【作者】 张荣；

【导师】 姚雪；

【作者基本信息】 山东大学 ， 外科学， 2024， 硕士

【摘要】 研究背景脊髓损伤(spinal cord injury,SCI)作为以高死亡率为特征的中枢神经系统疾病,给患者的生理和心理都带来了毁灭性的打击。脊髓损伤发生后会引发一系列的继发性损伤反应,引起炎症微环境和营养微环境失衡,最终导致神经元和神经胶质细胞的坏死和凋亡。人脐带间充质干细胞(Human Umbilical Cord Mesenchymal Stem Cells,hucMSCs)在脊髓损伤治疗方面具有抑制炎症、调节免疫反应、释放神经营养因子等作用,应用前景广泛。但是由于脊髓损伤后抑制性的微环境,人脐带间充质干细胞在进行细胞移植后存活的时间较短,具有一定的局限性。8—溴腺苷酸(8-bromo-cAMP,8-Br-cAMP)是一种细胞渗透性cAMP类似物,cAMP信号对神经突生长和轴突引导至关重要。近年来,与cAMP相关的联合治疗已被证明对脊髓损伤的恢复具有协同作用。因此,本研究通过8-bromo-cAMP预处理hucMSCs在脊髓损伤后进行细胞移植,促进hucMSCs神经营养因子的分泌,增强免疫调节能力,提高在损伤部位存活时间,进一步促进脊髓损伤的修复。实验目的探究 8-溴腺苷酸预处理的 hucMSCs(8-bromo-cAMP Preconditioning hucMSCs,APC-hucMSCs)与普通hucMSCs在修复脊髓损伤功能上的差异,阐明预处理之后hucMSCs修复脊髓损伤的机制。研究方法1.应用不同浓度的8-溴腺苷酸预处理hucMSCs,通过EDU增殖实验和CCK8细胞活性实验检测其对细胞的影响。2.通过迁移实验(Transwell实验)检测8-溴腺苷酸预处理hucMSCs后迁移能力的改变,以及通过免疫荧光实验检测hucMSCs的趋化因子血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)的表达。3.通过免疫荧光检测8—溴腺苷酸预处理hucMSCs后脑源性神经营养因子(Brain-derived neurotrophic factor,BDNF)和胶质细胞源性神经营养因子(Glial cell line-derived neurotrophic factor,GDNF)的分泌。利用 Western Blot 检测其 PKA/CREB信号通路的调控情况。4.建立体外人脐带间充质干细胞与巨噬细胞共培养体系,在脂多糖(Lipopolysaccharides,LPS)诱导之后,通过免疫荧光对巨噬细胞诱导型一氧化氮合酶(Inducible Nitric Oxide Synthase,iNOS)和精氨酸酶 1(Arginase 1,Arg1)的检测确定细胞的分化情况。5.建立C57小鼠脊髓在T10节段的损伤模型,在小鼠脊髓损伤1周后,通过原位注射在脊髓损伤中心移植感染过Luciferase的人脐带间充质干细胞。通过小动物活体成像实验检测细胞在体内的存活情况。6.通过BMS评分和Catwalk等行为学实验和电生理实验检测APC-hucMSCs对小鼠脊髓损伤后的恢复作用。通过对小鼠脊髓胶质纤维酸性蛋白(Glial Fibrillary Acidic Protein,GFAP)和神经丝蛋白-200(Neurofilament-200,NF200)的表达情况进行免疫荧光染色检测脊髓损伤后APC-hucMSCs抑制胶质瘢痕形成,促进轴突生长的能力。研究结果1.8-bromo-cAMP在浓度为1 mM时能够提高hucMSCs的迁移能力,促进其趋化因子的表达:1 mM 8-bromo-cAMP预处理24小时后可明显提高transwell小室中hucMSCs穿透薄膜的数量并且促进趋化因子VEGF的表达。2.8-bromo-cAMP预处理激活PKA/CREB信号通路,促进hucMSCs神经营养因子的分泌,同时增强hucMSCs免疫调控能力:8-bromo-cAMP预处理后可激活hucMSCs下游的PKA/CREB信号通路,进而促进BDNF和GDNF的分泌。应用PKA抑制剂H89后可抑制该信号通路,减少BDNF和GDNF的分泌。将APC-hucMSCs与巨噬细胞共培养之后可明显抑制巨噬细胞M1标志物iNOS的表达,促进M2标志物Argl的表达。3.APC-hucMSCs在脊髓中的存活时间明显延长,促进脊髓损伤的修复:小鼠脊髓损伤1周后进行细胞移植,普通hucMSCs可存活3-7天,8-bromo-cAMP预处理之后,hucMSCs可存活7-10天。小鼠脊髓损伤在经过细胞移植治疗7周后,通过行为学实验包括BMS和Catwalk实验,结果显示APC-hucMSCs组小鼠后肢的运动能力恢复好。小鼠脊髓后的电生理实验显示APC-hucMSCs能够促进小鼠神经传导功能的恢复。脊髓组织通过免疫荧光实验发现APC-hucMSCs能够进一步抑制胶质瘢痕的形成,促进轴突再生。研究结论1.8-bromo-cAMP预处理可促进hucMSCs趋化因子VEGF的表达,提高其迁移能力,同时激活PKA/CREB信号通路,促进神经营养因子BDNF和GDNF的表达。2.8-bromo-cAMP预处理的hucMSCs可进一步抑制巨噬细胞向M1极化,促进巨噬细胞向M2极化,并且在脊髓损伤后,明显延长移植的细胞在损伤部位的存活时间。3.hucMSCs在经过8-bromo-cAMP预处理之后,能够进一步的促进小鼠神经传导功能和后肢运动功能的恢复,同时能够进一步在脊髓损伤后抑制脊髓形成胶质瘢痕,促进神经元轴突的再生。更多还原

【Abstract】 BackgroundSpinal cord injury(SCI)as a disease of the central nervous system characterized by high mortality rates,which brings devastation to patients both physically and psychologically.The occurrence of SCI triggers a series of secondary injury responses,causing an imbalance in the inflammatory and trophic microenvironment,which ultimately leads to necrosis and apoptosis of neurons and glial cells.In the treatment of SCI,Human Umbilical Cord Mesenchymal Stem Cells(hucMSCs)have the ability to inhibit inflammation,regulate the immune response,and promote the release of neurotrophic factors,so they have a wide range of applications.However,due to the inhibitory microenvironment after SCI,hucMSCs have certain limitations in terms of their short survival time after cell transplantation.8-bromo-cAMP(8-Br-cAMP)is a cell-permeable cAMP analog,and cAMP signaling is essential for neurite growth and axon guidance.In recent years,combination therapies associated with cAMP signaling have been shown to have synergistic effects on recovery from SCI.Therefore,in this study,hucMSCs were induced by 8-bromo-cAMP precondition to enhance their immunomodulatory ability,improve survival time at the injury site,and promote their secretion of trophic factors to further better achieve functional recovery from SCI.ObjectiveTo explore the difference between 8-bromo-cAMP preconditioned hucMSCs and normal hucMSCs in repairing the function of SCI,and to elucidate the mechanism of hucMSCs repairing SCI after precondition.Methods1.hucMSCs were preconditioned with different concentrations of 8-bromo-cAMP,and the effects on cell proliferation were detected using EDU and CCK8 to determine the optimal induction concentration.2.Transwell assay was used to detecte the migration ability of hucMSCs as well as their expression of vascular endothelial growth factor(VEGF)associated with migration by immunofluorescence.3.Secretion of brain-derived neurotrophic factor(BDNF)and glial cell line-derived neurotrophic factor(GDNF)from APC-hucMSCs was detected by immunofluorescence.The PKA/CREB signaling pathways were detected using Western Blot.4.An in vitro co-culture system of hucMSCs and macrophages was established,and the cell differentiation was examined by immunofluorescence of macrophage inducible nitric oxide synthase(iNOS)and arginase 1(Argl)expression after induction with lipopolysaccharides(LPS)expression for immunofluorescence to detect cell differentiation.5.An injury model of C57 mouse spinal cord at T10 segment was established,and hucMSCs infected with Luciferase were transplanted at the center of the spinal cord injury by in situ injection 1 week after the mouse SCI.Cell survival in vivo was observed by small animal live imaging.6.Behavioral experiments such as BMS score and Catwalk,and electrophysiological experiments were performed to detect the recovery effect of APC-hucMSCs on mice after SCI.The capacity of APC-hucMSCs to impede glial scar formation and facilitate axonal growth following spinal cord injury was discerned through immunofluorescence staining of the expression of glial fibrillary acidic protein(GFAP)and neurofilament-200(NF200)in mice.Results1.Preconditioning with 8-bromo-cAMP at 1 mM increased the migration ability of hucMSCs and promoted the expression of their chemokines:1 mM 8-bromo-cAMP pretreatment for 24 hours significantly increased the number of hucMSCs penetrating the membrane in transwell chambers and promoted the expression of their chemokine VEGF.2.8-bromo-cAMP preconditioning activated PKA/CREB signaling pathway,promoted the secretion of neurotrophic factors in hucMSCs,and also enhanced the immunoregulatory ability of hucMSCs:8-bromo-cAMP preconditioning activated hucMSCs downstream PKA/CREB signaling pathway,which in turn promotes the secretion of BDNF and GDNF.Application of the PKA inhibitor H89 inhibited this signaling pathway,and secretion of BDNF and GDNF was reduced.Co-culture of APC-hucMSCs with macrophages significantly inhibited the expression of the macrophage Ml marker iNOS and promoted the expression of the M2 marker Arg1.3.APC-hucMSCs transplantation significantly prolonged the survival time in the spinal cord and promoted the repair of SCI:After cell transplantation at 1 week of SCI,normal hucMSCs could survive for 3-7 days,APC-hucMSCs could survive for 7-10 days.After 8 weeks of SCI in mice,BMS scores and Catwalk results showed that motor function recovery was more pronounced in the APC-hucMSCs group.Electrophysiological experiments after mouse spinal cord showed that APC-hucMSCs could promote the recovery of nerve conduction function in mice.Spinal cord tissues revealed by immunofluorescence experiments that APC-hucMSCs were able to further inhibit glial scar formation and promote axonal regeneration.Conclusions1.8-bromo-cAMP preconditioning of hucMSCs promoted the expression of chemokine VEGF and improved migration ability,while activating the PKA/CREB signaling pathway and promoting the expression of neurotrophic factors BDNF and GDNF.2.8-bromo-cAMP preconditioning of hucMSCs inhibitd macrophages polarization to Ml,promoted macrophages polarization to M2,and prolonged survival time at the site of spinal cord after SCI.3.After preconditioning with 8-bromo-cAMP,hucMSCs were able to further promote the recovery of behavioral functions and neurophysiological signaling after SCI in mice than normal hucMSCs.Additionally,it has been demonstrated to inhibit the formation of glial scarring in the spinal cord following a spinal cord injury,while simultaneously promoting the regeneration of neuronal axons.更多还原