全基因组表达谱芯片在新疆维汉族胶质母细胞瘤患者中的应用研究

目的：运用基因表达谱芯片,筛选有意义的新疆维、汉族患者胶质母细胞瘤的差异表达基因,从而探讨差异基因在新疆维、汉族患者胶质母细胞瘤发病机制及预后转归中的作用及临床意义。方法运用全基因组表达谱芯片检测3例维族患者胶质母细胞瘤、3例汉族患者胶质母细胞瘤的基因表达情况,并通过GenomeStudio软件,R语言lumi包进行标准化处理筛选差异表达基因（P＜0.05）;运用Web Gestalt软件,对差异基因做GO生物信息学分析（P＜0.05）。结果维、汉族患者胶质母细胞瘤全基因组表达谱的比较分析发现,筛选出显著差异表达的基因共1475个,上调669个,下调807个（其中1个基因（STRC）对应2个转录本,1个转录本表达上调1个转录本表达下调）,差异基因中通过GO分析有1175个关键基因分布在小G T P酶调节信号通路、Ras信号通路、神经元反应蛋白调节、中枢神经系统髓鞘形成等多个生物学过程的功能节点中。结论通过对维、汉族患者胶质母细胞瘤基因表达谱的研究,筛选出差异表达的相关基因,并对其功能及定位信息进一步分析,从多基因相互作用的角度探究胶质母细胞瘤发生发展分子机制及维、汉族胶质母细胞瘤的发病机制差异性。     

大鼠前脑缺血再灌注后GFAP、S-100表达的变化

目的 探讨胶质纤维酸性蛋白（GFAP）和S-100蛋白在大鼠前脑缺血再灌注后反应性星形胶质细胞的活化情况.方法 利用免疫组织化学方法检测前脑缺血再灌注模型的细胞活化情况.结果 脑缺血再灌注后第1d,顶叶皮层和海马可见少量GFAP阳性细胞表达 脑缺血再灌注第3d及第5d后GFAP阳性表达明显增加,并与对照组比较有统计学意义（P＜0.01）.S-100蛋白在脑缺血再灌注后第1d即有增加,并随着时间延长表达明显增强,各时间点与对照组有显著性差异（P＜0.01）.结论 脑缺血再灌注后GFAP、S-100蛋白表达增加,说明反应性星形胶质细胞的活化参与了脑缺血损伤后神经元的修复过程.     

cDNA芯片和Oligo芯片结合Meta分析筛选高低级别星形胶质细胞瘤特异性靶标研究

目的 筛选高级别与低级别星形胶质细胞瘤之间的特异性差异基因,并结合Meta分析法对不同样本在两种不同芯片和不同平台研究的结果进行综合.方法 (1)采用eDNA芯片建立33张星形胶质细胞瘤基因表达谱,Oligo芯片建立17张星形胶质细胞瘤表达谱芯片,筛选低级别(Ⅰ、Ⅱ级)和高级别(Ⅲ、Ⅳ级)星形胶质细胞瘤间的差异基因,并对其生物信息进行分析;(2)利用挖掘出的基因数据采用Fisher判别、支持向量机法和贝叶斯混合协变量分析法对高、低级别胶质瘤进行判别检验;(3)对两种不同芯片得出的数据采用Meta分析法进行综合分析.结果 cDNA表达谱芯片星形胶质瘤Ⅰ、Ⅱ级与Ⅲ、Ⅳ级间的分型基因148条;Oligo芯片数据找到高、低级别之间判别基因46条,对胶质瘤样本可达到85%以上预测率;生物信息分析发现靶基因与众多生物功能相关,对两种芯片的表达谱数据的Meta综合分析结果发现一些功能还尚不清楚的差异基因,值得深入研究.结论 不同样本在不同平台、不同芯片中的数据不尽相同,Meta分析能较好地减少芯片数据的误差,筛选出的基因可作为进一步研究的靶基因.     

利用基因芯片研究与胶质母细胞瘤侵袭性相关的基因

目的探讨利用基因表达谱芯片筛选人脑胶质母细胞瘤与侵袭性相关基因的表达及功能。方法用含13 939种人类基因的BioStarH140S型芯片,以成人脑及6例胶质母细胞瘤组织总RNA制备的探针杂交芯片;ScanArray4 000扫描芯片荧光信号,提取脑及胶质母细胞瘤组织差异基因,并进行生物信息分析及功能研究。结果表达谱芯片筛选出胶质母细胞瘤差异基因198条(1.42％),与细胞信号和传递蛋白、细胞骨架、代谢、蛋白翻译合成、细胞周期蛋白类、癌基因和抑癌基因等多类基因密切相关;与侵袭性相关的8条细胞骨架和细胞外基质基因表达谱相似,均在胶质母细胞瘤中显著上调,生物信息分析为α-连环素基因、钙粘附素1基因、层粘连蛋白、纤连蛋白1基因、基质金属蛋白酶2、Ⅲ型胶原基因、组织金属蛋白酶抑制1基因和血小板衍生生长因子受体A基因。结论表达谱芯片是高通量筛选胶质瘤相关基因的生物高新技术,侵袭性相关基因为判断胶质母细胞瘤患者的预后提供了分子生物学指标,有助于临床诊治。     

槲皮素对缺血缺氧损伤星形胶质细胞基因表达的影响

目的 应用高密度寡核苷酸(Oligo)基因芯片技术研究槲皮素对缺血缺氧损伤的星形胶质细胞基因表达的影响.方法 体外原代培养星形胶质细胞分为缺血缺氧组和缺血缺氧+槲皮素处理组.2组细胞厌氧培养4h后缺血缺氧+槲皮素处理组加入含50 μmol/L槲皮素的培养液,缺血缺氧组加入等量培养液,培养24 h后应用基因表达谱芯片筛选2组细胞表达差异的基因并用实时荧光定量PCR检测进行验证.结果 基因表达谱芯片分析显示缺血缺氧+槲皮素处理组与缺血缺氧组比较表达差异的基因共180个,其中上调基因49个,下调基因131个;实时荧光定量PCR结果 显示缺血缺氧+槲皮素处理组细胞与缺血缺氧组比较148个基因的表达发生变化,差异均有统计学意义(P＜0.05),其中上调基因34个,下调基因114个.实时荧光定量PCR与基因表达谱芯片结果 的符合率为82.2％(148/180).结论 基因表达谱芯片分析有助于从分子水平全面了解槲皮素对缺血缺氧的星形胶质细胞的作用机制,也为进一步研究槲皮素和星形胶质细胞在缺血缺氧脑损伤中的作用奠定了基础.     

远隔缺血后适应通过调节反应性星形胶质细胞的可塑性改善小鼠缺血后脑组织的恢复

目的星形胶质细胞的可塑性改变可以在脑缺血急性期引起脑水肿,在缺血恢复期改变胶质瘢痕的形成。本实验研究远隔缺血后适应(RIPC)在脑缺血再灌注损伤中对星形胶质细胞可塑性调节。方法脑缺血被诱导通过C57小鼠大脑中动脉短暂性的阻断1 h,在再灌注即刻给予RIPC。结果 RIPC能降低小鼠脑缺血再灌注3 d、14 d大脑半球的水肿、梗死面积,减少脑萎缩,提高神经功能恢复和生存率。而且RIPC能调节星形胶质细胞亚型的比例,在小鼠脑缺血再灌注3 d和14 d,RIPC能降低缺血侧纤维型星形胶质细胞(GFAP)及增加原浆型星形胶质细胞(GS)的表达,并且能够下调GFAPα的水平和上调GFAPδ/GFAPα的比例来调节GFAP的亚型。结论RIPC治疗能调节反应性星形胶质细胞的可塑性,提高缺血后神经功能的恢复。     

远隔缺血预处理诱导大鼠缺血脑保护的基因表达谱分析

目的检测远隔缺血预处理对大鼠缺血半暗带脑组织基因表达的影响,探讨远隔缺血预处理诱导的内源性脑保护机制。方法雄性SD大鼠24只随机分为常规缺血组(n=12)和远隔预处理组(n=12)。常规缺血组应用远端大脑中动脉阻塞术(dMCAO)建立局灶性脑缺血模型;远隔预处理组给予左侧股动脉15 min夹闭/15 min再通3个循环缺血预处理后,再予以dMCAO。两组大鼠分别在缺血1、3、6和24 h后取脑,采用Agilent大鼠全基因组芯片检测两组脑缺血半暗带区基因表达的变化,获取差异表达基因,并对差异基因进行GO分析和Pathway功能注释(KEGG数据库)。结果远隔缺血预处理后:①缺血半暗带区脑组织基因出现差异表达(P0.05且差异倍数2),随着缺血时间延长差异基因个数逐渐增加,在缺血6 h达高峰;②GO分析结果提示差异基因涉及刺激反应、生长、生物调节、细胞杀伤等多个生物过程;③对差异基因进行KEGG通路分析,结果提示Jak-STAT信号通路、p53信号通路、丝裂原蛋白激酶(MAPK)信号通路、谷氨酸突触信号通路、血管内皮生长因子(VEGF)信号通路等多条分子通路出现差异调节,差异调节通路的数目也在缺血6 h达高峰。结论远隔缺血预处理诱导半暗带基因差异表达,差异基因通过多条分子通路影响多个生物过程。基因表达谱芯片为远隔缺血预处理脑保护机制提供了全面的数据和信息,对各个靶点、各条通路的进一步深入研究具有全面、广泛的指导意义。     

胶质母细胞瘤放射治疗前后免疫相关基因研究

目的研究人脑胶质母细胞瘤组织放射治疗后基因表达谱的变化。方法采用BioStarH-141s（2004）型含13929条人类全长基因的cDNA表达谱芯片,对两例胶质母细胞瘤组织在直线加速器（60Gy）放射治疗前后相关基因表达情况进行检测,并分析它们之间的基因表达差异。结果胶质母细胞瘤放射治疗后与放射治疗前比较,组织表达差异基因共17个,其中上调10个,下调7个。表达差异基因中改变最明显的功能群是免疫系统相关的基因,如SPARC、ID3、HLA-DQA1和HLA-DOA等上调。细胞增殖、细胞调亡、细胞周期、DNA修复系统也有部分基因发生明显变化,如MLL5上调和POLR2B下调等。结论对胶质母细胞瘤组织经直线加速器照射（60Gy）后基因表达谱改变的研究可以更好地阐明其放射敏感性差异机制,为放射治疗前或放射治疗早期寻找预测肿瘤放射敏感性分子标志物提供理论依据。     

Dex基于CREB/PGC-1α信号通路调控星形胶质细胞线粒体功能在大鼠脑缺血再灌注损伤中的作用

目的 探究右美托咪定(Dex)基于CREB/PGC-1 α信号通路调控星形胶质细胞线粒体功能在大鼠脑缺血再灌注损伤中的作用。方法 采用改良的Zen-land法制作大鼠脑缺血再灌注损伤(middle cerebral artery occlusion,MCAO)模型,分别在造模后评估假手术(Sham)组、脑缺血再灌注模型(MCAO)组,脑缺血再灌注加药(MCAO+Dex)组大鼠神经功能损伤,脑梗死面积,实时荧光定量PCR(qPCR)和蛋白印迹检测环磷腺苷效应元件结合蛋白(CREB)、过氧化物酶体增殖物激活受体-γ共激活因子-1α(PGC-1α)的mRNA及蛋白表达;透射电子显微镜检测线粒体形态,免疫荧光法检测星形胶质细胞活性氧物质(ROS)及细胞凋亡。制备氧糖剥夺/复氧(OGD/R)星形胶质细胞模型,分为对照组(Control)、溶剂组(OGD/R-Dex0)、低剂量组(OGD/R-Dex-L)、高剂量组(OGD/R-Dex-H),qPCR检测CREB、PGC-lα的mRNA水平,免疫荧光和WB检测CREB、PGC-1α的蛋白表达。在OGD/R星形胶质细胞模型Dex处理的基础上过表达CR...     

低分子肝素对大鼠脑缺血后活化的小胶质细胞和巨噬细胞及TNF-α仅表达的影响

目的 研究低分子肝素（LMWH）对大鼠局灶性脑缺血再灌注后活化的小胶质细胞和巨噬细胞及TNF-α表达的影响。方法 建立大鼠大脑中动脉缺血再灌注模型，并将大鼠随机分为假手术组、对照组、LMWH治疗组，对照组及LMWH治疗组分别于脑缺血2 h再灌注3、24、48、72 h处死；应用免疫荧光染色检测脑组织中活化的小胶质细胞和巨噬细胞数量变化，应用免疫组化染色观察TNF-α阳性细胞。结果 治疗组与对照组比较24、48、72 h组梗死灶周围活化的小胶质细胞和巨噬细胞及TNF-α 阳性细胞明显减少（P＜0．05）。结论 LMWH对大鼠脑缺血再灌注后梗塞灶周围活化的小胶质细胞和巨噬细胞及TNF-α表达有明显抑制作用。     

MicroRNAs: a novel promising therapeutic target for cerebral ischemia/reperfusion injury?

To determine the molecular mechanism of cerebral ischemia/reperfusion injury, we examined the microRNA (miRNA) expression profile in rat cortex after focal cerebral ischemia/reperfusion injury using miRNA microarrays and bioinformatic tools to systematically analyze Gene Ontology (GO) function classifications, as well as the signaling pathways of genes targeted by these differentially expressed miRNAs. Our results show significantly changed miRNA expression profiles in the reperfusion period after focal cerebral ischemia, with a total of 15 miRNAs up-regulated and 44 miRNAs down-regulated. Target genes of these differentially expressed miRNAs were mainly involved in metabolic and cellular processes, which were identified as hub nodes of a miRNA-GO-network. The most correlated pathways included D-glutamine and D-glutamate metabolism, the renin-angiotensin system, peroxisomes, the PPAR signaling pathway, SNARE interactions in vesicular transport, and the calcium signaling pathway. Our study suggests that miRNAs play an important role in the pathological process of cerebral ischemia/reperfusion injury. Understanding miRNA expression and function may shed light on the molecular mechanism of cerebral ischemia/reperfusion injury.     

Mechanism underlying treatment of ischemic stroke using acupuncture:transmission and regulation

The inflammatory response after cerebral ischemia/reperfusion is an important cause of neurological damage and repair. After cerebral ischemia/reperfusion, microglia are activated, and a large number of circulating inflammatory cells infiltrate the affected area. This leads to the secretion of inflammatory mediators and an inflammatory cascade that eventually causes secondary brain damage, including neuron necrosis, blood-brain barrier destruction, cerebral edema, and an oxidative stress response. Activation of inflammatory signaling pathways plays a key role in the pathological process of ischemic stroke. Increasing evidence suggests that acupuncture can reduce the inflammatory response after cerebral ischemia/reperfusion and promote repair of the injured nervous system. Acupuncture can not only inhibit the activation and infiltration of inflammatory cells, but can also regulate the expression of inflammation-related cytokines, balance the effects of pro-inflammatory and anti-inflammatory factors, and interfere with inflammatory signaling pathways. Therefore, it is important to study the transmission and regulatory mechanism of inflammatory signaling pathways after acupuncture treatment for cerebral ischemia/reperfusion injury to provide a theoretical basis for clinical treatment of this type of injury using acupuncture. Our review summarizes the overall conditions of inflammatory cells, mediators, and pathways after cerebral ischemia/reperfusion, and discusses the possible synergistic intervention of acupuncture in the inflammatory signaling pathway network to provide a foundation to explore the multiple molecular mechanisms by which acupuncture promotes nerve function restoration.     

亚低温通过抑制两种凋亡通路对大鼠脑缺血再灌注损伤发挥保护作用

目的探讨亚低温对大鼠局灶性脑缺血再灌注后胱冬酶(caspase)依赖性及非依赖性两种凋亡通路的影响。方法线栓法建立大鼠大脑中动脉阻塞(M CAO)及再通模型,分为假手术组、常温及亚低温脑缺血再灌注组,应用RT-PCR技术检测再灌注后不同时相缺血侧皮层凋亡诱导因子(A IF)及caspase-3 mRNA的表达。结果脑缺血2h再灌注2~4h,A IF及caspase-3 mRNA表达开始增加,随着再灌注时间的延长表达逐渐增强,至再灌注24h达高峰。每一再灌注时间点亚低温组与常温组A IF及caspase-3 mRNA表达均有显著差异,亚低温组mRNA表达均低于相应常温组。结论亚低温不仅降低caspase依赖性通路中的关键蛋白酶—caspase-3的mRNA的表达,而且降低caspase非依赖性通路中的关键蛋白—A IF的mRNA的表达,亚低温通过抑制两种凋亡通路对大鼠脑缺血再灌注损伤发挥保护作用。     

Characterization of astrocytes and microglial cells in the hippocampal CA1 region after transient focal cerebral ischemia in rats treated with Ilexonin A

Ilexonin A is a compound isolated from the root of Ilex pubescens,a traditional Chinese medicine.Ilexonin A has been shown to play a neuroprotective role by regulating the activation of astrocytes and microglia in the peri-infarct area after ischemia.However,the effects of ilexonin A on astrocytes and microglia in the infarct-free region of the hippocampal CA1 region remain unclear.Focal cerebral ischemia models were established by 2-hour occlusion of the middle cerebral artery in rats.Ilexonin A(20,40 or 80 mg/kg)was administered immediately after ischemia/reperfusion.The astrocyte marker glial fibrillary acidic protein,microglia marker Iba-1,neural stem cell marker nestin and inflammation markers were detected by immunohistochemistry and western blot assay.Expression levels of tumor necrosis factor-αand interleukin 1βwere determined by enzyme linked immunosorbent assay in the hippocampal CA1 tissue.Astrocytes were activated immediately in progressively increasing numbers from 1,3,to 7 days post-ischemia/reperfusion.The number of activated astrocytes further increased in the hippocampal CA1 region after treatment with ilexonin A.Microglial cells remained quiescent after ischemia/reperfusion,but became activated after treatment with ilexonin A.Ilexonin A enhanced nestin expression and reduced the expression of tumor necrosis factor-αand interleukin 1βin the hippocampus post-ischemia/reperfusion.The results of the present study suggest that ilexonin A has a neuroprotective effect in the hippocampus after ischemia/reperfusion,probably through regulating astrocytes and microglia activation,promoting neuronal stem cell proliferation and reducing the levels of pro-inflammatory factors.This study was approved by the Animal Ethics Committee of the Fujian Medical University Union Hospital,China.     

Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes

JAK/STAT is one of the pathways bearing signals from the cell membrane to the nucleus in response to extracellular growth factors and cytokines. In the present study, we examined the cellular distribution of Jak1 and Stat3, and activation of the JAK/STAT pathway following transient focal cerebral ischemia in the rat. Jak1 was mainly seen in white matter astrocytes and in certain neurons. Notably, large pyramidal neurons of cortical layer V showed the highest neuronal Jak1 expression within cerebral cortex and, in addition, expressed Stat3 indicating that the JAK/STAT pathway is involved in signaling in the corticofugal projection system. Shortly following ischemia, Jak1 immunoreactive astrocytes located in the ipsilateral neighbouring white matter and ischemic cortex and striatum showed nuclear translocation of Stat3. These features were maintained in large reactive astrocytes that surrounded the infarct from 3 to 7 days. At these later times, the abundant reactive microglia/macrophages were strongly immunoreactive to Stat3 and, to a lesser extent, Jak1. Two main protein complexes showing DNA binding activity at the sis-inducible element site were found under basal conditions, followed by changes in this pattern following ischemia concomitant with neuronal cell loss and activation of glia. This study showed basal cerebral activity of JAK/STAT signaling pathway, involving Jak1 and Stat3 proteins, and selective activation following ischemia. It is suggested that the kinase activity of Jak1 mediates nuclear translocation of Stat3 in astrocytes, and that this signaling pathway is involved in the astroglial response to focal cerebral ischemia.     

Protein phosphatase 2A-negative regulation of the protective signaling pathway of Ca2+/CaM-dependent ERK activation in cerebral ischemia

Extracellular-signal-regulated kinase (ERK) undergoes rapid inactivation following the intense activation evoked by cerebral ischemia and reperfusion. However, the precise mechanism of this inactivation has not been elucidated. To investigate how phosphatases regulate the ERK cascade following ischemia, the PP2A inhibitors cantharidin and okadaic acid were administrated to the CA1 subregion of the rat hippocampus. The resulting sustained ERK activity implies that PP2A is a major phosphatase contributing to the rapid inactivation, but not activation, of ERK following cerebral ischemia. The increase in PP2A activity induced by ceramide has a weak effect on the activation of Raf via dephosphorylation of Ser259 in response to ischemia. In contrast, ketamine (Keta) and cyclosporine A (CsA), two chemicals that block calcium signal in ischemia, decrease ERK activity by blocking Raf dephosphorylation of Ser259. We also observed that activation of an upstream protein, Ras-GRF, leads to calcium/calmodulin-dependent activation of the ERK signaling cascade in response to ischemic stimuli. In addition, the activity of cyclic AMP response element-binding protein (CREB) and estrogen receptor alpha (ER alpha), target proteins of ERK and protective elements against ischemic lesion, parallels the activity of ERK. These data indicate that PP2A plays a significant role in blocking the protective effect induced by the ERK kinase pathway and that fast inactivation of ERK is the result of cross talk between calcium/calmodulin-dependent, positively regulated signal cascades and a ceramide-dependent negative signaling pathway.     

MicroRNA-670 aggravates cerebral ischemia/reperfusion injury via the Yap pathway

Apoptosis is an important programmed cell death process involved in ischemia/reperfusion injury. MicroRNAs are considered to play an important role in the molecular mechanism underlying the regulation of cerebral ischemia and reperfusion injury. However, whether miR-670 can regulate cell growth and death in cerebral ischemia/reperfusion and the underlying mechanism are poorly understood. In this study, we established mouse models of transient middle artery occlusion and Neuro 2 a cell models of oxygen-glucose deprivation and reoxygenation to investigate the potential molecular mechanism by which miR-670 exhibits its effects during cerebral ischemia/reperfusion injury both in vitro and in vivo. Our results showed that after ischemia/reperfusion injury, miR-670 expression was obviously increased. After miR-670 expression was inhibited with an miR-670 antagomir, cerebral ischemia/reperfusion injury-induced neuronal death was obviously reduced. When miR-670 overexpression was induced by an miR-670 agomir, neuronal apoptosis was increased. In addition, we also found that miR-670 could promote Yap degradation via phosphorylation and worsen neuronal apoptosis and neurological deficits. Inhibition of miR-670 reduced neurological impairments after cerebral ischemia/reperfusion injury. These results suggest that microRNA-670 aggravates cerebral ischemia/reperfusion injury through the Yap pathway, which may be a potential target for treatment of cerebral ischemia/reperfusion injury. The present study was approved by the Institutional Animal Care and Use Committee of China Medical University on February 27, 2017(IRB No. 2017 PS035 K).