The Preliminary Study About The Effects Of Extracellular Ubiquitin Accumulated In The Storage Of Whole Blood On Transfusion-related Immunomodulatory

Background:Multiple bioactive molecules accumulated in the bloodcomponents during storage regulated the immune response of patients after transfusion. In the substances causing immune response, extracellular ubiquitin in the plasma of storaged whole blood played an important role, which acted as the main material or coordination of other substances. In this study, we detected the changes of extracellular ubiquitin in the plasma of whole blood during storage, at the same time, which effected on the secretion of cytokines or mRNA expression of PBMNC in vitro were studied. We preliminary study the extracellular ubiquitin effected on the Thl/Th2cell differentiation, and analyzed its role in the mechanism of transfusion-related immunomodulatory(TRIM).Methods:(1) Using the special white blood cells filter of the blood bank to filter freshly collected whole blood as the leukocyte-reduction group, and non-leukocyte-reduction whole blood as the control group.(2) Collected the plasma of whole blood in different storage time, and detected the ubiquitin concentration using ELISA.(3) Detected the effect of extra Ub on cytokines secretion (IFN-γ, TNF-α, IL-2, IL-4, IL-8and IL-10) by adding exogenous Ub to day0plasma or closing day35plasma using Ub antibody. All the concentration of cytokines were detected by ELISA.(4) we detected the mRNA expression of proinflammatory cytokines(TNF-α、IL-8) by using fluorescence quantitative PCR. At the same time, Th1/Th2cytokine (IFN-γ and IL-4) and Th1/Th2-inducing transcription factors (T-bet/STAT6) were analyzed, for we can further observed the changes of differentiation of Th1/Th2cells.Results:With the extension of storage time, plasma Ub increased146-fold or73-fold on day35. Plasma Ub concentration of LR and NLR increased from (18.35±11.87)ng/mL to (865.11±83.58)ng/mL and (20.19±10.23)ng/mL to(631.01±97.05)ng/mL, respectively. Extra Ub inhibits the secretion of cytokines such as IFN-、TNF-α、IL-2, while promote the secretion of IL-4、IL-8. we further found that Both plasma of day35and addition of exogenous to day0simulated the cytokine production and mRNA expression of IL-8, but also inhibited TNF-α production apparently. Cytokines production and mRNA expression levels along with Thl/Th2-inducing transcription factors were biased in favor of Th2. The mRNA expression levels of IFN-γ and T-bet were inhibited, but IL-4and STAT6mRNA expression were increased.Conclusion:Extracellular ubiquitin in the stored whole blood played an important role in TRIM, which could influenced the differentiation of Th1/Th2cells, and promoted cytokine production of Th2and mRNA expression level of Th2-inducing transcription factor, and of Th1were suppressed simultaneously, and which mean that cellular immunity of the patients was suppressed and the ability of anti-tumor and anti-infection was reduced. This study suggested that it could be one of the factors arousing transfusion-related immunomodulation.