缺氧培养对大鼠神经干细胞体外增殖影响及其机制的研究

目的：研究应用缺氧对体外培养的大鼠神经干细胞增殖的影响。方法：将细胞分为4小时缺氧组、12小时缺氧组、促红细胞生成素（EPO）中和抗体组、IgG组以及对照组．测定大鼠神经干细胞经缺氧培养后的各组细胞克隆形成率以及EPO的表达变化。结果：单细胞培养条件下,与对照组相比,4小时缺氧组和IgG组克隆形成率明显增高;中和抗体组无明显变化;12小时组克隆形成率降低。但无统计学意义。缺氧4小时后,EPO蛋白在预处理后即刻出现表迭,4h达高峰,8h仍有部分表达。结论：短时间缺氧可以促进神经干细胞增殖．长时间缺氧则作用相反。缺氧对NSCs增殖作用的影响可能是由EPO介导产生。     

缺氧环境下大鼠骨髓间充质干细胞的凋亡及Fas、Fas-L的表达

目的探讨大鼠骨髓间充质干细胞(MSCs)在缺氧环境下是否存在凋亡及Fas和Fas-L蛋白在MSCs凋亡时是否表达及与细胞凋亡的相关性。方法将接种的P3细胞置于94%N2、1%O2和5%CO2缺氧箱中37℃孵育,分别于0.5h、1h、2h、4h、6h、8h和12h取出进行缺口末端标记(TUNEL)计算细胞凋亡指数(ApoptoticIndex,AI)和透射电镜观察细胞超微结构的改变,用Fas和Fas-L免疫组化试剂盒检测Fas-L和Fas蛋白的表达。结果1.培养的骨髓单个核细胞CD29、CD71、CD44免疫细胞化学染色均阳性,CD34染色阴性,5-氮胞苷诱导后表达TnT,提示其为MSCs。2.MSCs在缺氧前、缺氧0.5、1、2、4、6、8和12h时AI分别为0.11±2.03%、15.4±3.19%、16.7±2.51%、16.9±0.25%、17.7±2.50%、18.3±3.15%、18.4±4.22%、19.7±4.58%,缺氧不同时间点AI较缺氧前均显著性增高(P<0.01),并随着缺氧时间延伸,AI显著增加(P<0.05),不过,缺氧6h、8h和12h时AI没有统计学意义(P>0.05)。3.MSCs在不同缺氧时间点Fas、Fas-L蛋白表达较缺氧前均显著性增高(P<0.05),且随着缺氧时间延伸,表达显著增加,而在缺氧6h-12h时间点表达没有统计学意义(P>0.05)。3.缺氧0.5、1、2、4、6、8和12h,AI与Fas和Fas-L均显著正相关。结论缺氧是促进MSCs凋亡的重要因素,Fas和Fas-L蛋白可能在MSCs缺氧凋亡的调控中起着重要作用。     

凋亡诱导因子介导缺氧/复氧致肥大心肌细胞凋亡的作用

心肌细胞凋亡导致心肌组织合胞体功能丧失,最终使代偿性心肌肥大向心力衰竭转化。过去的研究已经确认天门冬氨酸特异性半胱氨酸蛋白酶(caspartate-specificcysteinylproteinase,caspase)依赖机制在心肌细胞凋亡中的作用,但对caspase非依赖机制即凋亡诱导因子(apoptosis-inducingfactor,AIF)在心肌细胞凋亡中的作用尚不明确。本研究应用血管紧张素Ⅱ(0.1μmol/L培养12h)诱导培养的小鼠肥大心肌细胞,利用三气孵箱建立缺氧/复氧模型以模拟缺血再灌注。应用RT-PCR、Westernblot、siRNA基因转染、Hoechst33258染色法检测AIF在mRNA和蛋白质水平的表达及细胞凋亡的变化,分析AIF在缺氧/复氧致肥大心肌细胞凋亡中的意义。结果如下:(1)与对照组比较,缺氧8h组(H8h)和缺氧12h组(H12h)AIFmRNA及蛋白表达水平均显著升高(mRNA:0.52±0.04及0.85±0.10vs0.29±0.08,P<0.05;蛋白质:2.07±0.15和3.12±0.19vs0.29±0.04,P<0.05),即随缺血时间的延长,AIFmRNA及蛋白表达水平均显著增加。(2)与对应单纯缺氧组比较,缺氧后给予复氧刺激,H8h/R组和H12h/R组AIFmRNA及蛋白表达水平均显著升高(mRNA:1.09±0.12和1.41±0.23,P<0.05;蛋白质:4.57±0.25和5.71±0.27,P<0.05)。仅在H8h/R及H12h/R组,可见AIF核转位显著增加。(3)AIFsiRNA转染可显著抑制肥大心肌细胞AIF的表达,对缺氧时细胞凋亡无明显影响(P>0.05),但可显著降低缺氧/复氧诱导的肥大心肌细胞凋亡率(P<0.05)。同时抑制AIF及caspase-3活性,可显著加强单一抑制剂对缺氧/复氧诱导的肥大心肌细胞凋亡的抑制作用。(4)抑制caspase-3活性对缺氧/复氧诱导的AIF核转位无明显影响。上述结果提示,缺氧/复氧时AIFmRNA、蛋白表达和核转位均显著增加,且在缺氧/复氧诱导肥大心肌细胞凋亡中具有重要的作用。     

KATP通道对缺氧海马CA1区神经元损伤保护作用的机制

目的:探讨KATP通道在缺氧中对海马CA1区神经元的保护作用机制。方法:比较对照组、单纯缺氧组、KATP通道激动剂 缺氧组、KATP通道阻断剂 缺氧组中神经元p53 mRNA的表达、DNA断裂、以及神经元存活情况。结果:将神经元暴露在氧浓度为0%的缺氧环境中12h,KATP通道的激动剂二氮嗪(diazoxide,100μmol/L)显著降低p53 mRNA的表达量及细胞的凋亡数量。KATP通道的阻断剂甲糖宁(tolbutamide,100μmol/L)使p53mR-NA表达量显著增加,细胞的凋亡数量也随之显著增加。p53的特异性阻断剂曲古抑菌素(trichostatin,TSA)可以逆转甲糖宁(tolbutamide,100μmol/L)的作用。结论:KATP通道可以通过下调p53 mRNA的表达水平,对缺氧中的海马CA1区神经元起到保护作用。     

缺氧预处理神经干细胞移植对大鼠急性脊髓损伤后神经胶质细胞凋亡及脊髓空洞形成的影响

目的:研究缺氧预处理神经干细胞(NSCs)移植对大鼠急性脊髓损伤(ASCI)后神经胶质细胞凋亡及脊髓空洞形成的影响.方法:将30只SD大鼠分为假手术对照组;脊髓损伤组;去铁敏组;普通NSCs组;缺氧预处理NSCs组.制成脊髓损伤模型,移植后观察脊髓神经胶质细胞凋亡情况及脊髓空洞形成情况.结果:经去铁敏缺氧预处理培养的NSCs与常规培养的NSCs无明显形态学变化.移植术后7d,缺氧预处理NSCs移植能显著减少脊髓损伤周围区神经胶质细胞的凋亡数量,减少脊髓空洞形成.结论:缺氧预处理NSCs移植能明显抑制大鼠急性脊髓损伤后神经胶质细胞凋亡,减少脊髓空洞的形成.     

缺氧预处理神经干细胞移植对大鼠急性脊髓损伤后神经胶质细胞凋亡及脊髓空洞形成的影响

目的:研究缺氧预处理神经干细胞(NSCs)移植对大鼠急性脊髓损伤(ASCI)后神经胶质细胞凋亡及脊髓空洞形成的影响。方法:将30只SD大鼠分为假手术对照组;脊髓损伤组;去铁敏组;普通NSCs组;缺氧预处理NSCs组。制成脊髓损伤模型,移植后观察脊髓神经胶质细胞凋亡情况及脊髓空洞形成情况。结果:经去铁敏缺氧预处理培养的NSCs与常规培养的NSCs无明显形态学变化。移植术后7d,缺氧预处理NSCs移植能显著减少脊髓损伤周围区神经胶质细胞的凋亡数量,减少脊髓空洞形成。结论:缺氧预处理NSCs移植能明显抑制大鼠急性脊髓损伤后神经胶质细胞凋亡,减少脊髓空洞的形成。     

Wnt 3a在神经干细胞向多巴胺能神经元分化过程中作用的研究

Wnt信号在中枢神经系统发育过程中起重要的作用,控制着细胞的生长及分化.Wnt3a是Wnt家族的成员之一,对神经干细胞的增殖及分化有一定的调控作用.将重组Wnt3a腺病毒转入神经干细胞中,研究Wnt3a在定向诱导神经干细胞向多巴胺能神经元分化过程中的作用.将神经干细胞分为4组,对照组(不加任何诱导因子组)、抗坏血酸诱导组(AA组)、Wnt3a重组腺病毒诱导组(Wnt3a组)以及Wnt3a重组腺病毒加抗坏血酸诱导组(Wnt3a AA组).结果显示,Wnt3a组细胞中的多巴胺能神经元前体细胞特异性标志Nurr1表达量显著增多,Wnt3a AA组多巴胺能神经元明显多于AA组,酪氨酸羟化酶(TH)在mRNA水平上的表达是AA组的1.86倍.蛋白质印迹及免疫细胞化学染色显示,各诱导组均有TH的表达,Wnt3a组和AA组多巴胺能神经元阳性细胞数比例分别为(5.76±3.34)%和(37.42±2.54)%,与Wnt3a AA组(73.96±2.61)%比较,差异有统计学意义(P<0.05).利用高效液相色谱法检测到诱导后的细胞可分泌多巴胺.结果表明,Wnt3a可促进神经干细胞向多巴胺能神经元前体细胞分化,再通过抗坏血酸的诱导作用,在体外可获得大量的多巴胺能神经元,这些神经元有分泌多巴胺的功能.     

IGF2BP1对白血病干细胞特性维持的作用

[目的]探究胰岛素样生长因子2 mRNA结合蛋白1(IGF2BP1)调控HOXB4、MYB、ALDH1A1表达在白血病干细胞特性维持中的作用。[方法] K562细胞分为3组:对照组、IGF2BP1组、siIGF2BP1组。通过转染过表达或沉默IGF2BP1。分别对各组细胞的增殖、凋亡、体外干细胞成球能力进行检测。检测HOXB4、MYB、ALDH1A1 mRNA和蛋白水平。[结果]三组细胞的上述指标比较差异显著(P<0.05)。IGF2BP1组细胞的IGF2BP1增殖(0.75±0.07,1.34±0.12)、成球数量(98.23±4.16)个和大小(205.97±9.54)μm、HOXB4、MYB、ALDH1A1 mRNA和蛋白水平显著高于对照组,凋亡率(3.62%±0.35%)显著低于对照组(P<0.05)。siIGF2BP1组细胞的IGF2BP1、增殖(0.46±0.04,0.81±0.08)、成球数量(25.76±1.35)个和大小(85.44±2.37)μm、HOXB4、MYB、ALDH1A1 mRNA和蛋白水平显著低于对照组,凋亡率(18.34±0.27)%显著高于对照组(P<0.05)。[结论]过表达IGF2BP1上调了HOXB4、MYB、ALDH1A1 mRNA和蛋白表达,促进白血病干细胞特性的维持。     

气体过饱和对长江鲟早期生活史的影响

为研究总溶解气体(Total Dissolved Gas, TDG)过饱和对长江鲟(Acipenser dabryanus Dumeril)早期生活史的影响, 以长江鲟受精卵和仔稚鱼为实验对象, 进行不同TDG饱和度的暴露试验。结果显示: 在不同饱和度的TDG过饱和水体中, 发育至神经胚阶段的长江鲟受精卵的孵化率存在显著差异。其中, 对照组中的受精卵孵化率为(79±3)%, 140%组的孵化率((78±2)%)与对照组无显著差异(P>0.05), 110%组、120%组130%组的孵化率分别为(84±0)%、(92±2)%和(91±1)%, 显著高于对照组(P<0.05)。将出膜1—2d的仔鱼置于不同梯度的TDG饱和度水体中暴露96h, 除对照组外, 各实验组均有仔鱼死亡, 但死亡率均未达到7%。其中130%组的死亡率最高, 为(5.59±1.86)%; 110%和120%组死亡率次之, 分别为(3.45±0.12)%和(3.45±0.00)%; 140%组的死亡率最低, 为(1.15±1.99)%。130%组仔鱼死亡率显著高于140%组(P<0.05), 其他各组之间无显著差异(P>0.05)。将50日龄的长江鲟稚鱼置于不同梯度的TDG饱和水体中暴露96h, 除对照组外, 各实验组均有稚鱼死亡, 且各组死亡率均在50%以上, 140%组死亡率达100%。除110%组和120%组差异不显著外(P>0.05), 其他各组之间差异显著(P<0.05), 且随着TDG饱和度增加, 死亡率增大。各实验组之间的半致死时间存在显著差异(P<0.05), 且呈现出TDG饱和度增加, 半致死时间呈减少趋势。总体来看, TDG饱和度为110%、120%和130%的水体对长江鲟受精卵的孵化率有显著促进作用, 140%的水体对其孵化率无影响。不同TDG饱和度的过饱和水体均可导致长江鲟仔稚鱼死亡, 但对仔鱼影响程度较小, 且死亡率与TDG饱和度之间无显著关联; 对稚鱼影响程度较大, 在研究范围内, 影响程度随饱和度的增加呈增大趋势。     

清开灵对小胶质细胞BV2缺氧再复氧损伤gp~(91phox)表达的影响

目的:建立小胶质细胞缺氧再复氧损伤模型,观察产生ROS的NADPH氧化酶的重要功能亚基gp91phox的表达变化及清开灵的干预作用,丰富清开灵基于解毒通络法以祛除内毒恢复脉络的作用内涵。方法:体外培养小鼠胶质细胞BV2,细胞分为正常组、模型组和清开灵高、中、低剂量组,在1%O2三气培养箱中缺氧12小时再复氧12小时模拟缺血再灌注损伤,正常对照组在培养箱中培养24小时,实时荧光定量PCR法检测gp91phoxmRNA的转录水平,Western blot法检测gp91phox蛋白表达。结果:缺氧再复氧损伤后,模型组gp91phox基因转录水平和蛋白表达提高(P0.05);与模型组比较,清开灵低、中、高剂量组都有明显改善作用,其中低剂量(0.0625%)对基因转录降低更明显,高剂量组(0.25%)对gp91phox蛋白表达的抑制更显著,具有统计学意义(P0.05)。结论:清开灵可通过降低缺氧再复氧后小胶质细胞gp91phox的表达,减少活性氧的产生而抑制脑缺血损伤氧化应激反应。     

The Effect of Hypoxic Preconditioning on Induced Schwann Cells under Hypoxic Conditions

Object

Our objective was to explore the protective effects of hypoxic preconditioning on induced Schwann cells exposed to an environment with low concentrations of oxygen. It has been observed that hypoxic preconditioning of induced Schwann cells can promote axonal regeneration under low oxygen conditions.

Method

Rat bone marrow mesenchymal stem cells (MSCs) were differentiated into Schwann cells and divided into a normal oxygen control group, a hypoxia-preconditioning group and a hypoxia group. The ultrastructure of each of these groups of cells was observed by electron microscopy. In addition, flow cytometry was used to measure changes in mitochondrial membrane potential. Annexin V-FITC/PI staining was used to detect apoptosis, and Western blots were used to detect the expression of Bcl-2/Bax. Fluorescence microscopic observations of axonal growth in NG-108 cells under hypoxic conditions were also performed.

Results

The hypoxia-preconditioning group maintained mitochondrial cell membrane and crista integrity, and these cells exhibited less edema than the hypoxia group. In addition, the cells in the hypoxia-preconditioning group were found to be in early stages of apoptosis, whereas cells from the hypoxia group were in the later stages of apoptosis. The hypoxia-preconditioning group also had higher levels of Bcl-2/Bax expression and longer NG-108 cell axons than were observed in the hypoxia group.

Conclusion

Hypoxic preconditioning can improve the physiological state of Schwann cells in a severe hypoxia environment and improve the ability to promote neurite outgrowth.     

Cx40.8, a Cx43-like protein, forms gap junction channels inefficiently and may require Cx43 for its association at the plasma membrane

In addition to having a Cx43 ortholog, the zebrafish genome also contains a Cx43-like gene, Cx40.8. Here, we investigate the expression of cx40.8 in zebrafish fins and the function of Cx40.8 in HeLa cells. We find that cx40.8 is present in the same population of dividing cells as cx43. Unlike Cx43, dye coupling assays suggest that Cx40.8 only inefficiently forms functional gap junction channels. However, co-transfection reveals that Cx40.8 can co-localize with Cx43 in gap junction plaques, and that the resulting plaques contain functional gap junction channels. Together, these data suggest the possibility that Cx40.8 may functionally interact with Cx43 to regulate cell proliferation in vivo.

Structured summary

MINT-7266123: cx40.8 (genbank_protein_gi:68354404) and cx43 (uniprotkb:O57474) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Hemichannel and junctional properties of connexin 50

Lens fiber connexins, cx50 and cx46 (alpha3 and alpha8), belong to a small subset of connexins that can form functional hemichannels in nonjunctional membranes. Knockout of either cx50 or cx46 results in a cataract, so the properties of both connexins are likely essential for proper physiological functioning of the lens. Although portions of the sequences of these two connexins are nearly identical, their hemichannel properties are quite different. Cx50 hemichannels are much more sensitive to extracellular acidification than cx46 hemichannels and differ from cx46 hemichannels both in steady-state and kinetic properties. Comparison of the two branches of the cx50 hemichannel G-V curve with the junctional G-V curve suggests that cx50 gap junctions gate with positive relative polarity. The histidine-modifying reagent, diethyl pyrocarbonate, reversibly blocks cx50 hemichannel currents but not cx46 hemichannel currents. Because cx46 and cx50 have very similar amino acid sequences, one might expect that replacing the two histidines unique to the third transmembrane region of cx50 with the corresponding cx46 residues would produce mutants more closely resembling cx46. In fact this does not happen. Instead the mutant cx50H161N does not form detectable hemichannels but forms gap junctions indistinguishable from wild type. Cx50H176Q is oocyte lethal, and the double mutant, cx50H61N/H176Q, neither forms hemichannels nor kills oocytes.     

Hypoxic preconditioning improves survival of cardiac progenitor cells: role of stromal cell derived factor-1α-CXCR4 axis

Background

Cardiac progenitor cells (CPCs) have been shown to be suitable in stem cell therapy for resurrecting damaged myocardium, but poor retention of transplanted cells in the ischemic myocardium causes ineffective cell therapy. Hypoxic preconditioning of cells can increase the expression of CXCR4 and pro-survival genes to promote better cell survival; however, it is unknown whether hypoxia preconditioning will influence the survival and retention of CPCs via the SDF-1α/CXCR4 axis.

Methods and Results

CPCs were isolated from adult mouse hearts and purified by magnetic activated cell sorting using c-kit magnetic beads. These cells were cultured at various times in either normoxic or hypoxic conditions, and cell survival was analyzed using flow cytometry and the expression of hypoxia-inducible factor-1α (HIF-1α), CXCR4, phosphorylated Akt and Bcl-2 were measured by Western blot. Results showed that the expression of pro-survival genes significantly increased after hypoxia treatment, especially in cells cultured in hypoxic conditions for six hours. Upon completion of hypoxia preconditioning from c-kit+ CPCs for six hours, the anti-apoptosis, migration and cardiac repair potential were evaluated. Results showed a significant enhancement in anti-apoptosis and migration in vitro, and better survival and cardiac function after being transplanted into acute myocardial infarction (MI) mice in vivo. The beneficial effects induced by hypoxia preconditioning of c-kit+ CPCs could largely be blocked by the addition of CXCR4 selective antagonist AMD3100.

Conclusions

Hypoxic preconditioning may improve the survival and retention of c-kit+ CPCs in the ischemic heart tissue through activating the SDF-1α/CXCR4 axis and the downstream anti-apoptosis pathway. Strategies targeting this aspect may enhance the effectiveness of cell-based cardiac regenerative therapy.     

Gap Junction Intercellular Communication Mediated by Connexin43 in Astrocytes Is Essential for Their Resistance to Oxidative Stress

Oxidative stress induced by reactive oxygen species (ROS) is associated with various neurological disorders including aging, neurodegenerative diseases, as well as traumatic and ischemic insults. Astrocytes have an important role in the anti-oxidative defense in the brain. The gap junction protein connexin43 (Cx43) forms intercellular channels as well as hemichannels in astrocytes. In the present study, we investigated the contribution of Cx43 to astrocytic death induced by the ROS hydrogen peroxide (H₂O₂) and the mechanism by which Cx43 exerts its effects. Lack of Cx43 expression or blockage of Cx43 channels resulted in increased ROS-induced astrocytic death, supporting a cell protective effect of functional Cx43 channels. H₂O₂ transiently increased hemichannel activity, but reduced gap junction intercellular communication (GJIC). GJIC in wild-type astrocytes recovered after 7 h, but was absent in Cx43 knock-out astrocytes. Blockage of Cx43 hemichannels incompletely inhibited H₂O₂-induced hemichannel activity, indicating the presence of other hemichannel proteins. Panx1, which is predicted to be a major hemichannel contributor in astrocytes, did not appear to have any cell protective effect from H₂O₂ insults. Our data suggest that GJIC is important for Cx43-mediated ROS resistance. In contrast to hypoxia/reoxygenation, H₂O₂ treatment decreased the ratio of the hypophosphorylated isoform to total Cx43 level. Cx43 has been reported to promote astrocytic death induced by hypoxia/reoxygenation. We therefore speculate the increase in Cx43 dephosphorylation may account for the facilitation of astrocytic death. Our findings suggest that the role of Cx43 in response to cellular stress is dependent on the activation of signaling pathways leading to alteration of Cx43 phosphorylation states.     

Pre-treatment with isoflurane ameliorates renal ischemic-reperfusion injury in mice

AimsPerioperative renal dysfunction is associated with a high mortality. The aim of this study was to investigate whether isoflurane preconditioning provides a protection against renal ischemic–reperfusion injury and whether hypoxia inducible factor 1α (HIF-1α) is responsible for the protection afforded by isoflurane in mice.Main methodsAdult male C57BL/6 mice received vehicle (PBS), scrambled siRNA or HIF-1α siRNA via hydrodynamic injection through tail vein. Twenty-four hours after injection, they were exposed to 1.5% isoflurane in oxygen enriched air for 2 h while controls without injection were exposed to oxygen enriched air. Twenty-four hours after gas exposure, mice were sacrificed and their kidney were harvested for western blot while other cohorts underwent renal ischemia–reperfusion injury induced by bilateral renal pedicle clamping for 25 min for renal histological or functional analysis 24 h after reperfusion or by unilateral clamping for 40 min for survival rate analysis.Key findingsSurvival rate and the expression of HIF-1α and erythropoietin were significantly increased while apoptosis, renal tubule score, blood plasma creatinine and urea were decreased by isoflurane preconditioning. HIF-1α siRNA but not scrambled siRNA injection abrogated the protective effect of isoflurane preconditioning.SignificanceOur data suggested that isoflurane preconditioning provided a protection against renal ischemic–reperfusion injury which is very likely due to hypoxia inducible factor-1α upregulation.     

缺氧诱导因子-1α在缺氧预处理预防心肌细胞损伤中的作用

本工作旨在研究缺氧预处理(hypoxic preconditioning,HPC)对于心肌细胞外信号调节激酶(extracellular signal-regulated proteinkinases,ERK)活性、缺氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)表达的影响,及其在缺氧复氧诱导心肌细胞损伤中的作用。通过在培养的SD乳鼠心肌细胞缺氧／复氧(H／R)模型上,观察HPC对于24h后H／R诱导心肌细胞损伤的影响,以台盼蓝排斥实验检测心肌细胞存活率、以TUNEL法检测细胞凋亡、并用荧光素染料Hoechst33258测定心肌细胞凋亡率：制备心肌细胞蛋白提取物,以磷酸化的ERK1／2抗体测定ERK1／2活性,以抗HIF-1α抗体检测HIF-1α的表达,并观察ERKs的上游激酶(MEK1／2)抑制剂PD98059对于HPC诱导的ERKs磷酸化、HIF-1α表达以及心肌细胞保护作用的影响,并分析细胞损伤与ERK1／2活性、HIF-1α表达量之间的相互关系。结果 显示缺氧复氧造成心肌细胞损伤,HPC可以增加心肌细胞H／R后存活率,降低凋亡率,并激活ERKll2,诱导HIF-1α表达：细胞凋亡与ERKs活性、HIF-1α表达量之间存在负相关,即ERKs活化、HIF-1α表达与预防细胞损伤有关：而ERKs活性与HIF-1α表达量之间存在正相关,ERKs的上游激酶MEK抑制剂PD98059可以消除HPC诱导的ERKs磷酸化、HIF-1α表达和心肌细胞保护作用。由此得出的结论是HPC可以提高乳鼠心肌细胞对于H／R的耐受性,其机制涉及ERKs介导的HIF-1α表达。     

Connexin43 regulates joint location in zebrafish fins

Joints are essential for skeletal form and function, yet their development remains poorly understood. In zebrafish fins, joints form between the bony fin ray segments providing essentially unlimited opportunities to evaluate joint morphogenesis. Mutations in cx43 cause the short segment phenotype of short fin (sof^b123) mutants, suggesting that direct cell-cell communication may regulate joint location. Interestingly, increased cx43 expression in the another long fin (alf^dty86) mutant appears to cause joint failure typical of that mutant. Indeed, knockdown of cx43 in alf^dty86 mutant fins rescues joint formation. Together, these data reveal a correlation between the level of Cx43 expression in the fin ray mesenchyme and the location of joints. Cx43 was also observed laterally in cells associated with developing joints. Confocal microscopy revealed that the Cx43 protein initially surrounds the membranes of ZNS5-positive joint cells, but at later stages becomes polarized toward the underlying Cx43-positive mesenchymal cells. One possibility is that communication between the Cx43-positive mesenchyme and the overlying ZNS5-positive cells regulates joint location, and upregulation of Cx43 in joint-forming cells contributes to joint morphogenesis.     

Atrial Fibrillation-Linked Germline GJA5/Connexin40 Mutants Showed an Increased Hemichannel Function

Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca²⁺]_o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.