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。

Effect of acetyl L-carnitine on human retinal pigment epithelium-19 cells in hypoxic conditions

AIM:To investigate the effect of acetyl-L-carnitine(ALCAR)on cell viability,morphological integrity,and vascular endothelial growth factor(VEGF)expression in human retinal pigment epithelium(ARPE-19)cells using a hypoxic model.METHODS:In the first set of experiments,the optimal CoCl_(2) dose was determined by exposing ARPE-19 cell cultures to different concentrations.To evaluate the effect of ALCAR on cell viability,five groups of ARPE-19 cell culture were established that included a control group,a sham group(200μM CoCl_(2)),and groups that received 1,10 and 100 mM doses of ALCAR combined with 200μM CoCl_(2),respectively.The cell viability was measured by MTT assay.The morphological characteristics of cells were observed by an inverted phase contrast microscope.The levels of VEGF and HIF-1α secretion by ARPE-19 cells were detected by enzyme linked immunosorbent assay(ELISA)assay.RESULTS:ARPE-19 cells were exposed to different doses of CoCl_(2) in order to create a hypoxia model.Nevertheless,when exposed to a concentration of 200μM CoCl_(2),a notable decrease in viability to 83% was noted.ALCAR was found to increase the cell viability at 1 mM and 10 mM concentrations,while the highest concentration(100 mM)did not have an added effect.The cell viability was found to be significantly higher in the groups treated with a concentration of 1 mM and 10 mM ALCAR compared to the Sham group(P=0.041,P=0.019,respectively).The cell viability and morphology remained unaffected by the greatest dose of ALCAR(100 mM).The administration of 10 mM ALCAR demonstrated a statistically significant reduction in the levels of VEGF and HIF-1α compared with the Sham group(P=0.013,P=0.033,respectively).CONCLUSION:The findings from the current study indicate that ALCAR could represent a viable therapeutic option with the potential to open up novel treatment pathways for retinal diseases,particular relevance for age-related macular degeneration(AMD).However,to fully elucidate ALCAR’s application potential in retinal diseases,additional investigation is necessary to clearly define the exact mechanisms involved.

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 。

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。

Hypoxia-inducible factor 1 alpha and vascular endothelial growth factor overexpression in ischemic colitis

AIM: To examine the etiology and pathophysiology in human ischemic colitis from the viewpoint of ischemic factors such as hypoxia-inducible factor 1 alpha (HIF-1 alpha and vascular endothelial growth factor (VEGF). METHODS: Thirteen patients with ischemic colitis and 21 normal controls underwent colonoscopy. The follow-up colonoscopy was performed in 8 patients at 7 to 10 d after the occurrence of ischemic colitis. Biopsy samples were subjected to real-time RT-PCR and immunohistochemistry to detect the expression of HIF-1 alpha and VEGF. RESULTS: HIF-1 alpha and VEGF expression were found in the normal colon tissues by RT-PCR and immunohistochemistry. HIF-1 alpha and VEGF were overexpressed in the lesions of ischemic colitis. Overexpressed HIF-1 alpha and VEGF RNA quickly decreased to the normal level in the scar regions at 7 to 10 d after the occurrence of ischemic colitis. CONCLUSION: Constant expression of HIF-1 alpha and VEGF in normal human colon tissue suggested that HIF-1 alpha and VEGF play an important role in maintaining tissue integrity. We confirmed the ischemic crisis in ischemic colitis at the molecular level, demonstrating overexpression of HIF-1 alpha and VEGF in ischemic lesions. These ischemic factors may play an important role in the pathophysiology of ischemic colitis.

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.

RNA interference against hypoxia inducible factor-1α inhibited growth of cervical cancer by limiting vascularization

Objective:Cervical cancer has become a major public health problem.The development of effective,systemic therapies for cervical cancer is highly desired.We show here that hypoxia inducible factor-1α(HIF-1α) was indicated as an attractive therapeutic molecular target for cervical cancer.Methods:Firstly,we observed the expressional level of HIF-1α in cervical cancer and Hela and Siha cell lines.Secondly,by constructuring HIF-1α shRNA targeting human HIF-1α mRNA common sequence and transfecting it with plasmid to cervical cell,we detected the changes of HIF-1α and its downstream genes levels VEGF.Then we injected selected stably transfected cell line into athymic nude mice to estimate its' antitumor effects.Results:We observed that HIF-1α inhibition was related to down-regulated VEGF resulting in prevention of angiogenesis,then leading to slower-growing tumors.Conclusion:The underlying concept of transfecting a HIF-1α shRNA expression vector to block the HIF-1α holds promise as the clinical potential of gene therapy for cervical cancer.

Electroacupuncture combined with intermittent pneumatic compression therapeutic apparatus for diabetic peripheral neuropathy and the effect on HIF-1α and VEGF levels

Objective： To observe the clinical efficacy of electroacupuncture combined with intermittent pneumatic compression therapeutic apparatus for treatment of diabetic peripheral neuropathy, and the effect on serum VEGF and HIF-lα levels of patients. Methods： Ninety-six patients were randomly divided into electroacupuncture treatment group （EA group）, intermittent pneumatic compression treatment group （IPC group）, electroacupuncture combined with intermittent pneumatic compression treatment group （EA ＋ IPC group） and cobamamide group （CM group）, with 24 cases in each group. Electroacupuncture treatment （once a day）, intermittent pneumatic com pression treatment （twice a day） and intramuscular injection with cobamamide （1 rag, once a day） were carried out in EA group, IPC group and CM group, respectively, and intermittent pneumatic compres- sion treatment （twice a day） was conducted on the basis of electroacupuncture treatment （once a day） in EA＋IPC group. After treatment for 2 consecutive weeks, the differences in subjective symptoms, mo- tor nerve conduction velocity, sensory nerve conduction velocity and serum HIF-lα and VEGF levels of patients in the four groups before and after treatment were observed and compared. Results： After treatment for 2 weeks, the differences in total effective rate between EA group and CM group, IPC group and CM group, as well as EA ＋ IPC group and CM group were all significant （all P 〈 0.05）, and the total effective rate in EA＋ IPC group was significantly higher than that in EA group and IPC group （both P 〈 0.05）. After treatment for 2 weeks, the motor nerve conduction velocity and sensory nerve conduction velocity of median nerve and common peroneal nerve of patients in EA group, 1PC group and EA＋IPC group were all higher than that before treatment （all P 〈 0.05）; the motor nerve conduction velocity of median nerve and the sensory nerve conduction velocity of common peroneal nerve in EA group were all higher than that in CM group （both P 〈 0.05）; the motor nerve conduction velocity and sensory nerve conduction velocity of median nerve in IPC group were also all higher than that in CM group （both P 〈 0.05）; the motor nerve conduction velocity and sensory nerve conduction velocity of median nerve and common peroneal nerve in EA＋IPC group were all higher than that in CM group （both P 〈 0.05）; the sensory nerve conduction velocity of common peroneal nerve in EA ＋ 1PC group was higher than that in EA group and IPC group （both P 〈 0.05）, and the motor nerve conduction velocity of median nerve in EA＋IPC group was higher than that in IPC group （P 〈 0.05）. The serum HIF-1α and VEGF levels of patients in EA group, IPC group and EA ＋ IPC group after treatment significantly reduced （all P 〈 0.05）. and were lower than that in CM group after treatment （all P 〈 0.05）; the serum HIF-lα and VEGF levels of patients in EA ＋ IPC group after treatment were lower than that in EA group and IPC group, and the difference in serum HIF-lα level was statistically significant （both P 〈 0.05）. Conclusion： Electroacupuncture combined with intermittent pneumatic compression therapeutic apparatus can effectively improve the clinical symptoms of patients with diabetic peripheral neuropathy, the efficacy were better than electroacupuncture, intermittent pneumatic compression treatment and cobamamide.

The effect of spironolactone on the levels of GATA4 protein and hypoxia inducible factor-1α in patients receiving coronary stent implantation

In the present study,we aimed to explore the protective effect of spironolactone on cardiac function in patients undergoing coronary stent implantation by determining the serum levels of GATA4 and hypoxia-inducible factor-1α(HIF-1α)proteins before and after the coronary stent implantation.A total of 134 patients undergoing coronary stent implantation in our hospital from March 2019 to March 2020 were retrospectively selected using the propensity score matching(PSM)method.Of the 134 patients,67 patients taking spironolactone were used as a test group,and the other 67 patients without taking spironolactone were used as a control group.In all patients,the levels of serum GATA4,HIF-1α,and troponin I proteins were determined before as well as 24 h and 6 months after the coronary stent implantation.Left ventricular ejection fraction(LVEF)and left ventricular systolic global longitudinal strain(GLS)were determined before and 6 months after the coronary stent implantation.There were no significant differences in the HIF-1αlevel between the two groups before and 6 months after the operation,while the HIF-1αlevel was significantly lower in the test group compared with the control group at 24 h after the operation(P<0.01).There were no significant differences in the GATA4 protein level between the two groups before and 24 h after the operation,while the GATA4 protein level was significantly lower in the test group compared with the control group at 6 months after the operation(P<0.01).There was no significant difference in LVEF between the two groups before and 6 months after the operation.GLS was significantly improved at 6 months after the operation compared with that before the operation in both groups,while GLS was significantly better in the test group compared with the control group at 6 months after the operation(P<0.01).Collectively,spironolactone could protect cardiac function probably by improving myocardial hypoxia and inhibiting myocardial remodeling.

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L

We recently report that the expression of polycomb chromobox 4(Cbx4)is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma(HCC)patients and it enhances hypoxia-induced vascular endothelial growth factor(VEGF)expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha(HIF-1α).Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L.Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner,and promotes the in vitro migration of the cancer cell,which can be effectively abrogated by anti-VEGF antibody.Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells,it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice.These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.