甲状腺激素对仔鼠大脑皮层缝隙连接蛋白表达的影响
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摘要
前言
先天性甲状腺功能减低症(先天性甲低)是儿科常见的内分泌代谢性疾病之一,是由于各种原因导致先天性甲状腺分泌的甲状腺激素(TH)不足引起,是造成儿童智力障碍的原因之一,一直是儿童内分泌医务人员关注的重点。随着分子生物学的发展,我们对TH的生理作用有了更深入的了解,TH在细胞核内与其受体(TR)结合,作用于靶基因启动子的甲状腺激素反应元件(TREs),在转录水平调节靶基因的表达,然而甲低具体通过影响哪些基因,从而导致儿童智力障碍的机制目前尚不十分清楚。信号蛋白和相关的功能蛋白是细胞各种信息传导及各种功能代谢活动的执行者,为神经细胞生长、分化等活动的基础,也是神经感觉、运动、学习和记忆等功能活动形成机制的重要环节,深入研究TH对脑组织信号系统相关蛋白的基因学调节,能更全面地揭示甲状腺调控脑发育机制和先天性甲低的发病机制。
神经系统经典的细胞信号连接主要指突触部位的神经递质传递,以往研究表明TH参与多种神经递质的合成、降解,以及递质受体表达和功能。事实上,神经系统还存在一些非经典的信号传导系统,如缝隙连接(GJ),它们也介导了神经系统的一些重要功能。GJ是构成相邻细胞间的细胞缝隙连接通讯(GJC)——一种电突触的结构基础。GJ关键成分是缝隙连接蛋白(Cx),其结构单位是由6个Cx亚单位构成连接子,相邻的细胞膜对应面上的一对连接子构成GJ通道-直径约1.5nm的亲水性通道。神经系统有丰富的GJ,至少有12种Cx在神经系统中表达,以Cx43最多,占90-95%,然而不同神经细胞上Cx分布存在差异,其中星形胶质细胞上主要是Cx43,少突胶质细胞上主要表达Cx32,神经元上主要表达Cx36,最近发现Cx45也有在中枢神经系统表达,数量也比较丰富。
研究表明,星形胶质细胞通过GJ相互连接形成功能合胞体,GJ还可在中枢神经系统内构成复杂的神经元-胶质细胞-神经元的信息网络,GJ连通相互耦联细胞的胞浆,允许一些小分子物质在细胞间通过,为细胞间物质传递和信息交流提供了直接通路,还有部分连接子并没有与相邻细胞形成GJ,直接与细胞外接触,是细胞内外物质交换和信息交流的一种直接通道。GJ在维持内外环境稳定、协调神经元功能方面起着非常重要的作用,在神经细胞的分化、发育和生理功能的调节中也起重要作用,而且是脑内长短信号转导的结构基础,已经证实许多疾病与GJ功能异常有关。研究表明GJ的通透功能可塑性比较大,先前研究发现甲状腺激素可能影响心肌细胞和睾丸支持细胞Cx43的表达,但结果也尚有争议。然而,至今尚无有关TH对中枢神经系统Cx表达影响的报道。
为此,我们首先通过建立先天性甲状腺功能减低症的动物模型,检测先天性甲低对大脑皮层Cx43、Cx32和Cx45表达的影响。再应用离体细胞培养技术,观察不同甲状腺素浓度对大脑皮层星形胶质细胞Cx43、Cx32和Cx45表达的影响,以进一步了解TH对星形胶质细胞Cx43、Cx32和Cx45表达的调节作用,为深入研究先天性甲低发病机制和保护干预措施等提供实验依据。
研究目的
通过检测先天性甲低仔鼠大脑皮层Cx43、Cx32和Cx45表达,以及不同甲状腺素浓度培养对大脑皮层星形胶质细胞Cx43、Cx32和Cx45表达变化,探讨先天性甲低对这些缝隙连接蛋白表达的影响。
动物模型部分
1材料与方法
1.1动物模型建立和分组
清洁级健康成年C57BL／6J合笼交配后自行产子,按处理条件不同分甲低组和对照组。
甲低组：合笼第10天开始给予母鼠含0.03%甲巯咪唑的饮用水,一直到仔鼠出生7天。
对照组：一直喂养清洁饮用水,其它实验控制因素同甲低组。
在仔鼠出生1、7、14和21天麻醉后处死,生理盐水灌注,留取大脑皮层标本。
1.2指标检测
采用Real time RT-PCR法检测Cx43、Cx32和Cx45的mRNA水平。采用AxyPrep总RNA小量制备试剂盒提取脑组织总RNA,自行设计引物,应用Takara荧光PCR试剂盒进行RT-PCR扩增(两步法)。目的基因mRNA水平采用ΔCt表示。
采用Western blot法检测Cx43、Cx32和Cx45的蛋白质水平。应用RIPA裂解液提取脑组织蛋白质,采用BCA试剂盒进行蛋白质定量,在丙烯酰胺电泳和转膜后,依次封闭、一抗和二抗孵育、放射自显影,保存胶片,最后统一将放射自显影条带进行扫描,对蛋白条带进行灰度分析,将Cx/β-actin的比值作为Cx的相对表达量。
新鲜脑组织予10%中性福尔马林固定和石蜡包埋,采用"EnvisionTM二步法”进行免疫组化检测。
2结果
对照组出生后Cx43表达上升,7天时达到高峰,此后基本维持稳定水平；而甲低组出生后Cx43表达上升较慢。与对照组比较,1、7和14天甲低组Cx43mRNA有所下降,但差异均不具有显著性意义。1和7天甲低组Cx43蛋白质有下降,其中1天时差异在边界水平,7天时差异具有显著性意义。免疫组化显示大脑皮层Cx43表达均非常丰富,广泛分布在神经细胞的胞体和神经纤维部位。
对照组大脑皮层Cx32表达水平相对较低,在出生后进一步呈逐渐下降趋势。甲低组在7、14和21天时Cx32 mRNA均有明显升高。不同日龄甲低组Cx32蛋白质均有上升,其中7日龄时差异具有显著性意义,14日龄时差异在边界水平。免疫组化显示大脑皮层的Cx32表达明显较Cx43低,部分神经元胞体和少量神经纤维阳性。
对照组大脑皮层Cx45水平处于Cx45和Cx32间,出生后也逐渐下降。两组间不同时间点Cx45mRNA和蛋白质表达差异均无显著意义。免疫组化显示大脑皮层Cx45表达比较丰富,也较广泛分布于神经细胞胞体和纤维部位。
细胞培养部分
1材料和方法
1.1培养基配制
购买活性炭吸附后低甲状腺激素胎牛血清(FBS)和未特殊处理的胎牛血清,同时购买T3配制T3终浓度为5 nmol/L的DMEM液。按下列方法配制各种培养液,并测定各组培养液总T3(TT3)和游离T3(FT3)甲状腺激素浓度。
培养基Ⅰ：普通DMEM+15%普通FBS(TT3:5.4nmol/L, FT3:22.0pmol/L);
培养基Ⅱ：普通DMEM+15%低TH的FBS(TT3:0.6 nmol/L, FT3:1.9 pmol/L);
培养基Ⅲ：含5 nmol/L T3的DMEM+15%低TH的FBS(TT3 4.3 nmol/L, FT3 23.2 pmol/L);
1.2细胞培养和分组
无菌条件下,取生后1～2d健康SD大鼠大脑皮层,置Hanks液中清洗、剪碎、胰蛋白酶消化后,置于培养基中培养。于培养8天后用抗神经胶质酸性蛋白(GFAP)抗体对星形胶质细胞进行鉴定。按照培养基不同,将培养的细胞分为4组：
对照组：培养基Ⅰ×2天+培养基Ⅲ×6天
甲低组：培养基Ⅰ×2天+培养基Ⅱ×6天
干预组1：培养基Ⅰ×2天+培养基Ⅱ×2天+培养基Ⅲ×4天
干预组2：培养基Ⅰ×2天+培养基Ⅱ×4天+培养基Ⅲ×2天
1.3检测指标和方法
采用四甲基偶氮唑蓝(MTT)方法绘制生长曲线。采用Takara Real time RT-PCR试剂盒对培养细胞的Cx43、Cx32和Cx45 mRNA进行检测,同时应用Western blot方法对Cx43、Cx32和Cx45的蛋白质水平进行检测,同时应用免疫荧光染色激光共聚焦扫描对蛋白质表达进行定位观察。
2结果
甲低组细胞增殖缓慢,指数增长期后移,干预后细胞增殖有所改善。两组星形胶质细胞Cx43表达均丰富。甲低组星形胶质细胞Cx43表达下降,正常甲状腺激素浓度干预后又有上升趋势,早期干预4天其mRNA水平即能恢复到正常水平,但蛋白质表达水平尚不能恢复到正常水平。激光共聚焦扫描显示离体培养星形胶质细胞Cx43蛋白质表达均非常丰富,分布于胞体和树突的胞浆内,正常组多数细胞间有荧光强度特别高的点状结构,甲低组荧光强度较低,点状结构少而且荧光强度较低,干预组1和干预组2荧光强度和高荧光强度的点状结构有恢复趋势,但干预组2高荧光强度的点状结构不均匀。
两组星形胶质细胞Cx32mRNA水平均较低。与对照组比较,甲低组Cx32mRNA更低,在干预后mRNA表达水平能较快上升,达到正常水平。然而,无论Western blot检测,还是激光共聚焦扫描,均显示各组离体培养星形胶质细胞的Cx32蛋白质表达均阴性。
两组星形胶质细胞Cx45表达均较丰富。甲低组Cx45表达水平明显下降,干预后有所上升,早期干预后2-4天可恢复到正常水平。激光共聚焦扫描显示各组离体培养星形胶质细胞的Cx45蛋白表达均非常丰富,分布于胞体和树突的胞浆内,少部分细胞间有荧光强度特别高的点状结构。
结论
1.先天性甲低可导致仔鼠大脑皮层总Cx43表达下降和总Cx32表达上升；
2.TH下降可导致离体培养的大脑皮层星形胶质细胞Cx43和Cx45表达下降,GJ分布异常；
3.TH对神经细胞Cx基因调控具有时空性；
4.短期的T3早期干预,能使星形胶质细胞Cx43和Cx45表达有恢复上升趋势,但Cx43不能恢复至正常水平。
Background
Congenital hypothyroidism (CH), due to insufficient secretion of thyroid hormone (TH) result from variety of reasons, is a common pediatric endocrine disease. It is also a main cause of childhood mental retarded and draws more and more attention from endocrine pediatrician. With the development of molecular biology, physiology of TH has been better understood. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoters. However, the candidate genes which be regulated by TH and cause mental retardation in children with CH are still unclear. The signaling proteins and related proteins have effect on the information transfer and the various metabolic activity, and then on nerve cell growth and differentiation. They also take part in the mechanisms of sensory, motor, learning, memory and other activities. Hence, study the effect of TH on the signaling proteins and its related proteins may help us to better understand the effect of TH on brain development and the mechanism of CH.
The classic information transfer in nervous system is the neurotransmitter transmittion throught the synapses, or the chemical synapses. Previous studies showed that TH had effect on the synthesis and degradation of neurotransmitters, and on the expression and function of their receptors. Recently, studies found several several non-classic information transfer pathway in the nervous system, such as gap junction (GJ) communication (GJC) signal transduction systems, which play important role on some nervous activities.
GJ is the morphological substrate of one type of electrical synapse and mediates the GJC among adjacent cells. The key component of GJ is connexin (Cx). GJ is composed of a pair of connexon that includes six Cx proteins and make a hemichannel with a diameter of about 1.5 nm. There is abundant GJ in the nervous system, including at least 12 types of Cx. Cx43 is the predominant type and accounts for 90-95% of the total Cx protein. Moreover, types of Cx on different nerve cells are different. Cx32 is mainly in oligodendrocytes while Cx43 is mainly in astrocytes. Recently, Cx45 is also found in the central nervous system.
Studies have shown that astrocytes interconnected as a syncytium through GJ and GJ may also constitute a complex of neurons-glial cells-neurons network. GJ allow the transfer of small molecules from cell to adjust cell, also information communication direct. Moreover, some Cx did not form a GJ with the adjacent cells, forming a direct channel for material and information exchange from the extracellular environments. Efficient intercellular communication at GJ is essential for normal electro-mechanical function of the brain. Compared with synapse, GJC has direct, fast and bidirectional characteristics. GJ has an important role on stability of the intercellular and extracellular environments, on nerve cell proliferation, differentiation, and development, also the signal transduction in brain. Several diseases have been associated with the disorder of GJ. Several studies showed that TH may affect the Cx43 expression on the myocardial and Sertoli cells, although the results are still controversial. However, no similar study about the effect of TH on the Cx in central nervous system was reported.
Here, we established a CH model firstly, and measured the total levels Cx43, Cx32 and Cx45 on cerebral cortex in this model. Then, cultuered in the astrocytes by different concentration of T3 and measured the levels of these Cxs.
Objectives
To investigate the effect of TH on Cx3 expression by measuring the Cx43, Cx32 and Cx45 expression in cerebral cortex in mice CH model, and these Cx in the astrocytes cultuered by different concentration of T3.
Mice CH Model
1 Materials and Methods
1.1 Animal Model
Health adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water. The CH group is definted as feeding 0.03% thiamazole contained water from the 10th day after mate to 7th day after the offsprings'birth. The offsprings were sacrificed on day 1,7,14 and 21. Saline perfusion was performed and the cerebral cortex was collected.
1.2 Parameter Measurements
The levels of Cxs mRNA were measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit. Self-designed primers and SYBR PrimeScriptTM PCR Kit were used real time RT-PCR. Two-step method was used for Cx43, Cx32, and Cx45. ACt was presented for target gene mRNA levels.
The levels of Cx protein were measured by Western blot. Proteins were extracted by RIPA and the concentration was measured by BCA Protein Assay Kit. A 50μg total protein was used for SDS-PAGE electrophoresis and transferred PVDF. Then blocked, combined with first and second antibodies, and ECL radioautography in sequence. Finally, scan the radiation autoradiography strip and analysis the protein bands with gray-scale. The Cx/β-actin ratio was calculated as the relative levels of Cx.
Tissue was fixed in 10% neutral formalin and embeded in paraffin. "EnvisionTM two-step" method was used for immunohistochemistry.
2 Results
In control group, the levels of Cx43 expression on the cerebral cortex increased after birth and reach the peak at day 7, while the levels of Cx43 increased slowly in CH group. Commpred with the control group, the Cx43 mRNA levels in CH group were lower on day 1,7 and 14 without significant difference. Moreover, the Cx43 protein in CH group was lower than these in controls with a marginal difference on day 1 and a significant difference on day 7. Immunohistochemistry showed Cx43 protein was abundant on the cerebral cortex in both groups, including the body of nerve cells and nerve fibers.
The levels of Cx32 were low on the cerebral cortex in the control and CH goups, and decreased after birth. Compared with the controls, the levels of Cx32 mRNA were significantly higher in the CH group on day 7,14 and 21, and the Cx32 protein increased with a significant difference on day 7 and a marginal difference on day 14. Immunohistochemistry showed that the expression of Cx32 was significantly less than the Cx43 on the cerebral cortex, and some nerve cells and few nerve fibers were positive.
The levels of Cx45 were less than Cx43 and higher than Cx32, and decreased after birth. Both mRNA and protein of Cx45 had no significant difference between the two groups. Immunohistochemistry showed that Cx45 expression is similar to the distribution Cx43, and most body of nerve cells and nerve fibers was positive
Cell Culture
1 Materials and Methods
1.1 Medium Preparation
Low TH fetal bovine serum (activated carbon adsorbed FBS), common fetal bovine and serum T3 were purchased. DMEM solution with 5 nmol/L of T3 was prepared. A variety of DMEM culture mediums were prepared as following and TH were measured.
MediumⅠ, common DMEM+ common FBS (TT3:5.4 nmol/L, FT3:22.0 pmol/L).
MediumⅡ, common DMEM+ 15% low TH FBS (TT3:0.6 nmol/L, FT3:1.9 pmol/L).
MediumⅢ, DMEM with 5 nmol/L T3+ 15% low TH FBS (TT3 4.3 nmol/L, FT3 23.2 pmol/L).
1.2 Cell Culture and Grouping
Cerebral cortex was obtained from 1-2 d age healthy SD rat under sterile conditions. Then, washed with Hanks solution, shredded, trypsin digestion and cultured in medium. The astrocytes were identified with anti-GFAP antibody at 8th day, and they were divided into 4 groups according to the different medium.
Control group:mediumⅠfor 2 days + mediumⅢfor 6 days
Hypothyroidism group:mediumⅠfor 2 days + mediumⅡfor 6 days
Intervention group 1:mediumⅠfor 2 days + mediumⅡfor 2 days + mediumⅢfor 4 days
Intervention group 2:mediumⅠfor 2 days + mediumⅡfor 4 days + mediumⅢfor 2 days
1.3 Parameter Measurements
Tetrazolium blue (MTT) method was used to draw the growth curve. The expression of Cx43, Cx32 and Cx45 mRNA in the cultured cells were detected by using the Takara Real time RT-PCR kit. The levels of Cx43, Cx32 and Cx45 protein were measured by Western blot, and laser scanning confocal microscope was used for locating the protein in the cells.
2 Results
The cell proliferation was slow and exponential growth phase was right shift in the hypothyroidism group, and improved after the intervention. The expression of Cx43 in astrocyte decreased in the hypothyroidism group. After T3 intervention, the Cx43 levels had an upward trend. They mRNA levels of Cx43 can recovry to normal levels while the protein levels did not return to normal levels. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were many well-distributed high- fluorescence intensity points between adjacent astrocytes in control group while the point structures in hypothyroidism group were less with low fluorescence intensity. After T3 intervention, the point structures were increased, but asymmetrical distribution.
The mRNA level Cx32 in the hypothyroidism group was lower than that in the controls, and it can be rapidly increased to near normal levels after T3 intervention. However, the protein was undetectable both using Western blot and laser scanning confocal microscopy.
A low Cx45 level was noted in the hypothyroidism group as well, and it can return to normal levels with early intervention. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were only few high- fluorescence intensity points between adjacent astrocytes.
Conclusions
1. The levels of Cx43 decrease and the levels of Cx32 increase in the cerebral cortex of CH mice.
2. Low TH induces low Cx43 and Cx45 expression, and the abnormality of GJ distribution in astrocytes.
3. The regulation of TH on gene transcription has a spatial and temporal control.
4. Short-term T3 early intervention increases the Cx43 and Cx45 expression, but the levels of Cx 43 does not recovery to normal levels.
先天性甲状腺功能减低症(先天性甲低)是儿科常见的内分泌代谢性疾病之一,是由于各种原因导致先天性甲状腺分泌的甲状腺激素(TH)不足引起,是造成儿童智力障碍的原因之一,一直是儿童内分泌医务人员关注的重点。随着分子生物学的发展,我们对TH的生理作用有了更深入的了解,TH在细胞核内与其受体(TR)结合,作用于靶基因启动子的甲状腺激素反应元件(TREs),在转录水平调节靶基因的表达,然而甲低具体通过影响哪些基因,从而导致儿童智力障碍的机制目前尚不十分清楚。信号蛋白和相关的功能蛋白是细胞各种信息传导及各种功能代谢活动的执行者,为神经细胞生长、分化等活动的基础,也是神经感觉、运动、学习和记忆等功能活动形成机制的重要环节,深入研究TH对脑组织信号系统相关蛋白的基因学调节,能更全面地揭示甲状腺调控脑发育机制和先天性甲低的发病机制。
神经系统经典的细胞信号连接主要指突触部位的神经递质传递,以往研究表明TH参与多种神经递质的合成、降解,以及递质受体表达和功能。事实上,神经系统还存在一些非经典的信号传导系统,如缝隙连接(GJ),它们也介导了神经系统的一些重要功能。GJ是构成相邻细胞间的细胞缝隙连接通讯(GJC)——一种电突触的结构基础。GJ关键成分是缝隙连接蛋白(Cx),其结构单位是由6个Cx亚单位构成连接子,相邻的细胞膜对应面上的一对连接子构成GJ通道-直径约1.5nm的亲水性通道。神经系统有丰富的GJ,至少有12种Cx在神经系统中表达,以Cx43最多,占90-95%,然而不同神经细胞上Cx分布存在差异,其中星形胶质细胞上主要是Cx43,少突胶质细胞上主要表达Cx32,神经元上主要表达Cx36,最近发现Cx45也有在中枢神经系统表达,数量也比较丰富。
研究表明,星形胶质细胞通过GJ相互连接形成功能合胞体,GJ还可在中枢神经系统内构成复杂的神经元-胶质细胞-神经元的信息网络,GJ连通相互耦联细胞的胞浆,允许一些小分子物质在细胞间通过,为细胞间物质传递和信息交流提供了直接通路,还有部分连接子并没有与相邻细胞形成GJ,直接与细胞外接触,是细胞内外物质交换和信息交流的一种直接通道。GJ在维持内外环境稳定、协调神经元功能方面起着非常重要的作用,在神经细胞的分化、发育和生理功能的调节中也起重要作用,而且是脑内长短信号转导的结构基础,已经证实许多疾病与GJ功能异常有关。研究表明GJ的通透功能可塑性比较大,先前研究发现甲状腺激素可能影响心肌细胞和睾丸支持细胞Cx43的表达,但结果也尚有争议。然而,至今尚无有关TH对中枢神经系统Cx表达影响的报道。
为此,我们首先通过建立先天性甲状腺功能减低症的动物模型,检测先天性甲低对大脑皮层Cx43、Cx32和Cx45表达的影响。再应用离体细胞培养技术,观察不同甲状腺素浓度对大脑皮层星形胶质细胞Cx43、Cx32和Cx45表达的影响,以进一步了解TH对星形胶质细胞Cx43、Cx32和Cx45表达的调节作用,为深入研究先天性甲低发病机制和保护干预措施等提供实验依据。
研究目的
通过检测先天性甲低仔鼠大脑皮层Cx43、Cx32和Cx45表达,以及不同甲状腺素浓度培养对大脑皮层星形胶质细胞Cx43、Cx32和Cx45表达变化,探讨先天性甲低对这些缝隙连接蛋白表达的影响。
动物模型部分
1材料与方法
1.1动物模型建立和分组
清洁级健康成年C57BL／6J合笼交配后自行产子,按处理条件不同分甲低组和对照组。
甲低组：合笼第10天开始给予母鼠含0.03%甲巯咪唑的饮用水,一直到仔鼠出生7天。
对照组：一直喂养清洁饮用水,其它实验控制因素同甲低组。
在仔鼠出生1、7、14和21天麻醉后处死,生理盐水灌注,留取大脑皮层标本。
1.2指标检测
采用Real time RT-PCR法检测Cx43、Cx32和Cx45的mRNA水平。采用AxyPrep总RNA小量制备试剂盒提取脑组织总RNA,自行设计引物,应用Takara荧光PCR试剂盒进行RT-PCR扩增(两步法)。目的基因mRNA水平采用ΔCt表示。
采用Western blot法检测Cx43、Cx32和Cx45的蛋白质水平。应用RIPA裂解液提取脑组织蛋白质,采用BCA试剂盒进行蛋白质定量,在丙烯酰胺电泳和转膜后,依次封闭、一抗和二抗孵育、放射自显影,保存胶片,最后统一将放射自显影条带进行扫描,对蛋白条带进行灰度分析,将Cx/β-actin的比值作为Cx的相对表达量。
新鲜脑组织予10%中性福尔马林固定和石蜡包埋,采用"EnvisionTM二步法”进行免疫组化检测。
2结果
对照组出生后Cx43表达上升,7天时达到高峰,此后基本维持稳定水平；而甲低组出生后Cx43表达上升较慢。与对照组比较,1、7和14天甲低组Cx43mRNA有所下降,但差异均不具有显著性意义。1和7天甲低组Cx43蛋白质有下降,其中1天时差异在边界水平,7天时差异具有显著性意义。免疫组化显示大脑皮层Cx43表达均非常丰富,广泛分布在神经细胞的胞体和神经纤维部位。
对照组大脑皮层Cx32表达水平相对较低,在出生后进一步呈逐渐下降趋势。甲低组在7、14和21天时Cx32 mRNA均有明显升高。不同日龄甲低组Cx32蛋白质均有上升,其中7日龄时差异具有显著性意义,14日龄时差异在边界水平。免疫组化显示大脑皮层的Cx32表达明显较Cx43低,部分神经元胞体和少量神经纤维阳性。
对照组大脑皮层Cx45水平处于Cx45和Cx32间,出生后也逐渐下降。两组间不同时间点Cx45mRNA和蛋白质表达差异均无显著意义。免疫组化显示大脑皮层Cx45表达比较丰富,也较广泛分布于神经细胞胞体和纤维部位。
细胞培养部分
1材料和方法
1.1培养基配制
购买活性炭吸附后低甲状腺激素胎牛血清(FBS)和未特殊处理的胎牛血清,同时购买T3配制T3终浓度为5 nmol/L的DMEM液。按下列方法配制各种培养液,并测定各组培养液总T3(TT3)和游离T3(FT3)甲状腺激素浓度。
培养基Ⅰ：普通DMEM+15%普通FBS(TT3:5.4nmol/L, FT3:22.0pmol/L);
培养基Ⅱ：普通DMEM+15%低TH的FBS(TT3:0.6 nmol/L, FT3:1.9 pmol/L);
培养基Ⅲ：含5 nmol/L T3的DMEM+15%低TH的FBS(TT3 4.3 nmol/L, FT3 23.2 pmol/L);
1.2细胞培养和分组
无菌条件下,取生后1～2d健康SD大鼠大脑皮层,置Hanks液中清洗、剪碎、胰蛋白酶消化后,置于培养基中培养。于培养8天后用抗神经胶质酸性蛋白(GFAP)抗体对星形胶质细胞进行鉴定。按照培养基不同,将培养的细胞分为4组：
对照组：培养基Ⅰ×2天+培养基Ⅲ×6天
甲低组：培养基Ⅰ×2天+培养基Ⅱ×6天
干预组1：培养基Ⅰ×2天+培养基Ⅱ×2天+培养基Ⅲ×4天
干预组2：培养基Ⅰ×2天+培养基Ⅱ×4天+培养基Ⅲ×2天
1.3检测指标和方法
采用四甲基偶氮唑蓝(MTT)方法绘制生长曲线。采用Takara Real time RT-PCR试剂盒对培养细胞的Cx43、Cx32和Cx45 mRNA进行检测,同时应用Western blot方法对Cx43、Cx32和Cx45的蛋白质水平进行检测,同时应用免疫荧光染色激光共聚焦扫描对蛋白质表达进行定位观察。
2结果
甲低组细胞增殖缓慢,指数增长期后移,干预后细胞增殖有所改善。两组星形胶质细胞Cx43表达均丰富。甲低组星形胶质细胞Cx43表达下降,正常甲状腺激素浓度干预后又有上升趋势,早期干预4天其mRNA水平即能恢复到正常水平,但蛋白质表达水平尚不能恢复到正常水平。激光共聚焦扫描显示离体培养星形胶质细胞Cx43蛋白质表达均非常丰富,分布于胞体和树突的胞浆内,正常组多数细胞间有荧光强度特别高的点状结构,甲低组荧光强度较低,点状结构少而且荧光强度较低,干预组1和干预组2荧光强度和高荧光强度的点状结构有恢复趋势,但干预组2高荧光强度的点状结构不均匀。
两组星形胶质细胞Cx32mRNA水平均较低。与对照组比较,甲低组Cx32mRNA更低,在干预后mRNA表达水平能较快上升,达到正常水平。然而,无论Western blot检测,还是激光共聚焦扫描,均显示各组离体培养星形胶质细胞的Cx32蛋白质表达均阴性。
两组星形胶质细胞Cx45表达均较丰富。甲低组Cx45表达水平明显下降,干预后有所上升,早期干预后2-4天可恢复到正常水平。激光共聚焦扫描显示各组离体培养星形胶质细胞的Cx45蛋白表达均非常丰富,分布于胞体和树突的胞浆内,少部分细胞间有荧光强度特别高的点状结构。
结论
1.先天性甲低可导致仔鼠大脑皮层总Cx43表达下降和总Cx32表达上升；
2.TH下降可导致离体培养的大脑皮层星形胶质细胞Cx43和Cx45表达下降,GJ分布异常；
3.TH对神经细胞Cx基因调控具有时空性；
4.短期的T3早期干预,能使星形胶质细胞Cx43和Cx45表达有恢复上升趋势,但Cx43不能恢复至正常水平。
Background
Congenital hypothyroidism (CH), due to insufficient secretion of thyroid hormone (TH) result from variety of reasons, is a common pediatric endocrine disease. It is also a main cause of childhood mental retarded and draws more and more attention from endocrine pediatrician. With the development of molecular biology, physiology of TH has been better understood. TH binds its receptor (TH receptor, TR) in the nucleus, which plays a role on regulation the target gene transcriptional level by recognition the thyroid hormone response elements (TREs) in the promoters. However, the candidate genes which be regulated by TH and cause mental retardation in children with CH are still unclear. The signaling proteins and related proteins have effect on the information transfer and the various metabolic activity, and then on nerve cell growth and differentiation. They also take part in the mechanisms of sensory, motor, learning, memory and other activities. Hence, study the effect of TH on the signaling proteins and its related proteins may help us to better understand the effect of TH on brain development and the mechanism of CH.
The classic information transfer in nervous system is the neurotransmitter transmittion throught the synapses, or the chemical synapses. Previous studies showed that TH had effect on the synthesis and degradation of neurotransmitters, and on the expression and function of their receptors. Recently, studies found several several non-classic information transfer pathway in the nervous system, such as gap junction (GJ) communication (GJC) signal transduction systems, which play important role on some nervous activities.
GJ is the morphological substrate of one type of electrical synapse and mediates the GJC among adjacent cells. The key component of GJ is connexin (Cx). GJ is composed of a pair of connexon that includes six Cx proteins and make a hemichannel with a diameter of about 1.5 nm. There is abundant GJ in the nervous system, including at least 12 types of Cx. Cx43 is the predominant type and accounts for 90-95% of the total Cx protein. Moreover, types of Cx on different nerve cells are different. Cx32 is mainly in oligodendrocytes while Cx43 is mainly in astrocytes. Recently, Cx45 is also found in the central nervous system.
Studies have shown that astrocytes interconnected as a syncytium through GJ and GJ may also constitute a complex of neurons-glial cells-neurons network. GJ allow the transfer of small molecules from cell to adjust cell, also information communication direct. Moreover, some Cx did not form a GJ with the adjacent cells, forming a direct channel for material and information exchange from the extracellular environments. Efficient intercellular communication at GJ is essential for normal electro-mechanical function of the brain. Compared with synapse, GJC has direct, fast and bidirectional characteristics. GJ has an important role on stability of the intercellular and extracellular environments, on nerve cell proliferation, differentiation, and development, also the signal transduction in brain. Several diseases have been associated with the disorder of GJ. Several studies showed that TH may affect the Cx43 expression on the myocardial and Sertoli cells, although the results are still controversial. However, no similar study about the effect of TH on the Cx in central nervous system was reported.
Here, we established a CH model firstly, and measured the total levels Cx43, Cx32 and Cx45 on cerebral cortex in this model. Then, cultuered in the astrocytes by different concentration of T3 and measured the levels of these Cxs.
Objectives
To investigate the effect of TH on Cx3 expression by measuring the Cx43, Cx32 and Cx45 expression in cerebral cortex in mice CH model, and these Cx in the astrocytes cultuered by different concentration of T3.
Mice CH Model
1 Materials and Methods
1.1 Animal Model
Health adult C57BL/6J mice mated together. They were divided into CH group and control group according to the different intervention. The control group is definited as feeding of clean drinking water. The CH group is definted as feeding 0.03% thiamazole contained water from the 10th day after mate to 7th day after the offsprings'birth. The offsprings were sacrificed on day 1,7,14 and 21. Saline perfusion was performed and the cerebral cortex was collected.
1.2 Parameter Measurements
The levels of Cxs mRNA were measured by real time RT-PCR. Total RNA was extracted using AxyPrep Mutilsoure Total RNA Miniprep Kit. Self-designed primers and SYBR PrimeScriptTM PCR Kit were used real time RT-PCR. Two-step method was used for Cx43, Cx32, and Cx45. ACt was presented for target gene mRNA levels.
The levels of Cx protein were measured by Western blot. Proteins were extracted by RIPA and the concentration was measured by BCA Protein Assay Kit. A 50μg total protein was used for SDS-PAGE electrophoresis and transferred PVDF. Then blocked, combined with first and second antibodies, and ECL radioautography in sequence. Finally, scan the radiation autoradiography strip and analysis the protein bands with gray-scale. The Cx/β-actin ratio was calculated as the relative levels of Cx.
Tissue was fixed in 10% neutral formalin and embeded in paraffin. "EnvisionTM two-step" method was used for immunohistochemistry.
2 Results
In control group, the levels of Cx43 expression on the cerebral cortex increased after birth and reach the peak at day 7, while the levels of Cx43 increased slowly in CH group. Commpred with the control group, the Cx43 mRNA levels in CH group were lower on day 1,7 and 14 without significant difference. Moreover, the Cx43 protein in CH group was lower than these in controls with a marginal difference on day 1 and a significant difference on day 7. Immunohistochemistry showed Cx43 protein was abundant on the cerebral cortex in both groups, including the body of nerve cells and nerve fibers.
The levels of Cx32 were low on the cerebral cortex in the control and CH goups, and decreased after birth. Compared with the controls, the levels of Cx32 mRNA were significantly higher in the CH group on day 7,14 and 21, and the Cx32 protein increased with a significant difference on day 7 and a marginal difference on day 14. Immunohistochemistry showed that the expression of Cx32 was significantly less than the Cx43 on the cerebral cortex, and some nerve cells and few nerve fibers were positive.
The levels of Cx45 were less than Cx43 and higher than Cx32, and decreased after birth. Both mRNA and protein of Cx45 had no significant difference between the two groups. Immunohistochemistry showed that Cx45 expression is similar to the distribution Cx43, and most body of nerve cells and nerve fibers was positive
Cell Culture
1 Materials and Methods
1.1 Medium Preparation
Low TH fetal bovine serum (activated carbon adsorbed FBS), common fetal bovine and serum T3 were purchased. DMEM solution with 5 nmol/L of T3 was prepared. A variety of DMEM culture mediums were prepared as following and TH were measured.
MediumⅠ, common DMEM+ common FBS (TT3:5.4 nmol/L, FT3:22.0 pmol/L).
MediumⅡ, common DMEM+ 15% low TH FBS (TT3:0.6 nmol/L, FT3:1.9 pmol/L).
MediumⅢ, DMEM with 5 nmol/L T3+ 15% low TH FBS (TT3 4.3 nmol/L, FT3 23.2 pmol/L).
1.2 Cell Culture and Grouping
Cerebral cortex was obtained from 1-2 d age healthy SD rat under sterile conditions. Then, washed with Hanks solution, shredded, trypsin digestion and cultured in medium. The astrocytes were identified with anti-GFAP antibody at 8th day, and they were divided into 4 groups according to the different medium.
Control group:mediumⅠfor 2 days + mediumⅢfor 6 days
Hypothyroidism group:mediumⅠfor 2 days + mediumⅡfor 6 days
Intervention group 1:mediumⅠfor 2 days + mediumⅡfor 2 days + mediumⅢfor 4 days
Intervention group 2:mediumⅠfor 2 days + mediumⅡfor 4 days + mediumⅢfor 2 days
1.3 Parameter Measurements
Tetrazolium blue (MTT) method was used to draw the growth curve. The expression of Cx43, Cx32 and Cx45 mRNA in the cultured cells were detected by using the Takara Real time RT-PCR kit. The levels of Cx43, Cx32 and Cx45 protein were measured by Western blot, and laser scanning confocal microscope was used for locating the protein in the cells.
2 Results
The cell proliferation was slow and exponential growth phase was right shift in the hypothyroidism group, and improved after the intervention. The expression of Cx43 in astrocyte decreased in the hypothyroidism group. After T3 intervention, the Cx43 levels had an upward trend. They mRNA levels of Cx43 can recovry to normal levels while the protein levels did not return to normal levels. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were many well-distributed high- fluorescence intensity points between adjacent astrocytes in control group while the point structures in hypothyroidism group were less with low fluorescence intensity. After T3 intervention, the point structures were increased, but asymmetrical distribution.
The mRNA level Cx32 in the hypothyroidism group was lower than that in the controls, and it can be rapidly increased to near normal levels after T3 intervention. However, the protein was undetectable both using Western blot and laser scanning confocal microscopy.
A low Cx45 level was noted in the hypothyroidism group as well, and it can return to normal levels with early intervention. Laser scanning confocal microscopy showed that abundant Cx43 protein expressed astrocytes, including the intracytoplasm of body and dendritic. There were only few high- fluorescence intensity points between adjacent astrocytes.
Conclusions
1. The levels of Cx43 decrease and the levels of Cx32 increase in the cerebral cortex of CH mice.
2. Low TH induces low Cx43 and Cx45 expression, and the abnormality of GJ distribution in astrocytes.
3. The regulation of TH on gene transcription has a spatial and temporal control.
4. Short-term T3 early intervention increases the Cx43 and Cx45 expression, but the levels of Cx 43 does not recovery to normal levels.
引文
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