EphA5在先天性甲状腺功能减低大鼠脑中的表达及DNA甲基化调控
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摘要
目的：
建立先天性甲状腺功能减低大鼠模型、培养原代胎鼠海马神经元及分组干预。
方法：
1.取妊娠日(Gestational day, GD) G0天孕大鼠，随机分为三组，甲减组从G9天开始给予母鼠含0.02%MMI的清洁饮用水直至仔鼠全部处死；T4治疗组给予母鼠含0.02%MMI的清洁饮用水同甲减组，且从分娩日(Postnatal day, PD)第7天即P7天开始每日对仔鼠实施腹腔皮下注射甲状腺素(T4)；对照组一直给予清洁饮用水。
2.观察母鼠及仔鼠一般生物学特性，动态监测其体重变化及血清甲状腺激素水平。
3.在P0,3,7,14,21天分别收集上述三组仔鼠海马、大脑皮层、小脑，一部分存放于-80℃冰箱中备用，待作基因及蛋白分析；另一部分行石蜡包埋切片，待作免疫荧光染色。
4.于G17天取甲减组和对照组胎鼠，断头分离海马，用含B27、谷氨酰胺的Neurobasal无血清培养液培养原代海马神经元，倒置相差显微镜下观察海马神经元的形态变化。取培养7d的海马神经元，用MAP-2进行鉴定及纯度计算。
5.按照培养液不同，将培养的甲减组胎鼠海马神经元分为三组：T3组用含T3的培养液，Azadc组用含5-氮杂-2’-脱氧胞苷(Azadc)的培养液，Hypo-组用无血清培养液；并将同期培养的对照组胎鼠海马神经元作为对照组。镜下观察不同培养液干预对甲减组海马神经元生长的影响，并用MTT比色法测定以上四组海马神经元的活力。
结果：
1.母鼠在G18天时，对照组的体重明显高于甲减组与治疗组(P<0.05)；仔鼠出生后，三组母鼠体重均骤然下降，随后在整个哺乳期体重逐渐增加，但对照组的母鼠体重增加速度高于甲减组与治疗组，且在P3,7,14,21天对照组的母鼠体重明显高于甲减组与治疗组(P<0.05)。母鼠血清甲状腺激素测定结果显示在哺乳期末，甲减组和治疗组母鼠的血清FT3、FT4水平无显著差异(P>0.05)，但均显著低于对照组水平(P<0.05)。
2.甲减组和治疗组较对照组仔鼠体形小、行动迟缓、反应迟钝、毛发萌出晚且光泽度降低。开始睁眼和完全睁眼的时间甲减组均迟于对照组和治疗组。对照组仔鼠出生体重显著高于甲减组和治疗组(P<0.05)；治疗组的仔鼠体重在P14天已显著高于甲减组但仍低于对照组(P<0.05)；直至P21天，治疗组仔鼠体重与对照组已无显著差异(P>0.05)。仔鼠甲状腺激素测定结果显示：治疗组的仔鼠血清FT3、FT4水平在P14天，显著高于甲减组水平(P<0.05)，但仍显著低于对照组水平(P<0.05)；直至P21天，治疗组的仔鼠血清FT3、FT4水平已达到对照组水平(P>0.05)，均显著高于甲减组水平(P<0.05)。
3.镜下观察海马神经元的形态变化，结果发现甲减组海马神经元与对照组比较，在同一时间点细胞胞体体积较小，突起形成的长度较短、突起的支数较少，突起形成的网络密集程度较低；但是细胞纯度均能达到96%以上。甲减组海马神经元经不同药液干预后突起增多增长、网络密集程度提高；MTT比色分析显示：Azadc组和T3组的细胞活力均显著高于Hypo-组，但这三组的细胞活力均显著低于对照组(P<0.05)。
结论：
母鼠、仔鼠甲状腺激素水平及一般生物学特性表明先天性甲减组及治疗组大鼠模型均成功建立。细胞形态学的观察、鉴定及活力测定表明原代正常和甲减胎鼠海马神经元培养及药物分组干预成功。
目的：
研究ephrin-A5/EphA5信号通道及下游关键分子NMDAR-1（NR1）、PSD95、CaMKII在先天性甲减大鼠脑中的表达特征，并初步探讨ephrin-A5/EphA5信号通道与甲状腺激素缺乏致突触发生障碍的关系。
方法：
1.分别于P0,3,7,14,21天，应用实时定量RT-PCR技术分析甲减组、治疗组、对照组海马、大脑皮层、小脑组织ephrin-A5/EphA5及该通道下游关键分子NMDAR-1（NR1）、PSD95、CaMKII的mRNA表达水平，通过Western Blot方法进行验证后，进一步运用免疫荧光染色检测其蛋白表达水平。
2.取培养7d的对照组和甲减组胎鼠海马神经元（包括T3组和Hypo-组），应用实时定量RT-PCR技术分析ephrin-A5/EphA5及该通道下游关键分子NMDAR-1（NR1）、PSD95、CaMKII的mRNA表达水平，通过Western Blot方法进行验证后，进一步运用免疫荧光染色检测其蛋白表达水平。
结果：
1.在先天性甲减大鼠脑中，ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白水平均下调且呈现显著的时空特征。（1）在不同实验组之间比较的结果表明：P0~P21，ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白的表达水平在甲减组海马、大脑皮层及小脑中均显著低于对照组(P<0.05)，且下降幅度最大的时间点集中在P7天（除了小脑中的NR1）；P0~P7，治疗组的表达水平在海马、大脑皮层及小脑与甲减组无显著差异(P>0.05)，P14~P21，治疗组的表达水平已显著高于甲减组但仍低于对照组(P<0.05)。（2）在不同脑组织之间比较的结果表明：P0~P21，甲减组、治疗组、对照组的ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白的表达水平在海马最高、大脑皮层其次、小脑最低，且在甲减组的海马中表达水平下降幅度最大。（3）在发育的不同时间点之间比较的结果表明：ephrin-A5、EphA5、NR1、PSD95、CaMKII在甲减组的海马、大脑皮层及小脑中的表达水平显著升高的时间点较对照组延迟，且峰值出现的时间点延迟或消失。
2.在原代培养的海马神经元中，Hypo-组ephrin-A5、EphA5、NR1、PSD95、
CaMKII基因和蛋白的表达水平显著低于对照组(P<0.05)；T3组的表达水平已显著高
于Hypo-组但仍低于对照组(P<0.05)。
结论：
Ephrin-A5/EphA5信号通道可能参与了先天性甲减大鼠神经元的突触发生障碍；
甲状腺素替代治疗不能完全逆转ephrin-A5/EphA5及该通道下游关键分子NR1、PSD95、CaMKII表达水平的下调。
目的：
检测先天性甲减大鼠海马中EphA5基因启动子DNA甲基化水平，探讨DNA甲基化与先天性甲减大鼠海马中EphA5表达调控的关系。
方法：
1.取P7天的甲减组及对照组大鼠海马组织各6份，及培养7d甲减组及对照组的海马神经元作为样本。
2.应用BSP克隆测序法检测样本的EphA5启动子CPG岛DNA甲基化水平；应用试剂盒测定样本的DNMT活力。
3.取培养7d的甲减组胎鼠海马神经元（包括Azadc组和Hypo-组），检测其EphA5启动子CPG岛DNA甲基化水平，同时应用实时定量RT-PCR技术和WesternBlot法分别检测EphA5基因mRNA和蛋白的表达水平。
结果：
1.先天性甲减大鼠海马和原代培养的甲减组海马神经元中，EphA5启动子CPG岛DNA甲基化水平均显著高于对照组(t=10.15,13.74; p<0.001)，与EphA5-mRNA表达水平均呈明显负相关(r=-0.957,-0.831; p<0.05)。
2.先天性甲减大鼠海马和原代培养的甲减组海马神经元中，DNMT活力均显著高于对照组(t=36.01,63.17; p<0.001)，与EphA5-mRNA表达水平均呈明显负相关(r=-0.998,-0.831; p<0.05)。
3. Azadc组与Hypo-组比较： EphA5基因DNA甲基化水平显著下降(t=7.33,p<0.05)，而EphA5基因mRNA和蛋白水平均显著上调(t=7.07,6.42; p<0.05)。
结论：
DNA甲基化参与了先天性甲减大鼠海马中EphA5表达的调控，去甲基化治疗可上调先天性甲减大鼠海马神经元中EphA5的表达。
Objective:
To establish the model of rat with congenital hypothyroidism, as well as to primarilyculture hippocampal neuron of fetal rat and further to intervene by group.
Methods:
1. Pregnant rats of G0(Gestational day, GD) were randomly divided into3groups:(1) the hypothyroid group, clean drinking water containing0.02%MMI was fed to thedams from G9until the pups were all executed,(2) the T4therapeutic group, cleandrinking water containing0.02%MMI was fed to the dams as same as the hypothyroidgroup, and the pups were treated with thyroxine by abdominal subcutaneous injectionseveryday from P7(Postnatal day, PD),(3) the control group, the dams were fed with cleandrinking water all the time.
2. We observed the general biological characteristics of all the dams and pups, anddynamically monitored their weight change and the level of serum thyroid hormone aswell.
3. The hippocampus, cerebral cortex and cerebellum of3groups were collected atP0,3,7,14and21individually. One part of the samples were stored in-80℃refrigeratorand reserved for the analysis of gene and protein. Another part were embedded withinparaffin and cut coronally for immunofluorescent staining.
4. The fetal rats of hypothyroid and control group were obtained at G17and isolatedfor hippocampus after decapitation. Hippocampal neurons were primarily cultured with theserum free medium which is called neurobasal containing B27and glutaminate. Weobserved the morphological changes of hippocampal neurons under inverted phase contrast microscope and took the hippocampal neurons of7day for identification and puritycalculation by MAP-2.
5. Hippocampal neurons of the hypothyroid group were divided into3groupsaccording to the medium:(1) T3group, the medium containing T3,(2) Azadc group, themedium containing Azadc,(3) Hypo-group, the serum free medium. Hippocampalneurons of the control group cultured during the same period were regarded as the controlgroup. Effects of different mediums on the growth of hippocampal neurons of thehypothyroid group were observed under the microscope. Activity of hippocampal neuronsof the above four groups were further determined by MTT colorimetric technique.
Results:
1. At G18, the weight of pregnant rats of the control group was significantly higherthan that of the hypothyroid group and the T4therapeutic group (P<0.05). The weight ofthe dams of3groups all dropped suddenly after the birth of the pups, then graduallyincreased during the whole location. But the acceleration speed of the weight of the controlgroup exceeded that of the hypothyroid group and the T4therapeutic group. The weight ofthe dams of the control group was significantly higher than that of the hypothyroid groupand the T4therapeutic group at P0,3,7,14and21(P<0.05). At the end of location, thelevel of serum FT3and FT4showed no difference between the hypothyroid dams and theT4therapeutic ones (P>0.05), but indicated marked difference in either the hypothyroidgroup or the T4therapeutic one and the control group (P<0.05).
2. Compared with the control group, pups in the hypothyroid group and the T4therapeutic one appeared smaller size, sluggish, unresponsive, hair growth late and glossdecrease. In addition, the time points of opening eyes initially and completely in thehypothyroid group were both later than that of the T4therapeutic group and the controls.The birth weight of the pups in the control group was significantly higher than that of thehypothyroid group and the T4therapeutic group (P<0.05). Although the weight of the T4therapeutic group at P14demonstrated significantly higher than that of the hypothyroidgroup, it was markedly lower than the control group (P<0.05). Until P21, there was nodifference of the weight between the T4therapeutic group and the controls (P>0.05). Thelevel of serum FT3and FT4in the T4therapeutic pups showed markedly higher than thehypothyroid pups, however lower than the controls (P<0.05). Until P21, the level of serumFT3and FT4in the T4therapeutic pups reached that in the controls, and the level of either
group was significantly higher than that of the hypothyroid pups.
3. The observation of morphological changes of the hippocampal neurons undermicroscope showed that hippocampal neurons in the hypothyroid group appeared smallercell body, shorter neurite, the fewer number of projections, and lower degree of intensivenetwork formed by projections when compared with the controls at the same time.However, the purity of hippocampal neurons in both groups reached more than96%.
Hippocampal neurons in the hypothyroid group presented longer neurite, the more number
of projections, higher degree of intensive network when intervened with different drugs.The analysis by MTT demonstrated that the activity of hippocampal neurons in eitherAzadc group or T3group was markedly higher than that in Hypo-group, but the above3groups were all significantly lower than the controls (P<0.05).
Conclusions:
The level of thyroid hormone and the general biological characteristics of both damsand pups indicate the successful production of model of congenital hypothyroid rat andtherapeutic rat. The observation of morphology and the identification and the activitydetermination of hippocampal neurons indicate the successful primary culture of normaland hypothyroid fetal rats and the successful grouping by drug intervention as well.
Objective:
To study the expressive characteristics of ephrin-A5/EphA5and several key moleculesin ephrin-A5/EphA5downstream signaling such as NMDAR-1（NR1）, PSD95andCaMKII in the brain of rat with congenital hypothyroidism. And to investigate therelationship between ephrin-A5/EphA5signal pathway and synaptogenesis disorder causedby thyroid hormone deficiency.
Methods:
1. The mRNA expression of ephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex and cerebellum of hypothyroid and therapeutic group and thecontrols at P0,3,7,14and21individually was measured by quantitative real time RT-PCR.And the protein expression was verified by Western Blot in these samples and furthermeasured by immunofluorescence.
2. Hippocampal neurons of7day in the control group and the hypothyroid groupincluding T3group and Hypo-group were taken as the samples. Real time PCR andWestern Blot assay were used to measure the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII. Immunofuorescent staining was performedto assess the protein expressive level of the above five molecules.
Results:
1. In the brain of congenital hypothyroid rat, the gene and protein expressive levelsof ephrin-A5, EphA5, NR1, PSD95and CaMKII were all down regulated with remarkablespatial and temporal characteristics.(1) The results were obtained from comparison amongthe different empirical groups: from P0to P21, the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex andcerebellum of hypothyroid group were all markedly lower than that of the controls(P<0.05), and the time point of the biggest decline was almost at P7except for NR1in thecerebellum; from P0to P7, there was no significant difference between the therapeutic andhypothyroid group in either the mRNA or protein expressive in the hippocampus, cerebralcortex and cerebellum (P>0.05), but the expressive level of the control group wassignificantly higher than that of the hypothyroid group and markedly lower than the controlgroup from P14to P21(P<0.05).(2) The results were obtained from comparison amongthe different brain tissues: the mRNA and protein levels of ephrin-A5, EphA5, NR1,PSD95and CaMKII were highest in the hippocampus, followed by the cerebral cortex andcerebellum the lowest in the hypothyroid group, the therapeutic group and the controlsfrom P0to P21; And the level of the biggest decline was in the hippocampus of thehypothyroid group.(3) The results were obtained from comparison among the differenttime points during the development: the time point when the expressive level ofephrin-A5、EphA5、NR1、PSD95、CaMKII in the hippocampus, cerebral cortex and cerebellum of the hypothyroid group markedly increased was postponed than that of thecontrols, and the time point of peak value delayed or vanished.
2. In hippocampal neurons of primary culture, the expressive levels of ephrin-A5,EphA5, NR1, PSD95and CaMKII in Hypo-group were significantly lower than that in thecontrols (P<0.05). The expressive level of T3group was significantly higher than that ofHypo-group but still markedly lower than that of control group (P<0.05).
Conclusions:
Ephrin-A5/EphA5signal pathway would participate in the synaptogenesis disorder ofneuron in congenital hypothyroid rat. The down regulation of expressive levels ofephrin-A5、EphA5、NR1、PSD95、CaMKII cannot be entirely reversible by the replacementtherapy with thyroxine.
Objective:
To detect the level of DNA methylation of EphA5promoter in the hippocampus ofcongenital hypothyroid rat. And to investigate the relationship between DNA methylationand the expressive regulation of EphA5in the hippocampus of rat with congenitalhypothyroidism.
Methods:
1. Both the hippocampus at P7and hippocampal neurons of7day in the hypothyroidgroup and the controls were regarded as the samples.
2. The level of DNA methylation of EphA5promoter CPG island in all the sampleswas detected by BSP cloning sequencing. And the activity of DNMT of all the samples wasdetected by kit.
3. Hippocampal neurons of7day in the hypothyroid group including Azadc groupand Hypo-group were taken as the samples. The level of DNA methylation of EphA5promoter CPG island of hippocampal neurons was detected by BSP cloning sequencing. Real time PCR and Western Blot assay were used to measure the gene and proteinexpressive levels of EphA5at the same time.
Results:
1. The levels of DNA methylation of EphA5promoter CPG island in both thehippocampus and hippocampal neurons of the hypothyroid group were significantly higherthan that of the controls (t=10.15,13.74; p<0.001), which were both negatively correlatedwith mRNA expression of EphA5(r=-0.957,-0.831; p<0.05).
2. The activities of DNMT in both the hippocampus and hippocampal neurons ofhypothyroid group were significantly higher than that of the controls (t=36.01,63.17;p<0.001), which were both negatively correlated with mRNA expression of EphA5(r=-0.998,-0.831; p<0.05).
3. The level of DNA methylation of EphA5gene was markedly dropped (t=7.33,p<0.05) while the levels of mRNA and protein of EphA5were both significantly upregulated (t=7.07,6.42; p<0.05) in Azadc group when compared with Hypo-group.
Conclusions:
DNA methylation of EphA5gene would participate in the expressive regulation ofEphA5in the hippocampus of congenital hypothyroid rat. The expression of EphA5inhippocampal neuron of rat with congenital hypothyroidism could be up-regulated bydemethylation therapy with Azadc.
建立先天性甲状腺功能减低大鼠模型、培养原代胎鼠海马神经元及分组干预。
方法：
1.取妊娠日(Gestational day, GD) G0天孕大鼠，随机分为三组，甲减组从G9天开始给予母鼠含0.02%MMI的清洁饮用水直至仔鼠全部处死；T4治疗组给予母鼠含0.02%MMI的清洁饮用水同甲减组，且从分娩日(Postnatal day, PD)第7天即P7天开始每日对仔鼠实施腹腔皮下注射甲状腺素(T4)；对照组一直给予清洁饮用水。
2.观察母鼠及仔鼠一般生物学特性，动态监测其体重变化及血清甲状腺激素水平。
3.在P0,3,7,14,21天分别收集上述三组仔鼠海马、大脑皮层、小脑，一部分存放于-80℃冰箱中备用，待作基因及蛋白分析；另一部分行石蜡包埋切片，待作免疫荧光染色。
4.于G17天取甲减组和对照组胎鼠，断头分离海马，用含B27、谷氨酰胺的Neurobasal无血清培养液培养原代海马神经元，倒置相差显微镜下观察海马神经元的形态变化。取培养7d的海马神经元，用MAP-2进行鉴定及纯度计算。
5.按照培养液不同，将培养的甲减组胎鼠海马神经元分为三组：T3组用含T3的培养液，Azadc组用含5-氮杂-2’-脱氧胞苷(Azadc)的培养液，Hypo-组用无血清培养液；并将同期培养的对照组胎鼠海马神经元作为对照组。镜下观察不同培养液干预对甲减组海马神经元生长的影响，并用MTT比色法测定以上四组海马神经元的活力。
结果：
1.母鼠在G18天时，对照组的体重明显高于甲减组与治疗组(P<0.05)；仔鼠出生后，三组母鼠体重均骤然下降，随后在整个哺乳期体重逐渐增加，但对照组的母鼠体重增加速度高于甲减组与治疗组，且在P3,7,14,21天对照组的母鼠体重明显高于甲减组与治疗组(P<0.05)。母鼠血清甲状腺激素测定结果显示在哺乳期末，甲减组和治疗组母鼠的血清FT3、FT4水平无显著差异(P>0.05)，但均显著低于对照组水平(P<0.05)。
2.甲减组和治疗组较对照组仔鼠体形小、行动迟缓、反应迟钝、毛发萌出晚且光泽度降低。开始睁眼和完全睁眼的时间甲减组均迟于对照组和治疗组。对照组仔鼠出生体重显著高于甲减组和治疗组(P<0.05)；治疗组的仔鼠体重在P14天已显著高于甲减组但仍低于对照组(P<0.05)；直至P21天，治疗组仔鼠体重与对照组已无显著差异(P>0.05)。仔鼠甲状腺激素测定结果显示：治疗组的仔鼠血清FT3、FT4水平在P14天，显著高于甲减组水平(P<0.05)，但仍显著低于对照组水平(P<0.05)；直至P21天，治疗组的仔鼠血清FT3、FT4水平已达到对照组水平(P>0.05)，均显著高于甲减组水平(P<0.05)。
3.镜下观察海马神经元的形态变化，结果发现甲减组海马神经元与对照组比较，在同一时间点细胞胞体体积较小，突起形成的长度较短、突起的支数较少，突起形成的网络密集程度较低；但是细胞纯度均能达到96%以上。甲减组海马神经元经不同药液干预后突起增多增长、网络密集程度提高；MTT比色分析显示：Azadc组和T3组的细胞活力均显著高于Hypo-组，但这三组的细胞活力均显著低于对照组(P<0.05)。
结论：
母鼠、仔鼠甲状腺激素水平及一般生物学特性表明先天性甲减组及治疗组大鼠模型均成功建立。细胞形态学的观察、鉴定及活力测定表明原代正常和甲减胎鼠海马神经元培养及药物分组干预成功。
目的：
研究ephrin-A5/EphA5信号通道及下游关键分子NMDAR-1（NR1）、PSD95、CaMKII在先天性甲减大鼠脑中的表达特征，并初步探讨ephrin-A5/EphA5信号通道与甲状腺激素缺乏致突触发生障碍的关系。
方法：
1.分别于P0,3,7,14,21天，应用实时定量RT-PCR技术分析甲减组、治疗组、对照组海马、大脑皮层、小脑组织ephrin-A5/EphA5及该通道下游关键分子NMDAR-1（NR1）、PSD95、CaMKII的mRNA表达水平，通过Western Blot方法进行验证后，进一步运用免疫荧光染色检测其蛋白表达水平。
2.取培养7d的对照组和甲减组胎鼠海马神经元（包括T3组和Hypo-组），应用实时定量RT-PCR技术分析ephrin-A5/EphA5及该通道下游关键分子NMDAR-1（NR1）、PSD95、CaMKII的mRNA表达水平，通过Western Blot方法进行验证后，进一步运用免疫荧光染色检测其蛋白表达水平。
结果：
1.在先天性甲减大鼠脑中，ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白水平均下调且呈现显著的时空特征。（1）在不同实验组之间比较的结果表明：P0~P21，ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白的表达水平在甲减组海马、大脑皮层及小脑中均显著低于对照组(P<0.05)，且下降幅度最大的时间点集中在P7天（除了小脑中的NR1）；P0~P7，治疗组的表达水平在海马、大脑皮层及小脑与甲减组无显著差异(P>0.05)，P14~P21，治疗组的表达水平已显著高于甲减组但仍低于对照组(P<0.05)。（2）在不同脑组织之间比较的结果表明：P0~P21，甲减组、治疗组、对照组的ephrin-A5、EphA5、NR1、PSD95、CaMKII基因和蛋白的表达水平在海马最高、大脑皮层其次、小脑最低，且在甲减组的海马中表达水平下降幅度最大。（3）在发育的不同时间点之间比较的结果表明：ephrin-A5、EphA5、NR1、PSD95、CaMKII在甲减组的海马、大脑皮层及小脑中的表达水平显著升高的时间点较对照组延迟，且峰值出现的时间点延迟或消失。
2.在原代培养的海马神经元中，Hypo-组ephrin-A5、EphA5、NR1、PSD95、
CaMKII基因和蛋白的表达水平显著低于对照组(P<0.05)；T3组的表达水平已显著高
于Hypo-组但仍低于对照组(P<0.05)。
结论：
Ephrin-A5/EphA5信号通道可能参与了先天性甲减大鼠神经元的突触发生障碍；
甲状腺素替代治疗不能完全逆转ephrin-A5/EphA5及该通道下游关键分子NR1、PSD95、CaMKII表达水平的下调。
目的：
检测先天性甲减大鼠海马中EphA5基因启动子DNA甲基化水平，探讨DNA甲基化与先天性甲减大鼠海马中EphA5表达调控的关系。
方法：
1.取P7天的甲减组及对照组大鼠海马组织各6份，及培养7d甲减组及对照组的海马神经元作为样本。
2.应用BSP克隆测序法检测样本的EphA5启动子CPG岛DNA甲基化水平；应用试剂盒测定样本的DNMT活力。
3.取培养7d的甲减组胎鼠海马神经元（包括Azadc组和Hypo-组），检测其EphA5启动子CPG岛DNA甲基化水平，同时应用实时定量RT-PCR技术和WesternBlot法分别检测EphA5基因mRNA和蛋白的表达水平。
结果：
1.先天性甲减大鼠海马和原代培养的甲减组海马神经元中，EphA5启动子CPG岛DNA甲基化水平均显著高于对照组(t=10.15,13.74; p<0.001)，与EphA5-mRNA表达水平均呈明显负相关(r=-0.957,-0.831; p<0.05)。
2.先天性甲减大鼠海马和原代培养的甲减组海马神经元中，DNMT活力均显著高于对照组(t=36.01,63.17; p<0.001)，与EphA5-mRNA表达水平均呈明显负相关(r=-0.998,-0.831; p<0.05)。
3. Azadc组与Hypo-组比较： EphA5基因DNA甲基化水平显著下降(t=7.33,p<0.05)，而EphA5基因mRNA和蛋白水平均显著上调(t=7.07,6.42; p<0.05)。
结论：
DNA甲基化参与了先天性甲减大鼠海马中EphA5表达的调控，去甲基化治疗可上调先天性甲减大鼠海马神经元中EphA5的表达。
Objective:
To establish the model of rat with congenital hypothyroidism, as well as to primarilyculture hippocampal neuron of fetal rat and further to intervene by group.
Methods:
1. Pregnant rats of G0(Gestational day, GD) were randomly divided into3groups:(1) the hypothyroid group, clean drinking water containing0.02%MMI was fed to thedams from G9until the pups were all executed,(2) the T4therapeutic group, cleandrinking water containing0.02%MMI was fed to the dams as same as the hypothyroidgroup, and the pups were treated with thyroxine by abdominal subcutaneous injectionseveryday from P7(Postnatal day, PD),(3) the control group, the dams were fed with cleandrinking water all the time.
2. We observed the general biological characteristics of all the dams and pups, anddynamically monitored their weight change and the level of serum thyroid hormone aswell.
3. The hippocampus, cerebral cortex and cerebellum of3groups were collected atP0,3,7,14and21individually. One part of the samples were stored in-80℃refrigeratorand reserved for the analysis of gene and protein. Another part were embedded withinparaffin and cut coronally for immunofluorescent staining.
4. The fetal rats of hypothyroid and control group were obtained at G17and isolatedfor hippocampus after decapitation. Hippocampal neurons were primarily cultured with theserum free medium which is called neurobasal containing B27and glutaminate. Weobserved the morphological changes of hippocampal neurons under inverted phase contrast microscope and took the hippocampal neurons of7day for identification and puritycalculation by MAP-2.
5. Hippocampal neurons of the hypothyroid group were divided into3groupsaccording to the medium:(1) T3group, the medium containing T3,(2) Azadc group, themedium containing Azadc,(3) Hypo-group, the serum free medium. Hippocampalneurons of the control group cultured during the same period were regarded as the controlgroup. Effects of different mediums on the growth of hippocampal neurons of thehypothyroid group were observed under the microscope. Activity of hippocampal neuronsof the above four groups were further determined by MTT colorimetric technique.
Results:
1. At G18, the weight of pregnant rats of the control group was significantly higherthan that of the hypothyroid group and the T4therapeutic group (P<0.05). The weight ofthe dams of3groups all dropped suddenly after the birth of the pups, then graduallyincreased during the whole location. But the acceleration speed of the weight of the controlgroup exceeded that of the hypothyroid group and the T4therapeutic group. The weight ofthe dams of the control group was significantly higher than that of the hypothyroid groupand the T4therapeutic group at P0,3,7,14and21(P<0.05). At the end of location, thelevel of serum FT3and FT4showed no difference between the hypothyroid dams and theT4therapeutic ones (P>0.05), but indicated marked difference in either the hypothyroidgroup or the T4therapeutic one and the control group (P<0.05).
2. Compared with the control group, pups in the hypothyroid group and the T4therapeutic one appeared smaller size, sluggish, unresponsive, hair growth late and glossdecrease. In addition, the time points of opening eyes initially and completely in thehypothyroid group were both later than that of the T4therapeutic group and the controls.The birth weight of the pups in the control group was significantly higher than that of thehypothyroid group and the T4therapeutic group (P<0.05). Although the weight of the T4therapeutic group at P14demonstrated significantly higher than that of the hypothyroidgroup, it was markedly lower than the control group (P<0.05). Until P21, there was nodifference of the weight between the T4therapeutic group and the controls (P>0.05). Thelevel of serum FT3and FT4in the T4therapeutic pups showed markedly higher than thehypothyroid pups, however lower than the controls (P<0.05). Until P21, the level of serumFT3and FT4in the T4therapeutic pups reached that in the controls, and the level of either
group was significantly higher than that of the hypothyroid pups.
3. The observation of morphological changes of the hippocampal neurons undermicroscope showed that hippocampal neurons in the hypothyroid group appeared smallercell body, shorter neurite, the fewer number of projections, and lower degree of intensivenetwork formed by projections when compared with the controls at the same time.However, the purity of hippocampal neurons in both groups reached more than96%.
Hippocampal neurons in the hypothyroid group presented longer neurite, the more number
of projections, higher degree of intensive network when intervened with different drugs.The analysis by MTT demonstrated that the activity of hippocampal neurons in eitherAzadc group or T3group was markedly higher than that in Hypo-group, but the above3groups were all significantly lower than the controls (P<0.05).
Conclusions:
The level of thyroid hormone and the general biological characteristics of both damsand pups indicate the successful production of model of congenital hypothyroid rat andtherapeutic rat. The observation of morphology and the identification and the activitydetermination of hippocampal neurons indicate the successful primary culture of normaland hypothyroid fetal rats and the successful grouping by drug intervention as well.
Objective:
To study the expressive characteristics of ephrin-A5/EphA5and several key moleculesin ephrin-A5/EphA5downstream signaling such as NMDAR-1（NR1）, PSD95andCaMKII in the brain of rat with congenital hypothyroidism. And to investigate therelationship between ephrin-A5/EphA5signal pathway and synaptogenesis disorder causedby thyroid hormone deficiency.
Methods:
1. The mRNA expression of ephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex and cerebellum of hypothyroid and therapeutic group and thecontrols at P0,3,7,14and21individually was measured by quantitative real time RT-PCR.And the protein expression was verified by Western Blot in these samples and furthermeasured by immunofluorescence.
2. Hippocampal neurons of7day in the control group and the hypothyroid groupincluding T3group and Hypo-group were taken as the samples. Real time PCR andWestern Blot assay were used to measure the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII. Immunofuorescent staining was performedto assess the protein expressive level of the above five molecules.
Results:
1. In the brain of congenital hypothyroid rat, the gene and protein expressive levelsof ephrin-A5, EphA5, NR1, PSD95and CaMKII were all down regulated with remarkablespatial and temporal characteristics.(1) The results were obtained from comparison amongthe different empirical groups: from P0to P21, the gene and protein expressive levels ofephrin-A5, EphA5, NR1, PSD95and CaMKII in the hippocampus, cerebral cortex andcerebellum of hypothyroid group were all markedly lower than that of the controls(P<0.05), and the time point of the biggest decline was almost at P7except for NR1in thecerebellum; from P0to P7, there was no significant difference between the therapeutic andhypothyroid group in either the mRNA or protein expressive in the hippocampus, cerebralcortex and cerebellum (P>0.05), but the expressive level of the control group wassignificantly higher than that of the hypothyroid group and markedly lower than the controlgroup from P14to P21(P<0.05).(2) The results were obtained from comparison amongthe different brain tissues: the mRNA and protein levels of ephrin-A5, EphA5, NR1,PSD95and CaMKII were highest in the hippocampus, followed by the cerebral cortex andcerebellum the lowest in the hypothyroid group, the therapeutic group and the controlsfrom P0to P21; And the level of the biggest decline was in the hippocampus of thehypothyroid group.(3) The results were obtained from comparison among the differenttime points during the development: the time point when the expressive level ofephrin-A5、EphA5、NR1、PSD95、CaMKII in the hippocampus, cerebral cortex and cerebellum of the hypothyroid group markedly increased was postponed than that of thecontrols, and the time point of peak value delayed or vanished.
2. In hippocampal neurons of primary culture, the expressive levels of ephrin-A5,EphA5, NR1, PSD95and CaMKII in Hypo-group were significantly lower than that in thecontrols (P<0.05). The expressive level of T3group was significantly higher than that ofHypo-group but still markedly lower than that of control group (P<0.05).
Conclusions:
Ephrin-A5/EphA5signal pathway would participate in the synaptogenesis disorder ofneuron in congenital hypothyroid rat. The down regulation of expressive levels ofephrin-A5、EphA5、NR1、PSD95、CaMKII cannot be entirely reversible by the replacementtherapy with thyroxine.
Objective:
To detect the level of DNA methylation of EphA5promoter in the hippocampus ofcongenital hypothyroid rat. And to investigate the relationship between DNA methylationand the expressive regulation of EphA5in the hippocampus of rat with congenitalhypothyroidism.
Methods:
1. Both the hippocampus at P7and hippocampal neurons of7day in the hypothyroidgroup and the controls were regarded as the samples.
2. The level of DNA methylation of EphA5promoter CPG island in all the sampleswas detected by BSP cloning sequencing. And the activity of DNMT of all the samples wasdetected by kit.
3. Hippocampal neurons of7day in the hypothyroid group including Azadc groupand Hypo-group were taken as the samples. The level of DNA methylation of EphA5promoter CPG island of hippocampal neurons was detected by BSP cloning sequencing. Real time PCR and Western Blot assay were used to measure the gene and proteinexpressive levels of EphA5at the same time.
Results:
1. The levels of DNA methylation of EphA5promoter CPG island in both thehippocampus and hippocampal neurons of the hypothyroid group were significantly higherthan that of the controls (t=10.15,13.74; p<0.001), which were both negatively correlatedwith mRNA expression of EphA5(r=-0.957,-0.831; p<0.05).
2. The activities of DNMT in both the hippocampus and hippocampal neurons ofhypothyroid group were significantly higher than that of the controls (t=36.01,63.17;p<0.001), which were both negatively correlated with mRNA expression of EphA5(r=-0.998,-0.831; p<0.05).
3. The level of DNA methylation of EphA5gene was markedly dropped (t=7.33,p<0.05) while the levels of mRNA and protein of EphA5were both significantly upregulated (t=7.07,6.42; p<0.05) in Azadc group when compared with Hypo-group.
Conclusions:
DNA methylation of EphA5gene would participate in the expressive regulation ofEphA5in the hippocampus of congenital hypothyroid rat. The expression of EphA5inhippocampal neuron of rat with congenital hypothyroidism could be up-regulated bydemethylation therapy with Azadc.
引文
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