目的 研究远隔缺血预适应(RIPC)血清外泌体对SH-SY5Y细胞基因表达的影响,探讨差异表达基因对神经细胞在糖氧剥夺(OGD)耐受中的作用.方法 RIPC前后分离人血清外泌体,取RIPC前外泌体(HuE-C)和RIPC后外泌体(HuE-RIPC).将SH-SY5Y细胞分为空白组(正常培养细胞),对照组(培养液加入HuE-C)和RIPC组(培养液加入HuE-RIPC),采用高通量测序各组细胞基因表达并进行生物信息学分析,将3组细胞置于OGD条件下,检测细胞活力,采用实时定量PCR及Western blot检测与神经低氧/缺血相关的基因表达.结果 生物信息学显示,RIPC组热休克蛋白(HSP)基因差异表达增加.MTS检测发现,OGD条件下,RIPC组细胞活力明显高于空白组(0.673±0.056 vs 0.481±0.027,P＜0.01).实时定量PCR和Western blot检测显示,OGD条件下,RIPC组HSP70 mRNA表达和蛋白表达明显高于空白组和对照组(P＜0.05).结论 人RIPC血清外泌体可引起众多基因表达变化,其中HSP70表达增加可能是其低氧耐受增加的一个原因.

目的 研究人远隔缺血预适应(RIPC)后产生的血清外泌体对人神经母细胞瘤细胞系(SH-SY5Y神经细胞)DNA甲基化及氧糖剥夺(OGD)耐受的影响.方法 6名男性在校大学生志愿者(身高:170～180 cm;体质量:60～80 kg;状态:上午、空腹)使用RIPC训练仪进行缺血预适应并采集肘中静脉血和血清,使用透射电镜方法对外泌体形态进行检测和鉴定.构建神经细胞OGD模型,当正常细胞培养至70％细胞密度时,撤去1640培养基改用无糖培养基,将OGD处理的SH-SY5Y细胞与外泌体孵育后进行处理,分为正常对照组(C组)、氧糖剥夺组(OGD组)、OGD+HuE-C组(SH-SY5 Y细胞经过OGD处理后与正常人血清外泌体共孵育)、OGD+HuE-RIPC组(SH-SY5 Y细胞经过OGD处理后和RIPC人血清外泌体共孵育).3-(4,5-二甲基噻唑-2-基)-5-(3-羧基喹啉)-2-(4-磺基苯基)-2 H-四唑(MTS)法检测细胞对OGD的耐受,亚硫酸氢钠限制性内切酶试验(COBRA)分析神经细胞基因组甲基化程度,实时聚合酶链反应(PCR)和Western Blot分析血管内皮生长因子(VEGF)和促红细胞生成素(EPO)的基因及蛋白表达水平.结果 透射电镜结果显示,超速离心法可以成功分离血清外泌体.显微镜下观察正常SH-SY5Y细胞胞体丰满,经历OGD后,大量细胞呈皱缩高亮的凋亡状态;加入HuE-C外泌体后细胞形态恢复有限;加入HuE-RIPC外泌体后,SH-SY5Y细胞形态有了较为明显的恢复.形态学分析结果显示,RIPC外泌体对缺血状态下神经细胞有保护作用.MTS结果显示,在OGD模型中,将人RIPC血清外泌体与SH-SY5Y细胞共孵育后,SH-SY5Y细胞活力较OGD组明显提高(P<0.01).COBRA实验结果显示,在SH-SY5Y细胞OGD模型中加入人RIPC血清外泌体后,SH-SY5Y细胞Alu甲基化程度下降(P=0.048),LINE-1非甲基化水平升高(P=0.040),LINE-1甲基化水平下降(P=0.004),细胞基因组甲基化呈显著下调趋势.实时PCR和Western Blot检测结果显示,加入RIPC血清外泌体后,OGD模型中,SH-SY5Y细胞中EPO、VEGF mRNA的基因表达水平明显上调(P=0.001、0.004)和EPO、VEGF蛋白表达水平明显升高(均P=0.008).结论 RIPC血清外泌体可对OGD下的神经细胞产生保护作用,可能是通过降低SH-SY5Y神经细胞基因组甲基化程度并上调VEGF、EPO等缺血耐受相关基因的表达,促进神经细胞缺血耐受.

缺血性卒中会对大脑神经细胞产生损伤,远隔缺血预适应(remote ischemic preconditioning,RIP)作为一种内源性的保护机制,通过远端器官或组织缺血/再灌注而产生的保护性物质经体液运输作用于神经中枢,发挥对神经细胞的保护作用,有望被应用于卒中的临床治疗.越来越多的研究发现,RIP过程中分泌的外泌体极可能是其产生的保护性物质,本文通过对外泌体在RIP中对神经细胞的保护作用进行综述,为探索远隔缺血预适应对神经元的保护机制提供新思路.

Ischemic tolerance in the brain can be induced by transient limb ischemia, and this phenomenon is termed remote ischemic preconditioning (RIPC). It still remains elusive how this transfer of tolerance occurs. Exosomes can cross the blood-brain barrier, and some molecules may transfer neuroprotective signals from the periphery to the brain. Serum miRNA-126 is associated with ischemic stroke, and exosomal miRNA-126 has shown protective effects against acute myocardial infarction. Therefore, this study aims to explore whether exosomal miRNA-126 from RIPC serum can play a similar neuroprotective role. Exosomes were isolated from the venous serum of four healthy young male subjects, both before and after RIPC. Exosomal miRNA-126 was measured by real-time PCR. The miRNA-126 target sequence was predicted by bioinformatics software. SH-SY5Y neuronal cells were incubated with exosomes, and the cell cycle was analyzed by flow cytometry. The expression and activity of DNA methyltransferase (DNMT) 3B, a potential target gene of miRNA-126, were examined in SH-SY5Y cells. The cell viability of SH-SY5Y cells exposed to oxygen-glucose deprivation (OGD) was also investigated. To confirm the association between miRNA-126 and DNMT3B, we overexpressed miRNA-126 in SH-SY5Y cells using lentiviral transfection. miRNA-126 expression was upregulated in RIPC exosomes, and bioinformatics prediction showed that miRNA-126 could bind with DNMT3B. DNMT levels and DNMT3B activity were downregulated in SH-SY5Y cells incubated with RIPC exosomes. After overexpression of miRNA-126 in SH-SY5Y cells, global methylation levels and DNMT3B gene expression were downregulated in these cells, consistent with the bioinformatics predictions. RIPC exosomes can affect the cell cycle and increase OGD tolerance in SH-SY5Y cells. RIPC seems to have neuroprotective effects by downregulating the expression of DNMTs in neural cells through the upregulation of serum exosomal miRNA-126.

Hypoxic preconditioning has been shown to improve hypoxic tolerance in mice, accompanied by the downregulation of DNA methyltransferases (DNMTs) in the brain. However, the roles played by DNMTs in the multiple neuroprotective mechanisms associated with hypoxic preconditioning remain poorly understood. This study aimed to establish an in vitro model of hypoxic preconditioning, using a cultured mouse hippocampal neuronal cell line (HT22 cells), to examine the effects of DNMTs on the endogenous neuroprotective mechanisms that occur during hypoxic preconditioning. HT22 cells were divided into a control group, which received no exposure to hypoxia, a hypoxia group, which was exposed to hypoxia once, and a hypoxic preconditioning group, which was exposed to four cycles of hypoxia. To test the ability of hypoxic preadaptation to induce hypoxic tolerance, cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. Cell viability improved in the hypoxic preconditioning group compared with that in the hypoxia group. The effects of hypoxic preconditioning on the cell cycle and apoptosis in HT22 cells were examined by western blot assay and flow cytometry. Compared with the hypoxia group, the expression levels of caspase-3 and spectrin, which are markers of early apoptosis and S-phase arrest, respectively, noticeably reduced in the hypoxic preconditioning group. Finally, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and western blot assay were used to investigate the changes in DNMT expression and activity during hypoxic preconditioning. The results showed that compared with the control group, hypoxic preconditioning downregulated the expression levels of DNMT3A and DNMT3B mRNA and protein in HT22 cells and decreased the activities of total DNMTs and DNMT3B. In conclusion, hypoxic preconditioning may exert anti-hypoxic neuroprotective effects, maintaining HT22 cell viability and inhibiting cell apoptosis. These neuroprotective mechanisms may be associated with the inhibition of DNMT3A and DNMT3B.

自噬是真核生物的一种自我保护机制,通过降解细胞内错误折叠的蛋白质以及细胞器,使细胞能够在应激状态下处于能量稳态和物质循环稳态.低氧/缺血预适应(H/IPC)是一种细胞内源性保护机制,其本质是低代谢/低能耗,使机体在低氧/缺血环境下维持相对稳定.本文就自噬在H/IPC神经保护中的关键分子及通路进行综述,为研究低氧/缺血神经保护提供思路.

为了探讨小分子非编码RNA-126-3p在低氧预适应小鼠血液外泌体中的表达变化.小鼠随机分为常氧组(对照组)和低氧预适应组(实验组),提取血液外泌体总RNA进行高通量测序,选取差异表达miRNA,运用生物信息学方法分析生物学过程、分子功能、生物途径、信号通路及其直接作用的靶基因预测和分析.采用实时荧光定量PCR (Real time-PCR)技术检测miR-126-3p和直接作用靶基因的表达水平变化.根据高通量测序结果筛选出差异表达miR-126-3p作为候选基因,实时荧光定量PCR结果显示,外泌体miR-126-3p表达趋势与测序结果一致,相对常氧组,低氧预适应组中的表达量下降,筛选出低氧神经保护相关靶基因(HIF1α)并检测不同处理组中相对表达量呈下降趋势.低氧预适应下调miR-126-3p的表达,正调控靶基因HIF1α的表达.

目的:研究高浓度葡萄糖促进神经细胞低氧耐受的机制.方法:在小鼠海马神经元细胞系(HT22细胞)低氧模型中加入不同浓度的葡萄糖,使用MTS检测神经细胞活力,Western Blot检测Hsp70和P53蛋白表达水平.结果:加入20 mmol/L和30 mmol/L的葡萄糖可以明显提升低氧状态下HT22细胞的细胞活力,同时Hsp70蛋白表达上调,P53蛋白表达下调(P<0.05).结论:高浓度的葡萄糖可以促进HT22细胞的低氧耐受,其保护机制可能是高浓度葡萄糖引起Hsp70表达上调从而抑制P53表达.

目的 探讨1,10-邻二氮杂菲双过氧钒酸钾〔BPV(phen)〕是否通过调节DNA甲基转移酶(DNMT)的表达,调控细胞周期相关基因表达,进而影响细胞周期.方法 BPV(phen)0.3和3.0μmol·L-1处理HT22细胞24 h,MTS法检测细胞存活;流式细胞术方法检测细胞周期;ELISA法检测DNMT活性;实时荧光定量PCR检测p21,DNMT1,DNMT3A和DNMT3B mRNA表达水平;Western印迹法分别检测相应蛋白表达水平.结果 与DMSO对照组相比,BPV(phen)0.3μmol·L-1对细胞存活率无显著影响,BPV(phen)3.0μmol·L-1组细胞存活率显著降低(P<0.05).细胞周期结果显示,与DMSO对照组相比,BPV(phen)0.3μmol·L-1组各细胞周期百分比无显著差异,BPV(phen)3.0μmol·L-1组S期细胞显著增加,为(76.1±1.6)％(P<0.05),G2期细胞显著降低(P<0.05),为(2.1±1.5)％.与DMSO对照组相比,BPV(phen)3.0μmol·L-1组细胞DNMT活性显著增加(P<0.05).实时荧光定量PCR结果显示,与DMSO对照组相比,BPV(phen)0.3μmol·L-1组p21,DNMT1,DNMT3A和DNMT3B mRNA表达水平无显著差异,BPV(phen)3.0μmol·L-1组各基因表达水平均显著增加(P<0.05,P<0.01).Western印迹结果显示,与DMSO对照组相比,BPV(phen)0.3μmol·L-1组各蛋白表达水平均无显著性差异,只有BPV(phen)3.0μmol·L-1组DNMT3B和P21蛋白表达显著增加(P<0.05).结论 BPV能够通过改变DNMT的表达,调节下游与细胞周期相关基因的表达,进而影响HT22细胞的生长和增殖.

Bisperoxo (1,10-phenanthroline) oxovanadate (BpV) can reportedly block the cell cycle. The present study examined whether BpV alters gene expression by affecting DNA methyltransferases (DNMTs), which would impact the cell cycle. Immortalized mouse hippocampal neuronal precursor cells (HT22) were treated with 0.3 or 3 μM BpV. Proliferation, morphology, and viability of HT22 cells were detected with an IncuCyte real-time video imaging system or inverted microscope and 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium, respectively. mRNA and protein expression of DNMTs and p21 in HT22 cells was detected by real-time polymerase chain reaction and immunoblotting, respectively. In addition, DNMT activity was measured with an enzyme-linked immunosorbent assay. Effects of BpV on the cell cycle were analyzed using flow cytometry. Results demonstrated that treatment with 0.3 μM BpV did not affect cell proliferation, morphology, or viability; however, treatment with 3 μM BpV decreased cell viability, increased expression of both DNMT3B mRNA and protein, and inhibited the proliferation of HT22 cells; and 3 μM BpV also blocked the cell cycle and increased expression of the regulatory factor p21 by increasing DNMT expression in mouse hippocampal neurons.

低氧/缺血会对神经系统产生损伤,减轻低氧/缺血状态对脑神经损伤的至关重要的一个方面就是抑制氧自由基和炎性因子的产生,这也是目前治疗的一个重要策略.低氧/缺血预适应可以通过抑制氧自由基和炎性因子来保护神经细胞.低氧/缺血预适应刺激的外泌体的转运可能是保护作用机制之一,本文通过对外泌体在低氧/缺血状态下发挥的神经保护作用和作用机制等方面进行综述,为低氧/缺血脑神经保护的治疗研究提供新思路.

Epigenetic processes such as DNA methylation are essential for processes of gene expression in normal mammalian development. DNA methyltransferases (DNMT) are responsible for initiating and maintaining DNA methylation. It is known that 5-Aza-CdR, an inhibitor of DNMT induces cytotoxicity by reducing DNMT activity in various tumor cell lines. However, disturbances in neuronal DNA methylation may also play a role in altered brain functions. Thus, it was of interest to determine whether alterations in DNA methylation might be associated with neuronal functions by using 5-Aza-CdR, on mouse hippocampus-derived neuronal HT22 cell line. In particular, the aim of this study was to investigate the effects of 5-Aza-CdR on cell growth inhibition, cell cycle arrest, apoptosis as well as the expression levels of DNMT in HT22 cells. HT22 cells were incubated with 5 or 20 ae mol/L 5-Aza-CdR for 24 h. Data showed that 5-Aza-CdR at both concentrations significantly inhibited proliferation of HT22 cells and exacerbated cytoplasmic vacuolization. Flow cytometry analysis demonstrated that 5-Aza-CdR treatment at both concentrations decreased early apoptosis but enhanced late apoptosis. Cell cycle analysis illustrated that 5-Aza-CdR treatment induced S phase arrest. Further, incubation with 5-Aza-CdR produced a down-regulation in expression of mRNA and protein DNMT1 and 3A but no marked changes were noted in DNMT 3B and p21 expression. In addition, DNMT1 activity was significantly decreased at both 5-Aza-CdR concentrations. Evidence indicates that 5-Aza-CdR induced cytotoxicity was associated with altered mRNA and protein expression of DNMT 1 and 3A associated with reduced DNMT1 activity in HT22 cells which might affect brain functions.

Purpose The aim of this study was to detect the expressions of DNA methyhransferase 3B isoforms and global methylation level in clear cell renal cell carcinoma (ccRCC) and to discuss the relationship between the changes of them and the ccRCC.Methods Fifteen pairs of tissue samples,which included ccRCC tissue and adjacent matched normal tissue from each patient,were used in this study.Real-time PCR was used to measure the mRNA expression levels of DNMT1,DNMT3A,total DNMT3B,and six kinds of DNMT3B variants.Western blot was used to detect the protein level of DNMT3B4.The repetitive sequences DNA methylation level of Alu and long interspersed nuclear elements (LINE-1) were measured by combine bisulfite restriction analysis (COBRA).Results Both mRNA and protein levels of DNMT3B4 were increased in ccRCC tissue compared with control tissue.Additionally,Alu elements and LINE-1 were hypomethylated in renal cell carcinoma tissue.Conclusion Overexpression of DNMT3B4 may play an important role in human kidney tumorigenesis through the decrease of global methylation.