miR-183/96/182簇靶向mTOR在氟铝联合致学习记忆损伤中的作用机制及雷帕霉素干预研究

Exposure of fluorine combined with aluminum had damaged intelligence of the indigenous children. But until now, mechanism on how fluorine combined with aluminum exposure affect learning and memory remains unclear, available treatments are scant and backward, too. The most key phase for learning and memory ability is from prenatal to postnatal. Our research team had found compared with the control group, fluorine combined with aluminum exposure damaged offspring rats’ learning and memory ability, miRNA expression spectrum (including miR-182, miR-183 and miR-96) in hippocampus was significantly different (P < 0.01); predicted by miRDB, the common target gene of the miR-182, miR-183 and miR-96 is mammalian target of rapamycin (mTOR). Therefore, it is speculated that the mir-186/96/182 cluster regulates mTOR to participate in the learning and memory impairment caused by fluorine combined with aluminum exposure. So in this study, firstly, we are going to explore the role of the mir-186/96/182 clustertargeting mTOR in the impairment effects of fluorine combined with aluminum on the learning and memory ability (1) cell lines relative to hippocampus will be exposed to fluorine combined with aluminum while the mir-186/96/182 cluster mimics/inhibitors being used to set up “gain of function”/ “Loss of function” model to detect mTOR, so as to verify the miRNA target gene prediction. (2) cell lines relative to hippocampus will be exposed to fluorine combined with aluminum, key molecules in the mTOR pathway will be determined, the relationship between the mTOR pathway and the learning and memory injury caused by the exposure of fluorine combined with aluminum will be analyzed. Secondly, hippocampus cell lines and offspring rats’ will be exposed to fluorine combined with aluminum while with or without adding rapamycin (R-ap), offspring rats’ behavior and key molecules in the mTOR pathway will be detected, so as to clarify protect mechanism of R-ap on the hippocampal cell and offspring rats’ learning and memory ability damage caused by fluorine combined with aluminum exposure. Aim of the study is to provide scientific basis for the prevention and treatment of the intelligence injury caused by the exposure of fluorine combined with aluminum.

氟铝联合暴露致世居儿童智力损伤，但防治落后。出生前后是影响学习记忆的关键时期。前期发现氟铝联合染毒组子鼠学习记忆受损且海马miR-183/96/182簇与对照组不同（P<0.01），靶基因预测到mTOR是该miR簇的共同靶基因，故推测该miR簇调控mTOR参与氟铝联合致学习记忆损伤。本研究第一部分探讨miR-183/96/182簇调控mTOR在氟铝联合致学习记忆损伤中的作用（1）构建miR-183/96/182簇功能获得和缺失的氟铝联合染毒海马细胞模型，检测mTOR，验证靶基因预测。（2）在氟铝联合染毒的海马细胞模型中，测定mTOR通路分子并分析其作用。第二部分构建添加和不添加雷帕霉素的氟铝联合染毒海马细胞模型及亲代孕哺期至子代成年前氟铝联合暴露子鼠模型，检测子鼠行为学及mTOR通路分子，阐明R-ap保护氟铝联合所致海马细胞和学习记忆损伤的机制。为病区世居儿童智力损伤防治提供科学依据。

氟铝联合暴露致世居儿童智力损伤。出生前后是影响学习记忆的关键时期。前期发现氟铝联合染毒组子鼠学习记忆受损且海马miR-183/96/182簇与对照组不同（P<0.01），靶基因预测mTOR是该miR簇的共同靶基因，推测该miR簇调控mTOR参与氟铝联合致学习记忆损伤。故本研究第一部分体内试验：复制氟铝暴露二代子鼠模型、Rno-miR- 183/96/182簇腺病毒干预与否的动物模型和雷帕霉素干预与否的动物模型，观察海马组织学及超微结构变化，检测子鼠行为及海马中miR-183、miR-96、miR-182表达水平和mTOR通路分子（PI3K、Akt、mTOR及P70S6K）的基因及蛋白表达水平；第二部分体外试验：复制氟铝联合染毒细胞模型、miR-183/96/182功能获得及功能缺失的氟铝联合染毒细胞模型及Rap干预与否的氟铝联合染毒细胞模型，检测NG108-15细胞凋亡情况及细胞中miR-183、miR-96、miR-182表达水平和mTOR通路分子（PI3K、Akt、mTOR及P70S6K）的基因及蛋白表达水平。结果表明：1.miR-183/96/182簇靶向调控mTOR的表达。2. 氟铝联合染毒致二代子鼠工作记忆及参考记忆损害及海马中miR-183/96/182簇上调和mTOR下调。3.氟铝联合染毒可诱导NG108-15细胞凋亡；上调miR-183/96/182簇表达后抑制mTOR及PI3K/Akt/mTOR/ eIF4E/ p70S6K/RPS6信号通路，可能是氟铝联合染毒致NG108-15细胞凋亡的机制之一。4.雷帕霉素干预后抑制mTOR及I3K/Akt/mTOR/ EIF4E/ P70S6K/S6信号通路表达增强自噬，从而发挥保护作用。本研究阐明了R-ap保护氟铝联合所致海马细胞和学习记忆损伤的机制，可为病区世居儿童智力损伤防治提供科学依据。