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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (2/3): 69-75    DOI: 10.13523/j.cb.2306035
技术与方法     
高效包载酪氨酸羟化酶mRNA外泌体的制备*
樊宇钦1,黎智康1,梁至轩1,赵紫涵1,谢秋玲1,2,**()
1 暨南大学生命科学技术学院 广州 510632
2 基因工程药物国家工程研究中心 广州 510632
Preparation of Exosomes Containing Highly Efficient Encapsulated Tyrosinase Hydroxylase mRNA
FAN Yuqin1,LI Zhikang1,LIANG Zhixuan1,ZHAO Zihan1,XIE Qiuling1,2,**()
1 School of Life Science and Technology, Jinan University, Guangzhou 510632, China
2 National Engineering Research Center of Genetic Medicine, Guangzhou 510632, China
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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。
关键词： 酪氨酸羟化酶;  外泌体;  古菌核糖体蛋白;  Kt环    
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.
Key words: Tyrosine hydroxylase    Exosome    Archaeal ribosomal protein    Kt-turn loop
收稿日期: 2023-06-27 出版日期: 2024-04-03
ZTFLH:  Q814  
基金资助: *广州市科技计划(20212210007)
通讯作者: **电子信箱:txql@jnu.edu.cn   
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樊宇钦
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引用本文:

樊宇钦, 黎智康, 梁至轩, 赵紫涵, 谢秋玲. 高效包载酪氨酸羟化酶mRNA外泌体的制备*[J]. 中国生物工程杂志, 2024, 44(2/3): 69-75.

FAN Yuqin, LI Zhikang, LIANG Zhixuan, ZHAO Zihan, XIE Qiuling. Preparation of Exosomes Containing Highly Efficient Encapsulated Tyrosinase Hydroxylase mRNA. China Biotechnology, 2024, 44(2/3): 69-75.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2306035    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I2/3/69

图2  重组质粒在HEK293F细胞中的瞬时表达 进行n=3的独立重复实验,并确保所有样品分析条件一致
图3  外泌体表征 A:粒径分析 B:透射电镜 C:免疫印迹。Control:未转染细胞外泌体;TH-Exo: pcDNA3.4-TH-Flag-Kt转染细胞外泌体;TH-Kt-Exo:pcDNA3.4hTH-Flag-Kt、pCMV-CD63-L7Ae-His共转染细胞外泌体
图4  外泌体包载TH mRNA检测 进行n=3的独立重复实验,并确保所有样品分析条件一致
图5  外泌体孵育HEK293T细胞后受体细胞中TH蛋白的相对mRNA和蛋白质表达情况 A:TH mRNA水平 B:TH蛋白水平。进行n=3的独立重复实验,并确保所有样品分析条件一致 C:Control 0.1:0.1×107 Exo/106 cell;0.25:0.25×107 Exo/cell;0.5:0.5×107 Exo/cell;1:1×107 Exo/cell
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