负载表没食子儿茶素没食子酸酯对血小板功能与凋亡等影响

Effects of Loaded Buffer with Epigallocatechin Gallate on Physiological Functions of Platelets

本研究探讨表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)负载后对血小板生理生化功能的影响。将血小板用不同浓度EGCG(浓度分别为:0、2.5、5、7.5、10、12.5、15、20、30mmol/L)的负载液处理,通过比对回收率和聚集活性,将实验分为2.5、5、10mmol/L负载液处理组和空白对照组,观察保存期内血小板生理生化功能的改变;血小板的回收率通过血细胞计数技术仪进行检测;血小板聚集活性通过比浊法测定;流式细胞术检测血小板的凋亡。结果表明,负载液中EGCG浓度为2.5、5、10mmol/L时负载入血小板的EGCG量分别为0.4006±0.12、1.0527±0.1503、1.6902±0.1112mmol/L。当负载液中EGCG浓度较低时(<15mmol/L),负载液中EGCG的浓度与血小板对EGCG的吸收量呈正相关。检测血小板回收率时发现,2.5mmol/L的负载液处理组回收率为(82.45±0.360)%,空白组为(90.33±1.115)%,负载EGCG血小板回收率下降(p<0.05);比较而言,回收率在5mmol/L组和10mmol/L组分别为(57.51±2.468)%和(47.45±2.030)%,下降更为明显(p<0.01)。当使用ADP作为诱聚剂时,空白对照组中血小板的最大聚集率(MAR)为(63.6±4.037)%,高于其它组(p<0.01),血小板的聚集活性与负载液的EGCG浓度呈明显的负相关。当使用THR作为诱聚剂时,对照组的MAR为(89.3±6.533)%,高于EGCG负载的实验组(p<0.05),其中10mmol/L EGCG负载液的血小板聚集活性为(70.1±5.400)%,受到的影响较为显著(p<0.01)。在细胞凋亡检测实验中发现,负载EGCG后的血小板容易呈现早期凋亡状态,说明EGCG对于血小板的程序性死亡有一定的促进作用。结论 :负载入血小板中的EGCG对血小板各种生理功能有着明显的影响,负载EGCG后的血小板更易发生细胞程序性死亡。

This study was aimed to explore the change of aggregation and activation of platelets loaded with epigallocatechin gallate （EGCG）. The platelets were treated by loading buffer with different concentrations of EGCG （0, 2.5, 5, 7.5, 10, 12.5, 15, 20 and 30 mmol/L） and were divided into 2.5, 5, 10 mmol/L groups and control group. The physiological and biochemical functions of platelets were observed, including recovery rate, aggregation and activation of platelets. The platelet counts were determined by Counter Cell - DYN 1200. The aggregation activities were tested through turbidimetry, the platelet apotosis was detected by flow cytometry. The results showed that the concentrations of EGCG loading in platelets of 2.5, 5 and 10 mmol/L groups were 0. 4006 ± 0.12, 1. 0527 ± 0. 1503, 1. 6902 ± 0.1112 mmol/L respectively. Along with the increasing of EGCG concentrations in loading-buffer, the EGCG absorbed by platelets increased too. When the concentration of EGCG in loading-buffer exceeded 15 mmol/ L, the EGCG absorbed by platelets did not increase. The recovery rate in 2.5 mmol/L loading buffer group was 82.45±0.360% which was lower than that in control group （90.33± 1. 115% ） （p 〈 0.05）. As compared with control group, the recovery rate in 5 mmol/L loading buffer group （57.51±2. 468）% and 10 mmol/L loading buffer group （47.45± 2. 030） % were even significantly lower （p 〈 0.01 ）. When ADP was used as the inducer, the maximal aggregation rate （MAR） in control group was （ 63.6±4. 037 ） % , which was higher than that in other EGCG-loading groups （p 〈 0. 01 ）. And the aggregation activity of platelets negatively correlated with the concentration of EGCG in loading-buffer. When THR was used as the inducer, the MAR in control group was （89.3 ＋6. 533） % and higher than that those in other groups （p 〈 0.05 ）, especially in groups with loading-buffer higher than 10 mmol/L EGCG（70. 1± 5. 400% ） （p 〈 0.01 ）. In the experiment of cellular apoptosis, the early apoptosis easy appeared in platelets loaded with EGCG. It is concluded that the EGCG ladding in platelets markedly enfluences the physiological and biochemical functions of platelets, the apoptosis easy occurs in platelest loaded with EGCG. The EGCG accelerates the course of platelet apoptosis.