摘要
通过PCR和免疫荧光技术实现生物素-链霉亲和素-异硫氰酸荧光素标记核酸,同时用0.1%(摩尔百分比)ATTO 647 DOPE标记LNP(SM-102∶DSPC∶Cholesterol∶PEG2000-DMG=50∶10∶38.5∶1.5,摩尔百分比),对制成的LNP-DNA制剂的理化性质及包封进行考察,并进行了体外细胞(Hela)实验,使用特异性抗体标记内涵体/溶酶体等内吞途径中相关囊泡,在倒置荧光显微镜或高内涵显微镜下考察LNP内吞过程及其在细胞内的转运情况。以GFP表达质粒作为递送货物,考察转染后绿色荧光蛋白表达效率。结果表明,祼DNA在被细胞内吞后,在细胞外周和表面形成内涵体颗粒且不会被进一步转运到细胞内。转染时间是影响LNP-DNA转染效率的主要因素之一,LNP-DNA通过内吞途径进入细胞,大部分被早期内涵体摄取形成LNP-DNA内涵体,随后转运至细胞核周围,部分转运至溶酶体,转运过程实现DNA的逃逸。
Biotin-streptavidin-fluorescein isothiocyanate labeled nucleic acid was achieved by PCR and immunofluorescence,while LNP was labeled with 0.1%(mole percentage)ATTO-647 DOPE(SM-102∶DSPC∶Cholesterol∶PEG2000-DMG=50∶10∶38.5∶1.5,mole percentage).Firstly,investigated the physical and chemical properties and encapsulation of the LNP-DNA preparations.Secondly,in vitro Hela cell experiments,specific antibodies were used to label relevant vesicles in endocytic pathways such as endosomes/lysosomes;LNP endocytosis and its intracellular transport were investigated under inverted fluorescence microscope or high-content imaging system.In addition,the GFP expression plasmid was used as the delivery cargo to examine the efficiency of green fluorescent protein expression after transfection.The results showed that the naked DNA,after endocytosis,forms endosomal granules in the periphery of the cell and does not be further transported into the cell.Transfection time is one of the main factors affecting the transfection efficiency of LNP-DNA.LNP-DNA enters cells through endocytosis,and most of LNP-DNA is taken up by early endosomes to form LNP-DNA endosomes,which are then transported around the nucleus and partially to lysosomes,during which DNA escape is realized.
作者
罗成枝
彭保卫
Luo Chengzhi;Peng Baowei(Dali University,Dali 671000,China)
出处
《广东化工》
CAS
2024年第20期89-91,共3页
Guangdong Chemical Industry
