本文選題：鉛 + 血腦屏障��； 參考：《第四軍醫(yī)大學》2013年博士論文

【摘要】：鉛是環(huán)境中普遍存在的重金屬污染物之一。人體經(jīng)由空氣、灰塵、食物、飲用水等接觸暴露，并可在體內長期蓄積，造成全身多系統(tǒng)和器官的損傷及功能障礙，其中以神經(jīng)系統(tǒng)損傷最為嚴重，即使是早期低劑量的鉛暴露也可導致中樞神經(jīng)系統(tǒng)出現(xiàn)不可逆損傷。兒童是鉛損傷的高危易感人群，鉛對兒童神經(jīng)系統(tǒng)的功能性損傷呈連續(xù)的劑量-效應過程，可導致兒童認知缺陷、心理及行為異常，并嚴重危害兒童生長和發(fā)育。作為確定的環(huán)境毒物，鉛已成為危害兒童健康的“隱形殺手”，一直是世界范圍內公共健康的主要危險因素和重要的社會問題。 血腦屏障（Blood-brain Barrier，BBB）是存在于腦和脊髓中的毛細血管與神經(jīng)組織之間的一個動態(tài)調節(jié)界面，是保持中樞神經(jīng)系統(tǒng)內環(huán)境平衡和穩(wěn)定的重要結構。BBB存在顯著的年齡差異，兒童由于BBB結構尚未成熟，致使外界化合物較成人更易進入腦組織，，對神經(jīng)系統(tǒng)的損害更嚴重。我們前期的研究結果顯示鉛暴露能夠破壞大鼠BBB的結構，從而損傷BBB的功能，為進一步系統(tǒng)闡明鉛損傷BBB結構和功能的機制，探索相應的防治措施，本研究擬通過建立鉛誘導BBB損傷的動物和細胞模型，觀察鉛誘導BBB緊密連接蛋白表達及其信號通路的改變，并利用體內實驗觀察補充鐵劑后對鉛損傷BBB的拮抗作用，探索鉛誘導BBB損傷的分子機制及其信號調控途徑和鐵劑的保護效應。 目的： 觀察鉛誘導BBB緊密連接蛋白表達及其信號通路的改變，為闡明鉛損傷BBB結構及功能的機制提供實驗支持；觀察補充鐵劑后對鉛神經(jīng)損傷的拮抗作用，闡明鐵劑對鉛損傷BBB的保護效應，為鉛中毒的預防及治療措施提供借鑒。 方法： 1.用醋酸鉛飲水法建立生長發(fā)育期大鼠鉛中毒模型，利用原子吸收分光光度法測定血鉛、腦組織鉛含量變化；利用電鏡硝酸鑭示蹤技術檢測BBB的滲漏；利用Morris水迷宮和曠場實驗確定染鉛大鼠行為學改變。 2.通過BBB毛細血管分離法富集染鉛大鼠腦毛細血管，Western blot分析TJ蛋白ZO-1、Occludin、Claudin-5的表達改變；利用磷酸化抗體蛋白芯片檢測調控BBB血管內皮細胞TJ相關蛋白的信號通路變化，并采用Western blot分析確證相應信號分子的變化。 3.在鉛暴露同時采用硫酸亞鐵灌胃大鼠補充鐵劑，與飲水組、單純染鉛組比較血鉛、血鐵水平的變化；通過電鏡硝酸鑭示蹤技術確定鐵對鉛誘導BBB損傷的保護效應；利用Morris水迷宮和曠場實驗確定補鐵對染鉛大鼠行為學改變的保護；通過Western blot分析補鐵對染鉛大鼠TJ蛋白ZO-1、Occludin、Claudin-5表達下降的拮抗作用及相應信號通路分子的調控作用。 結果： 1.生長發(fā)育期鉛暴露可破壞大鼠BBB的結構完整性 鉛暴露后大鼠血鉛、腦組織鉛含量逐漸增加，染鉛6周時趨于穩(wěn)定，與對照組相比差異顯著（p0.05）；電鏡顯示硝酸鑭由微血管滲透到了腦組織中；曠場實驗顯示染鉛組大鼠空間認知能力、興奮性、探索行為明顯減弱（p0.05）；平臺隱蔽實驗結果顯示染鉛組大鼠到達平臺的潛伏期與對照組相比明顯延長（p0.05）；空間探索實驗結果顯示染鉛組大鼠在目的象限的停留時間明顯縮短（p0.05），上述結果表明鉛可損傷BBB的結構完整性，導致大鼠學習記憶功能降低。 2.鉛暴露對腦血管內皮細胞TJ蛋白及其信號調控通路的影響 鉛暴露可導致TJ蛋白ZO-1、Occludin、Claudin-5的表達水平顯著下降（p0.05）；蛋白芯片檢測發(fā)現(xiàn)鉛暴露導致MAPK、Akt等信號分子磷酸化程度顯著增加，Westernblot結果進一步證實信號通路分子ERK1/2、JNK、p38、Akt473、Akt308磷酸化增加顯著（p0.05），這些結果表明鉛可活化MAPK、Akt信號通路分子和降低TJ蛋白的表達水平。 3.體內補充鐵劑可以拮抗鉛誘導的BBB通透性損傷及學習記憶功能影響 染鉛大鼠血鐵濃度較對照組顯著降低（p0.05），提示鉛暴露影響大鼠鐵的吸收和利用；利用灌胃法補充硫酸亞鐵后可以降低染鉛大鼠血鉛濃度，與單純染鉛組比較具有顯著性差異（p0.05），提示補鐵可以拮抗鉛的吸收；電鏡結果顯示補鐵后染鉛大鼠BBB通透性增加程度減弱，提示補鐵可能通過減少鉛的吸收或者拮抗鉛的毒性保護TJ完整性；補鐵后染鉛大鼠TJ蛋白ZO-1、Occludin、Claudin-5表達下降減少（p0.05），并可抑制鉛誘導的ERK1/2、JNK、p38、Akt473、Akt308等信號分子的磷酸化（p0.05），提示鐵通過抑制MAPK、Akt信號分子的磷酸化調控TJ通透性改變。 結論： 本研究通過建立染鉛大鼠BBB損傷模型和染鉛體外BBB細胞模型，發(fā)現(xiàn)TJ蛋白是鉛致BBB結構和功能損傷的重要靶分子，鉛可能通過活化MAPK、Akt信號傳導通路減少TJ蛋白的表達，從而使BBB通透性增加，而鐵可以拮抗鉛的神經(jīng)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錛

本文編號：1930300