激光共聚焦镜下观察天花粉蛋白进入瘤细胞的动态过程

Observation the dynamic process of trichosanthin ingression into neoplastic cells by laser scanning confocal microscopy

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。

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 eentrifugalization 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.