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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。
Objective To investigate the effect of glucose-hypertonic compound glucose, sucrose or mannitol pretreatment on TAT transmembrane efficiency. Methods The fluorescent labeled polypeptide TAT-FITC and non-significant peptide NCO-FITC were artificially synthesized and used to effect the in vitro culture of human cervical cancer cell line Siha, mouse fibroblast L929 and human hepatoma cell line HepG2. Fluorescence microplate reader was used to detect the effect of different preconditioning on uptake of fluorescent short peptide in each cell and the effect of endocytosis inhibitor amiloride on TAT transmembrane after different preconditioning.Fluorescence microscopy showed that TAT-FITC And its localization in Siha cells. The effect of different pretreatments on Siha cell survival rate was detected by MTT assay. Results TAT-FITC could penetrate into the cells with high osmotic compounds pretreatment, and evenly distributed in the cytoplasm and nucleus. The nucleus concentration was higher than that in the cytoplasm. No NCO-FITC penetrated the membrane cell. The fluorescence intensity of TAT-FITC uptake by 0.6mol·L-1 glucose was similar to that of 0.6mol·L-1 sucrose pretreatment and significantly higher than that of the control group (P <0.01). The fluorescence intensity of 0.5mol·L-1 mannitol The fluorescence intensity of alcohol pretreatment followed. After adding amiloride, the fluorescence intensity of TAT-FITC uptake in cells pretreated with saccharide osmotic significantly decreased (P <0.01). MTT data showed that appropriate concentrations of carbohydrate hypertonic compounds have a slight effect on cell viability. CONCLUSION: Pretreatment with 0.6 mol·L-1 glucose or 0.6 mol·L-1 sucrose or 0.5 mol·L-1 mannitol can increase the transmembrane efficiency of TAT cells, but have little effect on cell viability. After pretreatment Cells may take TAT-FITC short peptides endocytosed.