沙蚕蛋白酶的荧光标记及细胞摄取机制研究

Study on the Preparation and Cell Uptake Mechanism of Fluorescently-Labeled Nereis Active Protease

该文利用FITC对沙蚕蛋白酶(NAP)进行荧光标记,并通过薄层色谱法、红外光谱法、紫外光谱法及荧光光谱法对标记物进行表征,紫外分光光度法计算标记度。通过测定NAP与FITCNAP的酶促反应动力学参数来比较NAP在标记前后的活性,同时应用MTT法考察NAP标记前后对NCI-H1299细胞毒性的影响。流式细胞术研究NCI-H1299细胞摄取FITC-NAP的机制,荧光显微镜定性观察NCI-H1299细胞对FITC-NAP的摄取。结果表明,每分子标记物约含1.78分子FITC,其最佳荧光激发波长为490 nm、发射波长为515 nm;荧光标记后NAP的活性未受到显著影响; NCI-H1299细胞对FITC-NAP的摄取与药物浓度、作用时间呈正相关;氧化苯砷组的荧光强度低于FITC-NAP组且有极显著差异(P<0.01),因此NAP的摄取机制为内吞。进一步研究发现, NCI-H1299细胞摄取NAP受到网格蛋白依赖性内吞和小窝/脂筏蛋白介导的内吞共同作用。荧光显微镜观察发现,随着时间的增加,更多的细胞摄入FITC-NAP,且主要分布在细胞膜及细胞质中,细胞核中未见分布。

In this paper, fluorescent labeling of NAP was performed by FITC. FITC-NAP was characterized by thin-layer chromatography, infrared spectrum, ultraviolet spectrum and fluorescence spectrum.The fluorescence substitute ratio of FITC-NAP was measured by ultraviolet spectrophotometry. The activity of NAP and FITC-NAP were compared by measuring the enzymatic reaction kinetic parameters. Cell viability was evaluated by MTT assay. The uptake mechanism of NCI-H1299 cells to FITC-NAP was studied by flow cytometry. Fluorescence microscopy was used to observe the uptake of FITC-NAP qualitatively. The results indicated that each molecule of the labelled product approximately contained 1.78 molecules of FITC, The optimal ?uorescence excitation and emission wavelengths of FITC-NAP were 490 nm and 515 nm. The activity of NAP after fluorescent labeling was not significantly affected. the uptake of FITC-NAP was positively correlated with drug concentration and incubation time. The fluorescence intensity of the Phenylarsine oxide group was lower than that of the FITC-NAP group and there was a significant difference(P<0.01). This showed that the uptake mechanism of NCI-H1299 cells to FITC-NAP was endocytosis. Further studies found that the uptake of NAP was affected by clathrin-dependent endocytosis and caveolin/lipoprotein-mediated endocytosis.Fluorescence microscopy revealed that more cells uptaked FITC-NAP over time, and FITC-NAP mainly distributed in the cell membrane and cytoplasm, no distributed in the nucleus.