白细胞去除对悬浮红细胞上清中肿瘤相关因子蓄积改变及其对肿瘤细胞体外增殖影响的研究

Effect of Leukoreduction on Tumor-Associated Cytokine Accumutation in Supernatant of Stored Packed Red Cells and Its Effect on Tumor Cell Proliferation In Vitro

目的:本研究探讨悬浮红细胞(pRBC)储存前白细胞滤除是否会降低pRBC上清中肿瘤相关细胞因子的蓄积浓度,以及是否会抑制pRBC上清对Hep G2肝癌肿瘤细胞的体外增殖。方法:将来自相同献血员捐献的同1次全血制备成的pRBC均分成为2组,其中一组储存前滤除白细胞,另一组未滤除白细胞。两组pRBC置于2℃-6℃冰箱,在保存第0 d和35 d以1 006×g离心10 min并留取上清。采用ELISA方法测定正常T细胞的表达和分泌因子(RANTES/CCL5)、肿瘤坏死因子α(TNF-α)、血小板衍生生长因子(PDGF)、血管内皮生长因子(VEGF)和单核细胞趋化蛋白-1(MCP-1)在pRBC上清中的浓度。体外培养Hep G2肝癌细胞并观察其贴壁,加入保存第35天的两组pRBC上清,与Hep G2细胞共同孵育48 h,并用MTT法测定Hep G2细胞的体外增殖程度。结果:随着保存时间的延长,两组pRBC上清中5种细胞因子均有不同程度的浓度蓄积,即在35 d保存期末的浓度高于0 d;其中VEGF的浓度蓄积变化有统计学差异,在非去白细胞组35 d保存期末的浓度升高了549.61±299.43 pg/ml,明显高于去白细胞组95.46±110.87 pg/ml(P<0.05)。体外Hep G2肿瘤细胞增殖的MTT实验数据表明,保存35 d的非去白细胞pRBC上清孵育组的OD值为0.49(95%CI,0.43-0.55),明显高于去白细胞组的0.40(95%CI,0.38-0.42)(P<0.05),即去白细胞组pRBC上清可明显降低体外肿瘤细胞增殖。结论:储存前白细胞去除,通过去除pRBC中白膜层中的血小板,降低血小板源性生长因子特别是VEGF的蓄积,有可能降低pRBC上清对体外Hep G2细胞增殖的正向作用。

Objective： This study was to investigate whether prestorage leukoreduction could decrease the accumulative concentration of tumor-associated cytokines in supematant of stored packed red blood cells （pRBC） and to study the effect of prestorage leukoreduction on proliferation of HepG2 tumor cells by in vitro. The leukoreduced （LR） and non-leukoreduced （NLR） pRBC were equally obtained from one donation and were stored under 2 ℃ -6 ℃. The supematants of pRBC in these two group were performed by centrifugation with 1 006 × g for 10 min at day 0 and 35 d. The enzyme-linked immunosorbent assay （ELISA） was used to detect the expression of normal T cells and secretory factor（ RANTES/CCL5 ）, as well as the accumulative concentrations of tumor-necrosis factor （TNF-u）, platelet derived growth factor （PDGF）, vascular endothelial growth factor （VEGF） and monocyte chemotactic protein-1 （MCP-1） in pRBC supernantant of above-mentioned two groups. After HepG2 cells was cultured with the supernatant of NLR-pRBC and LR-pRBC at the end of day 35 together for 48 hours, the methyl thiazolil tetracolium （MTT） method was used to measure the proliferation of tumor cells in vitro. Results： The accumulative concentration of 5 cytokines in supernatants of above menthioned two groups increased in different degrees along with the prolongation of storage time, that is, the accumulative concentrations of 5 cytokines at 35 d were higher than that at day 0, in which the change of VEGF accumulative concentration showed statistical significance, its accumulative concentration in NLR group at day 35 elevated to 549.61 ±299.43 pg/ml, and was higher than that in LR group （95.46 ± 110.87 pg/ml） （ P 〈 0.05 ）. The experiment of HepG2 cell proliferation indicated that the supernatant of LR pRBC group produced less proliferation of tumor cells with OD value 0.40 （95 % CI, 0.38 - 0.42 ） than that of NLR pRBC group with OD value 0.49 （ 95 % CI, 0.43 - 0. 55 ） （ P 〈 0.05 ）. Conclusion： The prestorage leukoreduction has been confirmed to decrease the accumulative level of cytokines, particalarly decrease the accumulative level of VEGF, moreover, it may be a factor for inhibiting the proliferation of tumor cells in vitro.