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类型 基础研究 预答辩日期 2017-09-26
开始(开题)日期 2016-02-29 论文结束日期 2017-06-13
地点 东南大学附属中大医院2号楼502会议室 论文选题来源 国家自然科学基金项目     论文字数 7.5 (万字)
题目 椎间盘髓核细胞组成性表达低氧诱导因子-1α的发育基础以及类泛素化修饰调控机制
主题词 椎间盘,低氧,低氧诱导因子-1α,类泛素化修饰,椎间盘退变
摘要 第一部分 椎间盘形成、发育、退变整个自然史的组织学与影像学演变 目的:探明椎间盘从胚胎期形成到老化后退变的整个自然史,细化认识椎间盘形成、发育、退变过程中的组织病理学演变。 方法:获取胚胎第13天、14天、18天、刚出生、4周、12周、30周、60周共8个时间点大鼠脊柱正中冠状面切片。HE染色观察细胞成分及形态演变,阿利辛蓝+天狼星红复合染色观察软骨基质与纤维基质表达变化,马松染色观察肌纤维与胶原纤维表达变化,过碘酸雪夫染色评价糖元及其代谢产物糖蛋白与葡萄糖胺聚糖表达水平。免疫组织化学染色Ki67、PCNA表达分布,评价胚胎椎间盘不同解剖区域内细胞增殖活力。取刚出生、2周、6周龄三组大鼠行脊柱MRI检查;取12周和60周龄大鼠腰段脊柱行X线、CT、MRI检查。 结果:胚胎期大鼠椎间盘形成过程中脊索结构发育成髓核,轴旁中胚层分化成生骨节后其头侧的非致密区发育成椎体和软骨终板,而尾侧的致密区演变成纤维环;在此过程中脊索细胞快速空泡化,成对的生骨节非致密结构对称性的向中线融合并将空泡化的脊索挤压入椎间盘中央,而生骨节致密结构向外侧扩张最终形成纤维环。组织化学染色发现胚胎期内层纤维环与软骨终板具有相近的细胞密度和软骨基质表达,免疫组织染色见内层纤维环细胞的增殖活性也与软骨终板细胞相近。大鼠脊索细胞在空泡化之前由致密的脊索鞘包裹,大鼠脊索鞘富含软骨基质和纤维成分,其中胶原纤维染色强于肌纤维染色,糖蛋白及葡萄糖胺聚糖成分也呈阳性表达。在脊索细胞充分空泡化之前脊索鞘已开始变薄,在脊索结构被挤压入椎间盘过程中脊索鞘逐渐消失。随着年龄增加,髓核细胞的胞浆空泡数量和大小逐渐丢失,空泡化髓核细胞在30周时已大部分被软骨样髓核细胞取代。12周时次级骨化中心在软骨终板的外周成对出现并向内侧浸润,使得软骨终板内的软骨细胞层逐渐变薄;内层纤维环的软骨基质向外层纤维环浸润,使得纤维环外侧的纤维层厚度逐渐变薄。在影像学退变的老龄大鼠椎间盘内,细胞数量的整体下降与局部细胞丛性聚集共存,较大的纵向裂隙出现在内外层纤维环的交界区附近。 结论:椎间盘的形成、发育、自然退变涉及复杂而动态的细胞与基质成分调控;髓核与纤维环具有完全不同的胚层起源和组织细胞学演变过程。 第二部分 椎间盘细胞类泛素化修饰信号的低氧调控及其对低氧诱导因子-1α稳定性和低氧耐受能力的影响 目的:探明类泛素化(SUMO化)修饰信号是否在椎间盘细胞内表达。探讨低氧环境下髓核细胞与纤维细胞对SUMO化修饰信号分子的表达调控规律及调控差异;明确SUMO化修饰信号调控对髓核细胞低氧诱导因子-1α(HIF-1α)稳定性和低氧耐受力的影响。 方法:获取刚出生和10周龄大鼠脊柱正中冠状面切片,免疫组织化学染色SUMO化修饰信号成员:SUMO分子SUMO1和SUMO2/3,SUMO化E1激活酶SAE1和SAE2,SUMO化E2连接酶UBC9,SUMO化蛋白酶SENP1在椎间盘内的表达分布。平面培养大鼠腰椎髓核细胞与纤维环细胞,免疫荧光染色SUMO化修饰信号成员在体外培养椎间盘细胞内的表达与分布。髓核细胞与纤维环细胞分别低氧培养0-24小时,CCK-8法检测细胞增殖率变化;Annexin-V/PI染色后流式细胞仪检测细胞凋亡率变化,PI染色后流式细胞仪检测细胞周期分布变化,SA-β-gal染色并量化细胞老化率变化;Realtime-PCR和Western-blot检测并比较髓核细胞与纤维环细胞对SUMO1、SUMO2/3、SAE1、SAE2、UBC9、SENP1的转录和表达调控规律。SENP1特异性干扰RNA下调髓核细胞内SENP1表达,低氧培养0-24小时,Realtime-PCR和Western-blot检测HIF-1α的转录与蛋白稳定性变化,HIF-1α下游靶点VEGF、Glut-1、PDK1的转录和表达变化;SA-β-gal染色量化SENP1下调对低氧环境下髓核细胞老化的影响,Hoechst33342/PI染色后流式细胞仪检测SENP1下调后髓核细胞在低氧环境下的细胞凋亡率变化。 结果:SUMO化修饰信号成员在刚出生及10周龄大鼠椎间盘髓核、纤维环、软骨终板都表达,以胞核表达为主。SUMO化修饰信号成员在体外培养的髓核细胞与纤维环细胞内都表达,以胞核表达为主,但SUMO1仅在细胞核表达。游离的SUMO-2/3小分子在常氧与低氧下都表达,但游离的SUMO1小分子常氧下罕有表达,低氧后诱导性表达。椎间盘细胞内SUMO1主要与RanGAP1结合,Western-blot法未检测到被SUMO1修饰的HIF-1α。髓核细胞低氧4小时后SUMO1、SUMO2/3、SAE2、UBC9的转录与表达显著上调,随后恢复至常氧水平,SAE1低氧4-12小时表达下降。纤维环细胞低氧12小时后SUMO1转录上调,SUMO2/3、SAE1、SAE2、UBC9的转录与表达无显著改变。髓核细胞与纤维环细胞内SENP1随低氧培养时间延长逐渐上调。髓核细胞常氧和低氧环境下都表达HIF-1α,低氧时间延长并不上调髓核细胞HIF-1α表达。特异性下调SENP1表达后低氧环境下髓核细胞HIF-1α表达下降,下游靶点VEGF、Glut-1的转录激活减少,PDK1的蛋白表达下调。髓核细胞与纤维环细胞在低氧环境下的细胞增殖率、老化率、细胞周期分布以及细胞凋亡水平与常氧相比无显著改变。下调SENP1表达的髓核细胞尽管HIF-1α稳定性下降,但低氧环境下的细胞老化率和凋亡率无明显上调。 结论:SUMO化修饰信号机制在椎间盘细胞内存在且应答低氧微环境。低氧时髓核细胞与纤维环细胞差异性调控SUMO化修饰关键信号分子表达,其中SENP1的低氧诱导上调参与髓核细胞内HIF-1α的蛋白稳定性及下游转录激活调控。髓核细胞与纤维环细胞具有相近的低氧耐受性,HIF-1α的稳定性调控不是髓核细胞耐受低氧的唯一信号基础。
英文题目 Developmental basis and sumoylation mechanism of the constitutive expression of Hypoxia-Inducible Factor-1α in nucleus pulposus cells
英文主题词 intervertebal disc, hypoxia, Hypoxia-Inducible Factor-1α, sumoylation, intervertebral disc degeneration
英文摘要 Part 1 A histocytological and radiological overview of the natural history of intervertebral disc: From embryonic formation to age-related degeneration Objective:To characterize the histocytological change during intervertebral disc (IVD) formation, maturation, and age-related degeneration. Methods:Coronal sections of embryonic (E13、E14、E18、Neonatal) and postnatal (4W、12W、30W、60W) rat IVD were stained by a series of histological stainings: with HE staining to evaluate the change in cellular contents, Alcian blue staining to assess the accumulation of cartilagenous matrix, Picrosirius red staining for fibrotic matrix, Masson staining for collagenous fibers and myofibers, and PAS staining to identify the expression of glycoproteins and glycosaminoglycans. Growth kinemics within the different compartments of embryonic IVD were evaluated by immunohistochemical staining of Ki67 and PCNA. Postnatal maturation (Neonatal、2W、6W) and age-related degeneration (12W、60W) of rat IVD were visualized on radiology by X-ray, CT, and MR imaging. Results:Embryonically, non-condensed sclerotome developed into vertebrae and cartilaginous endplate (CEP), condensed sclerotome generated annulus fibrosis (AF), and axial notochord formed nucleus pulposus (NP). During the formation of IVD, notochordal cells increased and enlarged the cytoplasmic vacuoles, the paired vertebrae anlagen fused and compressed the vacuolating notochord into the forming NP, which was enwrapped by the outward-expanding AF anlagen. Noteworthy, inner AF shared similar histocytological contents and growth kinetics as the CEP, drawing distinct boundaries between the inner and the outer AF of neonatal IVD. At early development stages (E13.5–14.5) the axial notochord was surrounded by a thick notochord sheath that accumulated both cartilagenous and fibrotic matrix, in which the expression of glycoproteins and glycosaminoglycans and the accumulaton of more collagenous fibers than myofibers were also evident. While the notochord sheath was thick at E13.5, it became thinner and vanished as the axial notochord was compressed into the prospective NP. Postnatally, vacuolated notochord cells were reduced by devacuolation while chondrocytic NP cells increased; cartilaginous layers of CEP were narrowed by vertebrae growth and secondary ossification; fibrotic portion of AF decreased as cartilagenous matrix accumulated and infiltrated outwards. In aged and degenerated IVD, large longitudinal fissures were detected near the boundaries between inner and outer AF, whereas both reduced cellularity and accumulated cell clusters were evident within the dehydrated NP. Conclusion:From embryonic formation to age-related degeneration, the natural history of IVD is sophisticatedly orchestrated by a dynamic regulation of the histocytological components. NP is significantly different from AF in terms of embryonic origin and histocytological change. Part 2 Hypoxic regulation of sumoylation pathways in intervertebral disc cells: implications for Hypoxia-Inducible Factor-1α stability and hypoxic adaptations Objective:To identify the expression of sumoylation pathways within IVD. To explore the hypoxic regulation of sumoylation pathways and cell viability in NP and AF cells. Methods:Expression of SUMO molecules SUMO1 and SUMO2/3, SUMO E1 activating enzymes SAE1 and SAE2, SUMO E2 conjugating enzyme UBC9, and SUMO specific proteases (de-Sumoylation enzyme) SENP1 was immunolocalized in vivo within the IVD of neonatal and 10 weeks-old (W) rat, and in vitro in the rat IVD cells cultured in monolayers. NP and AF cells were maintained in hypoxia (1% O2) for 0-24 hours (h) and cell viability was evaluated by quantifying cell proliferation with CCK-8 analysis, cellular senescence with SA-β-gal staining, apoptosis with Annexin-V/PI staining and flow cytometry analysis, and cell cycle distribution with PI staining and flow cytometry analysis. Hypoxic regulation of sumoylation pathways was studied by analyzing the transcription and expression of SUMO molecules and sumoylation enzymes with Realtime-PCR and Western-blot. SENP-1 specific interfering RNA was used to silence the expression of SENP1 in NP cells. The transcription and stabilization of HIF-1α was evaluated by Realtime-PCR and Western-blot in the NP cells under hypoxia, while the transactivation of HIF-1α was evaluated by analyzing the expression of its downstream targets VEGF, Glut-1, and PDK1. SA-β-gal staining was used to quantify the hypoxia-induced senescence change, while Hoechst33342/PI staining and flow cytometry analysis were performed to evaluate the apoptosis in the NP cells with lowered SENP1 expression. Results:SUMO1, SUMO2/3, SAE1, SAE2, UBC9, and SENP1 were expressed in the NP, AF, and CEP of neonatal and 10W rat IVD. On immunocytofluocent staining, sumoylation pathways were also expressed in the cultured IVD cells and localized predominantly in nuclei, with SUMO-1 expressed exclusively in the nuclei. On western blotting, free SUMO1 (~12kD) was detected only in the NP cells cultured under hypoxia for 4h, whereas conjugated SUMO1 (~80kD) was promoted by hypoxia in both NP and AF cells. By contrast, free SUMO2/3 (~11kD) was detectable under both normoxia and hypoxia. RanGAP1 was detected in IVD cells at ~80kD under both normoxia and hypoxia. In NP cells, hypoxia for 4h significantly increased the transcription of SUMO1, SUMO2, and SUMO3, which returned to the normoxic level by 12h. In AF cells, the mRNA level of SUMO-1 was increased in hypoxia at 12h, while the transcription of SUMO2 and SUMO3 was decreased in hypoxic cultures. When NP cells were maintained in hypoxia for 4-12h, the transcription of SAE1 was decreased, followed by an increase to the nomoxic level by 24h; the mRNA expression of SAE2 and UBC9 was increased after hypoxic cultures for 4 h, followed by a return to the normoxic level by 12h. In contrast, AF cells reduced the transcription of SAE1, SAE2, and UBC9 after hypoxic cultures for 12-24h. In both NP and AF cells, the transcription and expression of SENP1 showed steady increase under hypoxic cultures.HIF-1α was constitutively expressed within rat NP cells under both normoxia and hypoxia. After SENP1 was silenced in NP cells, both the transcription and expression of HIF-1α were decreased under hypoxia. The expression of HIF-1α downstream targets GLUT-1, VEGF, and PDK1 was also decreased in the NP cells with lowered SENP1. CCK-8 analysis showed that oxygen deprivation had no significant effects on cell proliferation of NP and AF cells. Under hypoxia, no significant increase of SA-β-gal staining, elevation of apoptosis, or attenuation of cell cycle transition was induced in NP or AF cells. After SENP1 expression was silenced in rat NP cells, hypoxia induced no significant elevation of cellular senescence or apoptosis, although both the expression and transactivation of HIF-1α were downregulated therein. Conclusion:Sumoylation pathways are expressed in IVD cells and response to hypoxic microenvironment. NP and AF cells equally tolerate oxygen deficiency, but differently regulate the sumoylation pathways under hypoxia, with SENP1 involved in the regulation of HIF-1α expression and transactivation in NP cells. The constitutive expression and transactivation of HIF-1α is not the only factor essential for the survival of NP cells under hypoxia.
学术讨论
主办单位时间地点报告人报告主题
东南大学附属中大医院脊柱外科中心 2015-1-5 脊柱外科中心示教室 王锋 Sakai D新作讨论Exhaustion of nucleus pulposus progenitor cells with ageing and degeneration of the intervertebral disc
东南大学附属中大医院脊柱外科中心 2016-1-10 脊柱外科中心示教室 王锋 低氧诱导因子-1α(HIF-1α)的SUMO化修饰调控对椎间盘细胞低氧耐受的影响与机制研究(阶段小节)
东南大学附属中大医院脊柱外科中心 2016-3-9 脊柱外科中心示教室 王锋 胚胎早期脊柱发育过程与细节
东南大学附属中大医院脊柱外科中心 2016-11-6 脊柱外科中心示教室 王锋 斑马鱼脊索空泡化过程和信号基础
东南大学附属中大医院脊柱外科中心 2017-3-9 脊柱外科中心示教室 王锋 脊索细胞空泡形成、功能、消退的信号基础与功能
东南大学附属中大医院脊柱外科中心 2017-5-6 脊柱外科中心示教室 王锋 HIF的SUMO化修饰相关论文总结
东南大学附属中大医院脊柱外科中心 2017-2-21 脊柱外科中心示教室 王锋 Risbud课题组关于高渗感应信号TonEBP的研究进展
东南大学附属中大医院脊柱外科中心 2017-3-28 脊柱外科中心示教室 王锋 椎间盘内HIF表达、功能及调控机制讨论
     
学术会议
会议名称时间地点本人报告本人报告题目
2016 International Symposium on Life Science & Biological Engineering 2016-8-25~27 Tokyo, Japan The nature history of intervertebral disc: A histocytological and radiological observation from embryonic formation to age-related degeneration
中华医学会第18届骨科学术会议暨第11届COA国际学术会议 2016年11月17-20日 北京,中国 大鼠椎间盘自然史的组织学与影像学观察:从胚胎发育到自然退变的演变细节
     
代表作
论文名称
Hypoxia regulates sumoylation pathways in intervertebral disc cells: implications for hypoxic adapta
 
答辩委员会组成信息
姓名职称导师类别工作单位是否主席备注
蒋青 正高 教授 博导 南京大学附属南京鼓楼医院
殷国勇 正高 教授 博导 南京医科大学附属江苏省人民医院
沈孝兵 正高 教授 博导 东南大学公共卫生学院
陈陆馗 正高 教授 博导 东南大学附属中大医院
王运涛 正高 教授 博导 东南大学附属中大医院
      
答辩秘书信息
姓名职称工作单位备注
鲍军平 其他 主治医师 东南大学附属中大医院