糖类高渗化合物对TAT穿膜效率的影响

Penetrating efficiency of TAT pretreated by hyperosmotic saccharides

目的研究糖类高渗化合物葡萄糖、蔗糖或甘露醇预处理细胞对TAT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Siha、小鼠成纤维细胞L929及人肝癌细胞株HepG2。荧光酶标仪定量检测不同预处理对各细胞摄取荧光标记短肽的影响及内吞抑制药阿米洛利对不同预处理后TAT穿膜的影响;荧光显微镜观察不同预处理后,TAT-FITC的穿膜效率及其在Siha细胞内的定位;MTT检测不同预处理对Siha细胞存活率的影响。结果糖类高渗化合物预处理细胞后,TAT-FITC可高效穿膜进入细胞内,且在胞浆、胞核中均匀分布,胞核浓度高于胞浆浓度;未见NCO-FITC穿膜进入细胞。0.6mol·L-1葡萄糖预处理后,细胞摄取TAT-FITC的荧光强度较0.6mol·L-1蔗糖预处理后相近且较对照组明显增强(P<0.01),0.5mol·L-1甘露醇预处理的荧光强度次之。加入阿米洛利后,糖类高渗化合物预处理的细胞摄取TAT-FITC荧光强度均明显减弱(P<0.01)。MTT数据显示适当浓度的糖类高渗化合物对细胞的活力有轻微影响。结论0.6mol·L-1葡萄糖或0.6mol.L-1蔗糖或0.5mol·L-1甘露醇预处理均可提高TAT对培养细胞的穿膜效率,但对细胞活力影响较小;预处理后细胞可能以内吞方式摄取TAT-FITC短肽。

AIM To study the penetrating efficiency of TAT in cells pretreated by hyperosmotic saccharides in vitro. METHODS TAT-FITC （ FITC-labeled peptides） and NCO-FITC （ control nonsense peptides） used in this study were synthesized artificially. Human cervical carcinoma cell lines Siha, mouse fibroblast cell lines L929 and human hepatocarcinoma cell lines HepG2 cells were used in the in vitro experiments. The penetrating efficiency of TAT and the distribution of fluorescence within cytosol were observed under fluorescence microscope after the cells were exposed to hyperosmotic saccharides. Fluorescence intensity in cells was quantified by fluorescence spectrum analysis. Quantitation of cell fluorescence intensity after presenting endocytosis inhibitor, amiloride was also examined. Cell viability assay was carried out using MTT dye. RESULTS Treatment of cells with hyperosmotic saccharides significantly enhanced the cell-penetrating efficiency of TAT. The fluorescence distributed in nucleus was more than that in cytosol, whereas no fluorescence in NCO-FITC incubated cells was observed. After treated with 0.6 mol·L-1 glucose or 0.6 mol·L-1 sucrose, quantification by fluorescence spectrum from TAT-FITC uptake in cell showed that both of treatments enhanced with a significant increase than the NCO-FITC control cells （ P 0.01）,0.5 mol·L -1 mannitol treatment had a slight enhancement effect on TAT penetrating. The enhancement effect on TAT penetrating by treating with hyperosmotic saccharides was decreased after the presentation of endocytosis inhibitor amiloride （ P 0.01） . MTT data indicated that hyperosmotic saccharides had little toxic effects on cell viability. CONCLUSION The treatment of cells with hyperosmotic saccharides can improve the efficiency of TAT-FITC delivery with very little effects on cell viability, and the cellular uptake of TAT-FITC may be by way of endocytosis after the pretreatment.