【摘要】 目的：酪氨酸羟化酶(TH)是多巴胺合成的限速酶，其对于帕金森病发生有着重要意义。外泌体是由细胞分泌的直径30～200 nm的微囊泡，被认为是可通过血脑屏障的潜在药物载体。拟利用古菌核糖体蛋白L7Ae和Kt环的结合特性，获得高效包载酪氨酸羟化酶mRNA的外泌体(TH-Kt-Exo),以实现mRNA药物穿越血脑屏障的递送。方法：通过构建带有Kt环的TH mRNA重组质粒，以及外泌体膜蛋白CD63和L7Ae融合表达的重组质粒pCMV-CD63-L7Ae-His,共转染HEK293F细胞，通过超速离心方法收取细胞分泌的外泌体，通过qPCR检测外泌体中TH mRNA的含量并转染受体细胞。结果：与单一酪氨酸羟化酶质粒转染得到的外泌体TH-Exo相比，利用该方法获得的外泌体TH-Kt-Exo在TH mRNA的装载水平上有显著提高。此外，该外泌体能够将其装载的mRNA转运至受体细胞中。结论：通过L7Ae和Kt环的特异性结合，可以有效提高目的mRNA在外泌体中的包载。更多还原

【Abstract】 Objective: Tyrosine hydroxylase(TH), the rate-limiting enzyme in dopamine synthesis, plays a major role in the pathogenesis of Parkinson disease. Exosomes, vesicles with a diameter ranging from 30 to 200 nm secreted by cells, are considered potential carriers for drug delivery across the blood-brain barrier. To achieve efficient encapsulation of tyrosine hydroxylase mRNA in exosomes(TH-Kt-Exo), the binding properties of the archaeal ribosomal protein L7Ae and the Kt loop are exploited to enable delivery of mRNA-based therapeutics across the blood-brain barrier. Methods: The method involved constructing a recombinant plasmid containing TH mRNA with the Kt loop and a recombinant plasmid, pCMV-CD63-L7Ae-His, expressing the fusion of the exosomal membrane proteins CD63 and L7Ae. HEK293F cells were co-transfected with these plasmids, and exosomes secreted by the cells were harvested by ultracentrifugation. The content of TH mRNA in the exosomes was detected by qPCR, and the exosomes were then transfected into recipient cells. Results: Compared to TH-Exo obtained by transfection with the single tyrosine hydroxylase plasmid, the proposed method resulted in significantly higher levels of TH RNA encapsulation in exosomes(TH-Kt-Exo). In addition, the exosomes were able to deliver the loaded mRNA to recipient cells. Conclusion: The specific binding between L7Ae and the Kt loop effectively enhances the encapsulation of the target mRNA in exosomes.更多还原