P300/CBP基因定点诱变对神经病理性疼痛的靶向治疗作用

We have demonstrated in the last study that rats’ spinal P300/CBP proteins contributed to the development of neuropathic pain by upregulating the expression of several pain-related genes, and the neuropathic pain symptoms were relieved by the downregulation of P300/CBP proteins. However, since P300/CBP proteins play universe roles and they are essential for some vital cellular activities, the direct inhibition of these proteins can lead to severe side effects. Recent evidences indicated that the modification of amino acids in different sites of P300/CBP proteins resulted in the transcription of different downstream genes. Therefore, the intervention of the modification of amino acids in certain sites in order to regulating specific genes is a promising way for the targeted treatment of P300/CBP-related disease. In this project, first we will search for the site-specific modification of P300/CBP proteins which are related to neuropathic pain in the rats’ model of sciatic nerve injury. Then we will employ the technology of site-directed gene mutation to replace the modified amino acids separately so as to screen out effective sites of mutation for the treatment of neuropathic pain. Furthermore, we will evaluate the influence of site-directed mutations on the physiological parameters of rats’ spinal cord and the whole genomic changes downstreamed to the P300/CBP proteins. By completing the above studies, we expect to find out the site-directed mutations which are not only effective for the treatment of neuropathic pain but also have less disturbance on the physiological function of spinal cord, so as to create advantages for the genetic treatment of neuropathic pain.

在上一课题里我们获得了大鼠脊髓P300/CBP蛋白通过上调疼痛基因参与神经病理性疼痛发病的证据，也证实了控制P300/CBP表达可缓解疼痛。然而P300/CBP蛋白不仅作用广泛，而且还是维持细胞重要生理功能的蛋白，直接干预它们的表达可导致不良反应。实验表明P300/CBP蛋白不同位点氨基酸修饰介导不同的下游基因转录，通过对某位点氨基酸修饰的干预而特异性调控下游疾病基因的表达，可能为靶向治疗P300/CBP相关疾病提供新的思路。因此，首先我们将采用大鼠坐骨神经损伤模型筛选出与神经病理性疼痛相关的P300/CBP蛋白氨基酸修饰位点，再通过基因定点诱变改变其结构，寻找出有效缓解神经病理性疼痛的位点突变，并检测大鼠脊髓生理功能和P300/CBP下游全基因组表达，探索对疼痛治疗有效而对脊髓生理功能和相关基因影响微弱的P300/CBP基因位点突变，为开展神经病理性疼痛的基因治疗创造有利条件。

该项目为“青年-面上连续资助项目”。在上一课题里我们获得了大鼠脊髓P300/CBP蛋白通过上调疼痛基因参与神经病理性疼痛发病的证据，也证实了控制P300/CBP表达可缓解疼痛。然而P300/CBP蛋白不仅作用广泛，而且还是维持细胞重要生理功能的蛋白，直接干预它们的表达可导致不良反应。实验表明P300/CBP蛋白不同位点氨基酸翻译后修饰介导不同的下游基因转录，通过对某位点氨基酸修饰的干预而特异性调控下游疾病基因的表达，可能为靶向治疗P300/CBP相关疾病提供新的思路。在本项目中，我们在坐骨神经损伤大鼠模型中探索了P300/CBP不同位点修饰对下游基因转录的调控作用。结果显示，脊髓P300蛋白ser1834位点磷酸化可增强其组蛋白乙酰转移酶功能，进而增强H3K27ac表达，活化基因增强子，促进下游基因的转录。此外，通过基因测序等实验，进一步探索了P300/CBP蛋白及不同组蛋白修饰与差异基因表达变化的联系。通过蛋白质组学等实验，探索包括P300/CBP在内的蛋白质泛素化、巴豆酰化、琥珀酰化等氨基酸翻译后修饰改变，进一步明确P300/CBP等蛋白质不同位点氨基酸翻译后修饰对下游基因转录的调控及其在神经病理性疼痛发病机制中的作用。