论文部分内容阅读
目的研究二甲亚砜(DMSO)或蔗糖预处理细胞对TAT体外穿膜效率的影响。方法人工合成荧光标记多肽TAT-FITC和无意义肽NCO-FITC,将其作用于体外培养的人宫颈癌细胞株Caski、人肝癌细胞株HepG2及非洲绿猴肾细胞株COS7。荧光显微镜观察不同DMSO或蔗糖预处理后,TAT-FITC的穿膜效率及其在细胞内的定位;流式细胞仪及荧光酶标仪定量细胞对荧光标记短肽的摄取;10%DMSO或0.5mol·L-1蔗糖预处理后,荧光酶标仪定量内吞抑制剂NH4Cl和阿米洛利对荧光标记短肽摄取的影响。结果10%DMSO或0.5mol·L-1蔗糖预处理细胞后,TAT-FITC可高效进入细胞,且在胞浆、胞核中均匀分布,胞核中浓度高于胞浆;未见NCO-FITC穿膜进入细胞。10%DMSO预处理后,加入TAT-FITC的细胞荧光强度较0.5mol·L-1蔗糖预处理强,有显著差异(P<0.05)。加入2种抑制剂后,10%DMSO或0.5mol·L-1蔗糖预处理的、加入TAT-FITC的Caski细胞荧光强度均明显减弱(P<0.05)。结论10%DMSO或0.5mol·L-1蔗糖预处理均可提高TAT的体外穿膜效率,且预处理后TAT-FITC的穿膜途径仍为细胞内吞。
Objective To investigate the effect of dimethylsulfoxide (DMSO) or sucrose pretreatment on the transmembrane efficiency of TAT in vitro. Methods The fluorescent labeled polypeptide TAT-FITC and the nonsense peptide NCO-FITC were artificially synthesized and applied to human cervical cancer cell line Caski, human hepatoma cell line HepG2 and African green monkey kidney cell line COS7 in vitro. Fluorescence microscopy was used to observe the transmembrane efficiency of TAT-FITC and its localization in cells after pretreatment with different DMSO or sucrose. The uptake of fluorescently labeled short peptides was quantified by flow cytometry and fluorescence microplate reader. The concentration of 10% DMSO or 0.5 After pretreatment with mol·L -1 sucrose, the effects of endocytosis inhibitors NH4Cl and amiloride on the uptake of fluorescently labeled short peptides were quantified by fluorescence microplate reader. Results TAT-FITC could efficiently enter the cells after 10% DMSO or 0.5 mol·L-1 sucrose pretreatment, and were evenly distributed in the cytoplasm and nucleus with a higher concentration in the nucleus than in the cytoplasm. No NCO-FITC Through the membrane into the cell. After pretreatment with 10% DMSO, the fluorescence intensity of TAT-FITC-added cells was significantly higher than that of 0.5mol·L-1 sucrose pretreatment (P <0.05). After addition of two inhibitors, the fluorescence intensity of Caski cells pretreated with 10% DMSO or 0.5 mol·L -1 sucrose, TAT-FITC, was significantly decreased (P <0.05). CONCLUSION: Pretreatment with 10% DMSO or 0.5mol·L-1 sucrose can increase the transmembrane efficiency of TAT in vitro, and the transmembrane pathway of TAT-FITC remains the endocytosis after pretreatment.