【作者】 王琼；

【导师】 王伟；

【作者基本信息】 华中科技大学 ， 神经病学， 2018， 博士

【摘要】 目的:临床常见的髓鞘损伤原因主要包括缺血性,炎症性及遗传代谢性等。少突胶质细胞前体细胞的损伤、增殖及分化是髓鞘损伤后髓鞘再生的关键过程。星形胶质细胞参与该过程的发生,但具体的机制尚不清楚。以临床常见的慢性缺血性及炎症性脱髓鞘性损伤为切入点,分别以星形胶质细胞缝隙连接蛋白Connexin43和星形胶质细胞外泌体为研究靶点,研究在不同髓鞘损伤过程中星形胶质细胞对少突胶质细胞的增殖、分化及损伤的作用,探索不同病理条件下少突胶质细胞损伤的相关机制及可能的保护措施。方法:采用离体培养纯化星形胶质细胞及少突胶质细胞谱系细胞。一、构建星形胶质细胞-少突胶质细胞前体细胞共培养离体慢性缺氧模型,免疫荧光及western blot检测少突胶质细胞前体细胞增殖、分化及凋亡情况。给予缝隙连接蛋白抑制剂MFA及CBX,观察对少突胶质细胞的增殖、分化及凋亡情况。检测不同干预情况下Connexin43缝隙连接通道和半通道的活性及谷氨酸的释放。二、收集AQP4 IgG阳性视神经脊髓炎谱系疾病(Neuromyelitis optica spectrum disorders,NMOSD)患者血清,利用protein A纯化NMO IgG,构建离体NMO模型。利用梯度超速离心方法分离纯化星形胶质细胞来源的外泌体,干预分化成熟的少突胶质细胞,检测少突胶质细胞活性及相关髓鞘蛋白表达情况。结果:一、成功构建星形胶质细胞-少突胶质细胞前体细胞慢性缺氧模型,缺氧诱导因子(HIF-1α)表达上调及核内移。慢性缺氧条件下,星形胶质细胞明显活化,GFAP表达增加,Connexin43表达上调,少突胶质细胞前体细胞增殖但分化成熟减少;给予Connexin43抑制剂MFA及CBX干预后,星形胶质细胞活化降低,GFAP及Connexin43蛋白表达减少,少突胶质细胞前体细胞增殖减少而分化增加;MFA及CBX降低慢性缺氧引起的Connexin43缝隙连接通道及半通道的活性,降低慢性缺氧引起的谷氨酸的释放;给予AMPA谷氨酸受体拮抗剂NBQX改善慢性缺氧引起的少突胶质细胞分化成熟障碍。二、成功收集NMO患者血清,纯化NMO IgG,构建NMO离体模型。NMO患者血清干预后星形胶质细胞活性降低,AQP4蛋白表达下调,而纯化IgG干预后星形胶质细胞活性增加,GFAP表达上调,AQP4表达增加,TNF-α分泌增多。纯化星形胶质细胞来源的外泌体,电镜及western blot检测外泌体形态及特异性标记物;将纯化外泌体干预分化成熟的少突胶质细胞后,western blot及RT-PCR检测少突胶质细胞相关髓鞘蛋白表达,发现NMO IgG干预星形胶质细胞来源的外泌体使少突胶质细胞的髓鞘相关蛋白MBP、MAG、Olig2等表达减少,而对照组无明显变化。结论:一、慢性缺氧条件下,星形胶质细胞Connexin43半通道开放释放大量谷氨酸入细胞间隙,从而持续激活少突胶质细胞前体细胞AMPA谷氨酸受体影响前体细胞分化成熟;抑制Connexin43半通道功能,有望成为慢性缺氧性脑白质损伤的干预靶点;二、NMO IgG干预星形胶质细胞后来源的外泌体调控少突胶质细胞髓鞘形成相关蛋白的表达,说明星形胶质细胞外泌体可能参与NMO的髓鞘损伤,进一步阐明其机制,可能为明确NMO疾病的发生及治疗提供新的思路。更多还原

【Abstract】 Objective: Myelin injury are caused by ischemic,inflammatory or genetic metabolism.Oligodendrocyte precursor cells(OPCs)development are the key process of myelin regeneration after myelin injury.Astrocytes are involved in this process,but the mechanisms are still undefined.To investigate the role of astrocytes on the chronic ischemic and inflammatory mediated myelin injury,we analyzed the roles of astrocytic connexin43 and exosomes in the OPCs development respectively.To explore the mechanisms of myelin injury and the possible protective stratagems.Methods: Primary astrocytes and oligodendrocyte lineages were purified in vitro.Firstly,we performed an astrocytes-OPCs co-cultures chronic hypoxia model,and detected the proliferation,differentiation and apoptosis of OPCs by immunofluorescence and Western blot.Connexin43 inhibitors MFA and CBX were administrated to observe the effects on OPCs.Connexin43 gap junction and hemichannels activity were detected by cells scrape loading assay and dye uptake assay,respectively.The release of glutamate was detected underdifferent intervention conditions.Secondly,we collected serum from patients with Neuromyelitis optica spectrum disorders(NMOSD);sera were positive for AQP4 Ig G.We also used control sera collected from five healthy blood donors.Ig G were purified on ProteinA Sepharose 4 Fast FlowTM beads.We performed an NMO model by treated purified astrocytes with sera or NMO Ig G in vitro.The exosomes derived from astrocytes were isolated and purified by differential ultracentrifugation;then the exosomes were administrated to detect the effects on the oligodendrocytes.Results: Firstly,we successfully performed an astrocytes-OPCs co-culture chronic hypoxia model,which were verified by hypoxia inducible factor(HIF-1α)expression up-regulated and translocation from cytoplasm to nuclei.Chronic hypoxic conditions,astrocytes were activated with up-regulated expression of GFAP and connexin43 proteins,and OPCs proliferation but limited differentiation;connexin43 inhibitors MFA and CBX attenuated hypoxia-induced astrocytes activation,and significantly attenuate hypoxia-induced enhancement of GFAP and connexin43 expression at day 2 after hypoxia treatment,without affecting cells viability.Furthermore,connexin43 inhibitors rescued the limited OPCs maturation under chronic hypoxia;connexin43 inhibitors suppressed gap junction and hemichannels activity and glutamate release under chronic hypoxia.In addition,AMPA receptor inhibitor partially rescued the limited OPCs maturation under hypoxia.Secondly,sera were obtained from patients with a NMOSD or healthy donors.Prolonged exposure to seraNMO or Ig GNMO induced the functional changes of astrocytes in vitro.Exosomes derived from astrocytes were collected and using electron microscope and Western blot to detect the morphology and biomarkers of exosomes.Exosomes derived from astrocytes which treated with seraNMO or Ig GNMO regulated oligodendrocytes proteins expression detected by immunofluorescence,western blot and RT-PCR.Conclusion: Astrocytic Cx43 hemichannels could result in the increase of extracellular glutamate after chronic hypoxia,which could impair OPCs maturation and remyelination processes.Glutamate released via Cx43 hemichannels suppressed OPCs differentiationpartially mediated by AMPARs activation.Application Cx43 inhibitors could rescue hypoxia induced suppression of OPCs differentiation.Based on these findings,astrocytic Cx43 hemichannels could potentially be a therapeutic target for facilitating OPCs remyelination in hypoxia-induced white matter injury.In addition,our results confirm the pathogenic role of Ig G isolated from NMOSD patients with positive of AQP4 Ig G.Using models based on seraNMO or Ig GNMO-treated primary astrocytes,we showed that seraNMO or Ig GNMO induced alteration of astrocytes.Exosomes derived from astrocytes treated with seraNMO or Ig GNMO regulated oligodendrocytes myelin related proteins expression.To further elucidate the mechanism,which can provide new ideas for the pathogenesis of NMOSD and serve as a promising therapeutic target for NMO in clinic practice.更多还原