偶联穿膜肽携带外源蛋白进入眼球的研究

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,7d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.

Study on cell-penetrating peptides carrying foreign proteins into eyeball

Objective To investigate whether poly arginine as the carrier can carry foreign proteins to penetrate the cell membrane and even penetrate the eyeball barrier.Methods Poly-Args (R9) was used as a CPP in this study.R9-green fluorescent protein (GFP) and GFP were constructed.In vitro,human lens epithelial cells were treated with these two proteins.Then,MTT assay were used to detect whether the protein could affect the proliferation of the cells.Flow cytometry and laser confocal microscopy were used to detect the penetrability of CPPs on the cells.In vivo,eyes of mice were treated with protein in eye drops way for 7 days.Then total protein were extracted,ELISA were used to detect the penetrability of CPPs.Results The results of MTT,flow cytometry and laser confocal microscopy showed that CPPs could carry protein into cells in a dose dependent manner without affecting cell proliferation.In vivo,slit lamp showed that the mice eyeballs had no any abnormal after treated by GFP,R9-GFP,and ELISA results also showed that R9 could effectively get foreign protein into the eyeball.Conelusion R9 can carry foreign protein into the cell membrane and eyeball barrier.This study provides the basis for the eye medication and dosing mode improvement.