细胞外信号调节激酶1/2通路抑制剂PD98059对体外培养γδT细胞增殖和杀伤功能的影响

孙蕾清; 徐晶; 陈永强; 郑璐; 吕小婷; 刘军权; 陈复兴; 周忠海

doi:10.3969/j.issn.1001-5256.2016.07.038

细胞外信号调节激酶1/2通路抑制剂PD98059对体外培养γδT细胞增殖和杀伤功能的影响

DOI: 10.3969/j.issn.1001-5256.2016.07.038

解放军第九七医院中心实验室

基金项目:

南京军区医学科技创新研究基金(14MS032)；

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中图分类号: R329.2
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出版历程
- 出版日期: 2016-07-20

Influence of extracellular signal-regulated kinase 1/2 pathway inhibitor PD98059 on proliferation and killing function of γδT cells cultured in vitro

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摘要

摘要:
目的探讨细胞外信号调节激酶（ERK）1/2信号通路抑制剂PD98059对体外培养人γδT细胞增殖和杀伤功能的影响。方法从健康人外周血中分离单个核细胞,加入到含有唑来膦酸和白细胞介素2的RPMI 1640完全培养基中诱导培养获得γδT细胞。用ERK1/2特异性抑制剂PD98059阻断γδT细胞ERK1/2信号通路后,用细胞计数仪测定γδT细胞增殖和细胞计数试剂盒（CCK-8）法测定杀伤能力;采用流式细胞术检测γδT细胞颗粒酶B和穿孔素的表达。计量资料组间比较采用t检验。结果培养10 d后的γδT细胞纯度达到（87.94±2.36）%。PD98059作用后的γδT细胞数低于对照组[（6.74±0.36）×105/ml vs（9.42±0.31）×105/ml],而PD98059作用后γδT细胞颗粒酶B阳性表达率[（48.89±1.31）%vs（41.58±1.58）%]、穿孔素阳性表达率[（65.92±3.29）%vs（33.49±2.83）%]、对肝癌Hep G2细胞的杀伤率[（69.28±4.96）%vs（48.34±3.01）%]均高于对照组,差异均有统计学意义（t值分别为-12.7...
- 细胞外信号调节MAP激酶类 /
- 蛋白激酶抑制剂 /
- 受体,抗原,T细胞,γ-δ /
- 细胞增殖
Abstract:
Objective To investigate the effect of extracellular signal- regulated kinase 1/2( ERK1/2) pathway inhibitor PD98059 on the proliferation and killing function of γδT cells cultured in vitro. Methods Mononuclear cells were separated from peripheral blood in healthy subjects and placed in RPMI 1640 complete medium containing zoledronic acid and interleukin- 2 to obtain γδT cells through induction and culture. The ERK1/2 specific inhibitor PD98059 was used to block the ERK1/2 signaling pathway in γδT cells,and the proliferation of γδT cells was measured by cell counting,and the killing function of γδT cells was measured by CCK- 8 method. Flow cytometry was used to measure the expression of granzyme B and perforin in γδT cells. The t- test was used for comparison of continuous data between groups. Results After 10 days of culture,the purity of γδT cells reached 87. 94% ± 2. 36%. The number of γδT cells treated with PD98059 was significantly lower than that of control cells [( 6. 74 ± 0. 36) × 105/ ml vs( 9. 42 ± 0. 31) × 105/ ml,t =- 12. 708,P < 0. 001]. Compared with the control cells,those treated with PD98059 had significantly higher positive expression rates of granzyme B and perforin( granzyme B:48. 89% ± 1. 31% vs 41. 58% ± 1. 58%,t = 7. 582,P < 0. 001; perforin: 65. 92% ± 3. 29% vs 33. 49% ± 2. 83%,t = 15. 478,P < 0. 001) and a significantly higher killing rate of Hep G2 cells( 69. 28% ± 4. 96% vs 48. 34% ± 3. 01%,t = 11. 201,P < 0. 001). Conclusion The ERK1/2specific inhibitor PD98059 can inhibit the proliferation of γδT cells,but it can enhance the in vitro killing function of γδT cells.
- extracellular signal-regulated MAP kinases /
- protein kinase inhibitors /
- receptors /
- antigen

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