【作者】 李伟；

【导师】 杭春华；

【作者基本信息】 南京大学 ， 外科学（神经外科）， 2014， 博士

【摘要】 背景:大量研究证实无菌性炎性反应在创伤性脑损伤(traumatic brain injury, TBI)后继发性脑损伤中发挥着至关重要的作用,但其具体信号转导与调控机制目前尚不明确。我们前期临床及基础研究发现TBI后脑内核因子-κB (nuclear factor kappa B,NF-κB)炎性反应通路可被显著激活至某一高峰,然后逐渐下降。提示脑内可能存在某种重要介质或通路在TBI后被激活,从而负向调控炎性反应。因此,寻找此种可以下调TBI后脑内炎性反应的因子或因素显得愈发重要。国际最新研究表明CD24可以通过下调NF-κB的活性,从而负向调控无菌性炎性反应。TBI后脑内的炎性反应便是一种无菌性炎性反应,但关于CD24在TBI后脑内无菌性炎性反应中的作用机制至今未有报道。因此,本课题将针对TBI后脑内CD24的表达时程及其在无菌性炎性反应中的潜在作用机制进行研究。方法:本课题首先从临床研究出发,选取并收集临床TBI患者术中伤灶周围脑组织标本作为TBI组,同时收集颅底良性肿瘤手术入路上脑组织作为对照组,运用实时定量聚合酶链式反应(polymerase chain reaction, PCR)、Western blotting、免疫组织化学染色及免疫荧光技术检测CD24及炎性反应通路上关键因子髓样分化蛋白88 (myeloid differentiation factor 88, MyD88)的表达及分布情况;使用实时定量PCR对NF-κB下游炎性因子肿瘤坏死因子-α (tumor necrosis factor-α,TNF-α)及白介素-1β (interleukin-1β, IL-1β)的mRNA表达水平进行检测;同时对临床患者预后进行统计分析。随后使用C57BL/6小鼠,运用改良Feeney法建立体内TBI模型,分别采用实时定量PCR、Western blotting及免疫荧光双染方法检测CD24的表达及细胞定位情况。根据动物实验结果,采用重组高迁移率族蛋白 1 (recombinant high mobility group box 1,rHMGB1)处理的小鼠原代培养皮层神经元及星形胶质细胞建立体外神经细胞炎性反应模型,分别采用实时定量PCR、Western blotting及免疫荧光方法检测CD24的表达及分布情况。根据体内、体外实验中CD24的表达情况,分别运用RNA干扰技术针对CD24进行下调,系统评价NF-κB炎性反应通路的活性变化。采用实时定量PCR及Western blotting技术检测CD24的mRNA及蛋白表达水平变化;电泳迁移率实验(electrophoretic mobility shift assay, EMSA)检测NF-κB的DNA结合活性;实时定量PCR检测炎性因子TNF-α及IL-1β的mRNA表达水平,从而探讨CD24在TBI后脑内无菌性炎性反应中的具体作用。结果:临床实验结果表明:TBI后脑内CD24的mRNA及蛋白表达水平均较对照组明显升高,且具有时间依赖性,随致伤时间延长而不断增高(至72h后);同时CD24的表达可见于神经元、星形胶质细胞及小胶质细胞中。动物实验中,小鼠TBI后伤灶周围皮层内CD24的mRNA及蛋白水平均较假手术组明显增高,且表达高峰位于伤后7d。针对CD24予以RNA干扰后,CD24的表达水平得到显著抑制,同时NF-1κB活性及其下游炎性因子TNF-α和IL-1β的mRNA水平明显上调。体外实验中,小鼠原代皮层神经元及星形胶质细胞中施加rHMGB1后,可以显著诱导NF-κB炎性反应通路的激活,同时CD24的mRNA及蛋白水平也明显升高,并且其激活高峰位于rHMGB1施加后36h;针对CD24进行RNA干扰后,神经元及星形胶质细胞中的CD24表达水平可以显著抑制,同时能够明显上调NF-κB炎性反应通路的活性。结论:我们首先从临床研究发现TBI后脑内CD24表达水平明显增高,然后分别从体内、体外实验两方面验证此发现,并且运用RNA干扰技术发现CD24的针对性下调可以显著上调NF-κB炎性反应通路的活性。结果表明TBI后脑内CD24的高表达可能在无菌性炎性反应中发挥着至关重要的负向调控作用,高度提示CD24的靶向干预可能在TBI的临床治疗中具有潜在的应用价值。更多还原

【Abstract】 Backgroud: Increasing evidence indicates that non-infectious inflammatory response contributes to secondary brain injury following traumatic brain injury (TBI). However,the specific mechanisms of the signaling transduction and regulation remain largely unknown. We previously found that nuclear factor kappa B (NF-κB) dependent pathway could be activated to a peak in brain following TBI, and then it descended gradually. These findings suggested that some important factors or pathways could be activated to negatively regulate the non-infectious inflammatory response. Hence, it is becoming more and more important to find this kind of factor to inhibit the inflammatory response after TBI. Recently, it is reported that CD24 could down-regulate the activity of NF-κB, thus negatively regulating non-infectious inflammatory response. Moreover, the inflammatory response in brain after TBI is non-infectious. However, it remains obscure, as is whether CD24 plays a role in non-infectious inflammatory response after TBI. Hence, the aim of the current study is to explore the cerebral cortical expression pattern of CD24 and its potential role in non-infectious inflammatory response following TBI.Methods: Contused brain tissue biopsies were obtained from the pericontusinal cortex in TBI patients undergoing surgery, and samples of control group were from patients in the pathway during surgical removal of deep benign tumors. The expression and distribution of CD24 and myeloid differentiation primary response protein 88 (MyD88),a key regulator in inflammatory response,was assessed by quantitative real-time polymerase chain reaction (qPCR),Western blotting,immunohistochemistry and immunofluorescent double staining. The messenger RNA(mRNA) levels of tumor necrosis factor-a (TNF-a) and interleukin 1β (IL-1β),downstream pro-inflammatory cytokines of NF-κB dependent pathway, were measured by qPCR. Meanwhile, the outcomes of these patients were also investigated.Then we established an experimental TBI model in C57BL/6 mice using a modified Feeney’s model, and the expression and distribution of CD24 was detected by qPCR,Western blotting,and immunofluorescent double staining. According to the results in vivo, we treated mouse primary cortical neurons and astrocytes with recombinant high mobility group box 1 (rHMGB1) to simulate a neuronal inflammation model in vitro, and the expression and distribution of CD24 was measured by qPCR,Western blotting,and immunofluorescent staining. Based on the expression pattern of CD24, RNA interference was used to down-regulate the expression of CD24 both in vivo and in vitro. We detected the mRNA and protein levels of CD24 expression using qPCR and Western blotting; and NF-κB DNA-binding activity was assessed by electrophoretic mobility shift assay (EMSA);and mRNA levels of TNF-α and IL-1β were measured by qPCR. Thus, the specific effects of CD24 on inflammatory response were investigated after TBI.Results: The clinical study indicated that both the mRNA and protein levels of CD24 were markedly elevated after TBI, showing a time-dependent manner. The expression of CD24 was gradually enhanced with the extension of time after TBI, and it could be detected in neurons,astrocytes,and microglia. In animal study,we also found a higher level of CD24 mRNA and protein expression in TBI groups as compared to that in sham group, with the peak on day 7, and the elevation of CD24 could be detected in neurons, astrocytes, and microglia. Meanwhile, down-regulation of CD24 with RNA interference could induce a significant increase of NF-κB DNA-binding activity and mRNA levels of TNF-a and IL-1β. In vitro experiments, application of rHMGB1 could induce the activation of NF-κB dependent inflammatory pathway both in mouse primary cortical neurons and in astrocytes. Meanwhile, CD24 mRNA and protein expressions climbed to a peak level at 36 h after rHMGB1 addition. Similar to the animal study, NF-κB dependent inflammatory pathway could be significantly up-regulated with the RNA interference to CD24 both in neurons and in astrocytes.Conclusion: We first reported that CD24 could be markedly elevated in the brain following traumatic brain injury in patients, which was demonstrated by experiments both in vivo and in vitro. Meanwhile, down-regulation of CD24 with RNA interference could significantly up-regulate the activity of NF-κB dependent inflammatory pathway. Our findings suggested that the elevation of cortical CD24 might negatively regulate the non-infectious inflammatory response during secondary brain injury after TBI, thus providing a novel target for therapeutic intervention of TBI.更多还原