Objective To extract and identify primary culture rat pulmonary arterial smooth cells ( PASMCs) , and investigate the effects of hypoxia on the proliferation of PASMCs. Methods Rat PASMCs were separated by the method of tissue block anchorage, and the cellular morphology was observed under light microscope. The cells were identified by projection electron microscopy, and α-smooth muscle actin ( α-SMactin)in the cells was identified by immunohistochemistry and immunofluorescence. The primary cultured PASMCs were exposed to normoxic and/ or hypoxia condition for 2, 6, 12, 24, 48 hours respectively, thenMTT assay and PCNA ( proliferating cell nuclear antigen) immunohistochemistry were used to detect the proliferation of PASMCs. Results The cells tended to be long spindle and grew in the “peak-valley”mode under light microscope. Immunology results showed that endochylema was stained in brownish yellow, and the positive rate was beyond 96% . There were dense patch, dense body and many filaments in endochylema under projection electron microscopy. MTT assay demonstrated that the A values of PASMCs expose to hypoxia were higher than that of nomoxia. Comparing with normoxia, the A values of PASMCs exposed to hypoxia increased after 12 hours ( P lt;0. 05) , significantly increased after 24 hours ( P lt;0. 01) . Compared with 2 hours’exposure to hypoxia, the A values increased after 12 hours( P lt; 0. 05) , markedly increased after 24 hours ( P lt; 0. 01 ) , which after 48 hours was similar with 24 hours. The result of PCNA immunohistochemistry was consistent with that of MTT. Conclusions The tissue explants adherent method is simple and convenient, and can easily obtain rat PASMCs with high purity and stability. Hypoxia canpromote the proliferation of PASMCs.
Objective To explore effects of edaravone on apoptosis and expressions of apoptotic proteins Smac and XIAP in hippocampal CA1 pyramidal cell of rats under intermittent hypoxia. Methods A total of 96 adult male Wistar rats were randomly divided into control group, 5% intermittent hypoxic group and edaravone group, and each group was divided into 4 time groups at 7 d, 14 d, 21 d and 28 d, respectively, with 8 rats in each subgroup. The content of reactive oxygen species (ROS) in hippocampal tissues of the experimental rats was detected by the reactive oxygen species detection kit. Immunohistochemistry and Western blot were used to detect the expressions of Smac and XIAP protein in hippocampal CA1 region. The Tunel method detected the apoptosis of neurons. Results Compared with the control group, the content of ROS, the expressions of Smac and XIAP proteins and the neuronal apoptosis index in the hippocampus were increased in the 5% intermittent hypoxia group and the edaravone group at each time point (all P<0.05). The content of ROS, the Smac protein expression and the neuronal apoptosis index in the edaravone group were significantly lower than those in the 5% intermittent hypoxia group (all P<0.05). The expression of XIAP protein in the edaravone group was significantly higher than that in the 5% intermittent hypoxia group (P<0.05). Conclusion Edaravone may improve the antioxidant capacity of the body by scavenging oxygen free radicals and regulate Smac and XIAP- mediated apoptosis, thus playing a protective role on neurons.
Objective
To study the protective effects of bone marrow mesenchymal stem cells (BMSCs) of rhesus monkeys on porcine islets from hypoxia/reoxygenation (H/R)-induced injury.
Methods
BMSCs were isolated and cultured from the marrow of 5 adult rhesus monkeys (weighing, 6-10 kg) by adherent monocytes. Islets were isolated and purified from the pancreas of 5 neonatal porcine (3-5 days old) by collagenase V digestion method, and were cultured with or without BMSCs, and exposed to hypoxia (1%O2) for 12 hours and reoxygenation for 24 or 48 hours, respectively. The experiment was divided into 4 groups: normal islet group (group A), normal islet + BMSCs group (Group B), H/R islet group (group C), and H/R islet + BMSCs group (group D). The survival rate of islets was calculated by fluorescein diacetate/propidium iodide (PI) staining. The viability of the islet cells was detected by cell counting kit 8. Apoptotic rate of islet cells was tested using Annexin V-FITC/PI labeling and flow cytometry. The stimulation index (SI) of islet function was analyzed by glucose-stimulated insulin secretion assay.
Results
The islet cell cluster of group C was more dispersed than that of groups A and B, and group C had more death cells; and the islet cell cluster of group D was more complete and the survival rate was higher than those of group C. The survival rate of islet was 90.2% ± 9.1%, 88.3% ± 5.9%, 52.3% ± 12.1%, and 71.4% ± 11.5% in groups A, B, C, and D respectively, it was significantly lower in groups C and D than in groups A and B (P lt; 0.05), but it was significantly higher in group D than in group C (P lt; 0.05). After coculture of BMSCs and islet at the ratio of 1
∶
10 and 1
∶
20 in group D, the viability of islet cells was significantly higher than that in group C (P lt; 0.05). The apoptotic rate was 27.1% ± 3.2%, 24.0% ± 1.0%, 64.3% ± 1.8%, and 46.2% ± 1.4% in groups A, B, C, and D respectively, it was significantly higher in groups C and D than that in groups A and B (P lt; 0.05), but it was significantly lower in group D than in group C (P lt; 0.05). There was no significant difference in SI between groups A and B at each time point (P gt; 0.05), but it was significantly lower in group C than in groups A and B (P lt; 0.05); and it was significantly higher in group D than in group C at 24 and 72 hours (P lt; 0.05).
Conclusion
BMSCs of rhesus monkeys can protect islet vitality and function from H/R-induced injury.
ObjectiveTo observe the effect of conditional knocking out (KO) vascular endothelial growth factor (VEGF) gene on the mouse model of oxygen induced retinopathy (OIR).MethodsThe conditional VEGF KO mice were generated using Cre-Loxp technology, resulting in the deletion of VEGF in a portion of Müller cells permanently in mouse retina. Cre positive was CKO mice, Cre negative was NKO mice. OIR was induced by keeping mice in 75% oxygen at postnatal 7 days (P7) to P12 and in room air from P12 to P17 (each 20 mice for CKO and NKO, respectively). The mice mortality was analyzed. At day P17, the percentage of retinal avascular area was calculated using retinal flat-mounting with fluorescence angiography, the number of vascular endothelial cell nucleus breaking through retinal inner limiting membrane was counted with hematoxylin eosin (HE) staining of retinal sections, and the expression of hypoxia-inducible factor-1α (HIF-1α) was detected by immunofluorescence analysis. ResultsDuring the development of OIR, the mortality rate of CKO mice (65.00%) was higher than that of NKO mice (30.00%) with the significant difference (x2=4.912, P=0.027). At day P17, all the mice retinas were harvested. The retinal fluorescence angiography displayed that the normal retinal vascularization of CKO mice was delayed, and large avascular areas were observed. Meanwhile, rare new vascular plexus was found in CKO mice and the thickness of whole retina decreased dramatically. In contrast, NKO mice developed larger area of normal retinal vascular network structure with higher blood vessel density and more new vascular plexus with obvious fluorescein leakage. The percentage of avascular area in CKO mice [(28.31±11.15)%] was higher than NKO mice [(16.82±7.23)%] with the significant difference (t=2.734, P=0.014). The HE staining of retinal sections indicated smaller counts of vascular endothelial cell nucleus breaking through retinal inner limiting membrane in CKO mice (26.10±6.37) when compared to NKO mice (28.80±7.59) , the difference was significant (t=2.437, P=0.016). The immunofluorescence analysis showed stronger expression of HIF-1α in CKO mice than NKO mice, which was mainly located in the retinal ganglion cell layer.ConclusionsThe local VEGF gene knockout partially inhibits retinal neovascularization in OIR mice. However, it also suppresses the normal retinal blood vascular development with a decrease of OIR mice survival ability.
ObjectiveTo comprehensively analyze and compare the biological difference between bone marrow mesenchymal stem cells (BMSCs) and placenta-derived MSCs (PMSCs) in hypoxia and to extend the knowledge for seed cells selection.
MethodsThe domestic and foreign related literature about the effects of hypoxia microenvironment on proliferation, apoptosis, differentiation, paracrine secretion, migration, and homing ability of BMSCs and PMSCs were summarized and analysed.
ResultsPMSCs proliferated much faster and more sensitive to the hypoxia than BMSCs; in addition, PMSCs showed stronger survivability. Similar to BMSCs, PMSCs can home to hypoxic-ischemic tissues efficiently, secrete a lot of growth factors and differentiate into tissue-specific cells to stimulate tissue regeneration.
ConclusionPMSCs as the seed cells will have broad application prospects in the regenerative medicine.
ObjectiveTo investigate the role of PI3K/Akt/HIF-1αsignaling pathway in bleomycin-induced pulmonary fibrosis in mice.
MethodsFifty-six C57BL/6 mice were randomly divided into a control group and a bleomycin (BLM) group.The pulmonary fibrosis model was induced by single intratracheal instillation of BLM(2.5 mg/kg) in the BLM group.Similarly, 0.9% saline was instilled directly into the trachea in the control group.Then all mice were sacrificed on 21st day.The lungs were collected for morphometric analysis with HE and Masson staining.The degree of pulmonary fibrosis was evaluated with Ashcroft score and content of hydroxyproline.The activity of PI3K/Akt/HIF-1αsignaling pathway and pro-surfactant protein C (Pro-SPC) were measured by Western blot.The level of collagen3 mRNA was assessed with quantitative real time PCR analysis.Collagen3 protein and numbers of apoptosis cells were observed with immuno-histochemistry.
ResultsIt was exhibited that the thickening alveolar septa, accumulation of inflammatory cells, and fibrous obliteration in the BLM group but not in the control group.There was a significant difference in Ashcroft score and hydryoproline content in the BLM group.Meanwhile, the activity of PI3K/Akt/HIF-1αsignaling pathway was up-regulated and the protein of Pro-SPC was decreased in the BLM group.It was revealed that the numbers of apoptosis cells, expressions of Collagen3 protein and mRNA were increased in the BLM group.
ConclusionAberrant activity of PI3K/Akt/HIF-1αsignaling pathway may aggravate the pulmonary fibrogenesis.
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 investigate the expression of telomerase reverse transcriptase (TERT) and cell apoptosis in neonatal rats with hypoxia ischemia brain damage (HIBD). Methods A total of 42 7-day-old SD rats (12-18 g, male or female) were randomly allocated into sham-operation group (n=6) and hypoxia-ischemia (HI) group (n=36). In HI group, the rats were anesthetized with ethylether. The right common carotid artery (CCA) was exposed and permanently l igated with a 7-0silk suture through a midl ine cervical incision. A duration of 2.5 hours of hypoxia (8%O2 / 92%N2) was used to produce HIBD model. For sham-operation group, the CCA was exposed without l igation or hypoxia. The brain tissues were harvested at 4, 8, 12, 24, 48, and 72 hours after completion of an HI insult. The expressions of TERT and CC3 were detected by immunohistochemical staining. The apoptosis cells were detected with TUNEL staining method. Results The expression of TERT was increased at 4 hours after HI injury, significantly increased at 24-48 hours and then decreased at 72 hours. The expression of CC3 was increased at 4 hours after HI injury, significantly increased at 24 hours and still maintained high expression at 48 hours and 72 hours. However, in the sham-operation group, both the expressions of TERT and CC3 were extremely low. The expression of TERT and CC3 were higher in the HI group than in the sham-operation group at different time points, and the differences were significant (P lt; 0.05). The TUNEL staining showed that the positive cells in hippocampus and cortical areas were increased at 4 hours after HI injury, significantly increased at 24-48 hours and maintained a high level at 72 hours. However, there was few positive cells in the sham-operation group. There were significant differences between the HI group and the sham-operation group at different time points (P lt; 0.05). Conclusion TERT could be induced by HI in neonatal rats, and might have a protective role in regulating the cell apoptosis in the neonatal HIBD.
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.
ObjectiveTo investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation.
MethodLVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3FLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94%N2, 1%O2, and 5%CO2 for 0, 3, 6, 9, and 12 hours (group B1 and group C1) . The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3.
ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t=17.525, P=0.013) . The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P<0.05) . In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P<0.05) . The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P<0.05) except at 0 hour. MTT assay showed that absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P<0.05) ; the A value of group B was significantly lower than that of group C at each time point (P<0.05) . The A value of group B1 was significantly lower than that of group C1 at 6, 9, and 12 hours after hypoxia intervention (P<0.05) . Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P<0.05) .
ConclusionsAkt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.
