酸敏感离子通道1a介导细胞内钙离子内流调节椎间盘终板软骨细胞凋亡

目的:观察酸敏感离子通道1a(ASIC1a)介导的钙离子内流在酸诱导的终板软骨细胞凋亡的作用。方法:SD培养大鼠终板软骨细胞,慢病毒为载体转染终板软骨细胞沉默ASIC1a,钙离子成像分析细胞内Ca~(2+)浓度([Ca~(2+)]i)变化,采用四甲基偶氮唑蓝(MTT)实验分析酸诱导的细胞活力;ELISA测定凋亡率;荧光染料Hoechst 33342检测细胞凋亡。免疫印迹检测活化型半胱天冬酶-9(cleaved caspase-9)、活化型半胱天冬酶-3(cleaved caspase-3)等凋亡相关蛋白的表达。结果:阻断ASIC1a可以抑制酸诱导的[Ca~(2+)]i升高;用ASIC1a-siRNA或PcTX1特异性阻断ASIC1a,明显抑制酸诱导的终板软骨细胞凋亡;胞外钙离子浓度降低抑制酸诱导的终板软骨细胞凋亡,与阻断ASIC1a结果一致,提示ASIC1a介导的钙离子内流在酸诱导凋亡中的作用。阻断ASIC1a,抑制酸诱导的cleaved caspase-9、cleaved caspase-3蛋白的表达。结论:酸激活的ASIC1a可能通过钙超载促使大鼠椎间盘终板软骨细胞凋亡。     

大鼠破骨细胞的诱导及其酸敏感离子通道的表达

目的:检测酸敏感离子通道(ASICs)在大鼠破骨细胞中的表达情况,探讨其在破骨细胞中的作用.方法:采用1,25-(OH)2D3诱导大鼠骨髓单核细胞分化的破骨细胞.RT-PCR扩增ASICs基因;免疫印迹检测ASICs转录水平;免疫荧光细胞化学方法观察ASICs基因在破骨细胞中的表达和分布.结果:诱导5d后可见抗酒石酸盐酸性磷酸酶(TRAP)阳性多核细胞出现,大鼠破骨细胞在mRNA和蛋白水平均可检测到ASIC1、 ASIC2和ASIC3基因的表达产物.免疫荧光细胞化学显示ASIC1和ASIC2蛋白在细胞膜中有较强的表达,而ASIC3主要定位于细胞质中,少量定位于细胞膜上.结论:大鼠骨髓单核细胞可诱导分化成破骨细胞,能表达ASICs.     

AMP活化蛋白激酶在终板软骨细胞体外传代培养中的表达及意义

BACKGROUND: Endplate cartilage degeneration initiates intervertebral disc degeneration. AMP-activated protein kinase (AMPK) regulates the formation and degradation of cartilage. OBJECTIVE: To explore the role of AMPK in an in vitro natural degeneration model of chondrocytes derived from endplate of rat intervertebral discs. METHODS: Morphology of in vitro subcultured endplate chondrocytes of rat intervertebral discs at passages 0, 2, and 5 were observed under an inverted microscope following cytoskeleton staining. Chondrocyte phenotype, proliferation, and the cartilage marker genes (type II collagen, proteoglycan, SOX-9, matrix metalloproteinase-3 and -13), and AMPK phosphorylation were determined by toluidine blue staining, MTT assay, real-time PCR analysis, and western blot assay, respectively. RESULTS AND CONCLUSION: The altered morphology, decreased proliferation ability, and phenotype loss were observed in chondrocytes with increased passage number. Gene expression of type II collagen, proteoglycan, SOX-9 was significantly decreased; while gene expression of matrix metalloproteinase-3 and -13 was significantly increased in endplate chondrocytes at passage 5 compared with those at passages 0 and 2. AMPK phosphorylation in endplate chondrocytes at passage 5 was significantly decreased. These findings indicate that AMPK phosphorylation is involved in in vitro natural degeneration of chondrocytes derived from the endplate of rat intervertebral discs, and the degeneration of endplate chondrocytes and intervertebral discs can be inhibited through the regulation of AMPK activity. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

力学刺激在椎体软骨终板退变中的作用及机制

终板的重要功能是传递应力和供给营养。终板退变可能会诱发或促进椎间盘退变,引起一系列严重影响人们健康和生活质量的脊柱疾患。终板软骨细胞可以感受力学刺激。力学刺激是影响终板退变的重要因素,不适宜的力学刺激会加速终板退变。本文回顾力学刺激对终板软骨细胞凋亡、合成抑制、钙化及细胞外基质降解诸方面的影响,并总结力学刺激导致椎体终板退变的相关机制。力学刺激诱发的终板退变是由各种信号转导因子构成的复杂信号通路网络精细调节,包括NF-κB、Wnt、Hedgehog、MAPK、RhoA/Rock-1、AKT/mTOR、TGF-β、miRNA相关信号通路。同时,本文对这些通路相互联系进行梳理总结。多个信号通路可以共同作用调节终板软骨细胞代谢,并导致终板退变。本文希望通过相关机制系统回顾,给终板退变早期诊断及针对性治疗带来有益的启示。     

大鼠颈椎间盘老化及退变过程中髓核形态变化规律的实验研究

目的：研究颈椎间盘自然老化及退变过程中髓核软骨样细胞的来源和脊索性髓核向纤维软骨性髓核转化的规律及其与颈椎间盘退变的关系。方法：4周龄SD大鼠76只，随机分成两组。实验组40只大鼠通过截除前肢制备双后肢大鼠颈椎间盘退变的动物模型，按术后3、6、9、12个月4个时间段分组，每组10只；对照组36只大鼠未予处置，按实验开始后4、8、12、16个月分4组，每组9只。制备C4-5，C5-6和C6-7椎间盘中矢状面组织学切片，行HE、番红-O染色，研究观察不同老化及退变程度颈椎间盘髓核中软骨样细胞的起源和脊索性髓核向纤维软骨性髓核转化的规律。结果：随着颈椎间盘的不断老化，终板的软骨细胞向髓核迁移，脊索性髓核向心性皱缩并最终完全被纤维软骨性髓核取代，在此过程中，软骨终板的厚度逐渐变薄，进而出现缺损或断裂；在颈椎间盘退变的过程中，这一转化完成的更快、更早。结论：髓核中的软骨样细胞由终板的软骨细胞迁移而来，通过向心性的产生和沉积胶原纤维，脊索性髓核逐渐被纤维软骨性髓核替代，这一过程既是颈椎间盘成熟和老化的自然环节也可能是颈椎间盘退变的启动环节。     

动力失衡模型兔颈椎间盘病理改变及终板软骨细胞的迁移凋亡规律

背景:颈肌在维持椎体力学平衡以及颈椎病致病的每一阶段、每一环节都发挥着重要的作用,而且是临床多种颈部症状的主导病因,颈椎力学失平衡及颈椎间盘的退变可看作是以颈肌为主的软组织病变的结果。目的:观察颈前肌短缩痉挛所致颈椎动力失衡模型兔颈椎间盘终板软骨细胞的病理变化,以及异常应力下椎间盘各结构细胞的凋亡规律。方法:健康成年新西兰白兔28只,随机分成模型组(n=14)、假手术组(n=14)。模型组于甲状软骨外下方约1 cm处将双侧胸锁乳突肌用医用硅胶硬管垫起致其短缩,建立颈椎动力失衡模型;假手术组仅暴露双侧胸锁乳突肌分离其中部后,直接逐层缝合。于造模后2个月同一时间取材。颈椎间盘及终板软骨组织切片苏木精-伊红染色,光镜下观察颈椎间盘终板软骨细胞的迁移变化情况,运用TUNEL法检测颈间盘终板软骨、纤维环中细胞的凋亡情况。实验方案经北京中医药大学第三附属医院动物实验伦理委员会批准。结果与结论:(1)模型组颈椎间盘发生明显的病理学变化,而假手术组未见明显改变;模型组颈椎间盘终板关节软骨区与纤维环区界限明显,未发现明显细胞迁移现象,但椎间盘终板生长软骨区细胞已明显向关节软骨区迁移;模型组椎间盘关节软骨...     

β-连环素在人颈椎终板软骨中的表达及其意义

目的:观察β-连环素在人颈椎椎体终板软骨中的表达变化,探讨其与终板软骨退变的关系及意义.方法:选取2011年9月至2012年3月间住院接受颈前路椎体次全切手术的颈椎终板软骨标本,分为退变组(28例)和对照组(10例),运用RT-PCR和western-blot法检测终板软骨组织中β-连环素、蛋白多糖、Ⅱ型胶原及Sox9基因的表达.结果:退变组和对照组均可检测到β-连环素的表达,而退变组β-连环素表达量明显高于对照组(P＜0.01),蛋白多糖、Ⅱ型胶原及Sox9基因表达量明显低于对照组(P＜0.01).结论:人退变颈椎终板软骨组织内β-连环素表达明显升高,提示其与椎体终板软骨退变存在重要关系.     

酸敏感离子通道基因重组质粒的构建、表达及其活性测定

为了构建含有小鼠酸敏感离子通道(ASIC1a、ASIC2a)基因全长cDNA的重组质粒,并表达有生物学活性的ASIC1a和ASIC2a融合蛋白,本研究通过逆转录聚合酶链反应(RT-PCR)分别获得小鼠ASIC1a和ASIC2a全长cDNA,并将其克隆入真核表达载体pEGFP-N3中,构建含小鼠全长ASIC1a和ASIC2a基因的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,经DNA测序鉴定序列正确。脂质体法分别将其转染至中国仓鼠卵巢细胞(CHO),荧光显微镜下观察小鼠ASIC1a和ASIC2a融合蛋白在细胞内的表达分布,Westernblot检测其蛋白表达。酸性处理转染重组质粒细胞,并比较其细胞存活率及乳酸脱氢酶(LDH)的释放来评价融合蛋白的生物学活性。结果显示:本研究成功地分别将小鼠ASIC1a和ASIC2a全长cDNA克隆入pEGFP-N3载体中,并将其转染至CHO细胞,荧光显微镜下观察到CHO细胞膜周围呈强绿色荧光条带,Westernblot显示小鼠ASIC1a和ASIC2a融合蛋白分别在90kD和88kD处有表达。经酸性处理的转染ASIC1a和ASIC2a重组质粒的CHO细胞,分别较其在pH7.4条件下的细胞存活率明显降低,LDH释放量明显增高,且通道阻断剂可抑制该效应。上述结果提示,我们已成功构建出含小鼠ASIC1a和ASIC2a全长cDNA的重组质粒pEGFP-ASIC1a和pEGFP-ASIC2a,并使有生物学活性的ASIC1a和ASIC2a融合蛋白在CHO细胞中表达。     

ANK、 ENPP1及TGF-β1基因在大鼠终板软骨细胞传代培养中的表达变化及意义

目的:通过大鼠终板软骨细胞体外自然传代的退变模型,探讨传代过程中钙化相关基因ANK、ENPP1及内源性生长因子TGF-β1表达的变化及其意义.方法:取大鼠腰椎终板软骨细胞,采用酶消化法及自然传代法分离培养,选取P1、P5及P7代,均在体外培养6 d,分别用HE染色、甲苯胺蓝染色及Real-time RT-PCR法检测软骨标志性基因Ⅱ型胶原、蛋白多糖、转录因子Sox9变化,对终板软骨细胞表型进行鉴定.用Real-time RT-PCR及Western blot检测钙化相关基因ANK、ENPP1的变化,Real-time RT-PCR及ELISA检测生长因子TGF-β1的变化.结果:随着细胞传至P7代,细胞形态上有梭形变趋势.茜素红染色无明显变化.软骨标志性基因Ⅱ型胶原、蛋白多糖及转录因子Sox9表达均明显下调(P＜0.05),钙化相关基因ANK、ENPP1表达均明显下调(P＜0.05),内源性生长因子TGF-β1表达明显下调(P＜0.05).结论:终板软骨细胞传至P7代,终板软骨细胞发生退变,终板软骨细胞内未见钙盐沉积,钙化相关基因ANK、ENPP1下调可能由内源性TGF-β1下调引起,提示调节内源性TGF-β1及钙化相关基因ANK、ENPP1在终板软骨中的表达有可能会阻止椎间盘的退变.     

ANK、ENPPl及TGF-β1基因在大鼠终板软骨细胞传代培养中的表达变化及意义

目的：通过大鼠终板软骨细胞体外自然传代的退变模型,探讨传代过程中钙化相关基因ANK、ENPPl及内源性生长因子TGF—βl表达的变化及其意义。方法：取大鼠腰椎终板软骨细胞,采用酶消化法及自然传代法分离培养,选取P1、P5及P7代,均在体外培养6d,分别用HE染色、甲苯胺蓝染色及Real—timeRT—PCR法检测软骨标志性基因Ⅱ型胶原、蛋白多糖、转录因子Sox9变化,对终板软骨细胞表型进行鉴定。用Real·timeRT—PCR及Westernblot检测钙化相关基因ANK、ENPPl的变化,Re．al—timeRT—PCR及ELISA检测生长因子TGF-βl的变化。结果：随着细胞传至P7代,细胞形态上有梭形变趋势。茜素红染色无明显变化。软骨标志性基因Ⅱ型胶原、蛋白多糖及转录因子Sox9表达均明显下调（P〈0．05）,钙化相关基因ANK、ENPPl表达均明显下调（P〈0．05）,内源性生长因子TGF-B1表达明显下调（P〈0．05）。结论：终板软骨细胞传至P7代,终板软骨细胞发生退变,终板软骨细胞内未见钙盐沉积,钙化相关基因ANK、ENPPl下调可能由内源性TGF-βl下调引起,提示调节内源性TGF-βl及钙化相关基因ANK、ENPPl在终板软骨中的表达有可能会阻止椎间盘的退变。     

Regulation of acid-sensing ion channel 1a function by tissue kallikrein may be through channel cleavage

Recently, we have demonstrated that serine protease tissue kallikrein (TK) can protect cortical neurons against ischemia-acidosis/reperfusion-induced injury, and that this effect might be mediated by acid-sensing ion channels (ASICs). However, little is known about how TK regulates the function of ASICs. Here we provided evidence that the regulation of ASIC1a function by TK was probably correlated with its cleavage. High concentration of TK (3 μM) partially cleaved the extracellular loop of ASIC1a, followed by a marked decrease of LDH release and an increase of cell survival at pH 6.2. Pretreatment with a protease inhibitor aprotinin inhibited the cleavage of ASIC1a and prevented functional regulation by TK. However, the cleavage of ASIC2a, which was not functionally modified by TK, was not observed. Therefore, we propose that the limited proteolysis of extracellular loop within ASIC1a might be one of the potential regulatory mechanisms of ASIC1a function by TK.     

Acid-sensing ion channel 2 (asic 2) and trkb interrelationships within the intervertebral disc

The cells of the intervertebral disc (IVD) have an unusual acidic and hyperosmotic microenvironment. They express acid-sensing ion channels (ASICs), gated by extracellular protons and mechanical forces, as well as neurotrophins and their signalling receptors. In the nervous tissues some neurotrophins regulate the expression of ASICs. The expression of ASIC2 and TrkB in human normal and degenerated IVD was assessed using quantitative-PCR, Western blot, and immunohistochemistry. Moreover, we investigated immunohistochemically the expression of ASIC2 in the IVD of TrkB-deficient mice. ASIC2 and TrkB mRNAs were found in normal human IVD and both increased significantly in degenerated IVD. ASIC2 and TrkB proteins were also found co-localized in a variable percentage of cells, being significantly higher in degenerated IVD than in controls. The murine IVD displayed ASIC2 immunoreactivity which was absent in the IVD of TrkB-deficient mice. Present results demonstrate the occurrence of ASIC2 and TrkB in the human IVD, and the increased expression of both in pathological IVD suggest their involvement in IVD degeneration. These data also suggest that TrkB-ligands might be involved in the regulation of ASIC2 expression, and therefore in mechanisms by which the IVD cells accommodate to low pH and hypertonicity.     

酸敏感离子通道对海马神经元树突发育的影响

目的 探讨酸敏感离子通道（ASICs）在海马神经元树突发育中的作用。方法 在体外培养第5天的原代海马神经元中转染定位于膜上的绿色荧光蛋白（F-GFP）,随后在神经元培养液中加入ASICs拮抗剂Amiloride和ASIC1a 选择性拮抗剂Psalmotoxin 1（PcTX1）抑制ASICs的功能,观察体外培养8d和14d这两个时间点海马神经元的树突生长、分支复杂程度。结果Amiloride（10-5mol/L）和PcTX1（1∶20 000稀释）处理3d对海马神经元树突分支总长度、树突分支总数均无显著影响,表明在海马神经元发育早期短时间抑制ASICs功能不影响树突发育。Amiloride（10-5mol/L）处理9d可以显著降低树突分支总长度和树突分支总数; PcTX1（1∶20 000稀释）处理9d也可以显著降低树突分支总长度,但对树突分支总数无显著影响。实验结果表明,长时间抑制ASICs功能会影响树突发育。结论 ASICs参与调节树突的发育。     

Two types of acid-sensing ion channel currents in rat hippocampal neurons

Two types of acid-sensing ion channel (ASIC)-like currents in cultured rat hippocampal neurons were recorded and their characteristics were studied by using a whole-cell recording technique. The results revealed that the ASIC-like currents, induced by a quick drop of the extracellular pH, decayed with different time constants (τ) of 229 ± 16 (Type I) and 1209 ± 56 ms (Type II). The ASIC-like currents displayed different sensitivities to extracellular proton (pH_0.5 was 6.17 ± 0.04 for Type I and 5.70 ± 0.07 for Type II) and amiloride, a specific ASIC channel blocker (IC₅₀ was 1.19 ± 0.37 μM for Type I and 0.14 ± 0.02 μM for Type II). Among all the 360 recorded neurons, ASIC-like currents were induced in 314 neurons (87.2%). In the neurons expressing ASICs, Type I currents were evoked from 269 neurons (85.7%) and Type II currents were induced only from 45 neurons (14.3%). As these ASIC-like currents presented various electrophysiological and pharmacological properties, further experiments should be conducted to decipher the complex subunit composition of ASICs in the hippocampus.     

Effect of activation of the Ca2+-permeable acid-sensing ion channel 1a on focal cerebral ischemia in diabetic rats

We investigated the role of acid-sensing ion channel Ia (ASIC1a) expression and changes in intracellular Ca²⁺ concentration ([Ca²⁺]) in focal cerebral ischemia after middle cerebral artery occlusion (MCAO) in a rat model of diabetes mellitus (DM). Male Wistar rats (n = 108) were divided into three groups: the MCAO, DM + MCAO, and DM + MCAO + fasudil groups (n = 36 each). Samples were obtained 1, 3, 6, and 24 h after ischemia induction (n = 9). Rats in the DM + MCAO + fasudil group were treated with 1 mg/kg fasudil, a Rho-kinase inhibitor, by caudal vein injection 30 min after MCAO was performed. ASIC1a expression gradually increased with time in the MCAO and DM + MCAO groups (0.71 ± 0.10 nM, 0.80 ± 0.11 nM, 0.86 ± 0.08 nM, 0.93 ± 0.09 nM; 0.86 ± 0.11 nM, 1.05 ± 0.51 nM, 2.42 ± 0.08 nM, 2.78 ± 0.04 nM; pairwise comparisons at each time point, P < 0.05), and was higher in the DM + MCAO than the MCAO group (P < 0.05). [Ca²⁺] gradually increased in the DM + MCAO group (106.32 ± 18.6 nM, 137.84 ± 14.32 nM, 151.94 ± 18.38 nM, 183.61 ± 7.96 nM, P < 0.05). ASIC1a expression and calcium currents were reduced in the DM + MCAO + fasudil group. The overload of intracellular [Ca²⁺] caused by ASIC1a activation could be one mechanism for the aggravation of focal cerebral ischemia in diabetes.     

Proton binding sites involved in the activation of acid-sensing ion channel ASIC2a

Most acid-sensing ion channel (ASIC) subunits are activated by protons, but ASIC2b (a splice variant of ASIC2a) is acid-insensitive. Differences in protonatable residues between the extracellular loop regions of ASIC2a and ASIC2b may explain this difference. Site-directed mutagenesis, combined with immunocytochemistry and whole-cell patch clamp, demonstrated that mutating any one of five ASIC2a sites produces channels that traffic normally to the cell surface membrane but are insensitive to protons. One of the mutants forms functional heteromers with ASIC1a and ASIC2a, demonstrating that ion transport is intact in this mutant. These five sites may be involved in the activation of ASIC2a by protons.     

Acid-sensing ion channel 2 (ASIC2) in the intestine of adult zebrafish

Acid-sensing ion channels (ASICs) in mammals monitor acid sensing and mechanoreception. They have a widespread expression in the central and peripheral nervous system, including the gut. The distribution of ASICs in zebrafish is known only in larvae and at the mRNA level. Here we have investigated the expression and cell distribution of ASIC2 in the gut of adult zebrafish using PCR, Western blot and immunohistochemistry. ASIC2 mRNA was detected in the gut, and a protein consistent with predicted ASIC2 (64kDa molecular mass) was detected by Western blot. ASIC2 positivity was found in a subpopulation of myenteric neurons in the enteric nervous system, as well in enteroendocrine epithelial cells. These data demonstrate for the first time the occurrence of ASIC2 in the gut of adult zebrafish where it presumably acts as a chemosensor and a mechanosensor.     

Dependence of single channel properties of the N-methyl-d-aspartate ion channel on stereoisomer agonists

The cell-attached patch-clamp configuration has been used to determine the single channel properties of the N-methyl-D-aspartate (NMDA) ion channel with activation of the NMDA receptor by stereoisomer agonists. All of the agonists studied, including the L and d forms of N-methyl-aspartate and the L and d forms of homocysteate, activated a 42-pS conductance channel in cultured hippocampal neurons. For all agonists, the mean open times of the channel were diminished with increased patch hyperpolarization and exhibited an exponential dependence on potential over the range -40 mV to -120 mV. The mean open times, for patch potentials close to resting potential, and the mean frequencies of channel openings, at all patch potentials, were significantly different between each member of the stereoisomer pairs. For both L-homocysteate and NMLA, a fourfold increase in the patch pipette concentration caused an approximate quadrupling in the frequency of unitary events, with no significant change in mean open time. The open channel probability was used as a measure of agonist potency, and, at a concentration of 30 M, NMDA and L-homocysteate were significantly more potent (P open in excess of 1.5%) than the corresponding stereoisomer compounds NMLA and D-homocysteate (P open near 0.3%). The relative potencies of the stereoisomer pairs were in reasonable agreement with the potency ratios measured in binding studies.     

3D打印个体化人工椎体在胸腰椎肿瘤整块切除后脊柱稳定性重建中的应用

目的探索3D打印个体化人工椎体在胸腰椎肿瘤整块切除后脊柱稳定性重建中的临床疗效。方法回顾性分析华中科技大学同济医学院附属同济医院2015年7月至2020年6月收治的接受一期后路全脊椎整块切除术的胸腰椎肿瘤患者28例,分为2组,每组14例。其中3D组使用3D打印个体化人工椎体重建,常规组使用钛笼重建。对比两组的住院时间、手术时间、术中出血量,记录术前、术后7 d及末次随访时视觉模拟评分(VAS),Frankel分级情况,测量节段高度及角度,评估临床疗效。结果两组患者住院时间、手术时间、术中出血量和VAS评分比较,差异无统计学意义。共8例患者Frankel评分获得一个等级的改善(28.6%)。3D组椎间高度丢失(1.9±2.2)mm,内植物沉降2例,沉降率14.3%;常规组椎间高度丢失(6.6±5.5)mm,内植物沉降8例,沉降率57.1%;两组比较差异有统计学意义(<0.05)。在节段角度丢失方面组间差异无统计学意义(=0.571)。3D组所有患者内固定良好,常规组1例患者发生断棒情况。结论 3D打印人工椎体可以更好地维持节段高度,降低椎间隙塌陷和内固定失败的风险。