烫伤诱导大鼠心肌细胞缺氧诱导因子-1α定位表达的研究

目的 探讨 40 %Ⅲ度烫伤大鼠早期心肌组织中缺氧诱导因子 1α(HIF 1α)的蛋白表达的变化规律及其定位分布意义。方法 采用 40 %TBSAⅢ度烫伤大鼠模型 ,蛋白免疫印迹法 (Western blot)测定HIF 1α蛋白水平表达 ,免疫组化法定位显示HIF 1α表达。结果 烫伤后大鼠心肌组织内HIF 1α蛋白表达量明显升高。左、右心室HIF 1α蛋白表达不同 ,HIF 1α蛋白表达增加主要定位于细胞核。结论 严重烧伤可以诱导心肌组织HIF 1α蛋白表达增加。左心室对照组HIF 1蛋白含量明显高于右心室。HIF 1α蛋白表达增加主要分布于心肌细胞核内 ,诱导下游因子表达。

缺氧诱导因子(HIF-1α)与肿瘤病理的关系

缺氧是实体瘤生长中存在的普遍现象,它和肿瘤的发展、浸润和转移密切相关.缺氧诱导因子(hypoxia-in-ducible factor-1,HIF-1)是缺氧条件下广泛存在于哺乳动物及人体的一种转录因子,HIF-1是由α亚基和β亚基组成的异源二聚体,HIF-1α代表HIF-1的活性,其表达与肿瘤增殖、侵袭和转移、肿瘤新生血管生成等特征密切相关.HIF-1α对临床的诊断和对癌症治疗手段和新药物的研发有着重要的应用价值.

食管鳞癌细胞中缺氧诱导因子-1α与基质金属蛋白酶-2基因相关性研究

目的:观察食管鳞癌细胞中缺氧诱导因子-1α(HIF-1α)基因与基质金属蛋白酶-2(MMP-2)基因表达的关系以及与肿瘤的侵袭、转移行为的相关性。方法:以RNA干扰(RNAi)手段抑制HIF-1α的表达,构建HIF-1α基因沉默食管鳞癌细胞株;以稳定沉默HIF-1α基因的Eca-109细胞、100μmol/L氯化钴模拟缺氧细胞及未干预细胞为研究对象,通过Westernblot检测MMP-2基因在3种细胞中的表达差异,以了解其与HIF-1α基因的关系。结果:4个HIF-1α基因沉默Eca-109细胞组与空白对照细胞组相比HIF-1α蛋白表达均有明显下调,同时MMP-2的蛋白表达均出现不同程度的下降,灰度扫描提示差异有统计学意义(P<0.05)。结论:食管鳞癌中HIF-1α与MMP-2基因之间存在相关性。

人HIF1α基因重组逆转录病毒载体构建及鉴定

目的 构建人缺氧诱导因子 1α(HIF 1α)基因重组逆转录病毒载体 ,并观察其对NIH3T3细胞感染效率。方法 采用基因工程技术 ,经过 2次亚克隆将HIF 1α基因片段克隆至含IRES EGFP逆转录病毒载体上 ,鉴定后用脂质体法转染PT67细胞进行包装、扩增 ,最后用重组逆转录病毒感染NIH3T3细胞。其中采用PCR方法对重组载体进行鉴定 ,利用绿色荧光蛋白作为报告基因 ,对病毒滴度和感染效率进行检测。结果 酶切鉴定及PCR结果与HIF 1α基因重组逆转录病毒载体的预期结果一致 ,病毒滴度达 ( 1 4× 10 6pfu/ml) ,并对NIH3T3细胞有强感染能力。结论 应用基因工程技术 ,成功构建了含人HIF 1α基因重组逆转录病毒载体 。

Desferoxamine preconditioning protects against cerebral ischemia in rats by inducing expressions of hypoxia inducible factor 1α and erythropoietin

Objective To investigate whether desferoxamine （DFO） preconditioning can induce tolerance against cerebral ischemia and its effect on the expression of hypoxia inducible factor 1 α （HIF- 1α） and erythropoietin （EPO） in vivo and in vitro. Methods Rat model of cerebral ischemia was established by middle cerebral artery occlusion with or without DFO administration. Infarct size was examined by TTC staining, and the neurological severity score was evaluated according to published method. Cortical neurons were cultured under ischemia stress which was mimicked by oxygen-glucose deprivation （OGD）, and the neuron damage was assessed by MTT assay. Immunofluorescent staining was employed to detect the expressions of HIF-1 and EPO. Results The protective effect induced by DFO （decreasing the infarction volume and ameliorating the neurological function） appeared at 2 d after administration ofDFO （post-DFO）, lasted until 7 d and disappeared at 14 d （P 〈 0.05）; the most effective action was observed at 3 d post-DFO. DFO induced tolerance of cultured neurons against OGD： neuronal viability was increased 23%, 34%, 40%, 48% and 56% at 8 h, 12 h, 24 h, 36 h, and 48 h, respectively, post-DFO （P 〈 0.05）. Immunofluorescent staining found that HIF-1 α and EPO were upregulated in the neurons of rat brain at 3 d and 7 d post-DFO; increase of HIF-1 α and EPO appeared in cultured cortex neurons at 36 h and 48 h post-DFO. Conclusion DFO induced tolerance against focal cerebral ischemia in rats, and exerted protective effect on OGD cultured cortical neurons. DFO significant induced the expression of HIF- 1 α and EPO both in vivo and in vitro. DFO preconditioning can protect against cerebral ischemia, which may be associated with the synthesis of HIF- 1 α and EPO.

HIF-1对结直肠腺癌细胞凋亡的影响

【目的】探讨结直肠腺癌组织中HIF-1α、P53蛋白的表达及与凋亡的关系。【方法】采用免疫组化S-P法检测62例结直肠腺癌中HIF-1α、P53、MDM2蛋白的表达,用末端转移酶介导的dUTP切口末端标记法(TUNEI法)检测凋亡。【结果】结直肠癌组织中HIF-1α、P53、MDM2蛋白阳性率分别为46.77%(29/62),61.3%(41/62)和25.8%(16/62)。MDM2阳性率随组织学分级和临床分期有增高的趋势,并在B和C+D期之间有显著差异(P<0.05)。癌组织HIF-α阳性表达组的AI显著高于HIF-1α。阴性组(P<0.01);P53阴性组A1显著高于P53阳性组(P<0.05)。HIF-1α阳性P53阴性表达组AI显著高于HIF-1α阴性P53阳性组(P<0.01),HIF-1α与p53表达呈显著负相关(r=-0.31,P<0.01);HIF-1α与呈显著正相关(r=0.43,P<0.05)。MDM2阳性表达组AI显著低于其阴性组(P<0.01)。MDM2阳性HIF-1α阴性组AI显著低于MDM2阴性HIF-1α阳性组(P<0.05),MDM2与HIF-1α负相关(r=-0.24,P<0.05)。【结论】HIF-1α可诱导结直肠癌肿瘤细胞凋亡,其机制可能与P53和MDM2有关。

HIF-1α、VEGF及CD34在大鼠骨关节炎模型软骨中的表达及意义

目的：检测低氧诱导因子（HIF-1α）、血管内皮细胞生长因子（VEGF）及CD34在膝骨关节炎（OA）软骨中的表达。方法60只Wister大鼠，鼠龄3个月，体质量约200～250 g。随机分为实验组45只和对照组15只。实验组行单膝前交叉韧带切断术，对照组不做处理，作为空白组对照；术后9周处死动物，采用免疫组织化学苏木精-伊红（HE）染色对实验组和对照组HIF-1α、VEGF、CD34表达进行检测。结果 HIF-lα、VEGF、CD34在实验组中阳性表达率分别为66.67%（30/45）、73.33%（33/45）、71.11%（32/45），均明显高于对照组；两组数据之间的差异有统计学意义（P〈0.05）。实验组HIF-1α、VEGF、CD34阳性细胞计数显著高于对照组（P〈0.05）；HIF-1α与VEGF、CD34表达存在正相关（r=0.249、0.289，P〈0.05）。结论 OA软骨中HIF-1α、VEGF及CD34高表达，HIF-1α与VEGF、CD34表达存在正相关，且HIF-1α、VEGF与CD34在OA软骨退行性变进展中共同发挥着重要作用。

Oxidative stress and hypoxia-induced factor 1α expression in gastric ischemia

AIM:To investigate the relation of reactive oxygen species (ROS) to hypoxia induced factor 1α (HIF-1α) in gastric ischemia. METHODS:The animal model of gastric ischemia reperfusion was established by placing an elastic rubber band on the proximal part of the bilateral lower limb for ligature for 3 h and reperfusion for 0,1,3,6,12 or 24 h. Ischemic post-conditioning,three cycles of 30-s reperfusion and 30-s femoral aortic reocclusion were conducted before reperfusion. Histological and immunohistochemical methods were used to assess the gastric oxidative damage and the expression of HIF1-α in gastric ischemia. The malondialdehyde (MDA) content and superoxide dismutase (SOD),xanthine oxidase (XOD) and myeloperoxidase (MPO) activities were determined by colorimetric assays. RESULTS:Ischemic post-conditioning can reduce post-ischemic oxidative stress and the expression of HIF-1α of gastric tissue resulting from limb ischemia reperfusion injury. MDA,SOD,XOD and MPO were regarded as indexes for mucosal injuries from ROS,and ROS was found to affect the expression of HIF-1α under gastric ischemic conditions. CONCLUSION:ROS affects HIF-1α expression under gastric ischemic conditions induced by limb ischemia reperfusion injury. Therefore,ROS can regulate HIF-1α expression in gastric ischemia.

The involvement of hypoxia-inducible factor 1 alpha in Toll-like receptor 7/8-mediated inflammatory response

Toll-like receptors （TLRs） 7 and 8 are crucial in host defence against single-stranded RNA （ssRNA） viruses. Such viruses cause severe illnesses, which remain a serious medical burden in both industrialised and developing countries. TLR7/8 downstream signaling leads tO a dramatic cellular stress associated with energy consumption. However, the molecular mechanisms of cell survival and adaptation to TLR7/8-induced stress, which give the cells an opportunity to initiate proper inflammatory reactions, are not clear at all. Here we report for the first time that ligand-induced activation of TLR7/8 leads to the accumulation of hypoxia-inducible factor 1 alpha （HIF-1α） protein in THP-1 human myeloid macrophages via redoxand reactive nitrogen species-dependent mechanisms. MAP kinases and phosphoinositol-3K are not involved in TLR7/8-mediated HIF-1α accumulation. Experiments with HIF-1α knockdown THP- 1 cells have clearly demonstrated that HIF-1α is important for the protection of these cells against TLR7/8-induced depletion of ATP. Thus, HIF-1α might support both cell survival and the production of pro-inflammatory cytokines upon TLR7/8 activation.

HIF-1α siRNA Leads to Apoptosis of Pancreatic Cancer Cells under Hypoxic Conditions

OBJECTIVE To explore the role （HIF-1α） in the proliferation and cells under hypoxic conditions. of hypoxic inducible factor-1α apoptosis of pancreatic cancer METHODS A cassette encoding small interference RNA （siRNA） targeting HIF-1α mediated by recombinant adeno-associated virus （rAAV） was constructed, giving rAAV-siHIE rAAV-siHIF or rAAV- hrGFP was transfected into exponentially growing MiaPaCa2 cells under hypoxic conditions. Then, the expression of HIF-1α mRNA and protein, the proliferation and apoptosis of MiaPaCa2 cells were examined, using real-time PCR, Western Blot, MTT and TUNEL, respectively. RESULTS Under hypoxic conditions, rAAV-siHIF inhibited the expression of HIF-1α mRNA and protein in MiaPaCa2 cells. At the same time, rAAV-siHIF decreased MiaPaCa2 cell proliferation and induced apoptosis. However, rAAV-hrGFP had no effect on the expression of HIF-1α as well as the proliferation and apoptosis of MiaPaCa2 cells under hypoxic conditions. CONCLUSION Under hypoxic conditions, HIF-1α plays a key role in the proliferation of MiaPaCa2 cells, and inhibition of HIF- 1α expression can lead to MiaPaCa2 cell apoptosis.

Abnormal expression of hypoxia inducible factor-1α and clinical values of molecular-targeted interference in hepatocellular carcinoma

Objective:The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods:HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection,the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results:Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%,χ2=22.35,P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection,the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level,with VEGF and ANG-2 decreased to 54% and 36%,respectively.After RNA interference combined with anti-cancer drug,the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%,with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion:Abnormal expression of HIF-1α is associated with development of HCC,and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.

Inhibition of tumor angiogenesis by TTF1 from extract of herbal medicine

AIM:To study the inhibition of tumor angiogenesis by 5,2,4'-trihydroxy-6,7,5'-trimethoxyflavone(TTF1) isolated from an extract of herbal medicine Sorbaria sorbifolia.METHODS:Angiogenic activity was assayed using the chick embryo chorioallantoic membrane(CAM) method.Microvessel density(MVD) was determined by staining tissue sections immunohistochemically for CD34 using the Weidner capillary counting method.The mRNA and protein levels of vascular endothelial growth factor(VEGF),vascular endothelialgrowth factor receptor 2(VEGFR2,Flk-1/KDR),basic fibroblast growth factor(bFGF),cyclo-oxygenase(COX)-2 and hypoxia-inducible factor(HIF)-1α were detected by quantitative real-time polymerase chain reaction and Western blotting analysis.RESULTS:The TTF1 inhibition rates for CAM were 30.8%,38.2% and 47.5% with treatment concentrations of 25,50 and 100 μg/embryo × 5 d,respectively.The inhibitory rates for tumor size were 43.8%,49.4% and 59.6% at TTF1 treatment concentrations of 5,10,and 20 μmol/kg,respectively.The average MVD was 14.2,11.2 and 8.5 at treatment concentrations of 5 μmol/kg,10 μmol/kg and 20 μmol/kg TTF1,respectively.The mRNA and protein levels of VEGF,KDR,bFGF,COX-2 and HIF-1α in mice treated with TTF1 were significantly decreased.CONCLUSION:TTF1 can inhibit tumor angiogenesis,and the mechanism may be associated with the down-regulation of VEGF,KDR,bFGF,HIF-1α and COX-2.

Critical protein GAPDH and its regulatory mechanisms in cancer cells

Glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, initially identified as a glycolytic enzyme and considered as a housekeeping gene, is widely used as an internal control in experiments on proteins, mRNA, and DNA. However, emerging evidence indicates that GAPDH is implicated in diverse functions independent of its role in energy metabolism; the expression status of GAPDH is also deregulated in various cancer cells. One of the most common effects of GAPDH is its inconsistent role in the determination of cancer cell fate. Furthermore, studies have described GAPDH as a regulator of cell death; other studies have suggested that GAPDH participates in tumor progression and serves as a new therapeutic target. However, related regulatory mechanisms of its numerous cellular functions and deregulated expression levels remain unclear. GAPDH is tightly regulated at transcriptional and pnsttranscriptional levels, which are involved in the regulation of diverse GAPDH functions. Several cancer-related factors, such as insulin, hypoxia inducible factor-1 （HIF-1）, p53, nitric oxide （NO）, and acetylated histone, not only modulate GAPDH gene expression but also affect protein functions via common pathways. Moreover, posttranslational modifications （PTMs） occurring in GAPDH in cancer cells result in new activities unrelated to the original glycnlytic function of GAPDH. In this review, recent findings related to GAPDH transcriptional regulation and PTMs are summarized. Mechanisms and pathways involved in GAPDH regulation and its different roles in cancer cells are also described.

Hypoxia-inducible factor 1 alpha upregulates the expression of inducible nitric oxide synthase gene in pulmonary arteries of hypoxic rat

Objective To investigate the expression of hypoxia inducible factor 1 alpha (HIF 1α) and inducible nitric oxide synthase (iNOS) genes in rats’ pulmonary arteries in different phases of hypoxia induced pulmonary hypertension development Methods Models of chronic hypoxic pulmonary hypertension rat were duplicated by intermittent hypoxia Mean pulmonary arterial pressure (mPAP) was measured by right heart catheterization HIF 1α and iNOS messenger ribonucleic acid (mRNA) were detected by in situ hybridization HIF 1α and iNOS protein were measured by immunohistochemical analysis Results Expression of HIF 1α protein was upregulated in pulmonary arterial tunica intimae of all hypoxic rats In pulmonary arterial tunica media, the level of HIF 1α protein was markedly upregulated at days 3 and 7 of hypoxia ( P 【0 01), then tended to restore at 14 days and 21 days HIF 1α mRNA levels in pulmonary arteries of rats began to increase significantly at day 14 of hypoxia ( P 【0 01) Expression of iNOS mRNA and protein in pulmonary arteries of rats were upregulated by hypoxia for 3 days ( P 【0 01), then reached its peak and maitained the same level while the extension of hypoxia Linear correlation analysis showed that iNOS protein was associated with both mean pulmonary arterial pressure ( r =0 74, P 【0 01) and hypoxic pulmonary vascular remodeling ( r =0 78, P 【0 01), whereas the inverse was associated with HIF 1α protein ( r =-0 52, P 【0 01) Conclusions HIF 1α and iNOS are both involved in the pathogenesis of hypoxia induced pulmonary hypertension in rat HIF 1α protein may upregulate the expression of iNOS gene by transcriptional activation; in addition, iNOS protein may inhibit the expression of HIF 1α protein

Sumoylation of hypoxia inducible factor-1α and its significance in cancer

Hypoxia-inducible factor-1(HIF-1)is a key heterodimeric transcription factor for the cellular adaptive response to hypoxia,a common feature of the microenvironment in solid tumors.The transcriptional activity,protein stabilization,protein-protein interactions and cellular localization of HIF-1α,an oxygen-sensitive subunit of HIF-1,are mainly modulated by various post-translational modifications.Recently,we reported that polycomb chromobox 4(Cbx4)governs the transcriptional activity of HIF-1αby enhancing its sumoylation at K391 and K477,through which Cbx4 potentiates angiogenesis of hepatocellular carcinoma.This review summarizes the current knowledge of HIF-1α sumoylation and its roles in the pathogenesis of cancer.