低氧及低氧预处理时心肌细胞HIF-1α与凋亡相关蛋白表达的变化

目的:观察低氧时心肌细胞HIF-1α表达变化与凋亡相关蛋白表达关系.方法:采用体外心肌细胞培养的方法,将原代培养4～6 d的大鼠乳鼠心肌细胞随机分为对照组、低氧组与低氧预处理组.低氧预处理组在低氧培养箱中通入1%O2、5%CO2、94%N2的低氧混合气体,每天低氧12 h,低氧5 d,第6 d与急性低氧组一同放入0%O2、5%CO2、95%N2的低氧培养箱中进行低氧暴露.低氧48 h后,通过Western blot方法分别检测心肌细胞中HIF-1α、Bcl-2、P53及Bax的表达变化.结果:常氧时细胞不表达HIF-1α,低氧可增加HIF-1的表达,低氧预处理后,能降低HIF-1α的表达.低氧时,Bax的表达变化大致与此相同.p53在低氧时的变化也与其相同,但低氧预处理后似乎没有明显的改变.Bcl-2在低氧时表达下降,低氧预处理后可增加其表达.结论:HIF-1α的表达可协同Bcl-2家族凋亡相关蛋白的表达,在低氧导致的心肌细胞凋亡中发挥重要作用.     

内皮素-1致低氧心肌细胞凋亡的作用及其机制研究

目的:观察内源性内皮素-1(ET-1)在低氧所致心肌细胞凋亡中的作用及受体机制。方法:培养的新生乳鼠心肌细胞分为低氧对照组和内皮素受体拮抗剂预处理+低氧组,前者仅给DMEM液,后者给予ETA受体拮抗剂BQ610或BQ123及ETB受体拮抗剂BQ788后低氧培养24 h,TUNEL和AnnexinV-FITC/PI双标记流式细胞术检测心肌细胞凋亡情况。结果:TUNEL检测显示低氧24 h对照组心肌细胞凋亡百分数为24.2%±2.2%,BQ6105μmol/L+低氧24 h组为13.2%±3.7%,显著低于低氧24 h组(P<0.01)。流式细胞术检测显示BQ123(0.04,0.2,1.0μmol/L)浓度依赖性地抑制低氧引起的心肌细胞凋亡及浓度依赖性提高低氧培养心肌细胞存活率,而BQ788对低氧引起的心肌细胞凋亡及存活率的降低无明显影响。结论:内源性ET-1参与促进低氧引起的心肌细胞凋亡,该作用主要是通过ETA受体介导。     

欧前胡素通过ERK1/2信号保护心肌细胞低氧性损伤

目的:研究欧前胡素对低氧诱导的大鼠心肌细胞损伤的保护作用和机制。方法:采用CO2-95%和N2-5%的细胞培养箱诱导H9c2大鼠心肌细胞建立心肌细胞低氧损伤模型,采用不同浓度的欧前胡素孵育细胞12、24 h,检测上清液中乳酸脱氢酶(lactate dehydrogenase,LDH)、超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde MDA)的含量。采用MTT方法检测细胞的存活率,Annexin V/PI双标记流式细胞术检测细胞凋亡比例,蛋白质印迹法检测检ERK1/2蛋白的表达。结果:低氧处理H9c2心肌细胞12 h后,上清中LDH和MDA的含量分别为(523.28±90.29)U/L和(5.59±0.33)U/L,均明显高于对照组(P0.05),而SOD的含量[(12.23±1.38)U/mg]明显低于对照组(P0.05)。低、高浓度欧前胡素孵育12 h后,细胞存活率分别为(64.51±2.78)%和(73.22±3.56)%,低、高浓度欧前胡素孵育24 h后,细胞存活率分别为(80.21±4.67)%和(87.38±5.41)%,均较与模型组显著升高(P0.05)。高浓度欧前胡素孵育12 h和24 h后凋亡细胞比例分别为(39.67±4.11)%和(49.61±3.39)%,均较模型组显著降低(P0.05),10μmol/L的PD98059阻断ERK1/2信号通路后细胞存活率均较高浓度欧前胡素组显著降低,凋亡细胞比例较高浓度欧前胡素明显升高(P0.05)。高浓度欧前胡素孵育12 h和24 h后,ERK1/2蛋白相对表达量分别为(1.92±0.09)和(2.42±0.21),与模型组相比均显著增加(P0.05)。结论:欧前胡素可能通过活化ERK1/2信号通路保护低氧诱导的心肌细胞损伤。     

产藏红花素1(crocin)愈伤组织的诱导及其细胞系的筛选

对藏红花(cfocus sativus L)愈伤组织的诱导条件进行了优化.结果表明:Ms是藏红花芽愈伤组织的最佳诱导培养基,而B5是叶子和花愈伤组织的最佳培养基.藏红花芽、叶和花愈伤组织的最佳诱导温度分别是18℃、25℃和21℃.光照是叶子愈伤组织诱导的有利因素,但不利于芽和花愈伤组织的诱导.1.5～2.0 mg·L-1NAA和0.25 mg·L-6-BA是愈伤组织诱导的最佳激素组合.通过目视法和HPLC方法,从229株细胞系中筛选出细胞系Corml,其藏红花素1的含量是1 677 mg·g-,生长较快,且不易褐化.为采用植物细胞工程法解决藏红花素1资源短缺问题打下了基础.     

产藏红花素1(crocin)愈伤组织的诱导及其细胞系的筛选

对藏红花(Crocus sativus L.) 愈伤组织的诱导条件进行了优化。结果表明：MS 是藏红花芽愈伤组织的最佳诱导培养基,而B5是叶子和花愈伤组织的最佳培养基。藏红花芽、叶和花愈伤组织的最佳诱导温度分别是18℃、25℃和21℃。光照是叶子愈伤组织诱导的有利因素,但不利于芽和花愈伤组织的诱导。1.5～2.0 mg.L-1 NAA和0.25 mg.L-1 6-BA是愈伤组织诱导的最佳激素组合。通过目视法和HPLC方法, 从229株细胞系中筛选出细胞系Corm1,其藏红花素1 的含量是1 677 mg.g-1,生长较快,且不易褐化。为采用植物细胞工程法解决藏红花素1资源短缺问题打下了基础。     

内皮素-1预处理对培养乳鼠心肌细胞低氧损伤的保护作用

实验观察了 0 0 1- 1nmol/L内皮素 1(ET 1)预处理对低氧孵育 ( 3 %O2 5 %CO2 ,12h)的培养乳鼠心肌细胞乳酸脱氢酶 (LDH)释放量、培养液上清超氧化物歧化酶 (SOD)活性以及丙二醛 (MDA)含量的影响。用Fluo 3 /AM负载培养的心肌细胞 ,在激光扫描共聚焦显微镜下监测急性低氧的心肌细胞 [Ca2 +]i 的变化和ET 1预处理对低氧所致 [Ca2 +]i 变化的影响。结果如下 :( 1)心肌细胞低氧孵育 12h后 ,培养液上清LDH活力和MDA含量较常氧对照组明显升高 ,分别为 43 3 3± 1 2 1U/Lvs 19 3 3± 1 0 3U/L和 1 71± 0 0 2nmol/Lvs 0 91± 0 0 3nmol/L (P<0 0 1) ,SOD活性为 16 93± 1 11U/ml明显低于常氧对照组的 3 3 48± 1 15U/ml (P <0 0 1) ;0 0 1- 1nmol/LET 1预处理呈浓度依赖性抑制低氧培养心肌细胞LDH释放 ,减少培养液上清MDA含量、提高SOD活性 (P <0 0 1)。 ( 2 )低氧灌流后 2 9± 1 5s (n =2 3 )心肌细胞自发性钙瞬变完全终止 ,[Ca2 +]i 升高了 10 7± 13 2 % (P <0 0 0 1) ;0 0 1- 1nmol/LET 1能明显加快心肌细胞钙瞬变的频率 (P <0 0 1) ;ET 1预处理后低氧所致钙瞬变终止的时间较单纯低氧组明显推迟 ,[Ca2 +]i过度升高被明显减轻 (P <0 0 1)。上述结果表明 ,0 0 1- 1nmol/LET 1预处理可减轻培     

藏红花素对小鼠创伤性脑损伤保护作用的研究

目的:研究藏红花素对小鼠创伤性脑损伤的保护作用和可能的机制.方法:采用控制性皮层撞击法建立小鼠创伤性脑损伤模型,通过脑含水量测定和运动功能评分评价藏红花素对小鼠创伤性脑损伤的保护作用.结果:①藏红花素显著减轻创伤性脑损伤后脑水肿程度.②藏红花素显著减轻创伤性脑损伤造成的运动功能损伤.③藏红花素显著提高了脑组织SOD和GPX的活性,降低了MDA水平.结论:藏红花素通过抗氧化活性对小鼠创伤性脑损伤发挥保护作用.     

综述: 线粒体自噬在低氧适应中的作用

低氧是一种典型的应激环境,细胞在低氧条件下能量和氧化代谢发生改变,其中线粒体产生的大量活性氧严重威胁细胞的存活．线粒体自噬是近年来被发现的细胞适应低氧的一种适应性代谢反应．细胞在低氧条件下能通过上调低氧诱导因 子1(HIF-1),激活BNIP3/BNIP3L及Beclin-1介导的通路诱导线粒体自噬,最终减少ROS的产生,促进细胞的存活,使机体产生低氧适应．综述了线粒体自噬在低氧适应中的作用及其机制．     

同型半胱氨酸对心肌细胞的损伤作用及其信号转导机制探讨

目的：观察同型半胱氨酸（homocysteine,HCY)对心肌细胞的损伤作用,探讨该作用发生的信号转导机制及其关键调控环节。方法：分离培养Wistar乳鼠心肌细胞,经HCY作用后,以台盼蓝排斥实验测定细胞存活率,TUNEL法和流式细胞仪测定细胞凋亡率,免疫印迹法测定心肌细胞ERK2蛋白磷酸化水平,阻滞电泳法测定细胞NF－κB活化水平。结果：作用后,心肌细胞存活率显著降低,存活率降低程度与HCY的作用浓度、作用时间具有明确的剂量－效应关系及时间－效应关系;在10^-3mol/L HCY作用下,心肌细胞凋亡率升高,于4h达峰值,为7．56％;10^-3,10^-4、10^-5mol/L HCY均可抑制心肌细胞ERK2磷酸化,其中10^-3mol/L HCY作用于心肌细胞后,ERYK2蛋白磷酸化水平呈现迅速而明显的降低,4h降至对照组的3．04％（P＜0．01）;不同浓度HCY均明显阻抑NF－κB的活化。结论：HCY具有明显心肌细胞损伤作用,对心肌细胞凋亡的诱导是HCY心肌细胞损伤作用的形式之一;HCY可影响心肌细胞信号转导通路ERK,通过对转录因子NF－κB的活化抑制,导致心肌细胞损伤。     

HIF-1通路在低氧对成肌细胞增殖分化影响中的作用

氧是在环境中存在的并在发育过程中起作用的信号分子,调控着细胞的能量代谢、生长和分化。骨骼肌中存在着具有自我更新和分化为肌纤维能力的成肌细胞,出生后成肌细胞在骨骼肌的损伤修复和维持中起重要作用。尽管氧在几乎所有高级生命活动中具有重要作用,但有关氧水平对成肌细胞增殖和分化的影响的研究甚少。体内生理性和病理性微环境的氧浓度是处于低水平的,本文就低氧对成肌细胞增殖和分化的影响,以及HIF-1信号通路在其中的作用进行了综述。     

Effects of hypoxia on lipolysis in isolated rat myocardial cells

Summary The effect of hypoxia on myocardial lipolysis (glycerol release) was investigated in freshly isolated, calcium-tolerant rat ventricular myocytes. Hypoxia was produced by gassing the incubation medium (Joklik-minimum essential medium, supplemented with 1.2 mM MgSO₄, 1 mM DL-carnitine, 1.5 mM CaCl₂ and 0.6 mM palmitate bound to 0.15 mM fatty acid free bovine serum albumin) with 95% N₂–5% CO,. Control (normoxic) incubations were carried out under air-5% CO₂ atmosphere. Basal glycerol release increased from 46.6 ± 3.0 nmol/106 cells · 30 min in normoxia to 64.5 ± 4.3 nmol/10⁶ cells · 30 min in hypoxia (p < 0.05). Addition of isoprenaline (10 M) resulted in a significant (p < 0.05) stimulation of the glycerol release both in normoxia and in hypoxia, but the enhancement above basal rates was apparently lower in hypoxia (8.7 ± 2.5 nmol/10⁶ cells · 30 min) than in normoxia (12.2 ± 2.7 nmol/106 cells · 30 min). Furthermore, whereas the isoprenaline-induced rise in lipolysis both in normoxia and hypoxia was prevented by inclusion of propranolol (10 M), propranolol did not affect the hypoxia-induced increase in lipolysis. Thus, the above findings suggest that myocardial lipolysis may be stimulated by local non-adrenergic mechanisms during hypoxia.     

低氧对胚胎干细胞增殖的影响

目的:观察间歇性低氧和持续性低氧对体外培养的胚胎干细胞(ES细胞)增殖的影响.方法:利用细胞记数法和BrdU (5-溴脱氧尿苷)掺入的流式细胞分析检测细胞增殖,并用RT -PCR的方法检测低氧诱导因子(HIF-1a)的表达变化.结果:①将ES细胞分别放在低氧(3%～10% O2)和常氧(20% O2)的环境中培养24 h后,在低氧环境中培养的ES细胞数较常氧组明显减少;②将ES细胞分别给予间歇性低氧刺激(3%～10% O2),每天10 min,连续4 d后,发现3%低氧组较常氧对照组的细胞增殖明显升高.③用RT-PCR方法观察HIF-1a的表达与细胞增殖的关系,发现在常氧环境中培养的ES即有HIF-1a的表达,ES细胞在持续低氧24 h或间歇性低氧(3%～10% O2)刺激4 d后对HIF-1a的表达均无明显影响.结论:间歇性低氧(3% O2)可明显促进体外培养的ES细胞增殖,而持续性低氧抑制ES细胞增殖,间歇性低氧(3% O2)刺激促进ES细胞增殖的机制尚有待于进一步的研究.     

Hyperlipidemia does not prevent the cardioprotection by postconditioning against myocardial ischemia/reperfusion injury and the involvement of hypoxia inducible factor-1α upregulation

Hyperlipidemia is regarded as an independent risk factor in the development of ischemic heart disease, and it can increase the myocardial susceptibility to ischemia/reperfusion （I/R） injury, lschemic postconditioning （Postcon） has been demonstrated to attenuate the myocardial injury induced by I/R in normal conditions. But the effect of ischemic Postcon on hyperlipidemic animals is unknown. Hypoxia inducible factor-1 （HIF-1） has been demonstrated to play a central role in the cardioprotection by preconditioning, which is one of the protective strategies except for Postcon. The aim of this study was to determine whether Postcon could reduce myocardial injury in hyperlipidemic animals and to assess whether HIF-1 was involved in Postcon mechanisms. Male Wistar rats underwent the left anterior descending coronary occlusion for 30 min followed by 180min of reperfusion with or without Postcon after fed with high fat diet or normal diet for 8 weeks. The detrimental indices induced by the I/R insult included infarct size, plasma creatine kinase activity and caspase-3 activity. Results showed that hyperlipidemia remarkably enhanced the myocardial injury induced by I/R, while Postcon significantly decreased the myocardial injury in both normolipidemie and hyperlipidemic rats. Moreover, both hyperlipidemia and I/R promoted the HIF-αL expression. Most importantly, we have for the first time demonstrated that Postcon further induced a significant increase in HIF-αL protein level not only in normolipidemie but also in hyperlipidemic conditions. Thus, Postcon reduces the myocardial injury induced by I/R in normal and hyperlipidemic animals, and HIF-αL upregulation may involve in the Postcon-mediated cardioprotective mechanisms.     

缺氧诱导因子-1在非小细胞肺癌胸水中的表达及其与肿瘤血管生成的关系

目的探讨缺氧诱导因子-1(hypoxia inducible factor-1,HIF-1)在非小细胞肺癌胸水细胞中的表达及其与肿瘤血管生成的关系。方法利用免疫组织化学、免疫细胞化学分别检测1180例非小细胞肺癌标本中HIF-1α和HIF-1β蛋白的表达以及非小细胞肺癌胸水细胞中血管生成标记物CD34和VEGF的表达情况,其中包括1060例非小细胞肺癌胸水标本以及用于对照的120例肺穿刺非小细胞肺癌组织标本。结果 HIF-1α在非小细胞肺癌穿刺组织中的表达率72.50%(87/120)明显高于非小细胞肺癌胸水中表达率17.55%(186/1060),差异有统计学意义(P0.05);HIF-1β在非小细胞肺癌穿刺组织中的表达率为77.50%(93/120),而在非小细胞肺癌胸水中表达率为81.42%(863/1060),差异无统计学意义(P0.05);CD34、VEGF在非小细胞肺癌胸水细胞中阳性表达率分别为77.92%(826/1060)、82.92%(879/1060)。HIF-1α与HIF-1β的表达呈显著正相关(r=0.093,P=0.002),HIF-1α与VEGF的表达呈显著正相关(r=104,P=0.001),HIF-1β与VEGF的表达无相关性(P0.05)。结论 HIF-1α在非小细胞肺癌穿刺组织与非小细胞肺癌胸水中的差异性表达,可能与非小细胞肺癌胸水细胞缺氧适应性调节有关,HIF-1与肿瘤血管生成密切相关。     

低氧预适应对小鼠海马组织HIF-1与EPO低氧应答元件结合活性的影响

目的：观察低氧预适应对小鼠海马组织HIF-1与EPO的低氧应答元件（HRE）结合活性的变化,探讨这种变化与低氧预适应形成的关系。方法：小鼠低氧0次（H0）,1次（H1）,4次（H4）后取海马组织,应用凝胶迁移改变试验（EMSA）,染色体免疫共沉淀（ChIP）试验和荧光定量PCR（real—time PCR）技术,检测小鼠海马组织内HIF-1与EPO的低氧应答元件结合能力的变化。结果：EMSA体外结合实验及ChIP体内结合实验发现。H0、H1和H4组结合活力依次增强。结论：HIF-1与EPO的低氧应答元件结合增强可能参与预适应的形成。     

Hypoxia and cancer cell metabolism

The past decade has witnessed a rapid accumulation of evi- dence showing that hypoxic microenvironment, which is typical during cancer development, plays key roles in regu- lating cancer cell metabolism. In this review, we will focus on the role of hypoxic response, particularly, its master regulator hypoxia-inducible factor-I, in regulating glucose, lipid, as well as amino acid metabolism in cancer cells. We will also discuss the therapeutic opportunities by targeting specific pathways that facilitate metabolic reprogramming in cancer cells.     

The Shc protein Rai enhances T-cell survival under hypoxia

Hypoxia occurs in physiological and pathological conditions. T cells experience hypoxia in pathological and physiological conditions as well as in lymphoid organs. Indeed, hypoxia-inducible factor 1α (HIF-1α) affects T cell survival and functions. Rai, an Shc family protein member, exerts pro-survival effects in hypoxic neuroblastoma cells. Since Rai is also expressed in T cells, we here investigated its role in hypoxic T cells. In this work, hypoxia differently affected cell survival, proapoptotic, and metabolic programs in T cells, depending upon Rai expression. By using Jurkat cells stably expressing Rai and splenocytes from Rai^−/−mice, we demonstrated that Rai promotes T cell survival and affects cell metabolism under hypoxia. Upon exposure to hypoxia, Jurkat T cells expressing Rai show (a) higher HIF-1α protein levels; (b) a decreased cell death and increased Akt/extracellular-signal-regulated kinase phosphorylation; (c) a decreased expression of proapoptotic markers, including caspase activities and poly(ADP-ribose) polymerase cleavage; (d) an increased glucose and lactate metabolism; (e) an increased activation of nuclear factor-kB pathway. The opposite effects were observed in hypoxic splenocytes from Rai^−/−mice. Thus, Rai plays an important role in hypoxic signaling and may be relevant in the protection of T cells against hypoxia.     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。