偶联穿膜肽携带外源蛋白进入眼球的研究

发布时间：2018-01-21 18:31

  本文关键词： 细胞穿膜肽 聚精氨酸 眼球屏障 晶状体上皮细胞　出处：《眼科新进展》2017年06期 　论文类型：期刊论文

【摘要】：目的探讨以多聚精氨酸为载体,是否能够将外源蛋白质穿透细胞膜甚至穿透眼球屏障。方法本研究以9聚精氨酸(R9)为穿膜肽,构建R9-绿色荧光蛋白(green fluorescent protein,GFP)、GFP两种重组蛋白。体外实验中,将GFP、R9-GFP与人晶状体上皮细胞(human lens epithelial cell,HLEC)共培养后,经流式细胞仪、激光共聚焦显微镜及MTT法检测二者进入HLEC的能力及对HLEC的影响。体内实验中,将GFP、R9-GFP分别通过滴眼的方式作用于小鼠眼球,7 d后裂隙灯下观察后提取眼球总蛋白,ELISA法检测眼球中重组蛋白的含量。结果 MTT结果显示,R9-GFP、GFP都不影响HLEC的增殖活力。流式细胞仪和激光共聚焦显微镜检测结果显示,R9-GFP可以快速有效地进入HLEC内部且效果与给药剂量呈正相关。体内实验中,裂隙灯观察结果示,GFP、R9-GFP作用后小鼠眼球均未发现异常;ELISA检测结果显示,R9-GFP可以进入眼球内部。结论 R9具有携带蛋白质进入细胞膜甚至穿透眼球屏障的能力。本研究为眼部用药及给药模式的进一步改良提供了基础。
[Abstract]:Objective to investigate whether polyarginine can penetrate the membrane and even the eyeball barrier with polyarginine as the carrier. Methods in this study, 9 polyarginine R9 was used as the transmembrane peptide. Two recombinant proteins of R9-green fluorescent protein (GFP) were constructed. In vitro, GFP was prepared. R9-GFP was co-cultured with human lens epithelial cell line HLEC. Laser confocal microscope and MTT method were used to detect the ability of HLEC entry and its effect on HLEC. In vivo, GFP R9-GFP was applied to mouse eyeball by dropping eyes. After 7 days observation under slit lamp, the content of recombinant protein in the eyeball was detected by Elisa. Results the results of MTT showed R9-GFP. GFP did not affect the proliferative activity of HLEC. The results of flow cytometry and laser confocal microscopy showed. R9-GFP could enter the HLEC quickly and effectively, and the effect was positively correlated with the dosage. In vivo, the slit lamp observation results showed that GFP. After R9-GFP treatment, no abnormal eyeballs were found in mice. The results of ELISA test showed that. Conclusion R9 has the ability to carry protein into the cell membrane and even penetrate the eyeball barrier. This study provides a basis for the further improvement of drug administration and administration pattern.
【作者单位】： 爱尔眼科医院;
【基金】：黑龙江省卫生计生委科研课题资助(编号:2014-044)~~
【分类号】：R77
【正文快照】： 随着医学及生物技术的发展,以抗体为代表的重组蛋白药物越来越多地出现在眼科的临床应用中。由于角膜屏障及血-眼屏障的存在,分子量大、结构复杂且渗透性较差的蛋白药物不论是通过传统的滴眼给药还是全身注射,都不能有效到达眼球内的病灶区,因此临床往往选择玻璃体内注射方式，

本文编号：1452249