p38MAPK在海藻糖保护血小板低温贮存损伤中的机制研究

Storage at room temperature for platelet is limited to 5 days. Due to the change of morphology and loss of functionality, the platelet stored at low temperature could cause its clearance quickly by hepatic cells after transfusion. Hence, preservation of platelet for a long time is a key step in the field of blood transfusion. In the previous studies, we found that trehalose could decrease the activation of platelet in vitro and the phagocytosis of cold platelet in vivo. In addition, we also confirmed primarily that trehalose can influence phosphorylation of p38MAPK for cold platelet. Other researchers showed that the activation in vitro and clearance in vivo of platelet at 22 and 37?C temperature could slow down by inhibiting p38MAPK. Therefore, we speculate about p38MAPK could mediate the cold-activation of platelet, and trehalose can prevent the loss of functionality for platelet by inhibiting the phosphorylation of p38MAPK. We have successfully developed a method to get a mount of platelets in vitro by fusing megacaryocyte and gastric cancer cells. Based on the method, we will study the expression of p38MAPK in the fused cells for platelet production, observe the storage lesion of the platelet in different situation, determine the related proteins in the signal path of p38MAPK and test the clearance of platelet in vitro by co-culture cells method. We wish to make clear the protection mechanism of p38MAPK on trehalose to protect platelet and present theoretical data for future use of platelet stored with trehalose at low temperature.

血小板制剂在常温下只能保存5天。低温保存血小板因形态和功能变化可致输注后被受者肝细胞快速清除。因此，血小板的长期保存是输血领域关注的重要课题。本项目组前期研究发现海藻糖可抑制低温保存血小板的活化及在动物体内的清除，且可抑制低温保存血小板的p38MAPK磷酸化。而常温保存的血小板通过抑制p38MAPK可显著降低其损伤及体内的清除，结合这一研究进展，我们提出p38MAPK介导了血小板的低温活化，海藻糖可能通过调控p38MAPK信号通路减少其损伤。拟通过巨核细胞与胃癌细胞融合后在体外培养获得血小板，采用转基因技术对融合细胞的p38MAPK基因表达进行过表达和下调，获得转基因血小板，对常温和低温保存血小板进行p38MAPK信号通路相关蛋白的检测及利用细胞共培养技术观察血小板的清除变化，对阐明p38MAPK信号通路对海藻糖低温保护血小板的机制具有重要意义，为海藻糖低温保存血小板的应用提供理论依据。

血小板制剂在常温下仅保存5天。低温保存血小板因形态和功能变化致输注后被受者肝细胞清除。因此，血小板的长期保存是输血领域关注的重要课题。本项目组前期研究发现海藻糖可抑制低温保存血小板的活化及在动物体内的清除。文献报道通过抑制p38可显著降低其损伤及体内的清除，结合这一研究进展，我们提出海藻糖可能通过调控p38信号通路减少其损伤。原计划通过转基因技术对融合细胞的p38MAPK基因表达进行过表达和下调得到转基因血小板。结合iPSCs诱导成血小板的成功报道，本项目先后尝试了iPSC及CD34+诱导分化为血小板，由于具体实施过程中遇到暂不能解决的问题，目前该部分工作实现了表达质粒pCDH-CMV-MCS-EF1-GFP-T2A-Puro-p38a 构建， iPScs培养平台成功建立，但未能成功包装出高滴度病毒液转染iPScs及诱导其分化转基因血小板。为了加快项目进展，获得更多海藻糖作用于低温血小板的数据，我们从代谢水平、蛋白组学水平及miRNA水平变化阐述海藻糖通过p38保护低温血小板的分子机制。研究结果显示，保存第5d血小板回收率比较，海藻糖低温冷藏组（93.18+/-2.47）或冷冻保存组（87.71+/-7.31）与22度正常保存组（89.74+/-8.57）之间差异不具统计学意义，血小板PS外翻并未明显增加，因此海藻糖对血小板凋亡发生过程影响不明显。蛋白质谱分析，海藻糖可引起血小板48种蛋白表达变化，177种蛋白磷酸化水平变化，将这些蛋白富集分析它们属于包括p38MAPK信号通路在内的多条信号通路，主要涉及的蛋白有SNARP23（与血小板a颗粒分泌有关），FERMT3（与血小板黏附相关），p38等血小板活化相关通路蛋白，同时WB证实海藻糖抑制了p38MAPK信号通路相关蛋白如Akt，CaMKIIa，p38，Erk等蛋白磷酸化。血小板miRNA分析也证实海藻糖可引起miR-22-3p上调（靶向MKK3，可激活p38），miR-4707-3p上调（靶向p38），miR-227-5p上调（靶向ERK，p38下游激酶），结果与蛋白组学结果一致，因此，本项目从血小板代谢水平、蛋白水平及miR水平研究得出海藻糖可通过p38MAPK信号通路保护低温血小板，同时也发海藻糖可影响其他血小板功能相关蛋白，下一步将继续挖掘数据，探索新型血小板低温保存方法。