【摘要】 目的:观察FITC标记的天花粉蛋白(TCS)进入瘤细胞的过程。方法:通过低温离心、丙酮分级沉淀、等电点沉淀及透析技术由新鲜栝楼根原汁中提取TCS;用FITC标记TCS,在激光共聚焦显微镜下动态观察FITC-TCS进入瘤细胞的过程。结果:纯化的蛋白经SDS-聚丙烯酰胺凝胶电泳分析为单一带,且与标准TCS泳率相同;终浓度50μg/ml FITC-TCS的孵育3h时已经进入黑色素瘤B-16细胞,6h时FITC-TCS进入细胞量达到最高,12h时已轻度下降,组间差异有统计学意义(P<0.01)。FITC-TCS组与对照组(FITC-BSA)荧光强度差异有统计学意义(P<0.01)。结论:采用FITC-TCS,可在激光共聚焦显微镜下观察到进入瘤细胞的动态过程。更多还原

【Abstract】 Objective:To extract trichosanthin from fresh trichosanthis natural juice,label trichosanthin with fluorescein isothiocyanate,and study the dynamic process of trichosanthin ingression into cells by laser scanning confocal microscopy.Methods:Trichosanthin was extracted from fresh trichosanthis natural juice by centrifugalization at low temperature,acetone fractional precipitation,isoelectric precipitation and dislysis;labeling trichosanthin with fluorescein isothiocyanate,the dynamic process of trichosanthin ingression into cells was studied by laser scanning confocal microscopy.Results:Purified trichosanthin displayed a single band in sodium dodecyl sulfate polyacrylamide gel electrophoresis,and was identical with the normal trichosanthin;labeled trichosanthin at a final concentration of 50 μg/ml entered B-16,after 3 incubation hours;FITC-TCS was at a high concentration in endochylema when incubating 6 hours;subsequently,the concentration of FITC-TCS fell-off in endochylema;statistical significance was conspicuous among groups(P<0.01).The fluorescence intensity of the FITC-TCS group was higher than that of the FITC-BSA group(P<0.01).Conclusion:The dynamic process of trichosanthin labelled with FITC ingression into neoplastic cells can be observed by laser scanning confocal microscopy.更多还原