核黄素光化学法对人外周血淋巴细胞灭活作用的研究

Study on the inactivation of human peripheral blood lymphocytes using riboflavin photochemical treatment

目的研究核黄素光化学法对人外周血淋巴细胞的增殖及细胞因子分泌活性的抑制作用,探讨该方法对淋巴细胞凋亡的诱导作用。方法实验分为实验组:从随机采集的全血中分离淋巴细胞,将其悬浮于核黄素终浓度为100μM的自体血浆中,注入PVC袋,在波长为420 nm的可见光下照射,总照射剂量为40 J/cm2;对照组:使用相同来源的淋巴细胞,悬浮于不含核黄素的自体血浆中,不进行光照处理。用植物凝集素(PHA)同步刺激两组淋巴细胞,用BrdU细胞增殖检测试剂盒检测淋巴细胞的增殖活性,并计算核黄素光化学法对淋巴细胞的增殖抑制率;用酶联免疫试剂盒检测淋巴细胞细胞因子的分泌量;使用Annexin V-PE细胞凋亡试剂盒和流式细胞技术检测细胞凋亡。结果与对照组比较,经过核黄素光化学法处理的实验组淋巴细胞对PHA刺激的增殖抑制率为(94.69±7.61)%;实验组淋巴细胞接受PHA刺激后,细胞因子IFN-γ、IL-10和IL-12的分泌量与对照组比较分别降低了(89.54±22.75)%、(77.83±22.3)%和(88.57±15)%,IL-4的分泌量较对照组差异不明显;流式细胞术检测结果表明,实验组绝大多数淋巴细胞发生了凋亡,其早期凋亡率和晚期凋亡或坏死率均明显高于对照组。结论核黄素光化学法能够抑制人外周血淋巴细胞的增殖和一些细胞因子的分泌,同时可诱导淋巴细胞凋亡。该方法有望成为预防TA-GvHD的新途径。

Objective To study the effects of riboflavin photochemical treatment on the inhibition of lymphocyte prolif- eration and cytokine secretion, and also explore the induction of the apeptosis of lymphocytes. Methods Lymphocytes sepa- rated from whole blood were suspended in autologous plasma. For experimental groups, riboflavin was mixed with the cell suspensions in a final concentration of 100μM. The mixture was transferred into PVC bags, and then exposed to 420 nm visi- ble light at a final dose of 40J/cm2. The control group lymphocytes didn＇ t recriboflavin photochemical treat- ment. Both lymphocytes aforementioned were tested for the proliferative ability and the level of several cytokines after the stimulation of phytoagglutinin（PHA）. The apoptosis of lymphocytes was detected by flow cytometer. Results The prolifera- tion inhibition rate of riboflavin photochemical treated lymphocytes （RPT-lymphocytes） after the stimulation of PHA was （94. 69 ± 7.61 ） %, and the productions of IFN-γ, IL-10 and IL-12 were decreased by （ 89.54 ± 22. 75 ） %, （77. 83 ± 22. 3） % and （88.57 ±15 ） %, and the production of IL-4 was not obvious difference. After the riboflavin photochemical treatment,the number of lymphocytes apotosis increased obviously. Conclusion The riboflavin photochemical treatment could inhibit lymphocyte proliferation and cytokine production, and induce lymphocyte apoptosis. Consequently, this method could be a promising method to prevent TA-GvHD.