不同饮食钙条件下饮水型氟暴露对子代脑发育和功能的影响

The project intends to investigate scene epidemiology of fluorosis in drinking water,take subchronic fluorosis experiment animals as vectors,and detect hippocampal neurons exposed to fluoride in vitro, in order to further explore the influence of fluoride exposure in drinking water on offspring brain development and function under the conditions of different dietary calcium..(1) Fluorosis scene epidemiology investigation:Intend to respectively selecte 50 one year old children, 50 two years old children and 50 six to eight years old school age children (evenly divide into male and female)in elects potable water fluorosis regions.Use Denver nerve psychology growth screening children development business,and appraise the children intelligence quotient with Swedish..(2) subchronic fluorosis animals experiment:Plan to select 200 female Wistar rats (raise 100 male rats to use in copulation at the same time).Divide into control group, low calcium group, high fluorine group and low calcium high fluorine group,50 rats in each group,and the high fluorine group will drink fluorine water.After raising for three months, test the replicates of chronic animal model of drinking water fluorosis if success,then copulate,pregnant,and birth the first generation rats.Take fetal rats by putting the pregnant rats to death in pregnancy of 15-17 days and 20-22 days (10 rats each group at a time). Randomly choose 30 female rats and 30 male rats from the descendant of the mother rats in each group, and continue raising. Take fetal rats, tsai rats as the research object. Observe the mice in different day age (after birthing 14, 28 and 56 days) based on learning-memory behavior. Use immunohistochemistry, Western blot, RT-PCR and laser scanning confocal microscopy to detect the expression level of a series of hippocampus endoplasmic reticulum cells stress partner protein/gene,the relevant apoptosis signaling pathways molecules,apoptosis involved in nerve cells and special proteins for nerve growth in brain development process.. (3) The experiments of hippocampus nerve cells with the fluorine in vitro culture:Plan to take reference methods reported in the literature.Take 24h new birth Wistar fetal rat, after a series of experimental steps,hippocanlpal neurons are trained 3d by using NaF solution(end concentration of 0.625,1.25,2.5, 5, 10, 20, 40 and 80 mg/L)to continue fluorine handling to the training neurons. after 8d,and take the research methods above, then detect the expression level of above proteins / genes in hippocampal cellof rats..Through researches, it will take some academic value and social significance in free radical damage theory of fluorine,and can explore the intervention or preventive measures of the normal devdevelopment of the offspring's brain in endemic fluorosis regions.

氧化应激诱发神经元凋亡被证实为氟致脑发育和功能影响关键机制，但氧化应激如何引起脑细胞凋亡机理尚未明了。基于我们前期研究发现，氟中毒与海马突触体内Ca2+超载和NF-κB表达水平过高有关，借鉴一些脑疾病/损伤发生发展与内质网应激（ERS）有关研究报道。本项目拟动物实验、体外神经元培养和流行病调查相结合，在脑功能观测基础上，用Western Blot、RT-PCR和激光扫描共聚焦显微镜等技术，检测饮水型氟暴露对子代脑海马细胞内质网应激伴侣蛋白/基因表达水平、相关凋亡信号通路分子、神经发育特异蛋白和突触及树突可塑性影响，率先探讨ERS及相关凋亡信号通路在不同饮食钙条件下氟致脑发育和功能影响中的作用，寻找确定氟致脑发育和功能影响的最关键凋亡信号通路和最关键因子，评价饮水型氟暴露对后代脑发育和功能的潜在影响，这对完善氟致自由基损伤学说，探寻氟病区后代正常脑发育干预措施，具有一定的学术价值和科学意义。

氧化应激诱发神经元凋亡可能是氟致脑损伤的关键，“钙矛盾”可能是发病机制之一。选用初断乳SD雌性大鼠100只，随机分为对照组、高氟组、低钙组、低钙高氟组和高钙高氟组，饲养3个月后，雌雄鼠合笼交配产仔，取胎鼠、14日龄及28日龄雌、雄仔鼠，在脑功能观测基础上，用Western Blot、RT-PCR等技术，探讨不同饮食钙条件下饮水型氟暴露对子代鼠脑海马细胞内质网应激伴侣分子和相关凋亡信号通路分子基因/蛋白、神经发育特异蛋白表达水平和突触可塑性等的影响。同时还探讨了氟暴露致体外培养PC12细胞内质网应激机制和地氟病区调查。结果表明，过量氟会透过胎盘屏障而蓄积在子代体内，透过血脑屏障，诱导氧化损伤，导致内质网过度氧化应激，继而使其脑海马神经内质网应激伴侣分子BIP、CHOP、CRT和凋亡信号通路相关蛋白凋亡Caspase12及JNK分子基因/蛋白表达水平明显升高，凋亡信号通路相关蛋白Bcl-2表达水平显著降低，引起大脑凋亡细胞增多；氟中毒还导致其脑海马突触界面结构异常，并能引起其脑发育特异蛋白双皮质素DCX表达上升，突触素p38表达下降，从而影响子代脑发育，最终损害脑功能。低钙（0.063% CaCO3）饲料与高氟溶液同喂通过加剧子代鼠脑海马细胞上述分子基因/蛋白异常表达，对子鼠脑功能损伤产生协同毒性效应，高钙（7% CaCO3）饲料则通过逆转上述分子基因/蛋白异常表达而产生拮抗效应。与非氟病区相比，氟病区儿童饮水钙和饮食钙摄入量、儿童智商无明显差异，但智力低下率仍明显偏高。这些结果既为探寻氟病区后代脑发育干预措施提供了新思路， 也首次系统探讨了内质网伴侣分子、相关凋亡信号通路分子在氟致神经损伤中的作用，有较高学术价值。