成体齿状回神经干细胞microRNA-132/212在阿片成瘾中的作用及分子机制

Previous studies indicate that adult hippocampal neurogenesis contributes to memory trace clearance. Non-coding nucleic acid microRNA-132/212 family can regulate the neural stem cell development through silencing other genes by inhibiting their mRNA translation. However, it still remains unclear how microRNA-132/212 within adult dentate gyrus neural stem cells control neurogenesis and addiction memory. In the present work,we plan to: ? observe the changes in dentate gyrus neurogenesis as well as the expression level of CREB signaling members, microRNA-132/212 and their putative target genes in the sub-population of neural stem cell-derived cells, based on morphine self-administration by rats; ? using the μ-opioid receptor over-expressing stable cell line (Neuro2a MOR), investigate whether the neural stem cell developmental outcome after morphine exposure is mediated by the CREB-induced microRNA-132/212 and their target molecules; ? verify that the over-expression of microRNA-132 and its novel target in a time-specific and cell type-restricted manner can affect the acquisition, extinction/clearance and reinstatement of morphine self-administration in rats. We aim to demonstrate that addiction memory can be controlled by CREB-induced microRNA-132/212 and their target molecules through the regulation of adult hippocampal neurogenesis.

研究表明，成体海马齿状回神经元新生可参与记忆痕迹的清除。非编码核酸microRNA-132/212家族可直接抑制其它基因的mRNA翻译，参与调控齿状回神经干细胞发育。然而，成体齿状回神经干细胞内的microRNA-132/212，如何调控海马齿状回神经元新生及成瘾记忆，仍不清楚。本课题计划：①观测阿片自给药大鼠齿状回神经干细胞发育，胞内CREB通路、microRNA-132/212及靶基因水平变化；②构建稳定高表达μ-阿片受体的N2a细胞系，确定吗啡处理后N2a细胞发育变化，是否受胞内CREB诱导的microRNA-132/212水平及其主要靶基因影响；③在体验证，时空特异性地过表达microRNA-132及其靶基因对大鼠阿片自身给药形成、消退/清除和重建的影响。以期证明CREB诱导性的microRNA-132/212家族及其靶基因通过调控成体海马齿状回神经元新生，参与药物成瘾记忆。

近来研究表明，成瘾性药物不仅诱发奖赏相关脑区神经元的可塑性，而且修饰成体神经元新生，包括神经干细胞的增殖和分化。神经干细胞增殖后一部分快速凋亡，另一部分分化发育为成熟神经元。因此，相对于神经干细胞的增殖，成瘾性药物对其分化的影响更持久。已知脑内多种信号分子参与调控成体神经干细胞的分化，包括微小RNAs。其中，miR-132/212已被报道能促进啮齿类动物胚胎和成体神经干细胞的分化。目前仍不清楚miR-132/212是否参与调控成体大鼠神经干细胞的分化，进而介导吗啡成瘾，如干预miR-132/212是否对吗啡成瘾行为产生影响。本项目采用大鼠吗啡自我给药（self-administration, SA）和离体培养的神经干细胞模型，证明了神经干细胞内的miR-132/212通过调控干细胞的分化，参与介导吗啡成瘾行为。主要结果如下：1）吗啡和miR-132均能促进离体培养的神经干细胞的分化；2）吗啡SA促进成体大鼠齿状回神经干细胞的分化；3）大鼠齿状回神经干细胞过表达miR-132也能够促进神经干细胞的分化；4）大鼠齿状回神经干细胞时空特异性过表达miR-132可促进其吗啡SA行为的形成，而不影响SA的消退过程。本项目研究首次证明，大鼠海马齿状回神经干细胞的miR-132通过调控干细胞的分化贡献于吗啡成瘾行为的形成，该结果对揭示吗啡成瘾的分子机制提供了新的依据。