【作者】 孙毅娜；

【导师】 阎玉芹；

【作者基本信息】 天津医科大学 ， 内科学， 2009， 博士

【摘要】 碘缺乏疾病(Iodine Deficiency Disorders, IDD)是全世界导致智力缺陷最常见的可预防性疾病,其对人类大脑的损伤主要发生在脑发育的关键期-胎儿期和生后早期,妊娠期间适宜的碘营养和甲状腺激素(TH)水平是保证胎儿正常的生长发育、尤其是脑发育的必要条件。目的：本研究旨在从整体、细胞、分子水平深入探讨母亲及其联系胎儿的胎盘组织对碘缺乏和碘过量的代偿能力(即对胎儿的保护能力),为孕期适宜补碘和进行孕期碘营养监测及甲状腺功能筛查提供实验依据。方法：按体重将4-6周龄健康Wistar雌性大鼠随机分为5组：重度缺碘组(S1D)、中度缺碘组(MoID)、轻度缺碘组(MiID)、正常碘组(NI)、30倍碘过量组(ExI),其中NI组作为正常对照。每组大鼠均食用低碘鼠粮,SID饮用自来水,每日总碘供给量为1.24μg；中、轻度缺碘组和正常碘组、碘过量组饮用含不同剂量碘化钾的自来水,每日总碘供给量分别是2.5μg、5μg、10μg和300μg。喂养3月后与按正常组条件饲养的Wistar雄性鼠交配,以妊娠16、20天孕鼠及其胎鼠为研究对象。砷铈催化分光光度法检测孕鼠和胎鼠的(尿、血、羊水、胎盘组织)碘含量,化学发光免疫分析法检测孕鼠血清和胎鼠羊水甲状腺激素及促甲状腺激素(TSH)水平,观察甲状腺形态；应用实时荧光定量PCR、Western blotting、免疫组织化学、激光捕获显微切割等技术,对胎脑TH代谢脱碘酶(D2、D3)和TH转运体(MCT8)、发育中神经元标志物(DCX)和突触发育标志物(p38)、胎盘组织和纯化的滋养层细胞的碘转运体(NIS、pemdrin)和TH转运体(MCT8、OATP14)以及TH代谢脱碘酶(D2、D3)进行了基因表达水平及部分蛋白表达水平检测。酶联免疫吸附双抗体夹心法测定孕鼠血清绒毛膜促性腺激素(rCG)水平。结果：1不同碘营养对孕鼠甲状腺功能的影响1.1轻、中、重度碘缺乏①机体碘水平降低尿碘和血碘水平与对照组相比显著降低,随缺碘加重,降低更加明显；但血碘变化的幅度小于尿碘。②甲状腺摄碘增强甲状腺NIS免疫阳性反应明显上调,主要沿滤泡细胞基底膜呈线性分布,并与缺碘程度呈正相关。③甲状腺功能减退症血清TT4、FT4呈降低趋势,TSH呈增高趋势,随缺碘加重,激素的变化趋势更加明显。④甲状腺质量增加随缺碘加重,甲状腺绝对和相对质量呈升高趋势,与对照组比较,SID组有统计学意义,MoID和MiID组无统计学意义。⑤甲状腺解剖和组织学变化SID组甲状腺明显充血肿大,呈明显的小滤泡增生性甲状腺肿改变；MoID和MiID组甲状腺肿大不明显,MoID组呈轻度的小滤泡增生性改变,MiID甲状腺与NI组甲状腺滤泡大小相似,但胶质较少。1.2碘过量①机体碘水平增高尿碘和血碘水平与对照组相比显著升高,但血碘升高的幅度低于尿碘。②甲状腺摄碘减少甲状腺NIS免疫阳性反应下调,主要位于滤泡上皮细胞胞浆内。③甲状腺功能不足血清TT4、FT4呈轻度降低趋势,TSH呈增高趋势。④甲状腺质量甲状腺绝对和相对质量与对照组接近。⑤甲状腺解剖和组织学变化甲状腺外观与对照组相似,组织学表现与对照组甲状腺滤泡大小相似,但胶质有增多的改变。2不同碘营养对胎鼠甲状腺功能和脑发育的影响2.1胎鼠甲状腺功能建立之前(孕16天)2.1.1轻、中、重度碘缺乏①机体碘水平未见降低羊水碘含量与对照组相似。②甲状腺激素不足(来自母鼠)SID和MoID组羊水FT4呈降低趋势,FT3/FT4比值呈升高趋势,仅在SID组FT3/FT4比值升高有统计学意义。而MiID组羊水TH与对照组无明显差别。③脑TH不足、发育受抑制各缺碘组脑组织D2mRNA表达显著降低；DCXmRNA和蛋白表达均呈升高趋势,DCXmRNA在MoID和MiID组升高有统计学意义,DCX蛋白升高无统计学意义。2.1.2碘过量①机体碘水平增高羊水碘含量与对照组相比显著升高,但其升高的幅度(约5倍)明显小于其母鼠碘供给量的升高幅度(30倍)。②甲状腺激素基本正常(来自母鼠)羊水FT4、FT3含量与对照组相似,FT3/FT4比值呈降低趋势,但无统计学意义。③脑TH及发育基本正常脑组织各项测定指标与对照组相似。2.2胎鼠甲状腺功能建立之后(孕20天)2.2.1轻、中、重度碘缺乏①机体碘水平降低SID和MoID组羊水碘水平降低明显,约为对照组的50%和36%；而MiID组几乎接近对照组(97%)。②甲状腺摄碘增强甲状腺NIS免疫阳性反应明显上调,主要沿滤泡细胞基底膜呈线状分布,并与缺碘程度呈正相关,与孕鼠相似。③甲状腺激素不足(来自母鼠和胎鼠两方面)SID羊水FT4降低,TSH、FT3/FT4比值升高,二者均有统计学意义。而MoID和MiID组羊水TH与对照组比较无显著差别,但TSH较对照组有升高趋势。④甲状腺解剖和组织学变化SID)组甲状腺明显充血肿大,MoID、MiID组甲状腺肿大不明显,仅有充血表现；组织学SID组大多数细胞呈腺团样结构,但滤泡腔不明显,也未见胶质,毛细血管较丰富；MoID组甲状腺仅部分形成明显的滤泡腔,但腔内胶质极少,几乎看不到,仍有相当数量的细胞呈腺团样结构,小血管丰富；MiID组甲状腺形成腔滤泡的数量较MoID组明显增多,但比对照组仍少,腔内胶质仍较少,部分可见淡染胶质。⑤脑TH不足、发育受抑制各缺碘组脑组织D2和MCT8mRNA表达均显著降低,D3mRNA表达呈降低趋势。DCXmRNA表达在SID和MoID呈升高趋势(与对照组相比有统计学意义),但其蛋白表达无明显差异；MiID组DCXmRNA和蛋白表达则与对照组无明显差异。p38mRNA和蛋白表达在SID和MoID组均显著降低；而MiID组仅呈mRNA表达明显降低,蛋白表达与对照相似。2.2.2碘过量①机体碘水平增加羊水碘含量与对照组相比升高有统计学意义,但其升高的幅度(约25倍)小于其母鼠碘供给量的升高幅度(30倍)。②甲状腺摄碘减少甲状腺NIS免疫阳性反应下调,主要位于滤泡上皮细胞胞浆内,同孕鼠相似。③甲状腺激素基本正常(来自母鼠和胎鼠两方面)羊水TH水平与对照组比较差异无统计学意义。④甲状腺解剖和组织学变化甲状腺肿大不明显,仅有充血表现；组织学甲状腺滤泡大小与对照组相似,但小血管较丰富。⑤脑TH不足、发育受抑制脑组织D2mRNA水平显著降低；D3mRNA表达呈降低趋势；MCT8mRNA显著升高。DCXmRNA和蛋白表达与对照组相似。p38mRNA呈降低趋势,蛋白表达与对照相似。3不同碘营养对胎盘滋养层细胞碘转运体表达的影响3.1胎盘NIS及pendrin分布NIS主要表达在较大绒毛干的滋养层细胞(即细胞及合体滋养层细胞)靠近胎儿侧的胞质中,孕晚期的间充质细胞、母体的蜕膜细胞、子宫内膜腺体等多种细胞胞质中。滋养层细胞中功能活跃细胞(可见核分裂相)的细胞膜上也明显表达,孕早期可见全膜分布,晚期主要位于胎儿侧胞膜上。pendrin主要分布于较大绒毛干的滋养层细胞(即细胞及合体滋养层细胞)靠近母体侧胞质中。3.2碘缺乏时滋养层细胞NIS、pendrin mRNA水平SID组在孕16天NISmRNA水平呈升高趋势,pendrin mRNA显著升高；而孕20天NISmRNA呈降低趋势,pendrin mRNA呈升高趋势；MoID和MiID组在孕16和20天NISmRNA与对照组表达相似,pendrin mRNA呈升高趋势。3.3碘过量时滋养层细胞NIS、pendrin mRNA水平NISmRNA孕16天表达显著降低,pendrin mRNA呈降低趋势；在孕20天NISmRNA表达显著增高,pendrin mRNA呈升高趋势。4不同碘营养对胎盘组织TH代谢关键酶、转运体表达的影响4.1胎鼠甲功建立前(孕16天)①中、重度碘缺乏：在SID和MoID组,MCT8mRNA在胎盘组织表达显著减低,在纯化的滋养层细胞呈降低趋势；胎盘组织OATP14mRNA表达呈升高趋势；D2mRNA在胎盘组织呈降低趋势,在滋养层细胞呈升高趋势；D3mRNA在胎盘组织和滋养层细胞均呈降低趋势。②轻度碘缺乏：在MiID组,MCT8和OATP14mRNA的表达水平与SID和MoID组相似：D2、D3mRNA与对照组相似。③碘过量：在ExI组,MCT8 mRNA变化同各缺碘组相似,但胎盘组织OATP14mRNA呈降低趋势；D2mRNA在胎盘组织呈降低趋势,在滋养层细胞与对照组相似；D3mRNA表达与SID和MoID组相似。4.2胎鼠甲功建立后(孕20天)①中、重度碘缺乏：在SID和MoID组,MCT8 mRNA在胎盘组织表达显著减低(同孕16天),但SID组的滋养层细胞呈升高趋势,MoID组与对照组相似；胎盘组织OATP14mRNA呈降低趋势；D2mRNA在胎盘组织呈升高趋势,在滋养层细胞显著升高；D3mRNA在胎盘组织呈降低趋势。②轻度碘缺乏：在MiID组,MCT8mRNA在胎盘组织显著减低,在滋养层细胞与对照相似；胎盘组织OATP14mRNA变化同SID和MoID组相似：D2mRNA在胎盘组织呈降低趋势,在滋养层细胞呈升高趋势；D3mRNA表达与对照相似。③碘过量时：在ExI组,MCT8mRNA变化同各缺碘组相似；胎盘组织OATP14mRNA与对照相似；D2mRNA在胎盘组织和滋养层细胞均显著升高；D3mRNA表达与SID和MoID组相似。结论1.中、重度碘缺乏引起孕鼠、胎鼠体内碘水平显著下降,甲状腺功能减退,经母体-胎盘代偿后仍不能满足胎脑发育对TH的需求,导致明显的脑发育障碍。2.轻度碘缺乏引起孕鼠体内碘水平轻度降低,甲状腺功能轻度减退,在母体-胎盘的代偿下,胎儿体内碘水平和甲状腺功能基本接近正常,但胎脑发育所需要的TH仍相对不足,主要影响孕早期胎儿甲功建立之前(即依赖母体TH阶段)的胎脑发育。3.碘过量引起孕鼠体内碘水平升高,甲状腺功能不足,在母体-胎盘的代偿下,胎儿体内碘水平虽略有升高,但甲状腺功能基本接近正常,胎脑发育所需要的TH仍相对不足,主要影响孕晚期胎儿甲功建立之后的胎脑发育。总之,妊娠期间机体即使轻微的碘水平和(或)甲状腺功能的异常波动,都会不同程度地影响到胎儿脑发育。更多还原

【Abstract】 Iodine Deficiency Disorders (IDD) remains the most frequent cause worldwide, of preventable mental retardationin children. The damage to the human brain occurs mainly in the critical period of brain development-prenatal and early postnatal periods. Appropriate iodine nutrition and thyroid hormone (TH) level during pregnancy is an important condition to ensure normal growth and development of fetus, especially fetal brain development.Our study explores whether the pregnant dams and their placenta have the compensatory capacity to iodine deficiency or iodine excess (that is, the protection of the fetus) in different aspects, providing experimental basis for appropriate supplementation and monitoring of iodine nutrition during pregnancy and thyroid function screening of pregnant women.Methods:4-6 weeks healthy Wistar female rats were randomly divided into severe iodine deficiency (SID), moderate iodine deficiency (MoID), mild iodine deficiency (MiID), normal iodine (NI) and excessive iodine (ExI) groups by body weight. And NI was the control group. The male rats were fed as NI. All female rats were fed on an iodine deficient food and drinking water with different doses of KI for 3 months until to mating. Pregnant dams and their fetuses on 16 and 20 days after fertilization were studied. Iodine level in urine, blood, fetal amniotic fluid and placental tissue were detected in pregnant dams and their fetus by As-Ce catalytic spectrophotometry. Thyroid hormones (TH) and TSH in pregnant serum and their fetal amniotic fluid were detected by chemiluminascent immunoassay methods. Thyroid morphology and sodium-iodide symporter (NIS) expression of dams and their fetus were observed. The gene and protein expression levels of TH metabolism-related deiodinase(D2 and D3), TH transporter(MCT8 and OATP14), markers of the developing neurons and synapse(DCX and p38), iodide symporter(NIS and pendrin) were detected by real-time fluorescence quantitative PCR, Western blotting, immunohistochemistry in fetal brain, as well as in placental tissue and LCM purified trophoblast cells. The rat Chorionic gonadotropin (rCG) in serum of dams was measured by enzyme-linked immunosorbnent assay(ELISA).Results:1 the effects of different iodine nutrition on thyroid function in pregnant rats1.1 mild, moderate and severe iodine deficiency①The lower iodine level in vivo:The levels of urinary and blood iodine were significantly lower than that of normal iodine group and more significantly reduced with the increase of iodine deficiency. But the magnitude of changes in blood iodine was less than urinary iodine.②Enhanced thyroid iodine intake:Thyroid NIS immunoreactivity increased significantly and was localized mainly along the basement membrane of follicle cells and linear distribution. There was a positive correlation between thyroid NIS expression and the degree of iodine deficiency.③hypothyroidism:Serum TT4, FT4 displayed decreasing trend and TSH was increasing trend. Hormone changes were more apparent with decrease of iodine levels.④Enhanced thyroid weight:with iodine deficiency, absolute and relative thyroid weight was higher compared with the control group. There was statistical significance in SID group no statistical significance in MoID and MiID.⑤thyroid anatomy and histology changes:Thyroid of SID group showed clear signs of hyperaemia, swelling and a typical small follicular thyroid hyperplasia swollen; thyroid of MoID and MiID groups were not obvious goiter, a slight small follicular thyroid hyperplasia swollen was observed in MoID group; similar thyroid follicular size, but less thyrocolloid were observed in MiID group compared with NI Group.1.2 iodine excess①The high iodine level in vivo:The levels of urinary and blood iodine were significantly higher than that of normal iodine group. But the magnitude of changes in blood iodine was less than urinary iodine.②Reduced thyroid iodine intake:Thyroid NIS immunoreactivity decreased significantly and located mainly in the cytoplasm of follicular epithelial cells.③thyroid dysfunction:serum TT4, FT4 displayed slightly decreasing trend and TSH was increasing trend.④Normal thyroid weight:Thyroid absolute and relative weight closed to the control group.⑤Thyroid anatomy and histology changes:The appearance of the thyroid was similar to the control group, and similar thyroid follicular size, but more thyrocolloid were observed compared with NI Group.2 The impact of different iodine nutrition on fetal-rats thyroid function and brain development2.1 Before establishment of fetal thyroid function (gestation 16 day)2.1.1 mild, moderate and severe iodine deficiency①Normal iodine level in vivo:The content of iodine in amniotic fluid was similar with the control group.②Shortage of thyroid hormone (from dams):FT4 of amniotic fluid displayed decreasing trend and FT3/FT4 ratio was increasing trend in SID and MoID Group. There was a significant increase in FT3/FT4 ratio of SID. There was no significant difference between MiID and control group.③Inadequate TH of brain and depression of brain development:The expression of brain tissue D2mRNA significantly decreased in all iodine deficiency groups. DCX mRNA and protein expression showed a higher trend. There was statistically significant increase of DCXmRNA expression and was not significant increase of DCX protein expression in MiID and MoID group.2.1.2 Excessive iodine①The higher iodine level in vivo:The content of iodine in amniotic fluid significantly increased. But its rate of increase (about 5 times) was significantly less than their dams increased iodine supply (30 times).②Nearly normal thyroid hormone (from dams):FT4, FT3 of amniotic fluid were similar with the control group and FT3/FT4 ratio was decreasing, but not statistically.③Normal brain TH and tissue and development:The indicators were similar to the control group.2.2 after establishment of fetal thyroid function (gestation 20 day)2.2.1 mild, moderate and severe iodine deficiency①The low iodine level in vivo:The iodine level of amniotic fluid in SID and MoID group significantly reduced, respectively about 50% and 36% of the control group; and in MiID group it almost reached to normal level (97% of the control group).②Enhanced thyroid iodine intake:thyroid NIS expression and location was similar to the pregnant rats. ③Shortage of thyroid hormone (from dams and fetus):In SID group, FT4 of amniotic fluid significantly reduced and TSH, FT3/FT4 ratio significantly enhanced. But in MoID and MilD group, there was an increase trend in TSH of amniotic fluid and TH was similar to control group.④Tthyroid anatomy and histology changes:Thyroid hyperaemia and swelling showed clear in SID group and was not clear in MoID and MiID groups. In SID group, thyroid histological changes showed the majority cells clustered gland-like structure without the obvious follicular cavity and thyrocolloid, and was full of blood capillary. In MoID group, thyroid had formed partially obvious follicles with smaller cavity and few thyrocolloid that almost could not to be seen. A considerable number of cells were still gland-like structure and was full of blood capillary. In MiID Group, the number of thyroid follicles with cavity was more than that of MoID group and less than that of control group. But there were less thyrocolloid that showed partially Light staining in follicular cavity.⑤Inadequate TH of brain and depression of brain development:The expression of brain tissue D2 and MCT8 mRNA significantly decreased and D3mRNA was decreasing in all iodine deficiency groups. In SID and MoID group, DCX mRNA significantly increased and protein expression showed no difference compared with control group. In MiID group, there was no difference in DCX mRNA and protein expression. p38 mRNA and protein expression of SID and MoID group significantly decreased. And in MiID group, p38 mRNA significantly decreased while protein expression was similar to the control group.2.2.2Excessive iodine①The higher iodine level in vivo:The content of iodine in amniotic fluid significantly increased. But its rate of increase (about 25 times) was less than their dams increased iodine supply (30 times).②Reduced thyroid iodine intake:Thyroid NIS expression and location was similar to the pregnant rats.③Nearly normal thyroid hormone ((from dams and fetus)):TSH of amniotic fluid was higher than that in control group (without Statistical significance) while TH levels were similar to control group.④Thyroid anatomy and histology changes:Thyroid showed clear hyperaemia and unclear swelling. Similar thyroid follicular size and more blood capillary were observed. ⑤Inadequate TH of brain and depression of brain development:In all iodine deficiency groups, the expression of brain tissue D2 mRNA significantly decreased and D3mRNA was decreasing. But the MCT8 mRNA expression of brain tissue significantly increased. DCX mRNA and protein expression was similar with the control group. p38 mRNA was decreasing trend and protein expression was similar with the control.3 The effect of different iodine nutrition on iodide symporter expression of placental trophoblast cells3.1 The distribution of placental NIS and pendrinRat placental NIS immunoreactivity was mainly found in the cytoplasm facing the fetus of cyto-syncytiotrophoblast cells of the larger villous stem. The cytoplasm of intravillous mesenchyma, decidua and endometrial pregnancy glands showed NIS expression at gestation 20 day. The cell membrane of active trophoblast cells with mitotic phase could be seen. NIS immunoreactivity of active trophoblast cells was the whole film distribution at gestation 16 day and mainly located in the membrane facing the fetus at gestation 20 day.Rat placental pendrin immunoreactivity was mainly found in the cytoplasm facing the mother of cyto-syncytiotrophoblast cells of the larger villous stem.3.2 The trophoblast cells NIS, pendrin mRNA expression of iodine deficiencyIn SID group at gestation 16 day, NIS mRNA expression increased and pendrin mRNA expression significantly increased. NIS mRNA decreased and pendrin mRNA increased In SID group at gestation 20 day. In MoID and MilD group at both gestation 16 and 20 day, NIS mRNA expression was similar to control group, pendrin mRNA increased.3.2 The trophoblast cells NIS, pendrin mRNA level of iodine excessAt gestation 16 day, NIS mRNA expression significantly decreased and pendrin mRNA expression decreased. But NIS mRNA significantly increased and pendrin mRNA increased at gestation 20 day.4 The impact of different iodine nutrition on placental TH metabolism key enzyme and transporters expression4.1 Before establishment of fetal thyroid function (gestation 16 day)①Moderate and severe iodine deficiency:In SID and MoID group, MCT8mRNA expression significantly reduced in the placenta and was decreasing in the purified trophoblast cells. OATP14mRNA expression increased and D2mRNA expression decreased in placental tissue. D2mRNA expression in the trophoblast cells was increased in the trophoblast cells. D3mRNA expression showed a lower trend in the placental tissue and trophoblast cells.②Mild iodine deficiency:In MiID Group, MCT8 and OATP14 expression levels of mRNA were similar to SID and MoID group. D2, D3mRNA expressions were similar to the control.③Iodine excess:In ExI Group, MCT8 mRNA change was similar to the iodine deficiency groups, but OATP14 mRNA was decreasing in the placental tissue. D2 mRNA was decreasing in the placental tissue while was similar to the control group in the trophoblast cells. D3mRNA expression was similar to SID and MoID Group.4.2 after establishment of fetal thyroid function (gestation 20 day)①moderate and severe iodine deficiency:In SID and MoID group, MCT8mRNA expression significantly reduced in the placenta(with gestation 16 day). But in trophoblast cells, MCT8mRNA expression of the SID group was increasing and was similar to the control group in MoID group. Placental OATP14 mRNA expression was decreasing. D2 mRNA was increased trend in the placenta and significantly enhanced in trophoblast cells. D3mRNA in the placental tissue was decreasing.②mild iodine deficiency:in MiID group, MCT8 mRNA expression significantly reduced in the placenta and was similar to the control group in trophoblast cells. OATP14 mRNA change in placental tissue was similar to the MoID and SID group. Placental D2 mRNA was decreasing and and was a high-trend in the trophoblast cells. D3 mRNA expression was similar to the control.③Iodine excess:The ExI group, MCT8 mRNA change was similar to the deficiency groups. Placental OATP14 mRNA was similar to the control. D2 mRNA in placental tissue and trophoblast cells were significantly increased. D3 mRNA expression was similar to the SID and MoID group.Conclusions1. The levels of iodine of pregnant-fetal rats in moderate and severe iodine deficiency significantly decreased and displayed hypothyroidism. Fetal brain TH levels could not meet the needs of brain development after the mother-placenta compensation and led to significant brain development disorder.2. The level of iodine of dams in Mild iodine deficiency mildly decreased and displayed thyroid mild dysfunction. Fetal iodine levels and thyroid function are nearly close to normal after mother-placenta compensation, but brain THs that were required for fetal brain development were still relatively insufficient to mainly impact the early pregnancy when fetal brain development dependent on maternal TH before the establishment of fetal thyroid function.3. The level of iodine of pregnant rats in excessive iodine supply significantly increased and displayed thyroid mild dysfunction. Fetal iodine levels increased to some extent and thyroid function are nearly close to normal after mother-placenta compensation. But brain THs that were required for fetal brain development were still relatively insufficient to mainly impact the later pregnancy when fetal brain development dependent on maternal and fetal TH after the establishment of fetal thyroid function.In short, even if iodine levels and (or) thyroid function fluctuation during pregnancy are very slight, fetal brain development will be affected in varying degrees.更多还原