温度、血清以及肝素等因素对TAT穿膜效应的影响

Effects of TAT Penetrating by DMSO in Different Conditions of Temperature,Serum also Heparin

目的:探讨在不同实验条件下,二甲基亚砜(DMSO)预处理培养细胞对TAT穿膜效率的影响。方法:体外培养Caski、A549、HepG2及COS7细胞株在不同实验条件下与荧光标记多肽TAT或无意义肽NCO共孵育。荧光显微镜观察TAT-FITC的穿膜效率及其胞内定位;荧光酶标仪定量测定细胞内荧光强度。结果:10%DMSO预处理37℃和4℃下各细胞株后,TAT-FITC均可高效穿膜入胞,且胞浆、胞核中均匀分布,胞核浓度高于胞浆;相同条件下未见NCO-FITC穿膜进入细胞。无血清组(Caski:1881±66、HepG2:2112±74、A549:2126±59)血清组较有血清组(Caski:1312±90、HepG2:1308±11、A549:1370±22)细胞内荧光强度大,且有显著差异(P<0.05)。抑制剂Heparin存在时,Caski细胞荧光强度则明显减弱(加肝素组:1208±29,加肝素组:895±56;P<0.05)。结论:10%DMSO预处理不同培养细胞在37℃和4℃条件下均可提高TAT的穿膜效率,血清和Heparin可减弱DMSO的穿膜增强效应。

Objective：To study the effects of TAT penetrating into culturing cells pretreated by dimethyl sulfoxide（DMSO）with different culture conditions for optimization of the TAT penetrating condation.Method：Caski,A549,HepG2 and COS7 cells were treated with FITC-labeled peptides TAT and NCO.The penetrating efficiency of TAT and the distribution of fluorescence were observed under fluorescence microscopy after the cells were exposed to 10% DMSO incubating at the temperature of 37℃ or 4℃.Fluorescence in cells was quantified by fluorescence spectrum analysis.Quantitation of fluorescence intensity cell uptaken after adding antagonists or cultured with or without serum was examined.Result：Treatment of cells by 10% DMSO significantly enhanced the penetrating efficiency of TAT both at 37℃ and 4℃ and well-distribute in nucleus and in cytosol,fluorescence was distribute whereas no flourescence in NCO group was observed.After treated by 10% DMSO incubated with serum,quantification by fluorescence spectrum of TAT flourescence in cell showed that the enhancement effect of DMSO on TAT penetrating was significantly decreased than that without serum culturing（without serum group Caski：1881±66,HepG2：2112±74,A549：2126±59;serum group Caski：1312±90,HepG2：1308±11,A549：1370±22）P0.05.With Heparin TAT flourescence uptaken also decreased（without Heparin group：1208±29,Heparin group：895±56;P0.05）.Conclusion：Our data indicated that 10% DMSO enhanced different type cells uptake of TAT even culturing the cells at 4℃ and the fluorescence was uniformly distributed in cytosol and nucleus.And DMSO improved the penetrating efficiency of TAT could be inhibited by serum and Heparin.