二甲亚砜或蔗糖预处理对TAT体外穿膜效率的影响

Penetrating efficiency of TAT pretreated by dimethyl sulfoxide or sucrose in vitro

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。

AIM To study the penetrating efficiency of TAT pretreated by dimethyl sulfoxide （DMSO） or sucrose in vitro. METHODS FITC-labeled peptides TAT-FITC and NCO-FITC （control） were synthesized artificially. Human cervical carcinoma cell lines Caski, human hepatocarcinoma cell lines HepG2 and African green monkey kidney cell lines COS7 were used in this in vitro experiment. The penetrating efficiency of TAT and the distribution of fluorescence were observed under fluorescence microscope after the cells were exposed to 10% DMSO or 0.5 mol.L^-1 sucrose. Fluorescence in cell was quantified by FACS and fluorescence spectrum analysis. The effectiveness of the endocytosis inhibitor NH4C1 and amiloride on the uptake of fluorescent labeled peptide was measured. RESULT Pretreatment on cells by 10% DMSO and 0.5 mol.L^-1 sucrose significantly enhanced the penetrating efficiency of TAT, also fluorescence distribution in nucleus were more than that in cytosol, while no fluorescence in NCO group was observed. The fluorescence intensity in cells pretreated by 10% DMSO was stronger than that in cells pretreated by 0.5 mol.L^-1 sucrose （P 〈 0.05）. The fluorescence intensity was decreased significantly by the endocytosis inhibitor in cells pretreated with 10% DMSO or 0.5 mol .L^-1 sucrose （P 〈 0.05）. CONCLUSION TAT penetrating efficiency could be increased by 10% DMSO or 0.5 mol.L^-1 sucrose in vitro and the penetrating pathway of TAT-FITC was uptake.