Objective To investigate relationship between hypoxia microenvironment and occurrence and development of hepatocellular carcinoma (HCC). Method The relevant literatures on researches of the relationship between the hypoxic microenvironment and the HCC were review and analyzed. Results The hypoxia microenvironment played an important role in inducing the drug resistance and angiogenesis of the HCC cells, and it was an important factor of affecting the ability of tumor metabolism, invasion, and migration. The hypoxia microenvironment could up-regulate the expression of hypoxia-inducible factors (HIFs) and promote its transcriptional activity, promote the expression of the vascular endothelial growth factor gene, and regulate the neovascularization in the tumor. Among them, the HIF-1α played a major role in regulating the angiogenesis, immune escape, tumor invasion and metastasis-related gene expression, participating in the glycolysis, regulating lysyl oxidase 2 and thus regulated epithelial-mesenchymal transition, then promoted the invasion and metastasis of the HCC; HIF-2α was a key regulator of the malignant phenotype involving in the cell proliferation, angiogenesis, apoptosis, metabolism, metastasis, and resistance to chemotherapy. The hypoxia microenvironment posed some difficulties for the treatment of HCC, but it was also a potential therapeutic breakthrough. Conclusion Hypoxia microenvironment can promote invasion and metastasis of HCC through various mechanisms, which provides new targets and strategies for clinical treatment of HCC.
Objective To investigate the effect of hypoxia on expressions of erythropoietin（EPO）mRNA and protein in retinal Muuml;ller cells cultured in vitro. Methods Retina tissues from the new-born Wistar rats were dissected into cell suspension after digested by pancreatin.Muuml;ller cells were separated and purified by mechanical concussion and blowing and striking method.The expression of EPO mRNA and protein under the condition of hypoxia was detected by semi-quantitative reverse transcriptase（RT）-polymerase chain reaction（PCR）and immunocytochemical method. Results Retinal Muuml;ller cells were cultured successfully，95% of which were positively stained by glial fibrillary acidic protein（GFAP）.Positively stained EPO protein was located in the cytoplasm and protuberance.The expression of EPO mRNA and protein was faint in the normal retinal Muuml;ller cells，but increased obviously and time-dependently after hypoxia. Conclusion Expression of EPO mRNA and protein increases in Muuml;ller cells after hypoxia，which may be one of the protective factors for the nerves in anoxic retinopathy. (Chin J Ocul Fundus Dis, 2006, 22: 196-199)
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A), and to evaluate the effect of minocycline. Methods RF/6A was cultured and divided into four groups: control group, hypoxia group, hypoxia and low dose of minocycline group (0.5 mu;mol/L), hypoxia and medium dose of minocycline group (5 mu;mol/L), and hypoxia and high dose of minocycline group (50 mu;mol/L). Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2, respectively. Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F 24 h=0.17,F 48 h=1.53,F72 h=2.04;P＞0.05). Compared with hypoxia group, the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly downregulated (low minocycline, medium minocycline, high minocycline: t=4.69, 20.16, 17.12; P＜0.001). The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups, and the staining was relatively weak and mainly located in cell membrane and cytoplasm. The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points（F 24 h=0.251,F 48 h=0.340,F72 h=0.589;P＞0.05）. The VEGFR-2 positive staining cells were also observed in all groups, and the staining was relatively high. Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm. The staining density of VEGFR-2 was significantly higher in hypoxia group than control group. Compared with the hypoxia group, the protein expression of VEGFR-2 in minocycline treated groups was significantly lower（F 24 h=19.147,F 48 h=14.893,F72 h==11.984; P＜0.05). Conclusion The expression of VEGFR-2 is upregulated in RF/6A, and minocycline somewhat shows an inhibition effect.
ObjectiveTo investigate the expression and significance of CD73 in rats with intermittent hypoxia and high fat diet.MethodsThe rat model of chronic intermittent hypoxia combined with high fat diet was established. Twenty-four healthy male Wistar rats in the SPF level were randomly divided into 4 group, with 6 rats in each group, namely group A (normoxia and normal diet), group B (normoxia and high fat diet), group C (intermittent hypoxia and normal diet)and group D (intermittent hypoxia and high-fat diet). After 6 weeks of experiment, the serum lipid levels, myocardial morphological changes under microscope, the expression level of CD73 protein detected byimmunohistochemistry and Western blot in myocardial cells in rats were compared among these groups.ResultsThe serum lipid levels were significantly different among these groups (P<0.05). HE results showed that the myocardial cells of group A had no obvious abnormalities; disorganized visible myocardial fibers with focal necrosis in groups B and C; myocardial cell injury was most obvious in group D, in which visible muscle fibers arranged in disorder, and grain was not clear, part of the muscle fibers were dissolved predominantly. Compared with group A, CD73 protein expression levels in myocardial cells in groups B, C, and D were significantly elevated (P<0.01). Furthermore, CD73 protein expression level in myocardial cells in group D was significantly higher than those in groups B and C (P<0.01). Western blot showed consistent results as immunohistochemistry: compared with group A, CD73 protein expression levels in groups B, C, and D were significantly elevated (P<0.05), and CD73 protein expression level in myocardial cells in group D was significantly higher than those in groups B and C (P<0.01).ConclusionChronic intermittent hypoxia and high fat diet can cause myocardial cell damage and upregulate CD73 expression in the cardiomyocytes.
ObjectiveTo explore the relationship between the oxygen partial pressure of mice hindlimb muscles with normal blood supply or ischemia and expression of HIF-1αprotein, and to provide a theoretical basis for the study of angiogenesis in vitro hypoxia. MethodsMice hind limb ischemia model were established, tissue oxygen tension of gastrocnemius muscle and bone marrow were measured by micro electrode at different time points of ischemia (24 hours, 1 week, 2 weeks, 3 weeks, and unoperated as control). Protein level of hypoxia inducible factor-1αand histological examination were performed on gastrocnemius muscle as well. ResultsThe oxygen tension baselines of gastrocnemius muscle and femoral bone marrow was (47.78±4.37) mm Hg and (21±3.40) mm Hg, respectively. Muscle oxygen tension decreased significantly at all time points after modeling (P < 0.05), and reached lowest level in 1 week of ischemia. The inflammatory reaction was most serious and HIF-1αprotein reached highest level at the same time point. With the extension of ischemic time, the tissue oxygen tension recovered while HIF-1αlevel was down-regulated, however, There was no statistical correlation(r=-0.86, P > 0.05). Oxygen tension in bone marrow didn't show any significant change at all time points. ConclusionsThe expression level of HIF-1αprotein in ischemic tissue can reflect the degree of ischemic limb. The concept that physiological oxygen level differs in different tissue is highlighted, and may provide basis for ex vivo hypoxic research.
Objective： To observe the effect of estrogen on the expr ession of pigment epithelium derived factor (PEDF) in cultured retinal Muuml;ller cells under the anoxic condition． Methods：After the anoxic retinal Muuml;ll er cells were tre ated with estrogen (E2) with the concentration of 10-6、10-5 and 10-7 mmol/L, t he level of expression of PEDF mRNA and the protein was detected by reverse tran scriptionpolymerase chain reaction and Western blotting analysis. Results：Th e expression of PEDF mRNA and protein decreased 24 hours after anoxia. E2 with t he concentration of 10-5 and 10-6 mmol/L inhibited the decrease of expression of PEDF mRNA and protein induced by anoxia, which related to the concentration of E2． Conclusion：strogen can regulate the expression of PEDF, which ma y play an important role in the regulation of retinal neovascularization．
Objective To investigate the role of Kv1. 5 in the pathogenesis of pulmonary hypertension simulated by hypobaria and hypoxia, and the effects of dichloroacetate ( DCA) on the Kv1. 5 expression in pulmonary arterial smooth muscle cells ( PASMCs ) and mean pulmonary arterial pressure ( mPAP) . Methods Twenty-four SD rats were randomly divided into a normal group ( N group) , a high altitude group ( HA group) , and a DCA treated group ( DCA group) . The N group were fed in normalconditions, the HA group and DCA group were fed in a hypobaria and hypoxia chamber simulated to an altitude of 5000 meters. In addition, the DCA group rats were gastric gavaged with DCA ( 70 mg · kg - 1 · d - 1 ) .Twenty-one days later, percentage of wall thickness ( WT% ) and percentage of wall area ( WA% ) of the pulmonary arteriole, mPAP, and the ratio of right ventricle / left ventricle and septum ( RV/ LV + S) were evaluated. Real-time PCR, immunohistochemistry and Western blot were carried out to detect the Kv1. 5 expression in PASMCs. Results In the HA group, WT% , and WA% of pulmonary arteriole, mPAP and RV/ ( LV + S) all increased significantly compared with the N group ( P lt;0. 01) . These changes in the DCA group were significantly lower than those in the HA group( P lt; 0. 01) . Furthermore, the protein and mRNA expression of Kv1. 5 in the PASMCs deceased significantly in the HA group compared with the N group( P lt;0. 01) , but recovered in the DCA group ( P lt;0. 01) . Conclusions The expression of Kv1. 5 in PASMCs is tremendously inhibited in rats fed in high altitude, which might be a important role of pulmonaryhypertension. DCA can inhibit the remodeling of pulmonary arterials probably by recovering Kv1. 5 expression.
ObjectiveTo identify genes associated with resistance to programmed cell death protein 1 (PD-1) inhibitors in colorectal cancer and elucidate their underlying mechanisms using bioinformatics approaches. MethodsGenes expression datasets were downloaded from the Gene Expression Omnibus (GEO) database to screen hypoxia-related genes (HRGs) and differentially expressed genes (DEGs). The intersection of HRGs and DEGs was defined as hypoxia-related differentially expressed genes (HDGs). The gene expression data of patients with colorectal cancer from The Cancer Genome Atlas (TCGA) were analyzed using Pearson correlation to identify the PD-1-related genes, further the STRING analysis (minimum interaction score was greater than 0.7) and Cytoscape were subsequently employed to screen the key PD-1-related genes. The relation between the screened key PD-1-related genes and the prognosis of colorectal cancer patients was analyzed to screen out the target genes. The real-time fluorescence reverse transcription quantitative polymerase chain reaction was used to analyze the expression of the target genes in the cancer tissues and their corresponding adjacent tissues of 20 patients with colorectal cancer. The Kaplan-Meier Plotter database and the ROC Plotter database were used to analyze the relation between the high and low expression of the target genes and the prognosis in different patients. The significance level was set as α=0.05. ResultsA total of 651 HRGs and 329 DEGs were screened out. By taking the intersection of these two sets, 37 HDGs were obtained for subsequent analysis. Through Pearson correlation analysis, 25 key PD-1-related genes were screened out and 10 and 14 key PD-1-related genes were screened out by the MCC algorithm and the MCODE algorithm respectively. By taking the intersection of these three sets, 3 key PD-1-related genes were obtained, then survival analysis, the Aurora kinase A (AURKA) gene was finally screened out as the target gene. The expression level of the AURKA gene in the pan-cancer patients who responded to PD-1 inhibitor treatment was significantly higher than that in non-responders (P＜0.001), and was significantly lower in the six colorectal cancer cells treated with hypoxia than in six colorectal cancer cells treated with normoxia (P＜0.001). The AURKA expression in the colorectal cancer tissues was significantly higher than that in the corresponding adjacent colorectal tissues (P=0.008). The overall survival of pan-cancer patients with high AURKA expression was better than that of those with low AURKA expression [HR (95%CI)=0.67 (0.49, 0.93), P=0.015]. Among the colorectal cancer patients with MMR deficiency, the patients with low AURKA gene expression had worse overall survival [HR (95%CI)=2.596 (1.028, 6.332), P=0.043] and recurrence-free survival [HR (95%CI)=4.201 (1.092, 16.150), P=0.037] as compared with those with high AURKA gene expression. The low AURKA expression was associated with significantly worse overall survivals in the colorectal cancer patients harboring wild-type or mutant TP53, BRAF, and KRAS as compared with high AURKA expression (P＜0.05), while no statistically significant difference was found in the overall survival of the normal MMR patients between with high AURKA expression and low AURKA expression (P=0.307). ConclusionThe results of this bioinformatics analysis suggest that hypoxia down-regulated AURKA expression, and low AURKA expression is associated with worse prognosis in colorectal cancer patients, and worse reactivity and prognosis in patients treated with PD-1 inhibitors.
Objective To study the impact of chronic hypoxia on white matter (WM) injury and brain development delay using a neonatal rat model, and to explore its value in simulating chronic hypoxic brain damage in cyanotic congenital heart disease (CHD). Methods Three-day-old Sprague-Dawley (SD) rats were randomly distributed to an experiment group (n=36, FiO2 10.5%±1.0%) and a control group (n=36, FiO2 21.0%±0.0%) and were raised for 12 days. (1) Body weight of SD rats was recorded every day and fresh brain weight was measured on P14. (2) H&E staining was performed on sections of brain tissue to observe pathological changes and ventricular size. (3) Immunohistochemistry (IHC) was applied to reveal alterations of oligodendroglial progenitor cells (OPC), preoligodendrocytes (PreOL) and myelin basic protein (MBP) in brain WM area. (4) Protein was extracted from 50 mg of brain tissue in WM area and expression of MBP was determined using Western blotting. (5) Motor function and coordination of rats (P30) were assessed via rotation experiment. Results (1) Body weight and brain weight were significantly less in the experiment group compared with the control group on P14 (body weight 14.92±1.26 gvs. 30.26±1.81 g, t=7.51, P<0.01; brain weight 0.68± 0.05 gvs.0.97±0.04 g, t=13.26, P<0.01); (2) HE staining: Sections of brain tissue from the experiment group showed ventricular size enlargement with a statistical difference (P<0.01), disordered cell organization, local neuronal death and leukomalacia. (3) The number of OPC and PreOL in the experiment group were significantly less than those in the control group (64.8±6.3vs. 126.2±8.4, t=11.19, P<0.01; 19.1±7.6vs. 46.7±9.5, t=7.28, P<0.01, respectively). MBP distribution was sparse and disorganized in the experiment group. (4) Western blotting: Expression of MBP was less in the experiment group (P<0.01). (5) Behavioral test: Time on rotarod was less in the experiment group with a statistical difference (P<0.01). Conclusion Chronic hypoxia can result in WM injury and brain development delay in neonatal rats, with features comparable to those seen in infants with cyanotic CHD.
Objective To explore the difference between the hemorheology levels and the expression of hypoxia inducible factor 1α/2α (HIF-1α/2α) in the peripheral blood mononuclear cells of the Tibetan and Han patients with obstructive sleep apnea hypopnea syndrome (OSAHS). Methods This research recruited 30 high-risk Tibetan and Han patients with OSAHS, and 30 Tibetan and Han healthy volunteers at the same period. The whole blood viscometer was used to detect the high shear rate of whole blood viscosity, low shear rate of whole blood viscosity, plasma viscosity ratio, red blood cell aggregation index, and hematocrit in each group. RT-qPCR and Western blot assays were used to detect the mRNA and protein levels of phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), nuclear factor-κB (NF-κB) p65, HIF-1α and HIF-2α in peripheral blood mononuclear cells. Results The hemorheology level of Tibetan OSAHS patients was significantly higher than that of healthy Tibetans and Han OSAHS patients (P<0.05), and the hemorheology level of Han OSAHS patients was significantly higher than that of Han healthy people (P<0.05) . The mRNA and protein levels of PI3K, AKT, NF-κB p65 and HIF-1α in the peripheral blood mononuclear cells of Tibetan OSAHS patients were significantly higher than those of the healthy Tibetans or Han people, and these indexes of the Han OSAHS patients were significantly higher than those of the Han healthy people (all P<0.05), while HIF-2α mRNA and protein levels were significantly lower than those of healthy Han people (all P<0.05). Conclusion The upregulation of HIF-1α level and downregulation of HIF-2α expression in peripheral blood mononuclear cells of OSAHS patients depend on the activation of the PI3K/AKT/NF-κB p65 signaling pathway, and the hemorheological level of Tibetan OSAHS patients is higher than that of Han OSAHS patients.