氧诱导因子-1与恶性肿瘤

氧平衡是哺乳动物维持正常生长发育和生理过程的基础，是维持细胞生存的必要因素。缺氧诱导因子-1(hypoxia—induciblefactor1，HIF-1)是调节生理或病理状态下氧平衡的关键性转录调控因子，可介导涉及无氧糖代谢、氧输送、新生血管生成和血管扩张等数十种靶基因的转录。HIF-1在肿瘤     

低氧诱导因子-1类泛素化修饰在血液系统肿瘤中的潜在作用

低氧诱导因子-1(HIF-1)在人体内广泛存在,是氧稳态和缺氧反应调节的一种转录因子,它与血管生成、细胞存活、肿瘤侵袭和耐药形成等多种生理、病理生物学过程相关~([1]).HIF-1在肿瘤中的作用现已被广泛研究.而类泛素(Small Ubiquitin-related Modifier)作为翻译后修饰的重要调控因子,是近年基因表达研究的热点之一.     

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.     

HIF-1α与急性白血病关系的研究进展

低氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)是机体内低氧反应的重要转录调控因子,主要由HIF-1α和HIF-1β两个亚基组成.HIF-1β在组织中呈内在性表达,而HIF-1α蛋白受氧浓度调控,在常氧状态下通过泛素化作用被蛋白酶快速降解,但在低氧时进入细胞核中与HIF-1β结合,调控多种低氧反应性基因的转录与表达.近年的研究表明,HIF-1对肿瘤细胞的能量代谢及肿瘤组织内的血管生成也具有重要调节作用,但关于HIF-1α在急性白血病细胞的表达及功能研究较少.现将HIF-1与急性白血病的关系做一综述.     

低氧诱导因子及其他相关因子与缺血性卒中发病机制的研究进展

低氧是脑血管疾病尤其是缺血性脑卒中过程中一个重要的病理生理因素,是卒中过程中低氧应答时基因表达和恢复内环境平衡的调节中心。低氧诱导因子（HIF-1）对缺血坏死后新生血管的形成和低氧诱导的细胞凋亡过程都是非常关键的因素。调控HIF-1水平可能为缺血性疾病的治疗开辟新的途径。     

低氧对小鼠缺氧诱导因子-1α、P53和血管内皮生长因子表达的影响

目的：研究低氧时小鼠肺组织中缺氧诱导因子-1α（HIF-1α）、P53及血管内皮生长因子（VEGF）表达的变化,探讨三者之间的关系,以及低氧与血管新生的关系.方法：实验用雄性昆明小鼠,分为低氧组与对照组,低氧仓浓度分别为10%、7%、5%.低氧时间分别为3天、6天、9天.用免疫组织化学技术检测小鼠在低氧条件下肺组织中的HIF-1α、P53和VEGF蛋白表达的变化及微血管密度（MVD）.结果：低氧组HIF-1α、P53和VEGF蛋白表达均增加,并且随低氧时间的延长及低氧浓度的降低而增强,而对照组HIF-1α无表达,P53和VEGF有少量表达（P＜0.05）;低氧组的MVD也高于对照组;P53,VEGF表达与MVD均与HIF-1α表达呈正相关（r分别为0.609、0.730与0.691）.结论：HIF-1α/VEGF通路在低氧致小鼠肺组织血管新生过程中起重要作用,P53基因的失活可能经HIF-1α/VEGF通路促进血管新生.     

低氧诱导因子-1α在骨髓增生异常综合征患者血清中的表达及意义

目的检测低氧诱导因子-1α(HIF-1α)在骨髓增生异常综合征(MDS)血清中的表达,探讨其与MDS发生、发展的关系。方法采用双抗体夹心酶联免疫吸附试验(ELISA)检测30例MDS患者血清中HIF-1α水平及血管内皮生长因子(VEGF)水平,并与健康对照组进行比较。结果 MDS组血清中HIF-1α及VEGF较健康对照组明显增高(P<0.05);MDS高危组HIF-1α水平明显高于低中危组(P<0.05)。结论血清中HIF-1α增高与MDS不良预后相关。     

HIF-1α及其在视网膜缺氧相关疾病中的研究进展

HIF-1是Semenza GL等1992年发现的一种氧依赖转录激活因子，HIF-1的发现使人们对缺氧缺血状态下机体的各种应答反应机制的研究更加深入。HIF-1是细胞中调节血管生成主要缺氧反应信号蛋白，它能诱导多达70多种转录因子瞳一，包括促红细胞生成素（EPO）、糖酵解酶、诱导型一氧化氮合酶（INOS）和血管内皮生长因子（VEGF）等，这些基因涉及血管发生、红细胞生成、能量代谢、细胞凋亡和增殖等多方面，在细胞和机体的生理病理反应中至关重要。通过纯化证实HIF-1由120000的a亚单位和91～94000的8亚单位组成，其中HIF-1α则为HIF-1所特有，其活性和表达决定HIF-1的生理活性。HIF-1特别是HIF-1α在缺氧相关疾病中的作用越来越受到人们的重视。故本文对HIF-1α及其在视网膜相关疾病中的研究进展作一综述。     

缺氧诱导因子-1α和基质蛋白酶-9在鼻息肉嗜酸性粒细胞浸润过程中的作用

目的：探讨缺氧诱导因子-1α（HIF-1α）和基质金属蛋白酶-9（MMP-9）在鼻息肉组织中的表达及其与嗜酸性粒细胞（EOS ）浸润的关系。方法于2010年10月至2012年12月,以31例经鼻内窥镜手术治疗的鼻息肉患者作为实验组,以11例行下鼻甲部分切除术治疗的慢性肥厚性鼻炎患者作为对照组,分别采集鼻息肉组织和下鼻甲黏膜组织,检测并分析组织中 HIF-1α、MMP-9 mRNA表达水平,以及 HIF-1α和MMP-9阳性细胞数、阳性血管数和EOS浸润数。结果观察组HIF-1α和MMP-9 mRNA表达水平明显高于对照组（P＜0．05）。观察组HIF-1α和MMP-9阳性细胞数、阳性血管数和EOS浸润数均高于对照组（P＜0．05）。观察组 HIF-1α和MMP-9阳性细胞数与EOS浸润数均呈正相关（相关系数分别为0．56、0．88,P＜0．05）。结论 HIF-1α和MMP-9在鼻息肉组织中呈高水平表达,二者可能在鼻息肉在发病机制中具有重要作用。     

低氧诱导因子及其低氧调节机制

低氧诱导因子(hypoxis inducible factor，HIF)是介导哺乳动物细胞适应低氧状况的核转录因子，由α和β亚基组成的异二聚体，其中α亚基受氧调节，是调节HIF活性的功能亚单位。目前已知HIF-1α的氧调节途径主要包括两种：常氧下VHL蛋白与氧依赖降解结构域羟化的Pro-564．结合，介导HIF-1α的常氧下的泛素降解；常氧下天冬胺酰羟化酶使酶使HIF-1αAsp-803羟基化，抑制其募集共刺激因子p300／CBP的作用。从而抑制其转录活性。此外，还包括PAS结构蛋白抑制因子等低氧调节途径。深入研究HIF的低氧调节机制，有利于认识缺血缺氧性疾病的发病机制。     

Sirtuin 1 in lipid metabolism and obesity

Abstract

Sirtuin 1 (SIRT1), the mammalian ortholog of yeast Sir2, is a highly conserved NAD⁺-dependent protein deacetylase that has emerged as a key metabolic sensor that directly links environmental nutrient signals to animal metabolic homeostasis. SIRT1 is known to be involved in gluconeogenesis in the liver, fat mobilization in white adipose tissue, and insulin secretion in the pancreas. Recent studies have shown SIRT1 to regulate fatty acid oxidation in the liver, sense nutrient availability in the hypothalamus, influence obesity-induced inflammation in macrophages, and modulate the activity of the circadian clock in metabolic tissues. The activity of SIRT1 also appears to be under the control of AMPK and adiponectin. This review focuses on the involvement of SIRT1 in regulating metabolic diseases associated with obesity. It includes brief overviews of sirtuin signaling, with emphasis on SIRT1's role in the liver, macrophage, brain, and adipose tissue as it relates to obesity.     

HIF-1α、VEGF及MMP-9在非小细胞肺癌组织中的表达及其意义

[目的]探讨缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)及基质金属蛋白酶-9(MMP-9)在非小细胞肺癌(NSCLC)组织中的表达.[方法]采用免疫组化法检测91例NSCLC组织及相对应的癌旁正常组织中hIF-1α、VEGF及MMP-9的阳性表达,并分析其与NSCLC临床病理参数的关系.[结果]NSCLC中HIF-1α、VEGF及MMP-9阳性率均显著高于癌旁正常组织(P<0.05);低分化NSCLC中hIF-1α、VEGF及MMP-9阳性率高于中高分化(P<0.05);Ⅲ~Ⅳ期的NSCLC中hIF-1α、VEGF及MMP-9阳性率高于Ⅰ~Ⅱ期(P<0.05),有淋巴结转移的NSCLC中hIF-1α、VEGF及MMP-9阳性率高于无淋巴结转移的(P<0.05);而NSCLC中HIF-1α、VEGF及MMP-9阳性率与年龄、性别、肿瘤直径及病理类型无关(P>0.05);NSCLC中hIF-1α、VEGF及MMP-9阳性表达呈两两正相关(P<0.05).[结论]HIF-1α、VEGF及MMP-9在NSCLC组织中高表达,且与病情的发生发展相关,三者联合检测可能为NSCLC的预后评估及靶向治疗提供依据.     

SIRT1/FOXO1信号通路对小鼠脑缺血再灌注损伤的调控作用

目的 探讨沉默信号调节器1(SIRT1)/叉头转录因子(FOXO1)信号通路对小鼠脑缺血再灌注损伤的调控作用。方法 24只清洁级C57雄性小鼠随机分为假手术组、缺血/再灌注组(I/R组)、SIRT1FOXO1信号通路抑制组(实验组),每组8只小鼠。I/R组与实验组采用夹闭、开放双侧颈总动脉法建立脑缺血再灌注损伤小鼠模型,同时实验组给予EX527(SIRT1抑制剂)腹腔注射,I/R组和假手术组腹腔注射等剂量的生理盐水。比较三组的神经功能缺损评分、脑组织含水量、感觉功能和行走协调能力评分,血浆中白介素-6(IL-6)、白细胞介素1(IL-1β)、细胞间黏附分子-1(ICAM-1)和肿瘤坏死因子-α(TNF-α)水平,SIRT1、FOXO1、NF-E2相关因子2(Nrf2)和血红素加氧酶1(HO-1)蛋白表达量,B淋巴细胞瘤-2(Bcl-2)、半胱氨酸蛋白酶-3(Caspase-3)、BCL-2关联蛋白X(Bax)和半胱氨酸蛋白酶-9(Caspase-9) mRNA表达,超氧化物歧化酶(SOD)、一氧化氮(NO)、丙二醛(MDA)和谷胱甘肽(GSH)含量。结果与假手术组相比,I/R组经功能缺损评分、行走协调能力评分和感觉功能、脑组织含水量、炎症因子水平、FOXO1蛋白表达、BAX、Caspase-3和Caspase-9 mRNA表达以及氧化应激指标NO和MDA含量均显著增加(P <0. 05),Nrf2、SIRT1和HO-1蛋白表达、Bcl-2 mRNA表达以及SOD和GSH含量均显著降低(P <0. 05);与I/R组相比,实验组所有指标变化更显著。结论 抑制SIRT1/FOXO1信号通路后,脑缺血/灌注小鼠神经功能降低,脑损伤程度均加重,由此推测SIRT1/FOXO1信号通路的激活与抑制脑缺血/灌注后的炎症反应、减轻氧化应激作用以及减缓细胞凋亡进程相关。     

Sirtuin 1 in lipid metabolism and obesity

Sirtuin 1 (SIRT1), the mammalian ortholog of yeast Sir2, is a highly conserved NAD(+)-dependent protein deacetylase that has emerged as a key metabolic sensor that directly links environmental nutrient signals to animal metabolic homeostasis. SIRT1 is known to be involved in gluconeogenesis in the liver, fat mobilization in white adipose tissue, and insulin secretion in the pancreas. Recent studies have shown SIRT1 to regulate fatty acid oxidation in the liver, sense nutrient availability in the hypothalamus, influence obesity-induced inflammation in macrophages, and modulate the activity of the circadian clock in metabolic tissues. The activity of SIRT1 also appears to be under the control of AMPK and adiponectin. This review focuses on the involvement of SIRT1 in regulating metabolic diseases associated with obesity. It includes brief overviews of sirtuin signaling, with emphasis on SIRT1's role in the liver, macrophage, brain, and adipose tissue as it relates to obesity.     

Oxygen homeostasis and cancer: insights from a rare disease

Many aspects of physiology and anatomy are precisely adjusted so that the right amount of oxygen reaches cell throughout the body. Hypoxia-inducible factor-1 (HIF-1) is activated by low oxygen tension in all mammalian cells and underpins many aspects of the impressive ability to match oxygen supply and demand. As examples, HIF-1 regulates: local capillary architecture via angiogenic signalling red cell production via erythropoietin cellular metabolism via increased expression of glucose transporters and glycolytic enzymes. HIF-1 is also important in disease, for example in cancer where it is involved in angiogenesis. This review describes how HIF-1 is regulated by oxygen and the central role played by the von Hippel-Lindau tumour suppressor protein. The underlying oxygen sensor is provided by a family of enzymes which oxidize specific proline residues in HIF alpha subunits. Inhibiting these newly discovered enzymes provides a way of activating HIF-1 in the presence of oxygen--an exciting prospect for therapeutic intervention in ischaemic diseases.     

Deferoxamine preconditioning potentiates mesenchymal stem cell homing in vitro and in streptozotocin-diabetic rats

Objective: Today, cell therapy is considered a promising alternative in treatment of several diseases such as type 1 diabetes. Loss of transplanted stem cell and more importantly scarcity in the number of cells reaching to target tissue is a major obstacle in cell therapy. There is evidences showing that deferoxamine (DFO), an iron chelator, increases the mobilization and homing of progenitor cells through increasing the stability of hypoxia-inducible factor 1α (HIF-1α) protein. In this study, the effect of DFO on some factors involved in homing of bone marrow-derived mesenchymal stem cell was investigated, and the other objectives of this research were to determine whether DFO is able to increase migration and subsequent homing of mesenchymal stem cell (MSCs) both in vitro and in vivo in streptozotocin-diabetic rats.

Research design and methods: MSCs were treated by DFO in minimal essential medium α (αMEM) for 24 h. The expression and localization of HIF-1α were evaluated by western blotting and immunocytochemistry. The expression of C-X-C chemokine receptor type 4 (CXCR-4) and chemokine receptor 2 (CCR2) were assessed by western blotting and RT-PCR. The activity of matrix metalloproteinases (MMP) -2 and -9 were measured by gelatin zymography. Finally, in vitro migration of MSCs toward different concentrations of stromal cell-derived factor and monocyte chemotactic protein-1 were also evaluated. To demonstrate the homing of MSCs in vivo, DFO-treated chloromethyl-benzamidodialkylcarbocyanine-labeled MSCs were injected into the tail vein of rats, and the number of stained MSCs reaching to the pancreas were determined after 24 h.

Results: In DFO-treated MSCs, expression of HIF-1α (p < 0.001), CXCR4 (p < 0.001), CCR2 (p < 0.001), and the activity of MMP-2 (p < 0.01) and MMP-9 (p < 0.05) were significantly increased compared to control groups. Elevation of HIF-1α, upregulation of CXCR4/CCR2 and higher activity of MMP-2/MMP-9 in DFO-treated MSCs were reversed by 2-methoxyestradiol (2-ME; 5 μmol), a HIF-1α inhibitor. The in vitro migrations as well as in vivo homing of DFO-treated MSCs were also significantly higher than control groups (p < 0.05).

Conclusions: Preconditioning of MSCs by DFO prior to transplantation could increase homing of MSCs through affecting some chemokine receptors as well as proteases involved and eventually improving the efficacy of cell therapy.     

Sirtuin 1 inhibition delays cyst formation in autosomal-dominant polycystic kidney disease

Autosomal-dominant polycystic kidney disease (ADPKD) is caused by mutations in either PKD1 or PKD2 and is characterized by the development of multiple bilateral renal cysts that replace normal kidney tissue. Here, we used Pkd1 mutant mouse models to demonstrate that the nicotinamide adenine dinucleotide–dependent (NAD-dependent) protein deacetylase sirtuin 1 (SIRT1) is involved in the pathophysiology of ADPKD. SIRT1 was upregulated through c-MYC in embryonic and postnatal Pkd1-mutant mouse renal epithelial cells and tissues and could be induced by TNF-α, which is present in cyst fluid during cyst development. Double conditional knockouts of Pkd1 and Sirt1 demonstrated delayed renal cyst formation in postnatal mouse kidneys compared with mice with single conditional knockout of Pkd1. Furthermore, treatment with a pan-sirtuin inhibitor (nicotinamide) or a SIRT1-specific inhibitor (EX-527) delayed cyst growth in Pkd1 knockout mouse embryonic kidneys, Pkd1 conditional knockout postnatal kidneys, and Pkd1 hypomorphic kidneys. Increased SIRT1 expression in Pkd1 mutant renal epithelial cells regulated cystic epithelial cell proliferation through deacetylation and phosphorylation of Rb and regulated cystic epithelial cell death through deacetylation of p53. This newly identified role of SIRT1 signaling in cystic renal epithelial cells provides the opportunity to develop unique therapeutic strategies for ADPKD.     

SIRT1 RNAi knockdown induces apoptosis and senescence, inhibits invasion and enhances chemosensitivity in pancreatic cancer cells

The NAD(+)-dependent deacetylase, sirtuin 1 (SIRT1), has been recently been suspected to have a role in tumorigenesis. We investigated the expression of SIRT1 in pancreatic cancer and the effect of SIRT1-targeted RNA interference (RNAi) on cell proliferation and tumor formation in a pancreatic cancer cell line, PANC1. The expression of SIRT1 was investigated in 49 specimens of pancreatic cancer and adjacent normal pancreatic tissues. SIRT1 was overexpressed in pancreatic cancer tissues at both the mRNA and protein levels, with increased SIRT1 positivity associated with tumors from patients over 60 years old, tumors larger than 4 cm, higher TNM (extent of tumor (T), the extent of spread to lymph nodes (N), and presence of distant metastasis (M)) stage or the presence of lymph node or hepatic metastases. The PANC-1 was stably transfected with a SIRT1 small hairpin RNA (shRNA) expression plasmid and compared with untransfected and PANC-1-negative RNAi cells. Proliferation of PANC-1-SIRT1-RNAi cells was significantly reduced, accompanied by increased rates of apoptosis, G1 arrest and senescence. Furthermore, FOXO3a expression was markedly upregulated in PANC-1-SIRT1-RNAi cells, but no significant difference in p53 expression was observed. The invasive ability of PANC-1-SIRT1-RNAi cells was markedly reduced in vitro, which was linked to increased E-cadherin and reduced-MMP expression. Additionally, PANC-1-SIRT1-RNAi cells had a significantly reduced capacity to form tumors in vivo compared with untransfected and PANC-1-negative RNAi cells. These results suggest that SIRT1 may promote cell proliferation and tumor formation in pancreatic cancer, and downregulation of SIRT1 using shRNA could provide a novel therapeutic treatment.     

SIRT7生物学功能及其与肿瘤发生发展关系的研究进展

SIRT7是烟酰胺腺嘌呤二核苷酸(nicotinamide adenine dinucleotide,NAD+)依赖蛋白去乙酰化酶,对组蛋白H3的第18位赖氨酸残基(H3K18ac)有特异性去乙酰化作用。作为Sirtuins蛋白家族的成员之一,SIRT7是许多细胞活动的关键介质。越来越多的证据表明SIRT7的基本细胞程序功能对于致癌演变以及肿瘤生物学有重要的影响,因此SIRT7有望成为表观遗传药物治疗新靶点。     

Overexpression of pro-inflammatory genes and down-regulation of SOCS-1 in human PTC and in hypoxic BCPAP cells

Hypoxia-inducible factor-1α (HIF-1α) is frequently overexpressed and activated in many cancer types. However, its regulation and function in thyroid carcinomas are only partially known. Aim of our study was to demonstrate that adaptation to the hypoxic micro-environment by human papillary thyroid carcinoma (PTC) cells, in the absence of leukocyte infiltrate, induces a “molecular inflammation” process characterized by the expression of a large set of genes normally involved in inflammation. To address this, tumor, peritumor or normal host tissue from eleven human PTC surgical samples, were separated by laser capture microdissection (LCMD) and studied by real-time quantitative PCR and Western blot. In such condition, we observed an increased expression and activation of HIF-1α, NF-kB and pro-inflammatory genes only in tumor tissues. Importantly, an anti-inflammatory gene such as SOCS-1 was markedly down-regulated in tumor tissue compared to surrounding normal host tissue. Similar results were found in fine-needle aspiration biopsy (FNAB)-derived specimens from PTC and in hypoxic human papillary thyroid tumor cell line, BCPAP. Moreover, we also detected an elevated expression of metalloproteinase-9 (MMP9) both in solid tumor and in hypoxic-treated BCPAP cells. Our findings reveal that, in human PTC tumor, hypoxic conditions are accompanied by up-regulation of pro-inflammatory genes, down-regulation of anti-inflammatory genes and increased expression of MMP9. We propose that a better understanding of the pro- and anti-inflammatory pathways involved in the “molecular inflammation” process even in the absence of leukocyte, may help to clarify progression toward malignancy and may prove useful for new anti-tumor strategy.