紧密连接蛋白在铅诱导的血脑屏障损伤中的作用及调控机制

发布时间：2018-05-24 19:40

  本文选题：铅 + 血脑屏障　； 参考：《第四军医大学》2013年博士论文

【摘要】：铅是环境中普遍存在的重金属污染物之一。人体经由空气、灰尘、食物、饮用水等接触暴露，并可在体内长期蓄积，造成全身多系统和器官的损伤及功能障碍，其中以神经系统损伤最为严重，即使是早期低剂量的铅暴露也可导致中枢神经系统出现不可逆损伤。儿童是铅损伤的高危易感人群，铅对儿童神经系统的功能性损伤呈连续的剂量-效应过程，可导致儿童认知缺陷、心理及行为异常，并严重危害儿童生长和发育。作为确定的环境毒物，铅已成为危害儿童健康的“隐形杀手”，一直是世界范围内公共健康的主要危险因素和重要的社会问题。 血脑屏障（Blood-brain Barrier，BBB）是存在于脑和脊髓中的毛细血管与神经组织之间的一个动态调节界面，是保持中枢神经系统内环境平衡和稳定的重要结构。BBB存在显著的年龄差异，儿童由于BBB结构尚未成熟，致使外界化合物较成人更易进入脑组织，，对神经系统的损害更严重。我们前期的研究结果显示铅暴露能够破坏大鼠BBB的结构，从而损伤BBB的功能，为进一步系统阐明铅损伤BBB结构和功能的机制，探索相应的防治措施，本研究拟通过建立铅诱导BBB损伤的动物和细胞模型，观察铅诱导BBB紧密连接蛋白表达及其信号通路的改变，并利用体内实验观察补充铁剂后对铅损伤BBB的拮抗作用，探索铅诱导BBB损伤的分子机制及其信号调控途径和铁剂的保护效应。 目的： 观察铅诱导BBB紧密连接蛋白表达及其信号通路的改变，为阐明铅损伤BBB结构及功能的机制提供实验支持；观察补充铁剂后对铅神经损伤的拮抗作用，阐明铁剂对铅损伤BBB的保护效应，为铅中毒的预防及治疗措施提供借鉴。 方法： 1.用醋酸铅饮水法建立生长发育期大鼠铅中毒模型，利用原子吸收分光光度法测定血铅、脑组织铅含量变化；利用电镜硝酸镧示踪技术检测BBB的渗漏；利用Morris水迷宫和旷场实验确定染铅大鼠行为学改变。 2.通过BBB毛细血管分离法富集染铅大鼠脑毛细血管，Western blot分析TJ蛋白ZO-1、Occludin、Claudin-5的表达改变；利用磷酸化抗体蛋白芯片检测调控BBB血管内皮细胞TJ相关蛋白的信号通路变化，并采用Western blot分析确证相应信号分子的变化。 3.在铅暴露同时采用硫酸亚铁灌胃大鼠补充铁剂，与饮水组、单纯染铅组比较血铅、血铁水平的变化；通过电镜硝酸镧示踪技术确定铁对铅诱导BBB损伤的保护效应；利用Morris水迷宫和旷场实验确定补铁对染铅大鼠行为学改变的保护；通过Western blot分析补铁对染铅大鼠TJ蛋白ZO-1、Occludin、Claudin-5表达下降的拮抗作用及相应信号通路分子的调控作用。 结果： 1.生长发育期铅暴露可破坏大鼠BBB的结构完整性 铅暴露后大鼠血铅、脑组织铅含量逐渐增加，染铅6周时趋于稳定，与对照组相比差异显著（p0.05）；电镜显示硝酸镧由微血管渗透到了脑组织中；旷场实验显示染铅组大鼠空间认知能力、兴奋性、探索行为明显减弱（p0.05）；平台隐蔽实验结果显示染铅组大鼠到达平台的潜伏期与对照组相比明显延长（p0.05）；空间探索实验结果显示染铅组大鼠在目的象限的停留时间明显缩短（p0.05），上述结果表明铅可损伤BBB的结构完整性，导致大鼠学习记忆功能降低。 2.铅暴露对脑血管内皮细胞TJ蛋白及其信号调控通路的影响 铅暴露可导致TJ蛋白ZO-1、Occludin、Claudin-5的表达水平显著下降（p0.05）；蛋白芯片检测发现铅暴露导致MAPK、Akt等信号分子磷酸化程度显著增加，Westernblot结果进一步证实信号通路分子ERK1/2、JNK、p38、Akt473、Akt308磷酸化增加显著（p0.05），这些结果表明铅可活化MAPK、Akt信号通路分子和降低TJ蛋白的表达水平。 3.体内补充铁剂可以拮抗铅诱导的BBB通透性损伤及学习记忆功能影响 染铅大鼠血铁浓度较对照组显著降低（p0.05），提示铅暴露影响大鼠铁的吸收和利用；利用灌胃法补充硫酸亚铁后可以降低染铅大鼠血铅浓度，与单纯染铅组比较具有显著性差异（p0.05），提示补铁可以拮抗铅的吸收；电镜结果显示补铁后染铅大鼠BBB通透性增加程度减弱，提示补铁可能通过减少铅的吸收或者拮抗铅的毒性保护TJ完整性；补铁后染铅大鼠TJ蛋白ZO-1、Occludin、Claudin-5表达下降减少（p0.05），并可抑制铅诱导的ERK1/2、JNK、p38、Akt473、Akt308等信号分子的磷酸化（p0.05），提示铁通过抑制MAPK、Akt信号分子的磷酸化调控TJ通透性改变。 结论： 本研究通过建立染铅大鼠BBB损伤模型和染铅体外BBB细胞模型，发现TJ蛋白是铅致BBB结构和功能损伤的重要靶分子，铅可能通过活化MAPK、Akt信号传导通路减少TJ蛋白的表达，从而使BBB通透性增加，而铁可以拮抗铅的神经毒性作用。
[Abstract]:Lead is one of the common heavy metal pollutants in the environment. The human body is exposed through air, dust, food, drinking water and so on. It can be accumulated in the body for a long time, causing damage and dysfunction of the system and organs of the whole body. The most serious injury of the nervous system is that the early low dose of lead exposure can also lead to the central nervous system. There is an irreversible injury in the children. Children are high-risk groups of lead damage. Lead has a continuous dose effect process on the functional damage of the children's nervous system, which can lead to children's cognitive defects, psychological and behavioral abnormalities, and seriously harm the growth and development of children. As a determined environmental poison, lead has become a "stealth killing" of children's health. "Hand" has always been the main risk factor and important social problem of public health worldwide.
Blood-brain Barrier (BBB) is a dynamic adjustment interface between capillaries and nerve tissue in the brain and spinal cord. It is an important structure for maintaining the balance and stability of the central nervous system. There is a significant age difference between the.BBB and the central nervous system. Because the BBB node is not mature in children, the external compound is more easily entered than the adult. The damage to the nervous system is more serious in the brain tissue. Our previous study showed that lead exposure could destroy the structure of BBB, damage the function of BBB, further elucidate the mechanism of BBB structure and function of lead damage, and explore the corresponding preventive measures. This study is to establish the animal and cell model of lead induced BBB damage. The expression of BBB tight connexin induced by lead and the change of its signaling pathway were observed, and the antagonism of lead damage to BBB after supplementation of iron was observed in vivo, and the molecular mechanism of lead induced BBB damage and its signal regulation pathway and the protective effect of iron were explored.
Objective:
To observe the change of lead induced BBB close connexin expression and its signal pathway, provide experimental support for elucidating the mechanism of lead damage BBB structure and function, observe the antagonistic effect of supplemental iron on lead nerve injury, clarify the protective effect of iron on lead damage BBB, and provide reference for the prevention and treatment of lead poisoning.
Method锛

本文编号：1930300