视黄酸受体（retinoic acid receptors，RARs）mRNA及蛋白水平的表达；俭测Hoxa5基因启动子上游DNA甲基化的程度及位点分布，阐明Hox基因介导维生素A缺乏导致大鼠胚胎发育毒性的分子机制，为维生素A缺乏引起先天畸形的防治提供理论依据。2．方法2．1动物模型建立及分组：通过饮食诱导建立维生素A缺乏及边缘性维生素A缺乏的雌性大鼠模型。将初断乳SD雌性大鼠按体重随机分为正常对照组（AN）、维生素A缺乏组（VAD）、边缘性维生素A缺乏组（MVAD），分别喂饲维生素A含量为0 IU／g、0．4 IU／g、4 IU／g的饲料。亲代鼠喂养10w后采集尾血，用高效液相色谱法（High Performance Liquid Chromatography，HPLC）检测血浆视黄醇浓度。将雌雄大鼠2：1合笼交配，次日晨查到阴栓者记为妊娠第0．5d（E0．5d）。将维生素A缺乏、边缘性维生素A缺乏两组交配成功的雌鼠在各组内按体重随机分为维生素A缺乏组（AD组）、维生素A缺乏妊娠0．5d（E0．5d）补充组（DS组）、边缘性维生素A缺乏组（AM组）、边缘性维生素A缺乏妊娠0．5d（E0．5d）补充组（MS组）。AD、AM组饲料同前，DS、MS组于E0．5d起喂饲维生素A含量为10 IU／g的饲料。2．2亲代大鼠及其E12．5d、E19．5d胚胎生长发育情况：记录各组大鼠体重、身长、尾长、每日进食量、受孕率、流产率、胚胎存活情况（活胎数、吸收胎数、死胎数）；观察E12．5d胚胎的外部形态；观察E19．5d胚胎的外部形态、内脏和骨骼畸形情况。2．3 RT-PCR检测各组E12．5d胚胎组织中Hoxa1、Hoxa2、Hoxa5、Hoxa9、Hoxa10基因mRNA水平的表达。2．4应用RT-PCR及western blot检测各组E12．5d胚胎组织3种视黄酸受体（RARα、RARβ、RARγ）mRNA及蛋白水平的表达。2．5应用重亚硫酸盐测序方法（Bisufite Sequence PCR，BSP）检测各组E12．5d胚胎组织Hoxa5基因启动子上游DNA甲基化的分布及程度；应用RT-PCR及western blot分别检测E12．5d胚胎组织中Hoxa5 mRNA及蛋白水平表达的变化。3．结果3．1 AD组雌鼠在喂养10w后出现明显的维生素A缺乏症状，如生长缓慢、少食少动、反应淡漠、胡须脱落、后肢麻痹、双眼畏光，严重者眼周出现血性分泌物甚至失明。AM组未出现明显的维生素A缺乏症状。至交配时（10w末），AD组及AM组雌鼠血浆视黄醇浓度低于AN组，差异具有统计学意义（P＜0．05，P＜0．01），且AD组及AM组雌鼠血浆视黄醇浓度均分别低于0．07μmol／L、0．7μmol／L，表明维生素A缺乏及边缘性维生素A缺乏雌性大鼠模型已经建立成功。3．2至交配时，AD组亲代大鼠的体重、身长、食物利用率及受孕率均低于正常对照组，差异具有统计学意义（P＜0．01，P＜0．05）；AM组雌鼠的上述各指标与正常对照组相比无无统计学差异。与AN组相比，AD组E12．5d和E19．5d胚胎存活率降低，死胎率及吸收胎率增加，差异具有统计学意义（P＜0．01，P＜0．05），而DS组的上述各指标与AN组相比，差异无统计学意义。与AN组相比，AM组的死胎率增加，活胎率降低，差异有统计学意义（P＜0．01，P＜0．05），吸收胎率的差异无统计学意义，而MS组的上述各指标的差异与AN组相比差异无统计学意义。E12．5d时，AD组胚胎形态较小、发育不良，至E19．5d时全为吸收胎。AM组大鼠胚胎的各生长发育指标（t重、身长和尾长）显著性低于AN组（P＜0．01，P＜0．05），死胎率及畸胎率均高于AN组，差异有统计学意义（P＜0．01，P＜0．05），主要畸形包括脑水肿、突眼、小眼、皮下出血、内脏出血、骨骼发育不良（骨化迟缓）、脊柱侧弯、中轴骨骼向前同源转化等。DS组MS组胚胎各指标与AN组均无统计学差异。3．3 AD组E12．5d胚胎组织中Hoxa1、Hoxa2、Hoxa5、Hoxa9、Hoxa10基因mRNA水平的表达低于AN组（P＜0．01，P＜0．05）；AM组E12．5d胚胎组织中Hoxa1、Hoxa2、Hoxa5基因mRNA水平的表达低于AN组（P＜0．01，P＜0．05），而Hoxa9、Hoxa10基因mRNA水平表达与AN组比较无统计学差异。3．4 AD组E12．5d胚胎组织中3种视黄酸受体（RARα、RARβ、RARγ）基因mRNA及蛋白水平的表达均低于AN组（P＜0．01，P＜0．05）；AM组E12．5d胚胎组织中RARβ、RARγ基因mRNA及蛋白水平的表达低于AN组（P＜0．01，P＜0．05），而RARαmRNA及蛋白水平的表达与AN组比较无统计学差异。3．5 AD组及AM组E12．5d胚胎组织中Hoxa5基因启动子上游500 bp 34个甲基化位点的甲基化程度明显高于AN组与补充组（DS组和MS组），异常的甲基化位点多集中于9^th-10^th、12^th-16^th、20^th-21^st三个区域。4．结论维生素A对胚胎的正常发育至关重要。维生素A缺乏对胚胎具有显著的发育毒性，不能维持胚胎完整的发育过程，在妊娠中后期就可导致胚胎死亡；边缘性维生素A缺乏对胚胎具有较强的发育毒性，可导致胚胎生长发育迟缓和先天畸形，但亲代大鼠未见明显的缺乏症状，具有一定的隐蔽性；孕早期补充适量维生素A可有效改善维生素A缺乏及边缘性维生素A缺乏导致的胚胎发育毒性；维生素A缺乏及边缘性维生素A缺乏可能通过干扰视黄酸受体的正常表达，从而抑制Hox基因启动子上游区域在胚胎发育过程中的去甲基化作用，致使Hox基因在转录水平和翻译水平下调表达从而产生对胚胎的发育毒性。5．创新点5．1通过研究不同程度维生素A缺乏对大鼠胚胎视黄酸受体和多个Hox基因表达的影响，全面系统地探讨了维生素A缺乏特别是边缘性维生素A缺乏导致胚胎先天畸形的分子机理，完善了维生素A缺乏导致胚胎先天畸形的理论。5．2 DNA甲基化是胚胎发育过程中重要的一种表观遗传修饰。本次研究利用重亚硫酸盐测序法系统地检测了Hoxa5基因启动子上游区甲基化的程及度分布，首次揭示了维生素A缺乏可能干扰胚胎发育过程中去甲基化的作用，而导致胚胎多个系统的先天畸形；更多还原

【Abstract】 Objectives:Through the establishment of different levels of vitamin A deficiency animalmodel, we observed the the teratogenicity of complete vitamin A deficiency andmarginal vitamin A deficiency and the preventive effects of vitamin A supplement inearly pregnancy on embryonoic growth and developmet. We detected the effects ofdifferent levels of vitamin A deficiency and supplement on the expreesion ofhomeobox genes （Hox genes） at mRNA levels; we also detected the expression ofretinoic acid receptors （RARs） at mRNA and protein levels; meanwhile, we examinedthe DNA methylation pattern in the regulatory region of Hoxa5 gene. This studyaimed to elucidate the the molecular mechanism of the mediation of Hox genes in theembryonic teratogenicity of vitamin A deficiency and offered theoretical foundationof the prevention of congential malformation induced by vitamin A deficiency.Materials and Methods:1. We developed vitamin A deficient and marginal vitamin A deficient female ratmodel and experiment assignment. Weanling female SD rats were assigned into 3groups and given experiment diet containing different doses of vitamin A. Thevitamin A concentrations were 0 IU/g diet, 0.4 IU/g diet and 4 IU/g diet, respectively.10 weeks later, serum retniol concentration of female rat was examined. Then the rat were mated （female: male=2: 1） and the morning of vaginal plug positive was takenas embryonic day 0.5 （E0.5）. The pregnant rats were then subdivided into 5 groups:vitamin A normal group （AN）, completely vitamin A-deficient group （AD）, vitamin Adeficient-supplement at E0.5d group （DS）, marginally vitamin A-deficient group（AM）, and marginally vitamin A deficient-supplement at E0.5d group （MS）. Pregnantrats in group DS and group MS were fed diet containing vitamin A 10 IU/g dietstarted from E0.5 day.2. We observed the growth and development of both female rats and embryos ofE12.5d and E19.5d, including the conception rate, abortion rate and embryonicdevelopment. At E19.5d, gross weight of placenta, average placenta weight, the bodyweight, body length and tail length of the fetuses were measured; the malformation ofexternal appearance, internal organs and skeleton of the fetuses were examined.Meanwhile, the survival and morphogenesis of E12.5d and E19.5d embryos wererecorded.3. In order to shed the light on the important mediation of Hox genes in theeffects of vitamin A deficiency on embryonic development, E12.5 embryos were usedfor the analysis of Hox genes mRNA expression by real time RT-PCR.4. We use the real time RT-PCR and western blot to detect the mRNA andprotein expression of retinoic acid receptors in order to elucidate the possibility thatvitamin A deficency might disturb the regularly expression of RARs and thusdown-regulated the important target genes, Hox genes’ expression.5. To seek the association between the changes of DNA methylation pattern ofHox genes and the teratogenicity of vitamin A deficiency, we use the bisulfitesequence PCR to detect the DNA methylation of regulatory region of Hoxa5.Results:1. After 10 weeks, female rats of CVAD group exhibited an array of vitamin Adeficient symptoms, as were appetite loss, weight loss, loss of whiskers, patches offur, ocular porphyrin deposits and depression in response to external stimuli, whilerats of normal group and group MVAD exhibited normal appearance. Serum retinol concentration of pregnant rats of group CVAD and MVAD group was less than 0.7μmol/, indicating that both vitamin A-deficient and marginal vitamin A-deficient ratmodels were successfully developed.2. At 10 weeks, an obvious reduce of gross weight of placenta, average placentaweight, body weight, body length gain, food utilization rate and conception rate wasobserved in CVAD female rat （P＜0.01, P＜0.05）. There was no significantdifference in other growth and development index. No significant difference wasobserved in MVAD group compared to normal group. Compared to normal group,abortion rate of pregnant rat, incidence rate of dead or absorption of E12.5d andE19.5d embryos in CVAD group was significantly higher; while survival rate wassignificantly lower （P＜0.01）. There was no difference in supplement groupcompared to normal group. At E12.5d, the embryos in VAD group weremaldeveloped than normal group. At E19.5d, the embryos in CVAD group were allabsorbed. There was significant difference in the developmental index including bodyweight, body length and tail length, incidence rate of dead embryos and abnormalembryos in MVAD group compared to normal group and supplement group （P＜0.01, P＜0.05）, and obvious malformation of external appearance, internal organsand skeleton of the fetuses were observed, such as encephaledema, exophthalmos,microphthalmos, subcutaneous hemorrhage, internal organs hemorrhage, bonedeformity （dysostosis, short rib, et al）, scoliolosis. There was no significant differencein the abnormity rate of MVAD group compared to normal group. The embryos innormal group and supplement group appeared normal.3. The results of real tiome RT-PCR demonstrated that Hox genes near 3’ endmRNA expression level of E12.5 embryos was down-regulated by complete vitaminA deficiency and marginal vitamin A deficiency （P＜0.01, P＜0.05）, while the Hoxgenes near 5’ end exhibited no response to vitamin A deficiency.4. The results of real tiome RT-PCR and western blot demonstrated thatcomplete vitamin A deficiency could suppressed the mRNA and protein expression ofRARs （P＜0.01, P＜0.05） while the expression of RARβand RARγwere down-regulated by marginal vitamin A deficiency （P＜0.01, P＜0.05） while RARα’sexpression had no difference relative to vitamin A normal and supplement groups.5. The distrubition and level of DNA methylation in near 500bp nucleotide basesin regulatory region of Hoxa5 of complete vitamin A deficiency and marginal vitaminA deficiency were significantly different from the vitamin A normal and supplementgroups. The DNA methylation site in 34 methylated sites of vitamin A deficnecygroups were focused on 9^th-10^th、12^th-16^th、20^th-21^st regions.ConclusionsComplete vitamin A deficiency induces growth retardation, lower reproductivecapacity and teratogenicity of embryonic development. Marginal vitamin Adeficiency also induces embryonic congential malformation. Supplement withvitamin A at early stage of pregnancy could reverse these changes. Thedown-regulation of embryonic Hox genea near 3’ end mRNA expression is animportant molecular mechanism of abnormal embryonic development by vitamin Adeficiency. The interruption of mRNA and protein expreesion of RARs and DNAmethylation pattern of Hox genes at regulatory region might be further explanation ofthe down-regulation of Hox genes.更多还原