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目的利用人外周血单个核细胞(PBMC)研究用于血浆病毒灭活后残余的亚甲蓝是否对人体免疫细胞功能产生影响。方法采用Ficoll-Hypaque密度梯度离心法分离PBMC,在T细胞特异性刺激因子Anti-CD3/CD28存在的条件下,加或者不加不同浓度的亚甲蓝共培养,培养至72h,收集培养上清液,采用酶联免疫吸附试验(ELISA)检测细胞因子分泌情况;培养66h后,加入CCK-8染料继续培养4~6h,于A_(450)处测定细胞增殖情况。结果高浓度剂量亚甲蓝(1.25、2.5、5μmol/L组)对AntiCD3/28刺激PBMC的增殖均有明显抑制作用(P<0.01),其OD值由0.897±0.385分别降至0.632±0.334、0.524±0.254、0.445±0.287,呈一定的剂量依赖效应。高浓度亚甲蓝(1.25、2.5、5μmol/L组)可下调Anti-CD3/28诱导PBMC分泌细胞因子白细胞介素(IL)-17a、IL-10、γ-干扰素(IFN)-γ,且呈剂量依赖效应。1.25、2.5、5μmol/L的亚甲蓝影响PBMC分泌IL-17a,IL-17a水平由(406±57)pg/mL分别降至(276±38)、(192±31)、(134±24)pg/mL;影响PBMC分泌IL-10,IL-10水平由(184±15)pg/mL分别降至(132±13)、(110±12)、(42±8)pg/mL;影响PBMC分泌IFN-γ,IFN-γ水平由(4 512±187)pg/mL分别降至(2 876±143)、(2 234±153)、(1 988±112)pg/mL。结论高浓度亚甲蓝(≥1.25μmol/L)对人PBMC的增殖以及分泌细胞因子功能有显著抑制作用,换而言之,血浆病毒灭活后残余浓度(≤0.33μmol/L)的亚甲蓝对PBMC免疫功能无明显影响,但该浓度的亚甲蓝对人纯T细胞免疫功能是否有影响需要进一步评估研究。
OBJECTIVE: To investigate whether the residual methylene blue after inactivation of plasma virus has effects on human immune cell function using human peripheral blood mononuclear cells (PBMCs). Methods PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation and cultured in the presence of T-cell specific stimulator Anti-CD3 / CD28 with or without methylene blue at different concentrations for 72h. The culture supernatants were harvested Liquid, and the secretion of cytokines was detected by enzyme-linked immunosorbent assay (ELISA). After cultured for 66h, CCK-8 dye was added to continue the culture for 4 ~ 6h, and the cell proliferation was measured at A 450. Results High concentrations of methylene blue (1.25,2.5 and 5μmol / L) significantly inhibited the proliferation of PBMC stimulated by AntiCD3 / 28 (P <0.01), and their OD decreased from 0.897 ± 0.385 to 0.632 ± 0.334, 0.524 ± 0.254,0.445 ± 0.287, showing a dose-dependent effect. High concentrations of methylene blue (1.25,2.5 and 5μmol / L) could down-regulate the secretion of cytokines IL-17a, IL-10 and IFN-γ in PBMC induced by Anti-CD3 / And dose-dependent effect. The levels of IL-17a and IL-17a secreted by PBMC decreased from (406 ± 57) pg / mL to (276 ± 38), (192 ± 31) and ) pg / mL; the levels of IL-10 and IL-10 secreted by PBMC decreased from (184 ± 15) pg / mL to (132 ± 13), (110 ± 12) and (42 ± 8) pg / mL; The level of IFN-γ secreted by PBMC decreased from (4 512 ± 187) pg / mL to (2 876 ± 143), (2 234 ± 153) and (1 988 ± 112) pg / mL, respectively. Conclusion High concentrations of methylene blue (≥1.25μmol / L) significantly inhibited the proliferation and secretion of cytokines in human PBMCs. In other words, methylene blue with residual concentration (≤0.33μmol / L) Blue had no significant effect on the immunological function of PBMC. However, whether this concentration of methylene blue has any effect on the immune function of human pure T cells requires further evaluation.