Objective To evaluate effect of hypoxia condition (1% or 5% oxygen concentration) on proliferation, adhesion, migration, or viability ability of bone morrow-derived endothelial progenitor cells (EPCs). Methods The bone marrow mononuclear cells of SD rat were acquired with density gradient centrifugation method. They were cultured, induced, and differentiated to the EPCs. Then they were cultured respectively in three different oxygen concentrations (1%, 5%, or 21%). On the 3rd day and the 7th day, the effects of the different oxygen concentrations (1%, 5%, or 21%) on the EPCs’ neovascularization characteristics (including proliferation, adhesion, migration, and viability abilities) were evaluated. Results Whether cultured for the 3rd day or 7th day, the proliferation, adhesion, migration, and viability abilities of the cultured cells in the 1% and 5% oxygen concentrations were significantly better than those of the cultured cells in the 21% oxygen concentration (all P<0.05). Except for the proliferation ability of the cultured cells in the 5% oxygen concentration was significantly better than that of the cultured cells in the 1% oxygen concentration (P<0.05) on the 3rd day, and the adhesion ability on the 3rd day and the proliferation ability on the 7th day had no significantly differences, the other abilities (adhesion, migration, and viability abilities) of the cultured cells in the 1% oxygen concentration were significantly better than those of the cultured cells in the 5% oxygen concentration (allP<0.05). Conclusion Different oxygen concentration has an effect on proliferation, adhesion, migration, or viability ability of bone morrow-derived EPCs, appropriate hypoxia condition (1% or 5% oxygen concentration ) can enhance these abilities.
Objective To explore the effect and mechanism of rapamycin and deferoxamin on wound healing after ischemia and hypoxia. Methods The model of ischemia and hypoxia wound was made on the back of 40 SPF male adult Sprague Dawley rats, weight (300±20) g; they were randomly divided into 4 groups (n=10): the control group (group A), deferoxamine intervention group (group B), rapamycin intervention group (group C), and deferoxamine+rapamycin intervention group (group D). At 3, 6, and 9 days after model preparation, rats of groups A, B, C, and D were intra-peritoneally injected with normal saline, deferoxamin (10 mg/kg), rapamycin (3 mg/kg), deferoxamin (10 mg/kg)+rapamycin (3 mg/kg) respectively. The wound healing was observed and the healing time was recorded in each group; the wound healing tissue was harvested to test the mRNA and protein expressions of mammalian target of rapamycin (mTOR), hypoxia inducible factor 1α (HIF-1α), and vascular endothelial growth factor (VEGF) by real-time fluorescence quantitative PCR and Western blot at 2 days after wound healing. Results All rats survived to the end of the experiment, and wounds healed; the healing time of groups A, B, and D was significantly shorter than that of group C (P<0.05), but there was no significant difference between groups A, B, and D (P>0.05). Real-time fluorescence quantitative PCR showed that the expression of mTOR mRNA in groups C and D was significantly decreased when compared with the expressions in groups A and B (P<0.05); there was significant difference between groups A and B (P<0.05), but no significant difference between groups C and D (P>0.05). The expressions of HIF-1α mRNA and VEGF mRNA were signi-ficantly higher in groups B and D than groups A and C, and in group A than group C (P<0.05), but there was no signifi-cant difference between groups B and D (P>0.05). Western blot showed that the relative expressions of mTOR protein in groups C and D were significantly decreased when compared with the expressions in groups A and B (P<0.05), but there was no significant difference between groups C and D (P>0.05). The relative expressions of HIF-1α protein in groups A, B, and C were significantly increased when compared with expression in group D (P<0.05), but there was no significant difference between groups A, B, and C (P>0.05). The relative expression of VEGF protein were significantly lower in groups B, C, and D than group A, in group D than groups B and C, and in group C than group B (P<0.05). Conclusion Defe-roxamin can promote the wound healing of rats after ischemia and hypoxia, and the effect of rapamycin is opposite. It may be related to the existence of mTOR and HIF-1 signaling pathway in chronic ischemia-hypoxia wound.
Objective To study the effect of hypoxia on the prol iferation of hBMSCs and human placental decidua basal is-MSCs (hPDB-MSCs), and to provide the theoretical basis for discovering the new seed cells source for tissue engineering. Methods Density gradient centrifugation method was adopted to isolate and culture hBMSCs and hPDB-MSCs,flow cytometry (FCM) was appl ied to detect cell surface marker. After establ ishing the experimental model of CoC12 chemical hypoxia, MTT method was appl ied to evaluate the prol iferation of hBMSCs and hPDB-MSCs at different time points (6, 12, 24, 48, 72, 96 hours) with various CoC12 concentration (0, 50, 75, 100, 125, 150, 175, 200 μmol/L). Results FCM analysis revealed that hPDB-MSCs and hBMSCs expressed CD9, CD29, CD44, CD105, CD106 and human leucocyte antigen ABC (HLA-ABC), but both were absent for CD34, CD40L and HLA-DR. Compared with hBMSCs, hPDB-MSCs expressed stage-specific embryonic antigen 1 (SSEA-1), SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81 better. The prol iferations of hPDB-MSCs and hBMSCs were inhibited within the first 12 hours under hypoxia condition, but promoted after 12 hours of hypoxia. Compared with the control group, the hBMSCs were remarkably prol iferated 24 hours after hypoxia with CoC12 concentration of 150 μmol/L (P lt; 0.05), while hPDB-MSCs were significantly prol iferated 12 hours after hypoxia with CoC12 concentration of 75 μmol/L (P lt; 0.05). Conclusion Compared with hBMSCs, hPDB-MSCs express more specific surface antigens of embryonic stem cells and are more sensitive to the prol iferation effects of chemical hypoxia, indicating it may be a new seed cells source for tissue engineering.
ObjectiveTo explore the effect of five copies hypoxia-responsive element (5HRE) and carcinoembryonic antigen promoter (CEAp) element, and to explore the inhibition effect of lentiviral vectors targeted Ras association domain family 1 isoform A (RASSF1A) gene on SGC7901 human gastric cancer cells. Methods①Expressions of carcinoembryonic antigen (CEA) mRNA and its protein, and RASSF1A protein in SGC7901, MKN28, and MCF-10A cells were detect by real time-PCR (qRT-PCR), immunocytochemistry, and Western blot, to confirm the experimental and negative control cells.②The recombinant vectors of pGL4.20-5HRE-CEAp-Luc were constructed through molecular cloning technique to transfected SGC7901, MKN28, and MCF-10A cells. Each kind of cell was divided into 2 groups:one of them didn't add CoCl2 (normoxia group), and another group added CoCl2 (hypoxia group). Comparison of the fold of activation was performed.③SGC7901 cells were infected by lentiviral vectors of pLV-5HRE-CEAp-RASSF1A (infection group) and negative virus (negative control group), SGC7901 cells without any treatment as blank control group. Then SGC7901 cells of 3 groups were divided into 2 groups:one of them didn't add CoCl2 (normoxia group), and another group added CoCl2 (hypoxia group). The expression of RASSF1A protein was tested by Western blot, and the growth inhibition rate was confirmed by cell counting kit-8 (CCK-8) assay. Comparisons of expression of RASSF1A protein and growth inhibition rate of each group were performed. Results①Results of qRT-PCR, immunocytochemistry and Western blot showed that, SGC7901 cells showed higher expression of CEA mRNA and positive expression of RASSF1A protein than corresponding index of MKN28 cells and MCF-10A cells (P < 0.05), which were assigned as experimental cells; but MKN28 cells showed lower expression of CEA mRNA and negative expression of RASSF1A protein, which were assigned as negative control cells.②In SGC7901 and MKN28 cells transfected recombinant vectors of pGL4.20-5HRECEAp-Luc, compared with normoxia group in the same kind of cell group, the folds of activation in hypoxia group were higher (P < 0.01), but there was no significant difference between the normoxia group and hypoxia group in MCF-10A cells (P > 0.05). In the condition of with or without CoCl2, compared with SGC7901 cells in the same condition, the folds of activation in MCF-10A and MKN28 cells were both lower (P < 0.05); compared with MKN28 cells, the fold of activation in MCF-10A cells was lower (P < 0.05).③Western blot results showed that, in the condition with and without CoCl2, expressions of RASSF1A protein decreased in SGC7901 cells of blank control group and negative control group; weak expressions of RASSF1A protein was observed in SGC7901 cells of infection group when in condition of without CoCl2, but increased when adding CoCl2. But RASSF1A protein didn't expressed in MKN28 cells of blank control group, negative control group, and infection group, whether adding CoCl2 or not. CCK-8 assay result showed that, in SGC7901 cells, the growth inhibition rate of infection group which added CoCl2 was higher than those of other 5 groups (P < 0.05); in MKN28 cells, the growth inhibition rates of infection group and negative group were all higher than those of blank control group, whether adding CoCl2 or not (P < 0.05), but there was no significant difference among the infection group and negative group, whether adding CoCl2 or not (P > 0.05). ConclusionsA new hypoxia inducible and cea-positive tumor-targeting transcriptional regulatory element of 5HRE-CEAp is established successfully, and lentivirus vector of pLV-5HRE-CEAp-RASSF1A can significant inhibit the growth of SGC7901 cells under hypoxia condition.
ObjectiveTo investigate the correlations among the cadual homeobox gene 2 (CDX2), hypoxia inducible factor-1α (HIF-1α) protein expressions, and tumor budding in the colorectal cancer (CRC). MethodsIn this study, 63 CRC specimens surgically removed in the First Affiliated Hospital of Xi’an Jiaotong University from January 2012 to September 2015 were collected. The CDX2 and HIF-1α protein expressions were detected by immunohistochemical staining streptavidin-biotin peroxidase two-step method. The staining and the grade of tumor budding were observed under an optical microscope, and the correlation was analyzed using Spearman test. ResultsThe positive expressions of CDX2 and HIF-1α proteins in the CRC tissues were 35 (55.6%) and 47 (74.6%) cases, respectively, which was a negative correlation in the CRC (rs=–0.302, P=0.017). The positive expressions of CDX2 and HIF-1α proteins in the tumor budding of colorectal cancer were 21 (51.2%) and 26 (63.4%) cases, respectively, which was also a negative correlation in the tumor budding of CRC (rs=–0.336, P=0.031), but there was no statistic correlation between the grade of tumor budding and CDX2 or HIF-1α positive protein expression in the CRC (rs=0.113, P=0.370; rs=–0.026, P=0.838). ConclusionsThe positive expression between CDX2 and HIF-1α has a negative correlation in the same CRC specimen and which has a negative correlation in tumor budding. There is no statistic correlation between grade of tumor budding and CDX2 or HIF-1α protein expression in the CRC. Hypoxia environment may be involved in the downregulation of CDX2 level during the malignant progression of CRC.
ObjectiveTo investigate the mechanism of the early kidney injury in rats caused by intermittent hypoxia, and investigate the intervention effect of edaravone.MethodsEighty male Wistar rats were randomly divided into a control group (NC), an intermittent hypoxia group (IH), an intermittent hypoxia edaravone treatment group (IH+NE), and an intermittent hypoxia normal saline matched group (IH+NS). After 4 weeks of model establishment, serum urea nitrogen and creatinine concentration were determined. Pathological changes of kidney were observed under light microscope, and ultrastructural changes of glomeruli and renal tubules were observed under electron microscope. The kidney injury molecule 1 (KIM-1) protein was detected by immunohistochemistry. The levels of superoxide dismutase (SOD), malondialdehyde (MDA), hydroxyl radical and Bcl-2 mRNA, Caspase-3 mRNA, Bax mRNA in homogenate of kidney tissue were measured.ResultsSerum urea nitrogen in each group showed no significant difference. Serum creatinine increased significantly in IH group and significantly decreased after edaravone treatment. There were no significant pathological damages in NC group under light and electron microscopy. IH group showed varying degrees of renal tubule damages compared with NC group. Compared with NC group, the mean optical density of KIM-1 protein in IH group and IH + NS group significantly increased (P<0.01), and the mean optical density of KIM-1 protein in IH+NE group significantly decreased (P<0.01). Compared with NC group, the activity of SOD in IH group and IH+NS group significantly decreased, the content of MDA and hydroxyl radical increased, the expression of Bcl-2 mRNA decreased, the expression of Caspase-3 mRNA and Bax mRNA increased, Bcl-2/Bax?decreased.?After edaravone intervention, the activity?of SOD in?kidney tissue?of rats significantly?increased, the content of MDA and hydroxyl radical?significantly?decreased, the expression?of Bcl-2 mRNA?increased, the expression of Caspase-3 mRNA?and Bax mRNA decreased, Bcl-2/Bax increased.ConclusionsIntermittent hypoxia can cause kidney injury through oxidative stress and regulation of Bcl-2, Bax and Caspase-3. KIM-1 may be used as a sensitive indicator for monitoring early kidney injury. Edaravone can prevent kidney injury induced by intermittent hypoxia though scavenging oxygen free radical, improving antioxidant capacity, regulating cell apoptosis mediated by regulating Bcl-2/Bax and Caspase-3.
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 role of 17beta; estradiol on th e expressi on of vascular endothelial growth factor (VEGF) and on the releasing rate of lac tate dehydrogenase (LDH) in cultured anoxiainjured human retinal pigment epit h eliual (RPE) cells. Methods:Established the anoxiainjuried m odel of human RPE c ells with Cobalt Chloride (CoCl2) after RPE cells were pretreated with 17beta;E 2 and tamoxife, 17beta;E2 antagonist. The expression of VEGF mRNA was detecte d by re v erse transcriptionpolymerase chain reaction technique (RTPCR). The cultured RP E cells were divided into four groups: normal control group, anoxiainjured gro u p, 17beta;E2 pretreatment group and 17beta;E2 with tamoxifen pretreatment grou p. The releasing rate of LDH was detected by chromatometry. The expression of VEGF pro tein were detected by cellular immunohistochemistry. Results:T he expression of VEGF and LDH releasing rate were higher in anoxiainjured grou p than that in nor m al control group (P＜0.05), and were lower in 17beta;E2 pretreatment group than th at in anoxiainjured group （P＜0.05）. When the effect of 17beta;E2 was o bstructe d by tamoxifen, the expression of VEGF and LDH releasing rate increased but didn prime;t differ much from which in anoxiainjured group (P＞0.05). Conc lusion:The ex pression of VEGF increases in anoxiainjured human RPE cells. 17beta;E2 can do wnr egulate the expression of VEGF and decrease the releasing rate of LDH, which can be blocked by tamoxifen.
ObjectiveTo investigate the expression and correlation of hypoxia inducible factor 1α (HIF-1α) and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells under hypoxia in vitro.MethodsThe nucleus pulposus cells were extracted from the nucleus pulposus of healthy adult Sprague Dawley rats and passaged. The 3rd generation cells were identified by HE staining and collagenase type Ⅱ immunofluorescence staining and randomly divided into 4 groups. The cells in group A were cultured for 8 hours under normal oxygen condition (37℃, 5%CO2, 20%O2); the cells in group B were cultured for 8 hours under hypoxia condition (37℃, 5%CO2, 1%O2); the cells in group C were transfected with HIF-1α-small interfering RNA and cultured for 8 hours under hypoxia condition; and the cells in group D were cultured with autophagy inhibitor 3-MA for 8 hours under hypoxia condition. Western blot and real-time fluorescence quantitative PCR (qRT-PCR) were used to detect the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in all groups.ResultsHE staining of the 3rd generation nucleus pulposus cells showed that the cytoplasm was light pink and the nucleus was blue black, and the collagenase type Ⅱ immunofluorescence staining was positive. Western blot and qRT-PCR results showed that the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group B were significantly higher than those in group A (P<0.05); the relative expressions of HIF-1α, Beclin1, and LC3B proteins and genes in group C were significantly lower than those in group B (P<0.05). There was no significant difference in the relative expression of HIF-1α protein and gene between groups B and D (P>0.05); while the relative expressions of Beclin1 and LC3B proteins and genes in group D were significant lower than those in group B (P<0.05).ConclusionHypoxia can induce the expressions of HIF-1α and autophagy related molecules (Beclin1 and LC3B) in rat nucleus pulposus cells, and HIF-1α in rat nucleus pulposus cells under hypoxia is related to the expression of autophagy related molecules, that is, down-regulation of HIF-1α can significantly reduce the expression of autophagy related molecules, while the down-regulation of autophagy levels under hypoxia has no or little effect on the expression of HIF-1α.
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.