^125I-血管抑素内化及杀伤ECV304细胞的研究

Investigation of internalization and lethal effect of ^125I-angiostatin in ECV304 cell line

目的探讨人脐静脉内皮细胞系ECV304细胞内化血管抑素（AS）的能力及^125I-AS杀伤ECV304细胞的效应。方法采用放射配基结合分析法，37℃条件下AS受体阳性细胞ECV304与^125I-AS共同保温0．25，0．5，1，4，8，16，20和24h后，分别检测细胞的内化率；采用四甲基偶氮唑蓝（MTT）法测不同剂量^125I-AS、Na^125I及AS对ECV304细胞作用24，48，72，96h的杀伤作用。采用SPSS11．0软件对数据进行统计分析。结果37℃条件下保温，^125I-AS迅速与AS受体结合并使受体发生内化。0．5h内，ECV304细胞受体内化率、膜结合率及细胞结合率均随时间的延长而增加。0．5h时受体内化率为（12．6±1．1）％，膜结合率为（19．1±2．2）％，细胞结合率为（31．7±1．6）％，受体内化率与膜结合率呈正相关（r=0．814，P〈0．05）。0．5h后ECV304细胞受体内化率随时间延长继续增加，膜结合率随时间延长逐渐减少；至24h时受体内化率达（28．0±3．0）％，膜结合率为（10．8±1．4）％，细胞结合率为（38．8±2．3）％，受体内化率与膜结合率呈负相关（r=-0．681，P〈0．05）。^125I-AS对ECV304细胞杀伤作用呈剂量-效应、时间．效应关系，以370kBq／ml^125I-AS作用96h的杀伤作用最大，细胞抑制率达（79．4±3．7）％，明显强于Na^125I或AS（F=312．9013，P〈0．01）。结论^125I-AS可在AS受体介导下内化进入并杀伤ECV304细胞，呈剂量-效应、时间-效应关系。

Objective The aim of this study was to investigate the internalization of angiostatin （AS） receptors in ECV304 cell line （ an endothelial cell line of human placental vein） and the lethal effect of ^125I-AS on ECV304 cells in order to assess its therapeutic potential. Methods ECV304 cells were incubated together with ^125I-AS at 37℃ for different periods of time （0.25,0.5, 1,4,8,16,20 and 24 h） and the amount of internalized ^125I-AS were detected with γ counter. By using 3-（4,5-dimethylthiazol-2-yl）-2, 5-diphenyl-tetrazolium bromide （MTT） assay, the lethal effect of various doses of ^125I-AS, Na^125I and AS on ECV304 cells after 24, 48, 72, 96 h incubation was measured. SPSS 11.0 was used for data analysis. Resuits ^125I-AS bound rapidly to the AS receptors and induced the internalization of the membrane receptors of ECV304 cells at 37℃. The receptor internalization ratio, membrane receptor binding ratio and total binding ratio of ECV304 ceils showed time-dependent increase within 0.5 h. At 0.5 h, the receptor internalization ratio, the membrane receptor binding ratio and the total binding ratio of ECV304 cells were （ 12.6 ± 1.1）%, （19.1 ±2.2）% and （31.7 ± 1.6）%, respectively. The receptor internalization ratio showed positive correlation with the membrane receptor binding ratio （r = 0. 814, P 〈 0. 05 ）. After 0.5 h, the receptor internalization ratio continued to increase with time while the membrane receptor binding ratio decreased. At 24 h, the receptor internalization ratio, the membrane receptor binding ratio and the total binding ratio of ECV304 cells became （ 28.0 ± 3.0） %, （ 10.8 ± 1.4 ） % and （ 38.8 ± 2.3 ） %, respectively. The receptor internalization ratio showed negative correlation with the membrane receptor binding ratio （ r = -0. 681, P 〈 0.05 ）. The lethal effect of ^125I-AS measured by vital cell inhibition rate was dose-dependent and time-dependent, and was highest [ （79.4 ± 3.7 ） % ] at 96 h with a dose of 370 kBq/ml. The lethal effect of ^125I-AS was much stronger than Na^125I or AS （ F = 312. 9013, P 〈 0.01 ）. Conclusions ^125 I-AS can be internalized into ECV304 cells mediated by AS receptors and can thus effectively killed the cells. The lethal effect is dose-dependent and time-dependent.