OBJECTIVETo simulate the chemical microenvironment of injured brain tissue, and to explore the effect of this chemical microenvironment on temperature sensitive umbilical cord mesenchymal stem cells (tsUC).

METHODSRat models of traumatic brain injury (TBI) were made by fluid percussion injury, and then the brain tissue extracts of the injured regions were acquired. Human umbilical cord mesenchymal stem cells (UC) were isolated and cultured, and the tsUC were obtained through the infection of temperature-sensitive Simian 40 Large T- antigen (ts-SV40LT) retrovirus. After that, both the two kinds of cells were cultured on the polyacrylamide gels which mimicking the elastic modulus of brain. Four groups were included: UC cultured under normal temperature (UC group), UC cultured added brain tissue extract under normal temperature (UC plus extract group), tsUC cultured under mild hypothermia (tsUC group), and tsUC added brain tissue extract under mild hypothermia for 3 days, then normal temperature for 4 days (tsUC plus extract group). After 24 hours, the apoptosis level was checked. Cell growth and morphological changes in each group were given dynamic observation. Seven days later, cell immunofluorescences were implemented for examining neural differentiation level.

RESULTSCompared with UC plus extract group, the apoptosis and proliferation in UC plus extract group were significantly reduced (P < 0.01) and increased (P < 0.01) respectively. Cell immunofluorescence showed that the both GFAP and Neuron positive cells were significantly enhanced in UC plus extract group than those in tsUC plus extract group.

CONCLUSIONtsUC combining with mild hypothermia could significantly reverse injury induced cell apoptosis, improve cell proliferation and neural differentiation under chemical microenvironment after brain injury, which confirmed the adaptation and resistance of tsUC under mild hypothermia after TBI.

Animals ; Apoptosis ; Brain ; cytology ; pathology ; Brain Injuries ; pathology ; Cell Proliferation ; Humans ; Mesenchymal Stromal Cells ; chemistry ; Neurons ; cytology ; Rats ; Temperature ; Umbilical Cord ; cytology

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[摘 要] 目的：探究中链脂肪酸癸酸对CD8+ T细胞活化的影响，及其对CD8+ T细胞介导的抗肿瘤免疫反应的作用和机制。方法：建立C57BL/6小鼠黑色素瘤B16F10皮下荷瘤模型，随机分为癸酸组（10 mg/kg癸酸灌胃）和对照组（等量溶剂灌胃），观察癸酸对小鼠肿瘤生长以及生存率的影响，采用流式细胞术检测肿瘤微环境中浸润CD8+ T细胞的活化水平。建立B16F10-OVA和OT-I T细胞共培养体系，采用流式细胞术检测癸酸对CD8+ T细胞的肿瘤细胞杀伤能力的影响。采用α-CD8抗体清除B16F10荷瘤小鼠体内CD8+ T细胞，观察对小鼠肿瘤体积的影响。小鼠原代CD8+ T细胞经癸酸处理后，采用WB、ELISA及qPCR、流式细胞术检测T细胞受体（TCR）活化、效应细胞因子产生以及增殖和代谢水平。在B16F10荷瘤小鼠模型中，观察α-PD-1抗体联合癸酸给药对小鼠肿瘤生长以及生存率的影响。结果：在小鼠黑色素瘤荷瘤模型中，与对照组相比，癸酸组小鼠移植瘤体积显著降低且生存率显著提高（均P<0.05），肿瘤浸润CD8+ T细胞IFN-γ和TNF-α的表达水平显著升高（P<0.01）。经癸酸处理的OT-I T细胞对B16F10-OVA细胞的杀伤水平显著升高（P<0.01）。在荷瘤小鼠模型中用α-CD8抗体清除CD8+ T细胞后，癸酸对移植瘤的抑制作用显著降低（P<0.000 1）。小鼠原代CD8+ T细胞经癸酸处理后，TCR活化水平显著升高、细胞因子IL-2和IFN-γ的产生增多、线粒体代谢水平显著上调（均P<0.05）。在黑色素瘤荷瘤小鼠模型中，癸酸与α-PD-1抗体联用，能够显著抑制小鼠移植瘤生长并提高其生存率（均P<0.05）。结论：癸酸能够促进CD8+ T细胞活化、增强其抗肿瘤免疫反应能力。