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目的构建白细胞介素-1受体拮抗剂(IL-1RA)与细胞渗透肽(PEP-1)融合蛋白,探讨其对脑缺血再灌注(I/R)后神经细胞凋亡的抑制作用。方法构建IL-1RA与PEP-1融合基因,通过诱导表达和分离纯化,制备IL-1RA-PEP融合蛋白。建立大鼠大脑中动脉阻断模型(MCAo),形成I/R损伤,静脉注射IL-1RA-PEP,24 h后检测其在大鼠脑组织的分布,以及脑组织梗死体积,炎性细胞浸润和神经细胞凋亡情况,并分析相关凋亡蛋白的表达变化。结果 IL-1RA-PEP融合蛋白具有拮抗IL-1的生物学活性,其能有效地渗透进入I/R大鼠脑组织。给药24 h后,缺血半球脑梗死体积和神经细胞凋亡数量减少,胱天蛋白酶(caspase)3和9等凋亡蛋白表达水平下降,同时Bcl-x L抗凋亡蛋白表达水平升高。结论 IL-1RA-PEP融合蛋白能有效地渗透I/R大鼠脑组织,通过对相关凋亡蛋白的调控,抑制了I/R后脑组织神经细胞凋亡,对I/R损伤具有治疗作用。
Objective To construct a fusion protein of interleukin-1 receptor antagonist (IL-1RA) and cell penetrating peptide (PEP-1) and investigate its inhibitory effects on neuronal apoptosis after cerebral ischemia-reperfusion (I / R) Methods IL-1RA and PEP-1 fusion genes were constructed and induced by expression and purification. IL-1RA-PEP fusion protein was prepared. The model of middle cerebral artery occlusion (MCAo) was established in rats. I / R injury was induced and IL-1RA-PEP was intravenously injected. The distribution of IL-1RA-PEP was detected 24 h after operation and the infarction volume, inflammatory cell infiltration And apoptosis of nerve cells, and analyze the expression changes of related apoptosis proteins. Results The IL-1RA-PEP fusion protein could antagonize the biological activity of IL-1, which could effectively penetrate the I / R rat brain. After 24 h of administration, the volume of ischemic hemisphere infarction and the number of apoptotic neurons decreased, the expression of caspase 3 and 9 decreased, and the expression of anti-apoptotic protein Bcl-x L increased . Conclusion The IL-1RA-PEP fusion protein can effectively penetrate the brain tissue of I / R rats and inhibit the apoptosis of I / R neurons through I / R regulation of related apoptosis proteins, which may have a therapeutic effect on I / R injury.