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通讯作者:

刘晓梅,E-mail:lxmlxm_hi@xzhmu.edu.cn

中图分类号:R593.2

文献标识码:A

文章编号:1007-4368(2023)03-311-08

DOI:10.7655/NYDXBNS20230303

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参考文献 10
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参考文献 13
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参考文献 17
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参考文献 18
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参考文献 19
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参考文献 20
SONG Z,MAO H,LIU J,et al.Lanthanum chloride induces axon abnormality through LKB1 ⁃ MARK2 and LKB1 ⁃ STK25⁃GM130 signaling pathways[J/OL].Cell Mol Neu⁃ robiol,2022[2022 ⁃ 11 ⁃ 10].doi:10.1007/s10571 ⁃ 022 ⁃ 01237⁃0
参考文献 21
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参考文献 22
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目录contents

    摘要

    目的:探讨丝氨酸/苏氨酸蛋白激酶25(serine/threonine protein kinase 25,STK25)对星形胶质细胞活化及实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠疾病进程的影响。方法:髓鞘少突胶质细胞糖蛋白35-55 (myelin oligodendrocyte glycoprotein 35-55,MOG35-55)诱导小鼠EAE模型,Real-time PCR、Western blot和ELISA法分别检测脊髓组织中STK25、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)与干扰素诱导蛋白-10(interferon-inducible protein-10,IP-10)的转录水平与蛋白表达;免疫荧光双标法检测STK25在脊髓星形胶质细胞中的表达。干扰素(interferon,IFN)-γ体外刺激小鼠原代星形胶质细胞后不同时间,上述方法检测STK25的蛋白表达及细胞因子的转录水平变化。将特异性靶向星形胶质细胞的对照及STK25敲减的重组慢病毒悬液感染星形胶质细胞后,IFN-γ刺激,测定TNF-α与IP-10的转录水平;同时给予小鼠病毒悬液 7 d后,诱导EAE模型,Western blot检测脊髓组织STK25的表达;Real-time PCR检测脊髓炎性因子与趋化因子转录水平。HE 和劳克坚牢蓝染色(Luxol fast blue,LFB)观察脊髓炎症细胞浸润与髓鞘损伤。结果:与对照组相比,EAE组小鼠STK25的表达水平显著降低,炎性因子与趋化因子的转录与分泌水平明显升高;同时脊髓组织星形胶质细胞STK25的表达明显下降。IFN-γ 体外刺激星形胶质细胞STK25表达降低时,炎性因子生成增加;进一步敲减星形胶质细胞内源性的STK25基因,体外星形胶质细胞及体内EAE小鼠脊髓中炎性因子与趋化因子的生成显著升高,同时加剧了EAE小鼠脊髓炎症细胞浸润与髓鞘脱失。结论:STK25可能通过影响星形胶质细胞的活化,调节炎性与趋化因子的产生,从而参与EAE小鼠的病变进程。

    Abstract

    Objective:This study aims to investigate the effects of serine/threonine protein kinase 25(STK25)on the activation of astrocytes as well as the process of experimental autoimmune encephalomyelitis(EAE)in mice. Methods:The EAE mouse model was induced by myelin oligodendrocyte glycoprotein 35-55(MOG35 - 55)polypeptide. The transcription levels and the expression of STK25, tumor necrosis factor- α(TNF-α)and interferon inducible protein 10(IP-10)in spinal cord tissue and serum were detected by ELISA, Real -time PCR and Western blot,respectively. The expression of STK25 in astrocytes in spinal cord tissue of mice was detected by immunofluorescence double staining. Primary mouse astrocytes were stimulated in vitro with interferon-γ(IFN-γ),and the expression of STK25 and the transcription levels of cytokines were detected by the above methods at different times. Primary mouse astrocytes were transfected by recombinant lentiviral vector,and stimulated with IFN - γ,the transcription levels of TNF - α and IP - 10 were measured. Meantime,the EAE model was induced after injecting vectors 7 days,and the expression of STK25 in spinal cord tissue was detected by Western blot,the transcription levels of TNF - α and IP - 10 were detected by Real - time PCR,and the infiltration of inflammatory cells and the demyelination of spinal cord were observed by HE and Luxol fast blue(LFB)staining methods,respectively. Results:Compared with control group,the transcription level and the expression of STK25 in EAE group were significantly reduced while the levels of inflammatory cytokines and chemokines were significantly increased. Moreover,the expression of STK25 in astrocytes in spinal cord was significantly reduced. The expression of STK25 in astrocytes was decreased after stimulated by IFN-γ in vitro,and the production of inflammatory cytokines were increased. After specific knocking down endogenous STK25 in astrocyte,the production of inflammatory cytokines and chemokines in vitro astrocytes and in vivo EAE mice were increased significantly,which exacerbated the inflammatory cell infiltration and myelination of spinal cord tissue in EAE mice. Conclusion:STK25 may participate in the pathological process of EAE mice by affecting the activation of astrocytes and regulating the production of inflammatory cytokines and chemokines.

  • 多发性硬化(multiple sclerosis,MS)是中枢神经系统(central nervous system,CNS)的慢性进行性自身免疫性疾病,以免疫细胞浸润脊髓白质引起的炎症反应、轴突损伤、脱髓鞘病变为主要特征[1]。实验性自身免疫性脑脊髓炎(experimental autoimmune enceph⁃ alomyelitis,EAE)是公认的研究MS的理想模型[2]。研究表明,在 EAE 中,活化的星形胶质细胞通过释放炎症因子与趋化因子,募集炎症细胞浸润至CNS,加剧血脑屏障损伤,在疾病的发生发展中发挥关键作用[3],但其在MS/EAE中的具体作用机制尚不明确。

  • 丝氨酸/苏氨酸蛋白激酶 25(serine/threonine protein kinase25,STK25)是哺乳动物不育系20样激酶(mammalian sterile⁃20⁃like,MST)家族的一员,可使下游相关信号蛋白丝氨酸、苏氨酸残基发生磷酸化,激活相关转录因子,并参与协同刺激信号的转导,在神经系统发育、神经炎症中发挥重要作用[4-5]。研究表明,STK25与人类中枢神经系统的发育高度相关,敲除STK25会直接影响胚胎神经元的发育[6];而 STK25基因缺失会导致一系列神经功能障碍性疾病的发生[7]。研究指出,STK25 可在自身免疫性疾病及神经系统炎症中发挥重要作用[8],但STK25在MS/ EAE 中的作用及其对活化星形胶质细胞的影响尚无文献报道。本研究通过构建特异性靶向星形胶质细胞的 STK25 敲减的重组慢病毒载体,探讨 STK25对于活化的星形胶质细胞炎性因子和趋化因子生成的影响及其在EAE中的作用。

  • 1 材料和方法

  • 1.1 材料

  • 6~8周龄的C57BL/6雌性小鼠购自南京大学模式动物中心。研究中对实验动物所实施的方法均根据中国实验室协会的规定和建议进行,并获得徐州医科大学实验动物伦理委员会的批准。

  • TRIzol试剂(Thermo Fisher公司,美国);髓鞘少突胶质细胞糖蛋白35⁃55(myelin oligodendrocyte gly⁃ coprotein 35⁃55,MOG35-55)多肽(上海强耀生物公司);逆转录试剂(TaKaRa 公司,日本);Real ⁃time PCR试剂(南京诺唯赞生物技术有限公司);结核分枝杆菌H37Ra干粉(Difco公司,美国);完全弗氏佐剂(Sigma公司,美国);百日咳毒素(Invitrogen公司,美国);STK25抗体(Cell Signaling Technolog公司,美国);胶质纤维酸性蛋白(glial fibrillary acidic pro⁃ tein,GFAP)抗体和劳克坚牢蓝染色(Luxol fast blue, LFB)染液(Abcam 公司,英国);特异性靶向星形胶质细胞的STK25敲减(LV⁃STK25⁃shRNA)及阴性对照(LV⁃ctrl)的慢病毒载体的构建与包装,由上海吉凯公司完成。

  • 1.2 方法

  • 1.2.1 重组慢病毒载体的构建、包装及原代星形胶质细胞的慢病毒感染实验

  • 特异性靶向星形胶质细胞的慢病毒载体构建、包装参照本课题组刘晓梅等[9] 的方法。构建携带 GFAP 启动子的 STK25 敲减的重组慢病毒表达载体,并进行慢病毒的包装与滴度测定。将传至3代的星形胶质细胞接种到6孔板,细胞汇合度在80% 左右时,将慢病毒悬液 30 μL 加入到 1 mL 含 10% FBS无抗生素的DMEM/F12培养基,并加入polybrene (终浓度为5 μg/mL),移去细胞培养液,每孔加入病毒稀释液,37℃、5%CO2培养 12~24 h;移去细胞侵染后的病毒液,加入 1 mL 含 10%FBS 无抗生素的 DMEM/F12完全培养液,37℃、5%CO2条件下继续培养72 h,荧光显微镜观察病毒的感染效率。然后,干扰素(interferon,IFN)⁃γ刺激星形胶质细胞,检测相关指标的变化。

  • 1.2.2 质粒有效性验证

  • 参照本实验室已建立的方法[9],将敲减与对照慢病毒悬液按照1×107 TU/只,经尾静脉注射入小鼠体内,7 d后提取小鼠腰段脊髓组织总蛋白质,通过 Western blot检测STK25的表达。

  • 1.2.3 实验分组与EAE小鼠模型制备

  • C57BL/6 小鼠随机分为 4 组:正常对照(normal control,NC)组、EAE组、LV⁃ctrl+EAE组、LV⁃STK25⁃ shRNA+EAE组。参照本实验室已建立的方法[9],将敲减与对照慢病毒悬液按照1×107 TU/只,经尾静脉注射入小鼠体内,7 d后制备EAE小鼠模型:每0.1 mL无菌PBS溶液溶解300 μg MOG35⁃55多肽抗原,每0.1 mL 完全弗氏佐剂溶解 0.5 mg 灭活分枝杆菌 H37Ra 干粉,将二者充分混合,制成油包水抗原诱导乳剂,沿小鼠脊髓正中线多点注射入小鼠背部皮下。在注射抗原诱导乳剂的第 0 天和第 2 天,向小鼠腹腔注射200 ng百日咳毒素。在抗原诱导后,每日观察小鼠行为改变,用双盲法进行临床神经功能评分[9]: 0分,无临床症状;1分,小鼠尾部张力降低或麻痹; 2分,单侧或双侧后肢不完全麻痹(后肢轻瘫);3分,后肢完全瘫痪;4 分,后肢瘫痪和单/双侧前肢麻痹;5分,濒死或死亡。

  • 1.2.4 Western blot实验

  • 参考本实验室已建立的方法[10],提取小鼠腰段脊髓组织总蛋白质,BCA 法测定样品中蛋白质浓度,按每孔50 μg蛋白上样、电泳、转膜、封闭,一抗 4℃摇床孵育 12 h、洗膜,二抗室温孵育 90 min、洗膜,化学发光法显色,以GAPDH为内参。

  • 1.2.5 Real⁃time PCR实验

  • 用TRIzol试剂提取小鼠腰段脊髓组织总RNA,分光光度计检测RNA浓度,逆转录为cDNA,实时荧光定量 PCR 仪扩增目的基因,以β⁃actin 为内参,校正每个样本目的基因的Ct值,以2-ΔΔCt值计算基因的相对表达水平,每组实验重复3次,引物序列见表1。

  • 表1 Real⁃time PCR引物序列

  • Table1 Primer sequences used in real⁃time PCR

  • 1.2.6 免疫荧光

  • 将小鼠麻醉后用生理盐水与 4%多聚甲醛灌注,取腰段脊髓置于4%多聚甲醛中进行后固定,然后依次放入15%、30%蔗糖溶液进行梯度沉糖。待组织完全沉降后取出,用OCT包埋并冰冻切片(10 μm)。将脊髓切片依次用 0.4%Triton X⁃100 破膜,10%山羊血清封闭,加入抗GFAP和抗STK25抗体,4℃冰箱孵育过夜,次日室温复温,PBS 洗涤,再加入相应荧光二抗孵育 2 h、PBS 洗涤,加入 DAPI 染色 10 min,PBS 洗涤后滴加抗淬灭剂封片,荧光显微镜下观察。

  • 1.2.7 HE和LFB染色

  • 将小鼠麻醉后用生理盐水与 4%多聚甲醛灌注,取腰段脊髓置于4%多聚甲醛中进行后固定,依次置于70%、80%、90%、95%、100%乙醇中脱水,使用二甲苯透明后进行石蜡包埋切片(4 μm),苏木素⁃ 伊红(HE)及LFB染色,观察小鼠脊髓炎症细胞浸润与髓鞘脱失情况。

  • 1.3 统计学方法

  • 采用GraphPad Prism 8.3进行统计学分析,数据以均数±标准差(x-±s)表示。两组之间比较采用两独立样本 t 检验,多组之间比较采用单因素方差分析(one ⁃way ANOVA),两两比较采用 LSD ⁃t 检验, P <0.05为差异有统计学意义。

  • 2 结果

  • 2.1 EAE小鼠脊髓STK25表达下降,促炎因子与趋化因子转录水平升高

  • 为了探究EAE小鼠体内STK25的表达变化,使用 MOG35-55免疫 C57BL/6 小鼠制备 EAE 模型后,取小鼠腰段脊髓提取总 RNA 与总蛋白。Real ⁃time PCR 与Western blot结果显示,与NC组相比,EAE组小鼠脊髓中 STK25 mRNA 转录水平显著降低, STK25蛋白表达明显下调(图1A、B)。此外,EAE组小鼠脊髓组织及血清中炎性因子肿瘤坏死因子⁃α (tumor necrosis factor⁃α,TNF⁃α)与趋化因子干扰素诱导蛋白⁃10(interferon⁃inducible protein⁃10,IP⁃10) 的转录与分泌水平明显升高(图1C~F),提示, STK25 可能参与 EAE 的病变并负向调控炎性因子的生成。

  • 图1 EAE小鼠脊髓组织与血清中STK25表达及炎性因子和趋化因子的生成变化

  • Figure1 The expression of STK25 as well as the production of inflammatory cytokine and chemokine in spinal cord and serum of EAE mice

  • 2.2 EAE 小鼠脊髓组织星形胶质细胞 STK25 表达降低

  • 为了进一步确定 STK25 在 EAE 小鼠的表达变化是否与星形胶质细胞的功能密切相关,检测了小鼠脊髓组织星形胶质细胞中 STK25 的表达情况。小鼠腰段脊髓组织免疫荧光染色的结果显示,与NC 组小鼠相比,EAE组小鼠脊髓星形胶质细胞STK25 表达水平明显降低(图2),提示在炎症状态下,星形胶质细胞STK25的表达减少与疾病进展密切相关。

  • 2.3 IFN⁃γ刺激星形胶质细胞活化后,STK25 的表达与炎性因子生成的变化

  • 为了研究在炎症病理环境下,星形胶质细胞STK25的表达与炎性因子及趋化因子生成的关系,取新生乳鼠体外分离培养原代星形胶质细胞,用 IFN⁃γ诱导其活化。Western blot与Real⁃time PCR结果显示,IFN⁃γ刺激6 h后,STK25蛋白表达水平显著降低,促炎因子TNF⁃α及趋化因子IP⁃10的mRNA水平明显升高(图3)。结果表明,病理条件下活化的星形胶质细胞炎性因子的生成增加,其可能与 STK25的表达降低有关。

  • 图2 免疫荧光法检测小鼠脊髓星形胶质细胞中STK25的表达

  • Figure2 The expression of STK25 in astrocytes in the spinal cords of mice detected by immunofluorescence analysis

  • 2.4 敲减 STK25 增加了星形胶质细胞炎性因子与趋化因子的生成

  • 为了进一步验证 STK25 对星形胶质细胞炎性因子生成的影响,首先构建特异性靶向星形胶质细胞的STK25敲减慢病毒载体,转染小鼠原代星形胶质细胞,验证STK25的表达,STK25⁃shRNA⁃3的敲减效率最高,选择它进行后续实验(图4A、B)。之后,包装慢病毒悬液,命名为LV⁃STK25⁃shRNA,对照组为LV⁃ctrl,分别感染小鼠原代星形胶质细胞72 h后,用IFN⁃γ诱导星形胶质细胞活化。Real⁃time PCR结果显示,敲减 STK25 后,活化的小鼠原代星形胶质细胞炎性因子与趋化因子的转录水平显著升高(图4C、D),表明敲减STK25上调了星形胶质细胞中炎性因子与趋化因子的生成。

  • 2.5 敲减STK25加速了EAE小鼠的疾病进程

  • 前期研究已经证实,重组慢病毒悬液经尾静脉注射后,可以透过血脑屏障到达小鼠的中枢神经系统[9]。为了证实质粒干扰的有效性,将小鼠分为LV⁃ STK25⁃shRNA 组和 LV⁃ctrl 组,分别经尾静脉注射 STK25 敲减与对照慢病毒悬液。Western blot 结果证实,LV⁃STK25⁃shRNA 组小鼠脊髓 STK25 的表达水平显著降低(图5A、B)。为了探究敲减STK25对 EAE小鼠疾病进程的影响,本研究将特异性敲减星形胶质细胞 STK25 与对照的慢病毒悬液经尾静脉注射入小鼠体内,7 d后用MOG35⁃55诱导小鼠EAE模型,采用双盲法每日评分并记录小鼠发病情况。与 LV⁃ctrl 组比较,LV⁃STK25⁃shRNA 组EAE 小鼠发病时间提前,临床神经功能评分加重(图5C)。Real⁃ time PCR结果显示,敲减STK25后,EAE小鼠脊髓组织中炎性因子TNF⁃α与趋化因子IP⁃10的转录水平显著升高(图5D、E)。HE 和 LFB 染色发现,LV ⁃ STK25⁃shRNA 组 EAE 小鼠脊髓组织炎症细胞浸润明显增多,髓鞘脱失更加严重(图5F),提示 STK25 参与并调控了EAE的病变进程。

  • 图3 IFN⁃γ诱导原代星形胶质细胞活化后STK25与炎性因子、趋化因子的表达变化

  • Figure3 Changes of expression of STK25,inflammatory cytokine and chemokine in primary astrocytes activated by IFN⁃γ

  • 图4 敲减STK25促进了原代星形胶质细胞炎性因子与趋化因子的生成

  • Figure4 Knock⁃down of STK25 promotes the production of inflammatory cytokine and chemokine in primary astrocytes

  • 3 讨论

  • MS是以免疫细胞浸润引起炎症反应、白质脱髓鞘为主要特征的中枢神经系统的慢性进行性自身免疫性疾病[11]。作为影响青壮年残疾的主要非创伤性神经疾病,每年有数百万人受其影响,然而其致病机制尚不明确,治疗十分困难[12]。星形胶质细胞是中枢神经系统中数量最多的细胞群,在维持中枢神经系统内环境稳态、保持突触可塑性、保护神经细胞等方面具有重要生理功能[13-15]。已知,活化的星形胶质细胞在中枢神经系统的不同疾病模型中,发挥不同的生物学效应[16-18]。研究证实,星形胶质细胞活化与MS和EAE的病变密切相关[8],在脊髓损伤时,活化的星形胶质细胞可迅速迁移至损伤部位,释放大量的炎性因子加重脊髓损伤[19],但其在 MS和EAE中的具体作用机制尚不明确。

  • 图5 敲减STK25促进了EAE小鼠的病变进程

  • Figure5 STK25 knock⁃down exacerbates the process of EAE

  • STK25作为MST家族的一员,通过激活转录因子及协同刺激信号转导等方式,参与调节细胞的多种生理功能,包括增殖、凋亡、迁移和细胞骨架重排,在调控神经系统的正常生理功能中发挥重要作用[20]。研究表明,STK25与人类中枢神经系统的发育高度相关。敲除 STK25 会使胚胎神经元细胞中维持轴突生长的高尔基体定位错误,引起神经元异常树突形成[6];当 STK25 基因缺失时,会导致自闭症、多动症、癫痫等一系列神经功能障碍性疾病的发生[7]。然而,在阿兹海默症等神经退行性疾病中, STK25基因敲除可减少胚胎神经元中微管结合蛋白的过度磷酸化,从而缓解疾病进展[21]。但STK25在 MS及星形胶质细胞中的作用尚无文献报道。

  • 本研究首先证实了EAE小鼠STK25表达降低,炎性因子与趋化因子转录、分泌水平升高,同时 EAE 小鼠脊髓组织星形胶质细胞 STK25 表达下调。随后,在体外分离培养的原代星形胶质细胞中,用 IFN⁃γ诱导其活化后发现,活化的星形胶质细胞中STK25的表达降低与炎性因子生成增加有关。研究表明,星形胶质细胞活化后可分泌炎性因子与趋化因子,募集炎症细胞浸润到中枢神经系统,加剧脊髓的炎症损伤[22]。已证实在中枢神经系统中,STK25 在胚胎神经元表达并调控神经元发育[5-6]。为了进一步验证 STK25 对星形胶质细胞功能的调控及活化后炎性因子生成的影响,本研究使用STK25敲减的重组慢病毒载体并包装感染星形胶质细胞,经IFN⁃ γ诱导活化。结果显示,敲减 STK25 上调了星形胶质细胞炎性因子与趋化因子的生成;同时用慢病毒感染小鼠并诱导EAE,发现LV⁃STK25⁃shRNA+EAE 组小鼠脊髓组织中炎性因子和趋化因子的转录水平升高,炎症细胞浸润更加明显,髓鞘脱失更为严重,提示敲减STK25促进了EAE小鼠的病变进程。

  • 综上所述,STK25可能通过影响星形胶质细胞的活化,调节其炎性因子与趋化因子的产生,从而参与EAE小鼠的病变进程。

  • 参考文献

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    • [2] INOUE M,CHEN P H,SIECINSKI S,et al.An interferon⁃ β⁃ resistant and NLRP3 inflammasome ⁃independent sub⁃ type of EAE with neuronal damage[J].Nat Neurosci,2016,19(12):1599-1609

    • [3] BRAMBILLA R.The contribution of astrocytes to the neu⁃ roinflammatory response in multiple sclerosis and experi⁃ mental autoimmune encephalomyelitis[J].Acta Neuro⁃ pathol,2019,137(5):757-783

    • [4] THOMPSON B J,SAHAI E.MST kinases in development and disease[J].J Cell Biol,2015,210(6):871-882

    • [5] MENCARELLI C,NITARSKA J,KROECHER T,et al.RanBP1 couples nuclear export and Golgi regulation through LKB1 to promote cortical neuron polarity[J].Cell Rep,2018,24(10):2529-2539

    • [6] RAO S,KIRSCHEN G W,SZCZURKOWSKA J,et al.Re⁃ positioning of somatic Golgi apparatus is essential for the dendritic establishment of adult ⁃ born hippocampal neu⁃ rons[J].J Neurosci,2018,38(3):631-647

    • [7] TOHRU M,AKIO I,MASASHI U,et al.STK25 and MST3 have overlapping roles to regulate Rho GTPases during cortical development[J].J Neurosci,2021,41(43):8887-8903

    • [8] MATSUKI T,CHEN J,HOWELL B W.Acute inactive of the serine⁃threonine kinase STK25 disrupts neuronal mi⁃ gration[J].Neural Dev,2013,8:21

    • [9] LIU X,ZHOU F,YANG Y,et al.miR⁃409⁃3p and miR⁃ 1896 co⁃operatively participate in IL⁃17⁃induced inflam⁃ matory cytokine production in astrocytes and pathogene⁃ sis of EAE mice via targeting SOCS3/STAT3 signaling[J].Glia,2019,67(1):101-112

    • [10] 李小翠,王晓天,张清秀,等.MLK3信号通路在癫痫及脑缺血海马CA3区神经元中的不同作用[J].南京医科大学学报(自然科学版),2014,34(10):1329-1332

    • [11] OH J,VIDAL ⁃ JORDANA A,MONTALBAN X.Multiple sclerosis:clinical aspects[J].Curr Opin Neurol,2018,31(6):752-759

    • [12] LIL M,JULIAN B,FLORENT F,et al.SARS ⁃ CoV ⁃ 2 mRNA vaccinations fail to elicit humoral and cellular im⁃ mune responses in patients with multiple sclerosis receiv⁃ ing fingolimod[J].J Neurol Neurosurg Psychiatry,2022,93(9):960-971

    • [13] ISRAELOV H,RAVID O,ATRAKCHI D,et al.Caspase⁃1 has a critical role in blood⁃brain barrier injury and its in⁃ hibition contributes to multifaceted repair[J].J Neuroin⁃ flammation,2020,17(1):267

    • [14] LIU L R,LIU J C,BAO J S,et al.Interaction of microglia and astrocytes in the neurovascular unit[J].Front Immu⁃ nol,2020,11:1024

    • [15] JANNETH G,ANDRÉS P,ANDREA A R,et al.Advances in astrocyte computational models:from metabolic recon⁃ structions to multi⁃omic approaches[J].Front Neuroinfor⁃ matics,2020,14:35

    • [16] GOLLIHUE J,NORRIS C.Astrocyte mitochondria:cen⁃ tral players and potential therapeutic targets for neurode⁃ generative diseases and injury[J].Ageing Res Rev,2020,59:101039

    • [17] LI Z J,YU Y F,KANG J J,et al.microRNA⁃124 overex⁃ pression in schwann cells promotes schwann cell ⁃ astro⁃ cyte integration and inhibits glial scar formation ability [J].Front Cell Neurosci,2020,14:144

    • [18] ZAREI⁃KHEIRABADI M,HESARAKI M,KIANI S,et al.In vivo conversion of rat astrocytes into neuronal cells through neural stem cells in injured spinal cord with a sin⁃ gle zinc ⁃ finger transcription factor[J].Stem Cell Res Ther,2019,10(1):380

    • [19] HART C G,DYCK S M,KATARIA H,et al.Acute upregu⁃ lation of bone morphogenetic protein ⁃4 regulates endoge⁃ nous cell response and promotes cell death in spinal cord injury[J].Exp Neurol,2020,325:113163

    • [20] SONG Z,MAO H,LIU J,et al.Lanthanum chloride induces axon abnormality through LKB1 ⁃ MARK2 and LKB1 ⁃ STK25⁃GM130 signaling pathways[J/OL].Cell Mol Neu⁃ robiol,2022[2022 ⁃ 11 ⁃ 10].doi:10.1007/s10571 ⁃ 022 ⁃ 01237⁃0

    • [21] MATSUKI T,ZAKA M,GUERREIRO R,et al.Identifica⁃ tion of Stk25 as a genetic modifier of Tau phosphorylation in Dab1⁃mutant mice[J].PLoS One,2012,7(2):31152

    • [22] LIDDELOW S A,GUTTENPLAN K A,CLARKE L E,et al.Neurotoxic reactive astrocytes are induced by activat⁃ ed microglia[J].Nature,2017,541(7638):481-487

  • 参考文献

    • [1] LIU X M,ZHOU F,WANG W X,et al.IL⁃9⁃triggered lnc⁃ RNA Gm13568 regulates Notch1 in astrocytes through in⁃ teraction with CBP/P300:contribute to the pathogenesis of experimental autoimmune encephalomyelitis[J].J Neu⁃ roinflammation,2021,18(1):108

    • [2] INOUE M,CHEN P H,SIECINSKI S,et al.An interferon⁃ β⁃ resistant and NLRP3 inflammasome ⁃independent sub⁃ type of EAE with neuronal damage[J].Nat Neurosci,2016,19(12):1599-1609

    • [3] BRAMBILLA R.The contribution of astrocytes to the neu⁃ roinflammatory response in multiple sclerosis and experi⁃ mental autoimmune encephalomyelitis[J].Acta Neuro⁃ pathol,2019,137(5):757-783

    • [4] THOMPSON B J,SAHAI E.MST kinases in development and disease[J].J Cell Biol,2015,210(6):871-882

    • [5] MENCARELLI C,NITARSKA J,KROECHER T,et al.RanBP1 couples nuclear export and Golgi regulation through LKB1 to promote cortical neuron polarity[J].Cell Rep,2018,24(10):2529-2539

    • [6] RAO S,KIRSCHEN G W,SZCZURKOWSKA J,et al.Re⁃ positioning of somatic Golgi apparatus is essential for the dendritic establishment of adult ⁃ born hippocampal neu⁃ rons[J].J Neurosci,2018,38(3):631-647

    • [7] TOHRU M,AKIO I,MASASHI U,et al.STK25 and MST3 have overlapping roles to regulate Rho GTPases during cortical development[J].J Neurosci,2021,41(43):8887-8903

    • [8] MATSUKI T,CHEN J,HOWELL B W.Acute inactive of the serine⁃threonine kinase STK25 disrupts neuronal mi⁃ gration[J].Neural Dev,2013,8:21

    • [9] LIU X,ZHOU F,YANG Y,et al.miR⁃409⁃3p and miR⁃ 1896 co⁃operatively participate in IL⁃17⁃induced inflam⁃ matory cytokine production in astrocytes and pathogene⁃ sis of EAE mice via targeting SOCS3/STAT3 signaling[J].Glia,2019,67(1):101-112

    • [10] 李小翠,王晓天,张清秀,等.MLK3信号通路在癫痫及脑缺血海马CA3区神经元中的不同作用[J].南京医科大学学报(自然科学版),2014,34(10):1329-1332

    • [11] OH J,VIDAL ⁃ JORDANA A,MONTALBAN X.Multiple sclerosis:clinical aspects[J].Curr Opin Neurol,2018,31(6):752-759

    • [12] LIL M,JULIAN B,FLORENT F,et al.SARS ⁃ CoV ⁃ 2 mRNA vaccinations fail to elicit humoral and cellular im⁃ mune responses in patients with multiple sclerosis receiv⁃ ing fingolimod[J].J Neurol Neurosurg Psychiatry,2022,93(9):960-971

    • [13] ISRAELOV H,RAVID O,ATRAKCHI D,et al.Caspase⁃1 has a critical role in blood⁃brain barrier injury and its in⁃ hibition contributes to multifaceted repair[J].J Neuroin⁃ flammation,2020,17(1):267

    • [14] LIU L R,LIU J C,BAO J S,et al.Interaction of microglia and astrocytes in the neurovascular unit[J].Front Immu⁃ nol,2020,11:1024

    • [15] JANNETH G,ANDRÉS P,ANDREA A R,et al.Advances in astrocyte computational models:from metabolic recon⁃ structions to multi⁃omic approaches[J].Front Neuroinfor⁃ matics,2020,14:35

    • [16] GOLLIHUE J,NORRIS C.Astrocyte mitochondria:cen⁃ tral players and potential therapeutic targets for neurode⁃ generative diseases and injury[J].Ageing Res Rev,2020,59:101039

    • [17] LI Z J,YU Y F,KANG J J,et al.microRNA⁃124 overex⁃ pression in schwann cells promotes schwann cell ⁃ astro⁃ cyte integration and inhibits glial scar formation ability [J].Front Cell Neurosci,2020,14:144

    • [18] ZAREI⁃KHEIRABADI M,HESARAKI M,KIANI S,et al.In vivo conversion of rat astrocytes into neuronal cells through neural stem cells in injured spinal cord with a sin⁃ gle zinc ⁃ finger transcription factor[J].Stem Cell Res Ther,2019,10(1):380

    • [19] HART C G,DYCK S M,KATARIA H,et al.Acute upregu⁃ lation of bone morphogenetic protein ⁃4 regulates endoge⁃ nous cell response and promotes cell death in spinal cord injury[J].Exp Neurol,2020,325:113163

    • [20] SONG Z,MAO H,LIU J,et al.Lanthanum chloride induces axon abnormality through LKB1 ⁃ MARK2 and LKB1 ⁃ STK25⁃GM130 signaling pathways[J/OL].Cell Mol Neu⁃ robiol,2022[2022 ⁃ 11 ⁃ 10].doi:10.1007/s10571 ⁃ 022 ⁃ 01237⁃0

    • [21] MATSUKI T,ZAKA M,GUERREIRO R,et al.Identifica⁃ tion of Stk25 as a genetic modifier of Tau phosphorylation in Dab1⁃mutant mice[J].PLoS One,2012,7(2):31152

    • [22] LIDDELOW S A,GUTTENPLAN K A,CLARKE L E,et al.Neurotoxic reactive astrocytes are induced by activat⁃ ed microglia[J].Nature,2017,541(7638):481-487

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