氟铝联合致二代仔鼠海马miRNA表达谱改变及其与学习记忆的关系探讨

Both fluorine and aluminium have been testified neurotoxic. In many areas, such as coal-burning fluorosis areas where kaolin clays have been stired and mixed with coal, fluorine and aluminium co-exist and maybe enter human body through environmental media. In animal experiments done by other researchers and our project team, as well as in an epidemiological survey which was carried out in a endemic fluorosis area where fluorine and aluminium co-exist, results similarly indicated that fluorine combined with aluminium had done harm to intelligence of animal and the indigenous chlidren. But until now, mechanism on how fluorine combined with aluminium affect learning and memory remains unclear, available treatments are scant and backward, too. For central nervous system, the most fastest developmental and vunerable phase is from prenatal to postnatal. Maturity of neurodevelopment will affect children’s learning and memory ability. miRNAs regulate gene expression and play important roles in development, differentiation and function performance of nervous system, which are key to learning and memory..So in this study, first, the actual situation will to be stimulated to establish animal model of the second generation newborn mice by means of continued exposed to fluorine combined with aluminium from embryonic stage to adult period and lasting two generation , fluorine and aluminium in urine and blood will be assayed, maturity of neurogenesis and ability of learning and memory will be tested, pathological changes of hippocampus will be observed, miRNA expression profile of hippocampus will be detected and verified; target genes of miRNAs will be forecasted and verified. Then, HT22 hippocampal neuron cell line will be exposed to fluorine combined with aluminium while miRNA inhibitors being used to set up “Loss of function” model for preliminary study on the mechanism of how miRNAs participate in hippocampus toxicity induced by fluorine combined with aluminium. Aim of the study is to investigate the molecular mechanism of learning and memory damage caused by fluorine combined with aluminium from the aspects of gene regulation and post-transcriptional level, so as to provide scientific references for prevention and treatment of the indigenous children’s intelligence damage in fluorine and aluminium co-exist endemic areas.

氟、铝均有神经毒性。高岭土拌煤的燃煤型氟病区等环境皆有氟、铝共存，并可进入人体。他人及本项目组前期动物实验及地氟病区世居儿童调查均提示氟铝联合导致智力损伤，但机制尚不明了、治疗落后。出生前后是中枢神经系统发育最快也最易受损时期。神经发育成熟度受损会影响儿童学习记忆能力。miRNA在基因调控和神经系统的发育、分化及功能等方面作用重要，与学习记忆有关。.故本研究首先建立自亲代孕哺期、子代断乳至成年前氟铝联合持续暴露的二代仔鼠模型，测定或观察尿和血中氟及铝、神经系统发育成熟度、学习记忆能力，海马的病理改变和miRNA表达谱，预测miRNA靶基因并验证。然后以氟铝联合暴露HT22海马神经元细胞、用miRNA抑制剂建立“Loss of function”模型，探讨miRNA参与氟铝联合对海马的毒性机制。从基因调控及转录后水平探讨氟铝联合致学习记忆损伤分子机制，为病区世居儿童智力损伤防治提供科学依据。

氟铝联合致世居儿童智力损伤机制尚不明了、治疗落后。出生前后是中枢神经系统发育最快也最易受损时期。神经发育成熟度受损会影响儿童学习记忆能力。miRNA与学习记忆有关。本研究将SD孕鼠16只随机均分为4组。各组饮水中NaF、AlCl3分别为（0，0）、（60，600）、（120，600）、（240，600）mg／L，自由饮水染毒，建立亲代孕哺期至二代子鼠成年前持续暴露的二代子鼠模型，测定尿、血、脑中氟及铝、神经发育成熟度、空间学习记忆，海马结构和miRNA表达谱，预测miRNA靶基因并验证。再将海马相关细胞暴露于NaF、AlCl3分别为（0，0）、（2.5，200）、（5，200）、（10，200）mg/L的培养基24h、用miRNA的agomir and antagomir建立模型功能获得和缺失模型，探讨miRNA参与氟铝联合对海马的毒性机制。主要结果：持续氟铝联合暴露致二代子鼠生长发育减缓，血清/脑/尿氟、血清/脑/尿铝均明显升高，海马发生病理改变。中氟+铝组及高氟+铝组的二代子鼠黑箱停留时间延长、穿箱次数减少、白箱停留时间百分比减少、黑箱停留时间百分比增加。二代子鼠的逃避潜伏期均延长；首次达台时间延长、穿越平台次数减少。共筛选出253个miRNA，其中与对照组比较，氟铝联合暴露致二代子鼠海马中表达差异的miRNA占24.5%；与低氟+铝组比较，表达差异的miRNA占8.7%；与中氟+铝组比较，表达差异的miRNA占0.4%；其中任意两组表达无差异的miRNA占66.4%。验证筛选出的5个差异miRNA（miR-122-5p，miR-450a-3p，miR-183-5p，miR-130a-5p以及 miR-547-3p），结果与测序结果一致。预测到差异miRNA的共同靶基因为152个；GO分析可知，预测的靶基因功能各异，学习记忆功能在细胞组分、生物学过程以及分子功能中均有涉及；KEGG对部分差异miRNA预测的靶基因分析显示，与神经行为以及学习记忆相关的通路主要有：胆碱能突触、长时程增强/抑制、多巴胺能突触。结论：本实验条件下，氟铝联合可致二代子鼠空间学习记忆能力降低，机制与miRNA及其靶基因的协同调控密切相关。本研究从基因调控及转录后水平探讨了氟铝联合致空间学习记忆损伤分子机制，可为病区儿童智力损伤防治提供科学依据。