缺氧诱导因子1α在大鼠急性缺血心肌组织中的变化

目的:了解缺氧诱导因子1αmRNA和蛋白质在大鼠急性缺血心肌组织中表达的变化规律。方法:实验在南通大学生物技术系,江苏省神经生物学重点实验室完成。①实验分组:雄性SD大鼠78只,其中42只大鼠随机分为7组,每组6只,分别设为空白对照组(0h)和缺血0.5h,1h,1.5h,2h,3h,4h组,检测缺氧诱导因子1αmRNA表达变化。另外36只大鼠随机分为6组,每组6只,分别设为空白对照组(0h)和缺血1h,2h,4h,5h,6h组,检测缺氧诱导因子1α蛋白表达变化。②实验方法:通过结扎大鼠左冠状动脉前降支,构建左室心肌缺血模型。术中心电图等证明结扎成功后,分别在上述不同时间点取缺血心肌组织,分别运用反转录聚合酶链式反应法和免疫组织化学方法检测缺血心肌组织中缺氧诱导因子1αmRNA和蛋白质的表达变化。结果:缺氧诱导因子1αmRNA和蛋白质在正常大鼠心肌中微量表达,心肌缺血后表达明显增高,并随缺血时间变化而变化。0.5h缺氧诱导因子1αmRNA表达显著增加(P<0.05),1.0～2.0h达到高峰(P<0.01),以后逐渐下降并趋向基线。心肌缺血后1h缺氧诱导因子1α蛋白质表达明显上升(P<0.05),4h达到高峰(P<0.01),随缺血时间延长表达回降。结论:缺氧诱导因子1α在缺血心肌组织中高表达。缺氧诱导因子1α在大鼠缺血心肌组织中表达的上调,可能参加了心肌缺血的代偿机制,有助于改善心肌供血。     

退变颈椎间盘组织中缺氧诱导因子1α的表达及意义木

目的:已有研究表明缺氧诱导因子1α参与血管生成与重塑、糖酵解、细胞的增殖与凋亡等,并发现其在人类腰椎间盘组织的表达,但尚未明确其在人类退行性变颈椎间盘的表达和生物学作用.实验拟进一步验证缺氧诱导因子1α在不同类型人颈椎间盘突出组织中的表达.方法:①对象:选取2005-12/2006-09中国医科大学盛京医院脊柱与关节外科因颈椎间盘突出症而行前路手术切除的椎间盘组织标本40例.男23例,女17例;年龄45-73岁:病程1个月～11年.其中纤维环破裂型20例,纤维环完整型20例.同时选择颈椎骨折行前路减压内固定术患者的颈椎间盘组织标本20例作为对照组,两组患者均知情同意.②实验过程及评估:采用苏木精伊红和链霉亲和素-过氧化物酶复合物免疫组织化学方法及显微图像分析技术,观察突出颈椎间盘组织学变化并测定颈椎间盘突出症患者椎间盘组织中缺氧诱导因子1α的表达情况.结果:①形态学变化:可见纤维环破裂型椎间盘退行性变严重程度高于纤维环完整型.②退行性变椎间盘组织中缺氧诱导因子1α表达:纤维环破裂组缺氧诱导因子1α在髓核和纤维环的表达高于纤维环完整组和对照组,差异有显著性(P<0.05),尤其在髓核细胞中表达更为显著:各组软骨终板中缺氧诱导因子1α呈低表达,差异无显著性(P>0.05).结论:退行性变颈椎间盘组织中缺氧诱导因子1α的表达与突出类型相关,推测缺氧诱导因子1α可能在抑制椎间盘的退行性变中发挥重要作用.     

常氧条件下同时表达突变型低氧诱导因子1α目的蛋白和人源化绿色荧光蛋白报告分子腺病毒真核表达载体的构建

背景:低氧诱导因子1能够调控多种基因共同表达,在骨缺损部位可诱导生理功能完整的新血管生成.目的:构建能够同时表达突变型低氧诱导因子1α(hypoxia inducible factor 1 alpha,HIF-1α)目的蛋白和人源化绿色荧光蛋白(human renilla reniformis green fluorescent protein,hrGFP)报告分子的新型腺病毒真核细胞表达载体.方法:对目的基因供体质粒pCMV6-XL5-HIF1α携带的人低氧诱导因子1α基因进行测序和对其序列内部限制性内酶识别位点进行分析,利用PCR(poly-merase chain reaction,PCR)技术定点突变低氧诱导因子1α基因编码区的第402位、564位和803位氨基酸,酶切、测序检测突变情况,将正确突变后的低氧诱导因子1α基因(突变mutagenesis,突变后的HIF-1α基因写作HIF-1αmu)定向连入腺病毒穿梭载体pShuttle-CMV-IRES-hrGFP-1中.携带HIF-1αmu基因的重组腺病毒穿梭载体经测序鉴定、Pme I酶切线性化后转化BJ5183-AD-1电感受态细胞,利用细菌内同源重组机制将HIF-1αmu和人源化绿色荧光蛋白基因连同其顺势表达元件重组入腺病毒基因组质粒,通过Pac I酶切及测序鉴定获得重组体.结果与结论:经基因测序证实,HIF-1α基因编码区的第402位、564位和803位氨基酸均定点突变成丙氨酸.经酶切鉴定及测序证实,重组腺病毒表达载体构建成功.结果表明,成功构建了新型重组腺病毒突变型真核细胞表达载体pAd-HIF1αmu-IRES-hrGFP-1.     

Mitochondrial regulation by c-Myc and hypoxia-inducible factor-1 alpha controls sensitivity to econazole

Econazole is an azole antifungal with anticancer activity that blocks Ca(2+) influx and stimulates endoplasmic reticulum (ER) Ca(2+) release through the generation of mitochondrial reactive oxygen species (ROS), resulting in sustained depletion of ER Ca(2+) stores, protein synthesis inhibition, and cell death. c-Myc, a commonly activated oncogene, also promotes apoptosis in response to growth factor withdrawal and a variety of chemotherapeutic agents. We have investigated the role of c-myc in regulating sensitivity to econazole. Here, we show that c-myc-negative cells are profoundly resistant to econazole. c-Myc-negative rat fibroblasts failed to generate mitochondrial ROS in response to econazole and consequently failed to deplete the ER of Ca(2+). HL60 cells knocked down for c-myc expression also displayed decreased ROS generation and decreased econazole sensitivity. Addition of H(2)O(2) restored sensitivity to econazole in both c-myc-negative rat fibroblasts and c-myc knocked-down HL60 cells, supporting a role for ROS in cell death induction. c-Myc-negative cells and HL60 cells knocked down for c-myc have reduced mitochondrial content compared with c-myc-positive cells. The hypoxia sensor, hypoxia-inducible factor-1alpha (HIF-1alpha), interacts antagonistically with c-myc and also regulates mitochondrial biogenesis. Knockdown of HIF-1alpha in c-myc-negative cells increased mitochondrial content restored ROS generation in response to econazole and increased sensitivity to the drug. Taken together, these results show that c-myc and HIF-1alpha regulate sensitivity to econazole by modulating the ability of the drug to generate mitochondrial ROS.     

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.     

构建双突变型低氧诱导因子1α腺病毒载体的实验

目的:前期实验已成功构建无突变及564位是单突变腺病毒载体。在此基础上构建人双突变型低氧诱导因子1α腺病毒表达载体,为进一步研究低氧诱导因子1α基因及其突变体的临床应用奠定基础。方法:实验于2005-12/2006-12在南方医院心内科重点实验室完成。①实验材料:限制性内切酶PacI(NEB),PI-SceI、I-CeuI(Clontech);IPTG、X-Gal(Takara),转染试剂Lipofectamine2000(Invitrogen),凝胶回收试剂盒(Omega);DMEM(Omega);小牛血清(PAA);北京天为时代公司病毒基因组提取试剂盒;HIF-1α单克隆抗体(Chemicon International公司);羊抗鼠辣根过氧化物酶标记二抗(北京鼎国生物公司)。质粒、菌珠、细胞系等:Adeno-XTM-Adenoviral Expression Systems(BD.CLONTECH):包括有pShuttle2、pShuttle2-lacZ、Adeno-XTMViralDNA等;重组穿梭质粒pShuttle2-HIF-1α-Ala564-Ala803及pShuttle2-lacZ(本室已构建成功);大肠杆菌DH5α(本室保存);HEK293A细胞(中科院上海细胞所)。②实验方法:采用分子克隆技术,以PI-SceI和I-CeuI双酶切重组穿梭质粒pShuttle2-HIF-1α-Ala564-Ala803,然后切下含有低氧诱导因子1αcDNA表达的盒片段,通过体外连接法与线性化的腺病毒骨架质粒Adeno-XViralDNA连接,重组成pAdeno-HIF-1α腺病毒质粒,经酶切及测序鉴定正确后,在HEK293A细胞中包装成为重组腺病毒Adeno-HIF-1α-Ala564-Ala803。③实验评估:进行PCR鉴定及滴度测定。用重组腺病毒转染293A,采用Westernblot鉴定低氧诱导因子1α蛋白表达。结果:提取重组腺病毒质粒pAdeno-HIF-1α-Ala564-Ala803以引物A、B和引物1、2、3、4进行PCR鉴定,分别扩增出287,460,214bp的3种引物片段,与预期一致,经酶切鉴定及基因测序证实重组腺病毒质粒构建成功。pAdeno-lacZ转染293A细胞后X-Gal原位染色,显示约有近20%左右细胞染成蓝色,病毒滴度为6.8×107pfu/mL。Adeno-HIF-1α-Ala564-Ala803转染293A后稳定表达低氧诱导因子1α蛋白。结论:成功构建了重组腺病毒突变型的Adeno-HIF-1α-Ala564-Ala803。     

低氧诱导因子1α慢病毒载体转染骨髓间充质干细胞的成骨基因表达

背景：成骨作用和血管发生在骨的形成和修复过程中紧密结合,而低氧诱导因子1α被认为是促血管新生相关基因调控中最重要的核心转录因子,其可促进缺氧部位血管的形成,进而促进骨的形成。 目的：构建野生型和点突变型两个低氧诱导因子1α慢病毒真核表达载体 Lenti-HIF1α-IRES-EGFP,并比较其对兔骨髓间充质干细胞成骨基因表达的影响。 方法：首先根据野生型人源低氧诱导因子1α基因序列信息和确定酶切位点的点突变型序列信息构建低氧诱导因子1α基因野生型和点突变型两个慢病毒真核表达载体;然后采用制备的病毒液转染兔骨髓间充质干细胞。 结果与结论：免疫荧光显微镜观察显示,转染7 d后野生型组和点突变型组细胞均未见明显荧光,转染14 d后两组细胞均呈现明显的绿色荧光;qPCR定量分析结果表明,转染7 d后即有低氧诱导因子1α和骨形态发生蛋白2基因的显著表达,转染14 d后,两基因仍然显示较高的表达水平。说明实验成功构建野生型和点突变型低氧诱导因子1α基因慢病毒真核表达载体,转染后可以促进兔骨髓间充质干细胞成骨基因的表达。     

Hypoxia-induced sensitization of transient receptor potential vanilloid 1 involves activation of hypoxia-inducible factor-1 alpha and PKC

The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), acts as a polymodal detector of pain-producing chemical and physical stimuli in sensory neurons. Hyperglycemia and hypoxia are two main phenomena in diabetes associated with several complications. Although many studies on streptozotocin-induced diabetic rats indicate that early diabetic neuropathy is associated with potentiation of TRPV1 activity in dorsal root ganglion neurons, its underlying mechanism and distinctive roles of hyperglycemia and hypoxia have not been completely clarified. Here, we show that hypoxic and high glucose conditions (overnight exposure) potentiate the TRPV1 activity without affecting TRPV1 expression in both native rat sensory neurons and human embryonic kidney-derived 293 cells expressing rat or human TRPV1. Surprisingly, hypoxia was found to be a more effective determinant than high glucose, and hypoxia-inducible factor-1 alpha (HIF-1α) seemed to be involved. In addition, high glucose enhanced TRPV1 sensitization only when high glucose existed together with hypoxia. The potentiation of TRPV1 was caused by its phosphorylation of the serine residues, and translocation of protein kinase C (PKC)ε was clearly observed in the cells exposed to the hypoxic conditions in both cell types, which was inhibited by 2-methoxyestradiol, a HIF-1α inhibitor. These data suggest that hypoxia is a new sensitization mechanism for TRPV1, which might be relevant to diabetes-related complications, and also for other diseases that are associated with acute hypoxia.     

缺氧诱导因子-1和肿瘤放射敏感性的研究进展

人体实体肿瘤中存在低氧微环境是放射治疗预后较差的一个重要原因,肿瘤细胞在低氧微环境中会产生一系列生理、生化改变,其中缺氧诱导因子-1(HIF-1)起重要作用。HIF-1在实体肿瘤中呈普遍高表达,其调控下游多个靶基因,而这些靶基因在肿瘤发展、侵袭和转移中发挥着重要作用,导致肿瘤放射治疗预后较差。因此,研究HIF-1与肿瘤放射敏感性的关系可以改善肿瘤的预后。本文就HIF-1生物学特性、HIF-1降低肿瘤放射敏感性的机制及HIF-1抑制剂能提高放射敏感性等方面进行综述,旨在探讨HIF-1与放射敏感性的关系,为以后进行更多前瞻性研究提供基础资料。     

缺氧诱导因子-1α、血管内皮生长因子在胃癌中的表达及意义

目的：研究缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）蛋白在胃癌中的表达及意义。方法应用免疫组织化学EnvisionTM二步法检测172例胃癌和30例癌旁正常胃组织（距癌灶5 cm以上）中HIF-1α和VEGF蛋白的表达,应用卡方趋势检验对HIF-1α及VEGF蛋白表达与胃癌临床病理参数进行相关性检验;应用Spearman秩相关检验对HIF-1α和VEGF表达之间相关性进行分析。结果在172例胃癌中,HIF-1α和VEGF蛋白的阳性表达率分别为55.81％（96/172）和63.37％（109/172）,30例癌旁中HIF-1α和VEGF表达均阴性,两组比较,差异均有统计学意义（χ2分别=11.16、13.76,P均＜0.0,5）;卡方趋势检验显示,胃癌组织中HIF-1α表达和VEGF表达与胃癌的临床分期、组织学分级、淋巴结转移和肿瘤坏死呈正相关（χ2分别=25.53、24.99、11.20、30.35;25.43、13.13、11.31、23.29, P均＜0.05）,与患者年龄和术后5年生存率均无关（χ2分别=3.92、4.63、4.79、4.65,P均＞0.05）。Spearman结果显示,胃癌组织中HIF-1与VEGF蛋白表达之间的关系呈正相关（r=0.65,P＜0.05）。结论胃癌组织中HIF-1α及VEGF的蛋白表达水平明显增高,并与胃癌的侵袭性生物学行为有关。     

低氧诱导因子-1的生物学特性与胚胎发育

低氧诱导因子-1(hypoxia-induciblefactor-1,HIF-1)是由α,β两个亚单位形成的二聚体,其中的HIF-1α受低氧的调节,通过与靶基因特定序列DNA结合而调控制它们的转录,这些基因包括血管内皮生长因子,红细胞生成素和其他的一系列与机体氧代谢有关的基因。文章着重论述了HIF-1的生物学特性及其与胚胎发育的关系。     

Acute intensive insulin therapy exacerbates diabetic blood-retinal barrier breakdown via hypoxia-inducible factor-1alpha and VEGF

Acute intensive insulin therapy is an independent risk factor for diabetic retinopathy. Here we demonstrate that acute intensive insulin therapy markedly increases VEGF mRNA and protein levels in the retinae of diabetic rats. Retinal nuclear extracts from insulin-treated rats contain higher hypoxia-inducible factor-1alpha (HIF-1alpha) levels and demonstrate increased HIF-1alpha-dependent binding to hypoxia-responsive elements in the VEGF promoter. Blood-retinal barrier breakdown is markedly increased with acute intensive insulin therapy but can be reversed by treating animals with a fusion protein containing a soluble form of the VEGF receptor Flt; a control fusion protein has no such protective effect. The insulin-induced retinal HIF-1alpha and VEGF increases and the related blood-retinal barrier breakdown are suppressed by inhibitors of p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol (PI) 3-kinase, but not inhibitors of p42/p44 MAPK or protein kinase C. Taken together, these findings indicate that acute intensive insulin therapy produces a transient worsening of diabetic blood-retinal barrier breakdown via an HIF-1alpha-mediated increase in retinal VEGF expression. Insulin-induced VEGF expression requires p38 MAPK and PI 3-kinase, whereas hyperglycemia-induced VEGF expression is HIF-1alpha-independent and requires PKC and p42/p44 MAPK. To our knowledge, these data are the first to identify a specific mechanism for the transient worsening of diabetic retinopathy, specifically blood-retinal barrier breakdown, that follows the institution of intensive insulin therapy.     

兔耳瘢痕成熟过程中血管内皮细胞生长因子与缺氧诱导因子1α的表达

背景:尽管近20年来在组织、细胞和分子各个水平上展开了瘢痕的研究,但瘢痕形成的完整机制尚未阐明,探索瘢痕的形成的分子机制,寻求理想的瘢痕防治方法是医学研究和实践中一项紧迫而艰巨的任务。目的:观察兔耳瘢痕从形成到消退成熟过程中血管内皮细胞生长因子与缺氧诱导因子1α变化,探讨瘢痕形成的分子机制。方法:制作兔耳瘢痕从形成到消退成熟的动物模型,收集兔耳术后14,30,60和90d瘢痕和正常皮肤,应用苏木精-伊红染色方法观察兔耳术后各时间点瘢痕的组织形态学变化,免疫组织化学法方法检测术后各时间点血管内皮细胞生长因子、缺氧诱导因子1α在瘢痕组织中的表达。结果与结论:术后14d瘢痕组织中血管内皮细胞生长因子和缺氧诱导因子1α表达较其他时间点和正常组织均升高,而随着时间的延长,瘢痕的萎缩成熟,两者的表达水平逐渐降低,并在术后90d达到正常组织表达水平。结果表明缺氧诱导因子1α与血管内皮细胞生长因子在瘢痕组织内氧分压的变化过程中起到一定的调节作用。