【作者】 陈杰；

【导师】 蔡亚非；

【作者基本信息】 安徽师范大学 ， 生理学， 2019， 博士

【摘要】 脊髓损伤(Spinal cord injury,SCI)是中枢神经系统(Central nervous system,CNS)重大疾病的一类,也是临床上脊柱外科常见的严重疾患之一,分为初级阶段的原发性损伤和次级阶段的继发性损伤。继发性损伤是在原发性损伤基础上,逐级引发的一种可逆的细胞主动调节过程,探究次级阶段继发性脊髓损伤的分子机制,对脊髓损伤的认知及治疗具有特别重要的意义,因此降低脊髓继发性损伤是临床研究重点。ZBTB38,在人类基因中被表达的蛋白叫CIBZ,属于锌指蛋白家族,且具有典型的BTB结构域。这类蛋白家族多属转录因子,能够调节多种目的基因的转录活性,还能够参与细胞内多种信号通路的调控,在机体细胞分化、表达调控等生命过程中起到非常重要的作用。该基因在脊髓中高度表达,并且在SCI诱导的细胞凋亡中起到负调节剂的作用,但通过何种途径来调控这一事件的发生尚未见有相关报道。在真核生物中,内质网应激(Endoplasmic reticulum stress,ERS)和自噬(Autophagy)是机体必不可少的两道防御和保护机制。持续或严重的ERS可诱发机体意外的细胞凋亡,尽管对ERS反应的研究主要集中在大鼠脊髓损伤后神经元和少突胶质细胞的凋亡途径,但需要更多的体外研究来了解脊髓损伤后ERS相关凋亡的机制。广泛存在于真核细胞内的自噬,具有高度保守的溶酶体依赖性降解途径,是在真核生物细胞中区别于ERS的另一重要防御和保护机制,然而ZBTB38是否在中枢神经元细胞自噬性调控通路中发挥作用尚未有相关研究报道,脊髓损伤后自噬和凋亡可能共享一些信号通路共同参与了脊髓神经细胞的死亡过程,但自噬具体通过什么信号通路调控凋亡还需进一步的深入探讨。在本研究中,体外证明了ERS触发ZBTB38的表达下调,增加了SH-SY5Y细胞线粒体定位的促凋亡基因如Bak,Noxa,Puma和Bim的表达水平,当ERS发生时由ZBTB38表达抑制介导的细胞凋亡可能是导致创伤性SCI的继发性损伤的主要原因之一。而这一数据也进一步证实了本课题组前期的研究发现,认为ZBTB38在SCI后通过线粒体途径负调节细胞凋亡。ZBTB38基因被沉默下调后,神经母细胞瘤细胞SH-SY5Y的增殖与活力明显低于对照组,实验组的自噬标记蛋白LC3B表达水平较对照组显著性下降,p62表达有所上升,除此之外,LC3B免疫荧光染色及电镜下观察自噬小体的形态等均表明人骨髓源神经母细胞瘤细胞的自噬作用显然被抑制了,但同时观察到ERS调节自噬发生的重要调控元件eIF2α磷酸化水平(P-eIF2α)却显著增加了,表明ZBTB38在调控ERS触发的细胞凋亡的同时,很可能跳过ERS的诱导直接影响到细胞自噬的发生。为了探寻ZBTB38具体通过某一通路影响到自噬的发生,本研究基于体外RNA转录组高通量测序进一步探讨人神经母细胞瘤细胞在ZBTB38基因表达下调情况下的基因转录变化规律,根据测序结果筛选差异表达基因(Differentially expressed gene,DEG)2438个,其中下调基因占83.5%,并对这些差异表达基因进行功能聚类GO(Gene Ontology)和信号传导通路数据库KEGG(Kyoto Encyclopedia of Genes and Genomes)生物信息学分析,首次发现ZBTB38的表达抑制导致神经细胞凋亡最显著相关的信号通路是神经营养因子TRK受体信号传导途径(Neurotrophin TRK receptor signaling pathway),除此之外,ZBTB38的下调还可通过调控p53信号通路上关键基因以促进神经母细胞瘤细胞的凋亡;与自噬起始密切相关RB1CC1的表达被显著抑制,结合QRT-PCR的mRNA表达水平验证,结果显示ZBTB38的表达下调与自噬小体生物合成起始阶段关键因子RB1CC1的差异表达显著相关,ZBTB38基因敲低导致RB1CC1基因下调4.2倍;表明ZBTB38下调后使细胞阻断了程序性死亡的一种重要的保护机制-自噬的发生,加速了神经母细胞瘤细胞的凋亡,我们还观察到将过表达RB1CC1质粒再次转染至ZBTB38下调的SH-SY5Y细胞中,自噬标识相关因子LC3B和p62均恢复表达,自噬得以再次启动,共转染后细胞的增殖和活力也显著促进。在体内实验创伤性SCI小鼠中,再次证明了ERS诱导ZBTB38表达抑制并触发ZBTB38介导的细胞凋亡。另ChIP-QPCR分析结果显示ATF4作为一种ERS诱导型转录因子,是通过与ZBTB38启动子结合来直接激活ZBTB38转录调控,但在SCI后这种结合却呈现显著降低,从而导致ZBTB38的表达急剧下降。通过向SCI小鼠注射含有ZBTB38的慢病毒恢复受损脊髓中的ZBTB38功能,显著减轻脊髓继发性损伤,减少ERS相关凋亡和部分恢复脊髓功能,另外在脊髓继发性损伤的不同阶段RB1CC1与ZBTB38基因的表达趋势保持一致,且自噬体合成显著增强,促进了小鼠运动神经元功能的修复。这些研究结果均表明,ZBTB38表达的恢复可以减少脊髓损伤后的继发性组织损伤,并提示靶向ZBTB38的治疗策略可以促进脊髓功能恢复脊髓损伤患者。后期研究中通过调节ZBTB38基因的表达变化来探索自噬启动基因RB1CC1与神经性疾病功能恢复的分子机制,为治疗开辟一种新的策略。更多还原

【Abstract】 Spinal cord injury（SCI）is one of the major diseases of the central nervous system（CNS）which commonly appeared in spinal surgery.It can be divided into primary injury（primary stage）and secondary injury（secondary stage）.Secondary injury is a kind of positive regulation at cellular and molecular level caused by the primary injury.It is reversible and controllable.Exploration of the mechanisms of spinal cord injury at the secondary level is pivotal to the understating and treatment of this disease,so reducing the loss caused by the secondary injury of spinal cord is of great importance for clinical study.ZBTB38 encodes a protein called CIBZ,a member of the C2H2 zinc finger protein family that typically contains the BTB domain.The BTB domain is often contained in transcription factors,which regulate the transcription of multiple target genes and participate in several signaling pathways and plays an important role in gene expression regulation,cell differentiation,embryo development,and other biological processes.CIBZ is highly expressed in spinal cord and it functions as a negative regulator in SCI-induced apoptosis.In eukaryotes,endoplasmic reticulum stress（ERS）and autophagy are two important defense and protection mechanisms in the body More studies are needed to understand the mechanisms of ER stress-associated apoptosis in SCI,although ER stress responses seem to divert towards apoptosis pathways in neurons and oligodendrocytes following SCI in the adult rat.Autophagy is a lysosome-dependent degradation pathway highly conserved and widely spread in eukaryotic cells.It is also an important defense and protection mechanism which is different from endoplasmic reticulum（ER）stress of eukaryotic cells.However,whether ZBTB38 plays a role in the signaling pathways of autophagy in central neurons has not been reported.It is proposed that autophagy and apoptosis may share some signaling pathways and participate in the death of spinal cord nerve cells after spinal cord injury.However,which pathways are involved in the autophagy-induced apoptosis remain to be elucidated.In the present study,we demonstrated in vitro that ERS triggered down-regulation of ZBTB38 expression,increased the expression levels of proapoptotic genes,including Bak,Noxa,Puma and Bim,in mitochondria-localization of SH-SY5Y cells.Endoplasmic reticulum stress-related apoptosis mediated by ZBTB38 deletion may be one of the leading causes of secondary injury to traumatic SCI.This data further confirms our previous findings that ZBTB38 negatively regulates apoptosis through the mitochondrial pathway after SCI.When ZBTB38 gene was silenced,the proliferation and viability of neuroblastoma cell line SH-SY5Y was significantly lower than that of the control group.The expression level of autophagy marker protein LC3B in the experimental group was significantly lower than that in the control group,and the expression of p62 was increased.In addition,the immunofluorescence staining of LC3B and the morphology of autophagosomes under electron microscope showed that the autophagy of human bone marrow-derived neuroblastoma cells was obviously inhibited.However,it was observed that eIF2αphosphorylation level（P-eIF2α,an important regulatory element of ERS regulating autophagy）was significantly increased,the results indicate that ZBTB38 may play a role in the regulation of ERS-triggered apoptosis,and it is likely that skipping the induction of ERS directly affects the occurrence of autophagy.To have some insight into the role of ZBTB38 in neuroblastoma development,high throughput RNA sequencing was performed using the human neuroblastoma cell line SH-SY5Y with the deletion of ZBTB38.In the present study,2,438 differentially expressed genes（DEGs）in ZBTB38^-/-SH-SY5Y cells were obtained,83.5%of which was down-regulated functional annotation of the differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the neurotrophin TRK receptor signaling pathway.In addition,down-regulation of ZBTB38 also promotes apoptosis in neuroblastoma cells by regulating key genes in the p53signaling pathway,combined with gene expression on mRNA levels through qRT-PCR,our study first clarified the down-regulation of ZBTB38 was significantly related to the differential expression of key factor RB1CC1（a factor involved in the biosynthesis of autophagosome in the initial stage）.Expression of RB1CC1 was down-regulated 4.2 times after ZBTB38 knockdown.The results also indicated that the expression of autophagy-related proteins LC3 and p62 was rescued when transfected the vectors overexpressing RB1CC1 in the ZBTB38-downregulated SH-SY5Y cells.Moreover,through this way,autophagy was re-initiated and the proliferation and viability of co-transfected cells were increased significantly.In the traumatic SCI mice,ER stress presented in injured spinal cord induced repression of ZBTB38 expression and triggered ZBTB38-mediated apoptosis.ChIP-QPCR analysis revealed that ATF4,an ER-stress inducible transcription factor,directly activated ZBTB38transcription by binding to the ZBTB38 promoter.However,this binding was significantly reduced following SCI,leading to a sharp decrease in ZBTB38 expression.Restoring ZBTB38 function in injured spinal cord by injection of lentivirus containing ZBTB38 into SCI mice,significantly alleviated secondary damage of spinal cord with decreased ER stress-associated apoptosis and partially recovered spinal cord functions.The expression patterns of RB1CC1 and ZBTB38 were consistent at different stages of secondary spinal cord injury in vivo.At the same time,the biosynthesis of autophagosome as well as the ability of mouse motor neurones was enhanced significantly.These findings demonstrate that restoration of ZBTB38 expression can reduce secondary tissue damage after SCI,and suggest that a therapeutic strategy for targeting ZBTB38 promote functional recovery of spinal cord for patients with SCI.In the later studies,the molecular mechanism of autophagy initiation gene RB1CC1 and neurological disease recovery was explored by regulating the expression changes of ZBTB38 gene,opening up a new strategy for treatment.更多还原