ObjectiveTo observe the effects of A549 cells under hypoxicconditions on the migration of human umbilical vein endothelial cells (HUVECs) and microvascular formation. MethodsAfter cultured for 24 h in normoxia condition(21% O2),hypoxia condition (2% O2),and anaerobic condition (0% O2),respectively,morphology of A549 cells was observed with inverted phase contrast microscope,proliferation was detected by MTT assay,and intracellular hypoxia-inducible factor-1α (HIF-1α) protein was detected by immunocyto-chemical technique,for determining whether the hypoxia model is successful. Then A549 cells' supernatant in the normoxic group,the hypoxia group and HUVECs culture medium were taken to intervene HUVECs. The migration of HUVECs was observed with cell scratch test,pseudopodia formation of HUVECs was observed with microfilament green fluorescent staining method,and blood vessel formation was observed with three-dimensional culture techniques in vitro. ResultsCompared with the normoxic group,the growth of A549 cells was better in the hypoxia group with more proliferation,and was poor in the anaerobic group with decreased number of cells. A549 cells in the hypoxia group and the anaerobic group both expressed HIF-1α protein,which was more obvious in the anaerobic group. Compared with the HUVECs supernatant intervention group,the hypoxia supernatant intervention group and the normoxic supernatant intervention group both had varying degrees of migration,pseudopodia structure formation and vascular lumen sample structure formation,which were more obvious in the former group. ConclusionA549 cells in hypoxic environment grow very well,proliferated significantly,but anaerobic environment is not conducive to the growth of A549 cells which found to be apoptosis. A549 cells in hypoxic environment can promote HUVECs migration,pseudopodia formation and angiogenesis.
Objective To investigate the expressions of hypoxia-inducible factor-1α (HIF-1α) and caudal homeobox gene 2 (CDX2) in colorectal adenocarcinoma, and the relationships between them and the clinicopathologic factor of colorectal adenocarcinoma. Methods The expressions of HIF-1α and CDX2 were detected by immunohistochemistry in 62 specimens of colorectal adenocarcinoma and 20 specimens of normal colorectal mucosa tissue. The correlation between the expressions of HIF-1α and CDX2 was analyzed by Spearman rank correlation analysis. Results The positive rates of HIF-1α expression in normal colorectal mucosa tissue and colorectal adenocarcinoma were 5.0% （1/20） and 62.9% （39/62）, CDX2 were 95.0% （19/20） and 69.4% （43/62）, the differences of positive rate between different tissues were significant （Plt;0.05）. In colorectal adenocarcinoma, the expression of HIF-1α or CDX2 was related to tumor differentiation, lymph node metastasis, and Dukes staging (Plt;0.05). There was a negative correlation between HIF-1α and CDX2 expressions in colorectal adenocarcinoma （r=－0.293 2，Plt;0.05）. Conclusions The up-regulation of HIF-1α and down-regulation of CDX2 may be involved in the genesis of colorectal adenocarcinoma， and there is a negative correlation between the two kinds of protein. HIF-1α may participate in modulation of CDX2 expression and lead to accelerate the progression of colorectal carcinoma.
Objective To summarize the advance of bioenergetic metabolic mechanisms of cancer cell. Methods Literatures about the recent studies on the bioenergetic metabolic mechanisms of cancer cell were reviewed.Results Cancer cells required a steady source of metabolic energy in order to continue their uncontrolled growth and proliferation. Accelerated uptake of glucose and glycolysis was one of the biochemical characteristics of hypoxia cancer cells. Glucose transport and metabolism were essential for the survival of tumor cells, leading to poor prognosis. Conclusions The studies on relationships between hypoxia-inducible genes and cancer have come a new understanding of the bioenergetic metabolic mechanisms of cancer cell, become new and important supplementary means of diagnosis and treatment of cancer, and enhanced existing strategies so that the treatment could be more rationally applied and personalized for cancer patients.
Objective To construct the responsive plasmid PTRE-HIF-1αof Tet-on gene expression system and examine its expression. Methods RT-nested PCR was performed on the total RNA extracted from hypoxia HepG2 cells to obtain the cDNA of HIF-1α, which was inserted into the responsive plasmid PTRE2hyg. DNA sequencing was performed after the recombinant of responsive plasmid PTRE-HIF-1α was identified by endonuclease digestion. This recombinant vector was transfected into HepG2Tet-on cells by means of liposome and its expression was examined by RT-PCR and Western blot under the control of deoxycycline. Results The amplified products were confirmed as the cDNA of HIF-1α by DNA sequencing. The responsive plasmid PTRE-HIF-1α verified by edonuclease digestion, was capable of expression in HepG2Tet-on cells and could be controlled by deoxycycline. Conclusion The responsive plasmid PTRE-HIF-1α of Tet-on expression system is constructed successfully, and it can express under the regulation of deoxycycline in the HepG2Tet-on cells.
Objective To investigate the expression pattern of hypoxia-inducible factor 1α (HIF-1α) in experimental secondary spinal cord injury (SSCI) in rats and its potential effects on SSCI. Methods A total of 66 SD rats (female or male) with weight (250 ± 20) g were randomly divided into 3 groups: normal control group (group A, n=6), pseudo injury group (group B, n=6), and spinal cord injury (SCI) group (group C, n=54). In group A, no treatment was given as normal control. In groupB, only laminectomy was appl ied. In group C, laminectomy was appl ied and static compression model of SCI was built at T10 level. The expression of HIF-1α was measured with HE and immunohistochemical staining in groups A, B (1 hour after pseudo injury), and C (1, 3, 6, 12 hours and 1, 2, 3, 7, 14 days after SCI). Results All rats survived to the end of the experiment. HE staining showed that the spinal tissue of groups A and B were dense and the nucleus were round and big with l ight staining and clear nucleolus. The injured neuron at 1-12 hours after SCI of group C presented pyknosis and deep eosin staining. The swelling axon with bubbles and the disintegrated and disorganized medullary sheath in white matter appeared at 1-3 days after SCI. The hyperplasia of gl ial cells were obvious and gray matter cells were broken and apoptosis with cavities in injured spinal segment was observed at 7 and 14 days after SCI. Immunohistochemical staining showed that HIF-1α was poorly expressed in group A and increased a l ittle in group B. The positive expression in group C increased at 3 hours after SCI, which was found in spinal cord anterior horn neurons and a small amount of gangl ion cells. It reached peak at 1 day, maintained at a high level during 1-3 days and then decl ined. At 14 days, it appeared only in a small amount of gangl ion cells of white matter. There was no significant difference in the number of HIF-1α positive cells between groups A and B (t=1.325, P=0.137). The number of HIF-1α positive cells at each time point in group C was more than those in groups A and B (P lt; 0.05), and there were significant differences between all time points in group C (P lt; 0.05). Conclusion The expression of HIF-1α increases after SCI, it is related to the ischemia hypoxia after SSCI, and the expression pattern was correlated with the injury time.
Objective To study the effects of hypoxic preconditioning on the glucose metabol ism of rat BMSCs and its underlying mechanism so as to provide the theoretical basis for the optimization of the stem-cell based therapy. Methods Density gradient centrifugation method was adopted to isolate rat BMSCs from neonatal SD rats (aged 1-3 days). BMSCs were cultured to 4th passage and divided into 4 groups based on different culture conditions: group A in normoxia condition for 24 hours, group B in 1% O2 for 24 hours, group C in 2-methoxyestradiol (20 μmol/L) for 24 hours before hypoxic preconditioning, and group D in hypoxia-inducible factor 1 (HIF-1) specific siRNA (50 μmol/L) for 12 hours before hypoxicpreconditioning. MTT method was appl ied to evaluate the prol iferation of BMSCs. Biochemical analyzer and Real-timefluorescent quantitative PCR were appl ied to detect the glucose uptake, lactate production, and HIF-1α mRNA and Glut-1mRNA levels of BMSCs. Results MTT showed that the absorbance (A) values were 387.67 ± 58.92, 322.50 ± 50.60, 297.00 ± 53.00, and 286.00 ± 41.00 in groups A, B, C, and D, respectively, showing no significant difference among 4 groups (P gt; 0.05). The levels of glucose uptake and lactate production were higher in group B than in groups A, C, and D, showing significant differences (P lt; 0.05); the levels of groups C and D were higher than those of group A, but showing no significant difference (P gt; 0.05). The mRNA expressions of HIF-1α and Glut-1 elevated significantly in group B when compared with those in group A (P lt; 0.05); groups C and D were significantly lower than group B (P lt; 0.05) and were significantly higher than group A (P lt; 0.05). Conclusion Hypoxic preconditioning can stimulate the glucose uptake and metabol ism of rat BMSCs, whose mechanism is probably related to up-regulating the mRNA expressions of HIF-1α and Glut-1.
Objective To explore the change tendency of hypoxia-inducible factor-1α (HIF-1α) and extracellular signal-regulated kinase 1/2 (ERK1/2) in fetal rat cerebral cortex neurons cultured in vitro after hypoxia-ischemia reperfusion andto investigate their mutual relationship. Methods Cortical neurons obtained from cerebral cortex of 15 pregnant SD rats at16-18 days of gestation underwent primary culture. The primary neurons 5 days after culture were adopted to establ ish model of oxygen and glucose deprivation (OGD). The experiment was divided into 4 groups: the experimental group 1, culture medium was changed to neuron complete medium containing glucose after the preparation of OGD model to form reperfusion, and the neurons were observed 0, 2, 4, 8, 12 and 24 hours after reperfusion; the control group 1, the neurons were treated with normal medium; the experimental group 2, the neurons were pretreated with U0126 followed by the preparation of OGD model, and the neurons were observed 4 and 8 hours after reperfusion; the control group 2, the neurons were pretreated with DMSO, and other treatments were the same as the experimental group 2. Expressions of HIF-1α, VEGF protein, ERK1/2 and p-ERK1/2 were detected by Western blot. Expression and distribution of p-ERK1/2 and HIF-1α protein were detected by SABC immunocytochemistry method. Results Compl icated synaptic connections between cortical neurons processes were observed 5 days after culture. The expression of HIF-1α and VEGF were increased gradually, peaked at 8 hours, and decreased gradually after 12 hours in the experimental group 1, and there were significant differences between the experimental group 1 and the control group 1 (P lt; 0.05). There was no significant difference between the experimental group 1 and the control group 1 in terms of ERK1/2 protein expression (P gt; 0.05). The p-ERK1/2 protein expression in the experimental group 1 started to increase at 2 hours peaked at 4 hours, and started to decrease at 8 hours, showing significant differences compared with the control group 1 (P lt; 0.01). In the experimental group 2, the p-ERK1/2 protein decreased, and HIF-1αand VEGF protein expression subsequentlydecreased, showing significant differences compared with the control group 2 (P lt; 0.05). There was no significant difference between the experimental group 2 and the control group 2 in terms of ERK1/2 protein expression at each time point (P gt; 0.05). Immunocytochemistry staining showed that p-ERK1/2 and HIF-1α expression decreased, and the yellow-brown staining of the neurons was reduced. Conclusion Expressions of HIF-1α and its target-gene VEGF protein in the cortex neurons after OGD reperfusion are time-dependent. Their expressions decrease when ERK1/2 signal ing pathway is inhibited, indicating the pathway plays an important role in the regulation of HIF-1α and VEGF induced by OGD of cortical neurons
Objective To investigate the effects and mechanism of 17β-estradiol on the retinal neovasularization in rats with oxygen-induced retinopathy (OIR). MethodsA total of 48 SD rats were randomly divided into control group A, control group B, experimental group A and experimental group B with 12 rats in each group. The rats in control group A and experimental group A received a hypodermic injection of 0.1 ml PBS, and the rats in control group B and experimental group B group received an a hypodermic injection of 0.1 ml 17β-estradiol. At postnatal day 7 (P7) and P14, the mRNA expression of vascular endothelial growth factor (VEGF) and Hypoxia-inducible factor (HIF) -1α in the retina were measured by real-time polymerase chain reaction (RTPCR). At P14, endothelial cell nuclei breaking through the internal limiting membrane were counted after staining with hematoxylin and eosin (HE), and the protein expression of VEGF was measured after immunohistochemical staining. The changes of retinal ultrastructure were observed by transmission electron microscopy. ResultsAt P14, the difference of the number of endothelial cell nuclei among four groups was statistically significant(F=10.7, P＜0.05). The number of endothelial cell nuclei in experimental group A was increased greater than that in control group A (P＜0.05) and experimental group B(q=5.16，P＜0.05). But there was no difference between control group A and experimental group B (q=0.25，P＞0.05). The difference of VEGF protein expression among the four groups was statistically significant (P＜0.05). Comparing experimental group A with control group A, B and experimental group B, the difference was statistically significant (P＜0.05). In experimental group A there was ganglion cell swelling, pale staining cytoplasm and mitochondria vacuolizationin, while these were normal in other three groups. At P7 and P14, the differences of VEGF and HIF-1 mRNA expression among four groups were statistically significant(F=14.7,16.1, 13.4, 17.5; P=0.001, 0.005, 0.003, 0.009). At P7, the VEGF mRNA expression in control group B was more than that in control group A (q=5.22, P＜0.05). The VEGF mRNA expression in experimental group B was more than that in experimental group A (q=4.32, P＜0.05). At P14, the VEGF mRNA expression in control group B was more than that in control group A (q=3.72, P＜0.05), but there was no difference of HIF-1 mRNA expression between two groups. The VEGF and HIF-1 mRNA expression in experimental group B were both decreased more than those in experimental group A (q=5.12, 4.08;P＜0.05). Conclusions 17β-estradiol has the effect of two way regulation in VEGF mRNA, which increases VEGF expression in retina under hyperoxic conditions so as to develop the vascular system; which reduces VEGF and HIF-1α expression so as to prevent pathologic neovascularization under hypoxic conditions. It provides some protection from the damage of retinal neovascularization.
Objective To investigate the expression of hypoxia inducible factor 1(HIF1alpha;) in ratsprime; retinae during the embryonic and earlier postnatal period. Methods The retinal expression patterns of HIF-1alpha; protein and mRNA of embryonic day 12 (E12), E16, E20, and postnatal day 1(P1) and P5 rats were determined by immunohistochemical staining and reverse transcriptionpolymerase chain reaction (RT-PCR). Results HIF-1alpha; protein was detected in the neural epithelial layer and the pigment epithelial layer at all those 5 timepoints, with higher expression in the ganglion cell layer and the inner plexiform layer, and seems limited to the ganglion cell layer when re tina became more mature. Embryonic rat retina had higher expression of HIF-1alpha; protein and mRNA than postnatal retina, the difference was significant (P＜0.01）. Conclusion The expression of HIF1alpha; in ratsprime;retina e differs from embryonic to earlier postnatal stages.
Objective To observe the influence of the expression of CD18 on the neutrophile and the leukocyte adhesion to retinal vascular endothelium by hypoxia-inducible factor-1 alpha (HIF-1alpha;) in early diabetic retinopathy rats. Methods Male Sprague-Dawley rats received intraperitoneal injection of streptozotocin to induce diabetes model. 18 diabetic rats were divided into 3 groups randomly after 2 months of diabetes induction, including diabetic group (group B), HIF-1alpha; anti-sense oligonucleotides (ASODN) injection group (group C) and HIF-1alpha; sense oligonucleotides (SODN) injection group (group D), the age and weigh matched health rats were chosen as control group (group A), with 6 rats in each group. Then group A and B rats received 5% glucose solution caudalis veins injection, group C and group D rats received HIF-1alpha; ASODN and HIF-1alpha; SODN caudalis veins injection, respectively(025 mg/kg).The level of CD18 on the neutrophil isolated from the peripheral blood was measured by flow cytometry. Retinal leukostasis was quantified with acridine orange leukocyte fluorography. Results The percentage of CD18 positive neutrophil cell was（44.93plusmn;3.60）% in group B，（18.66plusmn;1.52）% in group A，（31.66plusmn;4.72）% in group C，（51.00plusmn;5.66）% in group D. Compared with each other groups，the differences are statistically significant （F=42.46, Plt;0.001）. The number of positive staining cells of retinal leukocyte was （46.16plusmn;10.68）in group A，（133.83plusmn;20.43）in group B,（99.83plusmn;9.28）in group C，（121.33plusmn;10.23） in group C. Compared group B with group C，the number of positive staining cells raised about 2.89 times；compared group B with group C and D，the differences are statistically significant （P=0.12，95% confidence interval -3.69~28.69）. Conclusions In vivo, HIF-1alpha; can decreased the expression of CD18 on neutrophils from diabetic ratsprime; peripheral blood and the collection of retinal leukostasis in the diabetic animals. HIF-1alpha; may serve as a therapeutic target for the treatment and/or prevention of early diabetic retinopathy. （Chin J Ocul Fundus Dis，2008，24：268-271）