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.
ObjectiveUnder hypoxic conditions, the survival and apoptosis of human amniotic mesenchymal stem cells (hAMSCs) were observed by transient transfection of hypoxia-inducible factor 1α (HIF-1α) gene, to investigate the effect of HIF-1α on hypoxic tolerance of hAMSCs.MethodsThe hAMSCs were isolated and cultured from amniotic membrane tissue from voluntary donors who were treated with cesarean section. And the morphological observation by inverted phase contrast microscope and immunofluorescence detection of the expressions of stem cell markers OCT-4 and NANOG were performed to identify the cultured cells. The third generation hAMSCs were treated with 200 μmol/L CoCl2, and transient transfection of plasmids were added according to the following grouping: group A was hAMSCs blank group; group B was pcDNA3.1 negative control group; group C was short hairpin RNA (shRNA) negative control group; group D was shRNA-HIF-1α interference group; group E was pcDNA3.1-HIF-1α over expression group. Cell survival rate of each group was measured by cell counting kit 8 (CCK-8) at 12, 24, 48 hours after hypoxia treatment. Flow cytometry was used to detect apoptosis rate of each group at 24 hours after hypoxia treatment. The expression levels of HIF-1α, vascular endothelial growth factor (VEGF), B-cell lymphoma 2 (Bcl-2), Bax, and cleaved Caspase-3 (C-Caspase-3) proteins were detected by Western blot at 24 hours after hypoxia treatment.ResultsCCK-8 assay showed that the cell survival rate of group D was significantly lower than those of groups A and C at all time points after hypoxia treatment; while the cell survival rate in group E was significantly increased than those in groups A and B, and the diffrences at 24 hours were significant (P<0.05). In group E, the cell survival rate at 24 hours was significantly higher than those at 12 and 48 hours (P<0.05). The results of flow cytometry showed that the apoptosis rate in group D was significantly higher than those in groups A and C (P<0.05), and the apoptosis rate in group E was significantly lower than those in groups A and B (P<0.05). Western blot showed that the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group D were significantly decreased when compared with those in groups A and C, and the expressions of Bax and C-Caspase-3 proteins were significantly increased (P<0.05). On the contrary, the expressions of HIF-1α, VEGF, and Bcl-2 proteins in group E were significantly higher than those in groups A and B, and the expressions of Bax and C-Caspase-3 proteins were significantly decreased (P<0.05).ConclusionOverexpression of HIF-1α gene can significantly improve hAMSCs tolerance to hypoxia, the mechanism may be related to up-regulation of VEGF and Bcl-2 expressions, and down-regulation of Bax and C-Caspase-3 expressions.
ObjectiveTo investigate the effects of 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), a hypoxia-inducible factor-1α (HIF-1α) inhibitor, on hypoxia induced rat pulmonary arterial adventitial fibroblasts (AFs) proliferation and collagen synthesis, and explore the molecular mechanism.MethodsUnder hypoxic condition, rat AFs were cultured in DMEM medium supplemented with 10% fetal bovine serum in vitro. The cells were divided into five groups, ie. a normoxia group, a hypoxia group and three hypoxia+YC-1 groups (treated with YC-1 at concentration of 0.01, 0.05 and 0.1 mmol/L, respectively). The cells proliferation was determined by MTT method. Collagen synthesis of AFs was measured by 3H-proline incorporation assay. The expression of HIF-1α in AFs in different conditions was measured by Western blot, and the mRNA expression of transforming growth factor-β1 (TGF-β1) was measured by reverse-transcription polymerase chain reaction.ResultsThe proliferation rate and the incorporation data of 3H-proline in the hypoxia group were significantly increased as compared with those in the control group (both P<0.01). YC-1 significantly reduced the proliferation rate and incorporation data of3H-proline induced by hypoxia in a dose-dependent manner. YC-1 could also down-regulate the expressions of HIF-1α and TGF-β1 mRNA significantly (both P<0.01). Compared with the hypoxia group, the expressions of HIF-1α and TGF-β1 mRNA decreased respectively by 65% and 61% in the hypoxia+YC-1 (0.1 mmol/L) group (bothP<0.01).ConclusionsYC-1 can inhibit hypoxia-induced AFs proliferation and collagen synthesis in a dose-dependent manner. The mechanism may relate to YC-1’s inhibitory effect on expressions of HIF-1α and TGF-β1 mRNA.
ObjectiveTo summarize the regulating mechanism of microRNA in tumor microenvironment. MethodThe literatures about the studies on the mechanism regulated by microRNA for tumor microenvironment were reviewed according to the results searched from PubMed in recent years. ResultsmicroRNA might be participated in regulation of tumor microenvironment factors such as hypoxia-inducible factor, tumor associated fibroblasts, extracellular matrix, which leaded to a change in biological behavior of tumor cells by reforming the microenviroment. ConclusionsmicroRNA has been participated in regulating many factors of tumor microenvironment. The change of neoplastic microenvironment has been recognized to play a critical role in the development of cancer. Therefore revealing microRNA mechanism for tumor microenvironment could not only help exploring the biological behavior of tumor cells, but also come an important insight for new means of diagnosis and treatment of cancer.
ObjectiveTo review the development and applications of hypoxia-inducible factor 1α (HIF-1α) in the strategy of tissue engineered angiogenesis and osteogenesis. MethodThe literature about HIF-1α in tissue engineering technology was reviewed, analyzed, and summarized. ResultsHIF-1α plays a key role in angiogenic-osteogenic coupling, and as an upstream regulator, HIF-1α can regulate the expressions of its target genes related with angiogenesis and osteogenesis. In addition, HIF-1α not only can control and improve the angiogenesis, but also has important significance in proliferation and differentiation of seed cells, especially stem cells, which is the foundation for bone healing. ConclusionsWith the development of tissue engineering technology, the problems in the applications of HIF-1α, such as the effective dose of targeting controlled-release, pro-inflammatory effect, and carcinogenicity, will be explored and solved in the future, so it can be used better in clinical.
The intervention therapy targeting vascular endothelial growth factor (VEGF) has become a specific and effective method for the treatment of diabetic retinopathy (DR). However, some patients did not respond or responded poorly to anti-VEGF therapy, and its effects of eliminating edema and improving vision appear to be unstable in the same patient. Hypoxia-inducible factor-1α (HIF-1α), an important upstream transcriptional regulator of VEGF, is an oxygen concentration-sensitive protein expressed in tissues under hypoxia. It can simultaneously target many downstream target genes except VEGF, such as placental growth factor and angiopoietin-like protein 4, to cause blood-retinal barrier damage and neovascularization, and thus participate in various pathological changes of DR to promote the occurrence and development of DR. Therefore, direct intervention of HIF-1α or targeting one or more downstream target genes regulated by HIF-1α to treat DR may have better efficacy. In the future, the development of effective and safe HIF inhibitors or anti-VEGF with HIF-1α other target gene inhibitors may have broader clinical application prospects.
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
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.
ObjectiveTo observe the expression and mechanism of hypoxia-inducible factor-1α (HIF-1α) and p53 protein at the altitude of 5000 meter plateau hypoxia environment in rats, as well as the effect of Astragalus injection. MethodsSixty Sprague Dawley rats were randomly divided into the Astragalus injection intervention group and normal saline control group, 30 rats in each group. Astragalus injection group rats were intraperitoneal injected of Astragalus injection (15 ml/kg) before 30 minutes into the plateau environment simulation cabin, normal saline group rats were intraperitoneal injected with the same volume of saline. 30 minutes after injection, rats in each group were reared in the plateau experiment cabin which simulated altitude of 5000 m (oxygen partial pressure 11.3 kPa) for 2, 6, 8, 12, 24 hours, each time period of 6 rats. When get out, the rats were executed immediately and eyes were harvested. Retinal sections were studied by hematoxylin eosin stain, and immunohistochemical method for HIF-1α and p53 expression. ResultsFor control rats, after 2 hours in the cabin, there was edema in retinal layers. HIF-1α and p53 were expressed mainly in the cytoplasm of retinal layers. When the periods in cabin extended, there was atrophy of retinal nerve fiber layer, swelling and degeneration of ganglion cells. The expression of HIF-1α and p53 was increased. Compared with the control group, the intervention group rat had similar but less severe retinal changes, and the expression of HIF-1α and p53 was significantly decreased (P<0.05). ConclusionAstragalus injection can reduce pathological retinal damage in rats at high altitude environment, and its mechanism may be associated with reduced HIF-1α, p53 expression.
ObjectiveTo investigate the effects of hypoxia-inducible factor 1α (HIF-1α) small interfere RNA construct pSUPERH1-siHIF1α on the expression of CD18 and ninjurin-1 by K562 (human chronic myelogenous leukemia cell line) cells cultured with serums from patients with early stage of diabetic retinopathy. MethodsK562 cells were cultured in 4 groups as control group (group A), diabetic group (group B), diabetes and pSUPERH1-siHIF1α transfect group (group C) and diabetes and pSUPER-retro transfect group (group D). The cells in group A were cultured in human serum from age-matched healthy control, and in group B, C and D, the cells were cultured in serum from the subjects of early stage of diabetic retinopathy. Twenty-four hours before the cells were cultured by the serum from the subjects of early stage of diabetic retinopathy, the HIF-1α specific siRNA expression vector pSUPERH1-siHIF1α and empty vector pSUPER-retro were transfected into the cells of group C and D, respectively. The percentages of CD18 and ninjurin-1 positive cell on the surface of K562 cells were measured by Flow Cytometry. The adherent rate between K562 and RF/6A was measured by the rose Bengal staining test. ResultsThe percentages of CD18 positive cell in the group A, B, C and D were significantly different (F=14.33, P=0.01). The percentage of group B was significantly higher than that in group A (P=0.001); the percentage of group C was significantly lower than that in group B (P=0.001) and group D (P=0.02); the difference between group C and A was not significant (95%CI=-14.89-2.13, P=0.12). The differences of the percentage of ninjurin-1 positive cell among the group A, B, C and D were significant (F=39.38, P=0.001). The percentage of group B was significantly higher than that in group A (P=0.00); the difference of the percentage between group C and B was not significant (P=0.06), that was also not significant between group C and D (P=0.49). The differences of the adherent rate between K562 and RF/6A (rhesus monkey retinal choroid blood vessel endothelial cell line) among the group A, B, C and D were significant (F=20.62, P=0.00). The adherent rate of group B was significantly higher than that in group A (P=0.00), the adherent rate in group C was significantly lower than that in group B (P=0.01), but it was still significantly higher than that in group A (P=0.002), the difference of adherent rate between group B and D was not significant (P=0.68). ConclusionUnder the early stage of diabetic retinopathy, HIF-1α small interfere RNA pSUPERH1-siHIF1α may significantly suppress the expression of CD18 on the surface of K562 cells, but it may not significantly influence the expression of ninjurin-1 on the surface of K562 cells.