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目的 研究相思豆毒素 (ABR)的内化入胞作用并探讨内化入胞的可能作用机制。方法 葡聚糖凝胶层析制备ABR的异硫氰酸荧光素 (FITC)标记探针 (FITC ABR) ;MTT比色法测定FITC ABR对体外培养人胚胎肾细胞 (HEK)增殖的抑制作用 ;激光共聚焦显微镜示踪FITC ABR的内化过程。结果FITC ABR在凝胶层析图谱中先于FITC洗脱 ,两者能较好的分离 ,所制备的FITC ABR浓度为 10 .0g·L- 1,FITC ABR与ABR在 10 0 0～ 0 .0 1μg·L- 1浓度范围内 ,以相同浓度作用于HEK细胞 ,对细胞增殖的抑制作用相似 ,两者的pIC50 均约为 16pmol·L- 1;分子荧光探针FM4 6 41.5 μmol·L- 1对体外培养HEK细胞连续标记 12h ,能特异性标记细胞膜及膜性细胞器如胞内体、溶酶体、高尔基体等 ,细胞膜及胞内分别可见连续以及散在的红色荧光 ;FITC ABR 10mg·L- 1单独标记细胞 15 ,30min ,即可见到胞内有绿色荧光 ,FITC ABR与FM4 6 4联合标记细胞 ,在胞内的某些红色荧光部位同时具有FITC ABR的绿色荧光 ,两种颜色的荧光叠加后产生棕褐色荧光 ;以0 .1mol·L- 1乳糖封闭细胞膜的ABR结合位点后 ,再以FITC ABR标记细胞 30min ,可见细胞膜的绿色荧光明显减弱 ,胞内几乎无绿色荧光。升高胞内体pH的药物NH4 Cl 1mmol·L- 1能明显增强FITC ABR对胞内各膜?
Aim To study the internalization and invasion of abrin and study the possible mechanism of abscisic acid (ABR). Methods FITC ABR (FITC ABR) was prepared by dextran gel chromatography. The inhibitory effect of FITC ABR on the proliferation of human embryonic kidney (HEK) cells was determined by MTT assay. Laser confocal microscopy traced the internalization of FITC ABR. Results The FITC ABR was eluted prior to FITC in the gel chromatogram. The FITC ABR concentration was 10 .0g · L -1 and the FITC ABR and ABR was between 100 ~ 0. In the concentration range of 0 1μg · L-1 HEK cells treated with the same concentration of the same concentration of inhibition of cell proliferation similar, both pIC50 were about 16 pmol·L-1; molecular fluorescent probe FM4 6 41.5 μmol·L- 1 to HEK cells cultured in vitro for 12h, which could specifically label the cell membrane and membranous organelles such as endosomes, lysosomes, Golgi apparatus and so on. Continuous and scattered red fluorescence were observed in the cell membrane and in the cells. FITC ABR 10 mg · L - 1 labeled cells 15, 30min, you can see the cells have green fluorescence, FITC ABR and FM464 labeled cells, some of the intracellular red fluorescent sites also have FITC ABR green fluorescence, two-color fluorescence After superimposed, the tan fluorescence was generated. After blocking the ABR binding sites of the cell membrane with 0 .1 mol·L-1 lactose, the cells were labeled with FITC ABR for 30 min. The green fluorescence of the cell membrane was obviously weakened and there was almost no green fluorescence in the cells. Elevated intracellular pH drug NH4Cl 1mmol·L-1 can significantly enhance the FITC ABR intracellular membrane?