胚胎期阿片类物质影响子代成瘾行为的中枢蛋白激酶C机制

Protein kinase C (PKC) plays an important role in the development of drug addiction. It has been domenstrated that prenatal opiate exposure can not only impair the physical and mental health of the offspring, but alter the individual susceptibility to addiction later in life.Based on the combination of the molecular biology and behavioral pharmacology technology and using the rodents and the day-old chicks as experimental objects, we will employ the alterations in individual addictive neurobehaviors produced by the injection of opiate in the embryonic periods as experimental modes to investigate: (1) the effects of morphine during different embryonic periods on the expression of PKC subtypes in IMM of chick brain and on the alterations of addictive behaviors in the day-old chicks prenatally exposured to morphine,and exploring the PKC subtypes sensitive to prenatal morphine exposure. (2) examing the effects of different PKC subtypes on the levels of GABA and GAT-1 and the affinity and density of GABAA/B receptors in IMM of chick brain, and on the cognitive function or the behavioral sensitivities to opiate dependence in the day-old chicks prenatally exposured to morphine during different embryonic peirods.(3) the effects of the excitement or inhibiton of sepcific PKC subtypes in the PFC of rodents exposed in utero to morphine on the function of GABAergic system, and on the addictive behaviors in the affected rodents.From the perspective of developmental biology, the main purpose of the present studies is to explore the molecular mechanism underlying the alterations in the addictive susceptibility induced by opiate exposure during the embryonic periods. The present investigations will provide new thoughts for the treatment of opiate addiction.

PKC在药物成瘾过程中发挥着重要作用。研究证实，孕期阿片类物质滥用不仅危害子代的身心健康，并可导致其成瘾易感性的变化。本研究应用分子生物学与行为药理相结合的技术手段，选用啮齿类动物和日龄小鸡为实验对象，以胚胎期注射阿片类药物对子代成瘾行为的影响为实验模型，考察（1）鸡胚发育不同阶段吗啡用药对日龄小鸡IMM内PKC各亚型表达及其成瘾行为变化的影响，探索对胚胎期吗啡暴露敏感的PKC亚型。（2）比较鸡胚发育不同阶段注射吗啡后，IMM内PKC各亚型的表达对GABA递质及GAT-1水平、GABAA/B受体亲和力与密度的影响，及其与脑认知功能或成瘾行为改变的关系。（3）通过脑定位给药方式，分析激动或抑制母源性吗啡暴露的仔鼠其前额叶皮层内PKC亚型的表达对GABA递质系统功能及成瘾行为变化的影响；从神经发育的角度，深入探讨胚胎期阿片类物质影响子代成瘾行为的分子机制，为阿片类药物成瘾的治疗提供新的思路。

蛋白激酶C(protein kinase C, PKC)参与对胚胎期吗啡暴露影响子代脑认知功能及成瘾行为的调节。运用分子生物学和行为药理学等技术，本项目对胚胎发育敏感期吗啡暴露影响子代成瘾相关行为的PKC调节机制进行了初步研究。首先通过比较鸡胚发育不同时期吗啡暴露对大脑中间腹内侧原皮质（intermediate medial mesopallium, IMM）中PKC亚蛋白表达的影响，寻找到了对胚胎期吗啡暴露敏感的PKC亚型（这些亚型包括α、β、δ、ε和γ）；继而通过Western Blot方法、免疫组化技术和质谱分析，筛选出了参与胚胎发育敏感期吗啡暴露导致日龄小鸡学习记忆能力受损（包括PKCα和PKCδ）及其对吗啡奖赏作用增强（PKCα）的PKC蛋白；而且本研究发现，这些蛋白对中枢IMM核团内GABA能递质系统功能的调节存在差异性。与此同时，考虑到本研究数据的可推广性，在鸡胚模型研究的基础之上，选用Wistar大鼠为参照，对胚胎期吗啡暴露敏感的PKC蛋白的调节机制进行了深入研究，结果显示：仔鼠大脑前额叶皮层（prefrontal cortex, PFC）神经元膜PKCδ表达增加可以下调PFC核团内GABA阳性神经元数量和GABA神经递质水平，并导致仔鼠的空间学习记忆能力受损；而膜PKCα表达减少有可能与PFC核团内GABA阳性神经元数量减少、GABA神经递质水平下降和仔鼠吗啡自身给药行为的敏感性增强高度相关。综合上述结果，通过对鸡胚和鼠胚两种不同物种模型之间的互补性研究，深入阐明了PKC蛋白在胚胎期吗啡暴露影响子代成瘾相关行为中的调节作用。本研究为阿片成瘾的生物学机制提供理论基础的同时，也为该疾病的治疗以及以PKC蛋白为靶点的新型药物的研发提供重要参考和理论依据。