Objective To elucidate whether glucose transporters-4 （GLUT-4） takes part in glucose uptake of mesenchymal stem cells （MSCs） and whether Akt gene improves translocation and expression of GLUT-4 in MSCs under hypoxic environment ex vivo. Methods MSCs, transfected by Akt gene and no, were cultured with normoxia (5% CO2) or hypoxia (94%N2, 1%O2 and 5% CO2) at 37 ℃ for 8 h. Glucose uptake was assayed by using radiation isotope 2-［3H］-deoxy-Dglucose (3H-G) and the expression of GLUT-4 protein and mRNA was assayed by immunocytochemistry, Western blot and RT-PCR, respectively. Results ①3 H-G intake of MSCs was significantly increased in hypoxiatransfection group than that in hypoxia-non-transfection 〔（1.39±0.13） fold, P＜0.05〕, but which was lower than that in normoxia-non-transfection group, P＜0.05. ②GLUT-4 was expressed by MSCs under any conditions. Compared with normoxia-non-transfection group, hypoxia decreased the expressions of GLUT-4 mRNA and protein significantly (P＜0.05). ③Compared with hypoxianontransfection group, the expression of GLUT-4 〔mRNA（1.756±0.152） fold, total protein in cell （1.653±0.312） fold, protein in plasma membrane （2.041±0.258） fold〕 was increased in hypoxia-transfection group significantly (P＜0.05), but which was lower than that in normoxianontransfection group (P＜0.05). ④There was significantly positive relation between 3H-G intake and GLUT-4 protein expression in plasma membrane (r=0.415, P=0.001).Conclusion GLUT-4 may take part in glucose uptake of MSCs, and the capability of Akt gene to improve MSCs anti-hypoxia may be finished by its role in increasing the expression and translocation of GLUT-4.
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.
ObjectiveTo investigate whether desferrioxamine (DFO) can enhance the homing of bone marrow mesenchymal stem cells (BMSCs) and improve neovascularization in random flaps of rats.MethodsBMSCs and fibroblasts (FB) of luciferase transgenic Lewis rats were isolated and cultured. Forty 4-week-old Lewis male rats were used to form a 10 cm×3 cm rectangular flap on their back. The experimental animals were randomly divided into 4 groups with 10 rats in each group: in group A, 200 μL PBS were injected through retrobulbar venous plexus; in group B, 200 μL FB with a concentration of 1×106 cells/mL were injected; in group C, 200 μL BMSCs with a concentration of 1×106 cells/mL were injected; in group D, cells transplantation was the same as that in group C, after cells transplantation, DFO [100 mg/(kg·d)] were injected intraperitoneally for 7 days. On the 7th day after operation, the survival rate of flaps in each group was observed and calculated; the blood perfusion was observed by laser speckle imaging. Bioluminescence imaging was used to detect the distribution of transplanted cells in rats at 30 minutes and 1, 4, 7, and 14 days after operation. Immunofluorescence staining was performed at 7 days after operation to observe CD31 staining and count capillary density under 200-fold visual field and to detect the expressions of stromal cell derived factor 1 (SDF-1), epidermal growth factor (EGF), fibroblast growth factor (FGF), and Ki67. Transplanted BMSCs were labeled with luciferase antibody and observed by immunofluorescence staining whether they participated in the repair of injured tissues.ResultsThe necrosis boundary of ischemic flaps in each group was clear at 7 days after operation. The survival rate of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Laser speckle imaging showed that the blood perfusion units of flaps in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). Bioluminescence imaging showed that BMSCs gradually migrated to the ischemia and hypoxia area and eventually distributed to the ischemic tissues. The photon signal of group D was significantly stronger than that of other groups at 14 days after operation (P<0.05). CD31 immunofluorescence staining showed that capillary density in groups C and D was significantly higher than that in groups A and B, and in group D than in group C (P<0.05). The expressions of SDF-1, EGF, FGF, and Ki67 in groups C and D were significantly stronger than those in groups A and B, and in group D than in group C. Luciferase-labeled BMSCs were expressed in the elastic layer of arteries, capillaries, and hair follicles at 7 days after transplantation.ConclusionDFO can enhance the migration and homing of BMSCs to the hypoxic area of random flap, accelerate the differentiation of BMSCs in ischemic tissue, and improve the neovascularization of ischemic tissue.
Objective To observe the effects of cobalt chloride (CoCl2)-simulated hypoxia on VEGF and TGF-β1 expression and to provide theoretical basis for deci phering the molecular mechanism of cl inical distraction osteogenesis. Methods The mandibular osteoblasts were obtained from newborn Wistar rats within 24 hours and cultured and purified through modified enzymatic digestion. The morphological and histological changes of cells were evaluated by the HE staining,the histochemical staining for ALP, the collagen I immunohistochemistry staining and the calcified nodules staining, and the growth curves were drawn. The best cells of the 3rd-passage rats were treated with CoCl2, and then immunofluorescence was used to detect the expressions of VEGF and TGF-β1 at 0, 3, 6, 9, 12 and 24 hours after culture. Results The HE staining demonstrated that the cellular forms were diverse, triangular, polygonal, circular and scaly and so on. The prominence varied in length and extended outwards. The nucleus was clearly discernible. The cytoplasma was rich and pink, with the nucleus royal purple. Sometimes 2 cell nuclei were seen. At the crowded place, cellular form was not clear, the dividing l ine was indistinct, and just the great-circle nuclear cells could be seen. The ALP immunohistochemistry staining demonstrated that the cell butcher nature appeared black pellets, the cell nucleus outl ine was unclear, and at the cell compact district, massive mascul ine cells could be seen clearly. The collagen I immunohistochemistry staining demonstrated that mascul ine cells were seen evenly, cytoplasma appeared yellowish brown especially around the nucleus. However, yellowish brown pellets were not seen in negative cells. The osteoblast calcium tubercle staining demonstrated that the cells gathered in the opaque region with the shape of tubercle after15 days of culture. After al izarin red staining, the reddish orange pigmentation appeared. At various time points, weak VEGF fluorescence was seen in the cells in the control group under the laser confocal microscope. As the hypoxia time prolonged, VEGF fluorescence of cells in the experimental group intensified, and reached the peak 9 hours after peration, and then dropped to the normal level. At various time points, TGF-β1 fluorescence was found in both groups under the laser confocal microscope, and fluorescence intensity in the control group was sl ightly ber than that in the VEGF control group. In the experimental group, TGF-β1 expression had short-term increase 3 hours after hypoxia, and reduced gradually with the prolonging of hypoxia time. Conclusion The method of culturing osteoblast from Wistar rats mandibular is practicable. The cells can be used for further studies. Moderate hypoxia can affect bone synthesis and turnover in distraction osteogenesis and up-regulate the expressions of VEGF and TGF-β1.
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.
Objective Isoflurane has an acute preconditioning effectiveness against ischemia in kidney, but this beneficial effectiveness can only last for 2-3 hours. To investigate whether isoflurane produces delayed preconditioningagainst renal ischemia/reperfusion (I/R) injury, and whether this process is mediated by hypoxia inducible factor 1α(HIF- 1α). Methods A total of 52 male C57BL/6 mice were randomly assigned to 4 groups (n=13 in each group): the controlgroup (group A), PBS/isoflurane treated group (group B), scrambled small interference RNA (siRNA)/isoflurane treated group (group C), and HIF-1α siRNA/isoflurane treated group (group D). In groups C and D, 1 mL RNase-free PBS containing 50 μg scrambled siRNA or HIF-1α siRNA was administered via tail vein 24 hours before gas exposure, respectively. Equivalent RNasefree PBS was given in groups A and B. Then the mice in groups B, C, and D were exposed to 1.5% isoflurne and 25%O2 for 2 hours; while the mice in group A received 25%O2 for 2 hours. After 24 hours, 5 mice in each group were sacrificed to assesse the expressions of HIF-1α and erythropoietin (EPO) in renal cortex by Western blot. Renal I/R injury was induced with bilateral renal pedicle occlusion for 25 minutes followed by 24 hours reperfusion on the other 8 mice. At the end of reperfusion, the serum creatinine (SCr), the blood urea nitrogen (BUN), and the histological grading were measured. Results The expressions of HIF-1α and EPO in groups B and C were significantly higher than those in group A (P lt; 0.01). The concentrations of SCr and BUN in groups B and C were significantly lower than those in group A, as well as the scores of tubules (P lt; 0.01), and the injury of kidney was amel iorated noticeably in groups B and C. The expressions of HIF-1α and the concentrations of SCr and BUN in group D were significantly lower than those in group A (P lt; 0.01). Compared with groups B and C, the expression of HIF- 1α and EPO in group D decreased markedly (P lt; 0.01), the concentrations of SCr and BUN were increased obviously, as well asthe scores of tubules (P lt; 0.01), and the renal injury was aggratived significantly. Conclusion Isoflurane produces delayed preconditioning against renal I/R injury, and this beneficial effectiveness may be mediated by HIF-1α.
ObjectiveTo summarize the research progress of the regulation effect of hypoxia inducible factor (HIF) on intervertebral disc.
MethodsThe domestic and foreign related literature about the regulation effect of HIF on intervertebral disc was reviewed, summarized, and analyzed.
ResultsHIF is a key transcription factor that is in response to hypoxia by cells, which is widely distributed in tissues and organs, including intervertebral disc. Hypoxia inducible factor is expressed highest in the nucleus pulposus which has the lowest oxygen concentration in the intervertebral disc. The effects of HIF include the regulation of nucleus pulposus differentiation and development, maintenance of the survival, energy metabolism, and anabolism of nucleus pulposus cells, and maintenance of the stability of extracellular matrix.
ConclusionHIF plays a vital role in the development and differentiation of intervertebral disc and maintenance of physiological function, which may become a target for the research of the mechanism and the treatment of intervertebral disc degeneration.
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.
Autophagy is a lysosome dependent, conservative material degradation process, which exists in all eukaryotic cells and plays import roles in many pathophysiology process. Erectile dysfunction (ED) is a common male disease with multiple etiology. In recent years, more and more evidences have demonstrated that autophagy has a close relation to ED, therefore, we combine previous study to classify ED by hypoxia, aging, diabetes and other causes, and review the advances of autophagy in ED.
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.
