Hypoxia stimulates osteopontin expression and proliferation of cultured vascular smooth muscle cells: potentiation by high glucose

We examined the effect of hypoxia on proliferation and osteopontin (OPN) expression in cultured rat aortic vascular smooth muscle (VSM) cells. In addition, we determined whether hypoxia-induced increases in OPN and cell proliferation are altered under hyperglycemic conditions. Quiescent cultures of VSM cells were exposed to hypoxia (3% O(2)) or normoxia (18% O(2)) in a serum-free medium, and cell proliferation as well as the expression of OPN was assessed. Cells exposed to hypoxia for 24 h exhibited a significant increase in [(3)H]thymidine incorporation followed by a significant increase in cell number at 48 h in comparison with respective normoxic controls. Exposure to hypoxia produced significant increases in OPN protein and mRNA expression at 2 h followed by a gradual decline at 6 and 12 h, with subsequent significant increases at 24 h. Neutralizing antibodies to either OPN or its receptor beta3 integrin but not neutralizing antibodies to beta5 integrin prevented the hypoxia-induced increase in [(3)H]thymidine incorporation. Inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein (MAP) kinase also reduced the hypoxia-induced stimulation of proliferation and OPN synthesis. Exposure to high-glucose (HG) (25 mmol/l) medium under normoxic conditions also resulted in significant increases in OPN protein and mRNA levels as well as the proliferation of VSM cells. Under hypoxic conditions, HG further stimulated OPN synthesis and cell proliferation in an additive fashion. In conclusion, hypoxia-induced proliferation of cultured VSM cells is mediated by the stimulation of OPN synthesis involving PKC and p38 MAP kinase. In addition, hypoxia also enhances the effect of HG conditions on both OPN and proliferation of cultured VSM cells, which may have important implications in the development of diabetic atherosclerosis associated with arterial wall hypoxia.     

A role for sodium in hypoxic but not in hypothermic injury to hepatocytes and LLC-PK1 cells

BACKGROUND: Hypothermia is considered to be responsible for sodium influx during cold hypoxic incubation. However, we have previously shown that hypothermia alone leads to a pronounced decrease in cellular sodium content when liver endothelial cells or hepatocytes are incubated under such conditions. In the research described here, we therefore studied the effects of hypothermia and hypoxia, alone or combined, on cellular sodium homeostasis and assessed the role sodium plays in the pathogenesis of hypoxic and hypothermic injury to cultured liver and kidney cells. METHODS: Isolated hepatocytes and LLC-PK1 cells were incubated in Krebs-Henseleit buffer or a sodium-free modification thereof under normoxic and hypoxic conditions at 4 degrees C as well as at 37 degrees C. Cytosolic sodium concentration was determined in isolated hepatocytes under both warm and cold conditions using digital fluorescence microscopy and the Na+-sensitive dye sodium-binding benzofuran isophthalate. RESULTS: When hepatocytes were incubated under cold normoxic conditions the cellular sodium concentration decreased. However, it increased strongly under hypoxic conditions at 4 degrees C and at 37 degrees C. When either hepatocytes or LLC-PK1 cells were incubated under hypoxic conditions at 4 degrees C or 37 degrees C, sodium-free medium provided protection. In contrast, sodium-free medium did not alleviate the hypothermic injury observed when cells were incubated under cold normoxia. CONCLUSIONS: The sodium influx observed during cold hypoxia is triggered by hypoxia and not by hypothermia. Sodium plays a prominent role in hypoxic injury to cultured liver and kidney cells, although hypothermic injury of these cells is independent of sodium homeostasis.     

Hypoxia/re-oxygenation injury induces apoptosis of LLC-PK1 cells by activation of caspase-2

Hypoxia/re-oxygenation injury induces apoptosis in renal tubule cells, but its underlying molecular pathways are not fully elucidated. Activation of caspase-2 has recently been proposed as a novel mechanism of apoptosis in fibroblasts. In this study we examined whether hypoxia/re-oxygenation injury induces apoptosis in proximal tubule cells by activation of caspase-2. Porcine proximal tubule (LLC-PK1) cells were subjected to hypoxia/re-oxygenation injury in the presence or absence of caspase inhibitors. Apoptosis was detected by DNA laddering, flow cytometry, and immunocytochemistry for Bax and cytochrome c. The activity of caspases-2, 8 and 9 was measured. Apoptosis was evident after hypoxia/re-oxygenation and was best prevented by pretreatment with caspase-2 inhibitor. Hypoxia/re-oxygenation resulted in a dramatic increase in caspase-2 activity (32-fold, in comparison with a 16-fold increase in caspase-8 activity and a tenfold increase in caspase-9 activity). Immunocytochemistry revealed Bax activation and translocation to mitochondria and cytochrome c release into the cytosol following hypoxia/re-oxygenation, both of which were significantly suppressed by pretreatment with caspase-2 inhibitor. These results indicate that hypoxia/re-oxygenation injury in cultured proximal tubule cells induced apoptosis by activation of caspase-2, which is required for the mitochondrial translocation of Bax.     

Oxygen tension is an important mediator of the transformation of osteoblasts to osteocytes

Osteocytes are derived from osteoblasts, but reside in the mineralized bone matrix under hypoxic conditions. Osteocyte-like cells show higher expression of ORP150, which is induced by hypoxia, than osteoblast-like cells. Accordingly, we hypothesized that the oxygen tension may regulate the transformation of osteoblasts to osteocytes. MC3T3-E1 cells and calvariae from 4-day-old mice were cultured under normoxic (20% O₂) or hypoxic (5% O₂) conditions. To investigate osteoblastic differentiation and tranformation to osteocytes, alizarin red staining was done and the expression of various factors was assessed. Hypoxic culture promoted the increased synthesis of mineralized matrix by MC3T3-E1 cells. Alkaline phosphatase activity was initially increased during hypoxic culture, but decreased during osteogenesis. Osteocalcin production was also increased by hypoxic culture, but decreased after mineralization. Furthermore, expression of Dmp1, Mepe, Fgf23, and Cx43, which are osteocyte-specific or osteocyte-predominant proteins, by MC3T3-E1 cells was greater under hypoxic than under normoxic conditions. In mouse calvarial cultures, the number of cells in the bone matrix and cells expressing Dmp1 and Mepe were increased by hypoxia. In MC3T3-E1 cell cultures, ORP150 expression was only detected in the mineralized nodules under normoxic conditions, while its expression was diffuse under hypoxic conditions, suggesting that the nodules were hypoxic zones even in normoxic cultures. These findings suggest that a low oxygen tension promotes osteoblastic differentiation and subsequent transformation to osteocytes.     

Osteopontin mediates hypoxia-induced proliferation of cultured mesangial cells: role of PKC and p38 MAPK

BACKGROUND: We previously reported that hypoxia induces the proliferation of cultured mesangial cells mediated by the stimulation of intracellular calcium and the activation of protein kinase C (PKC). In the present study, we examined the roles of mesangial cell specific growth factors (platelet-derived growth factor and endothelin-1) and osteopontin (OPN) in hypoxia-induced proliferation of mesangial cells. In addition, we determined the effect of hypoxia on p38 mitogen-activated protein (MAP) kinase activity and the roles of both PKC and p38 MAP kinase in hypoxia-induced alterations in OPN and mesangial cell growth. METHODS: Quiescent cultures of mesangial cells were exposed to hypoxia (3% O2) or normoxia (18% O2) in a serum-free medium, and [3H]-thymidine incorporation, OPN protein and mRNA expression, and p38 MAP kinase activity were assessed. RESULTS: Hypoxic-conditioned medium mimicked the effect of hypoxia on thymidine incorporation, suggesting the release of diffusable growth promoting factor(s) by hypoxia. Neither anti-endothelin-1 nor anti-platelet-derived growth factor-neutralizing antibodies had an effect on increased thymidine incorporation induced by hypoxia. However, blocking the effects of OPN either with anti-OPN antibody or its beta3 integrin receptor antibody completely prevented the hypoxia-induced increase in thymidine incorporation. Hypoxia also stimulated OPN protein and mRNA levels. Hypoxia caused an acute activation of p38 MAP kinase, which was inhibited by both verapamil and an inhibitor of PKC (calph C). PKC inhibitor and an inhibitor of p38 MAP kinase (SB203580) reduced the hypoxia-induced stimulation of both OPN and cell growth. CONCLUSIONS: These studies provide, to our knowledge, the first evidence demonstrating the role of OPN in hypoxia-induced proliferation of mesangial cells. In addition, hypoxia causes an activation of p38 MAP kinase in a calcium- and PKC-dependent manner, and the activation of PKC and p38 MAP kinase appears to be involved in the stimulation of both OPN and mesangial cell proliferation induced by hypoxia.     

Binding of the renal epithelial cell line LLC-PK1 to laminin is regulated by protein kinase C.

The alpha6beta1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of alpha6beta1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-beta1 integrin antibodies and by anti-alpha6 integrin antibodies. Antibodies against alpha2 integrin and a3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the alpha6beta1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-beta1 integrin and anti-alpha6 integrin antibodies. PKC activation did not alter expression of beta1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the alpha6betal integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface alpha6beta1 integrin.     

c-Jun氨基末端激酶信号通路对缺氧上调血管内皮细胞钙/钙调蛋白依赖性丝氨酸蛋白激酶表达的影响

目的 了解缺氧对钙／钙调蛋白依赖性丝氨酸蛋白激酶（CASK）表达的影响及c-Jun氨基末端激酶（JNK）信号通路在其中的作用。方法 将人血管内皮细胞株EA．hy926进行缺氧处理3h后继续常规培养0、12、24、48、72h，同时设常氧培养对照。采用蛋白质印迹法检测CASK的表达。构建CASK启动子区荧光素酶报告基因质粒。用其转染细胞后，于常氧及缺氧培养1、3、6、12h裂解细胞提取总蛋白，检测报告基因萤火虫荧光素酶及内参照海肾素荧光素酶活性，并用蛋白质印迹法检测JNK磷酸化情况。在培养的细胞中分别加入不同剂量（0、10、100nmo/L，1、10μmol／L）的JNK抑制剂SP600125预处理1h后再缺氧培养3h，观察其对CASK表达的影响。结果缺氧处理后常规培养0～72h，细胞CASK持续保持高表达，并明显高于常氧组。随着缺氧时间延长荧光素酶相对活性普遍增加，均高于常氧组（0．010±0．003，P〈0．01），且缺氧12h达峰值（0．192±0．023）。JNK的磷酸化随缺氧时间延长逐渐增强。加入SP600125后，CASK的表达显著降低，并呈剂量一效应依赖性，其浓度为10μmol／L时，抑制效率达高峰。结论 缺氧上调血管内皮细胞CASK的表达部分依赖于JNK信号通路活化。     

Hypoxic pulmonary vasoconstriction and pulmonary artery tissue cytokine expression are mediated by protein kinase C

Background. Pulmonary arteries exhibit a marked vasoconstriction when exposed to hypoxic conditions. Although this may be an adaptive response to match lung ventilation with perfusion, the potential consequences of sustained pulmonary vasoconstriction include pulmonary hypertension and right heart failure. The concomitant production of proinflammatory mediators by the pulmonary artery itself may exacerbate acute increases in pulmonary vascular resistance. We hypothesized that acute hypoxia causes pulmonary arterial contraction and increases the pulmonary artery tissue expression of proinflammatory cytokines via a protein kinase C (PKC) mediated mechanism. Methods. Isometric force displacement was measured in isolated rat pulmonary artery rings during hypoxia (95% N₂/5% CO₂, pO₂ = 30-35 mmHg) in the presence and absence of the protein kinase C inhibitor chelerythrine (1 μmol/L). Following 60 min of hypoxia, pulmonary artery rings were subjected to mRNA analysis for TNF-μ, IL-1β, and iNOS via RT-PCR. Data were analyzed using two-way analysis of variance (ANOVA) with post-hoc Bonferonni test or unpaired t tests with alpha level less than 0.05 considered statistically significant. Results. Hypoxia caused a biphasic contraction: an early and delayed contraction which occurred 1-3 and 15-20 min, respectively, after the onset of hypoxia. Hypoxic pulmonary artery tissue had increased expression of TNF-μ, IL-1β, and iNOS mRNA compared to normoxic controls. PKC inhibition significantly (P < 0.001) attenuated delayed hypoxic contraction (61.55 ± 3.91% versus 94.07 ± 5.94% in hypoxia alone) and prevented hypoxia-induced pulmonary artery tissue expression of TNF-μ, IL-1β, and iNOS mRNA. Conclusions. These findings demonstrate that hypoxia results in pulmonary artery contraction and promotes the expression of inflammatory mediators. Both processes are mediated by PKC. We conclude that there may be a therapeutic role for PKC inhibition in the treatment of acute hypoxic pulmonary vasoconstriction.     

Intracellular Ca<Superscript>2+</Superscript> regulation in hepatocytes under experimental transplantation conditions

Under transplant conditions excessive accumulation of intracellular calcium ([Ca2+](i)) is considered to be a mediator of cell injury during ischaemia and re-oxygenation. To clarify this consideration as well as the necessity of calcium-free preservation solutions, we used a well-known in-vitro model. Furthermore, a new application to mimic clinical situation was established, and we evaluated the correlation between [Ca2+](i) change and cell survival in monolayers of isolated rat hepatocytes undergoing cold hypoxia in defined solutions and during re-oxygenation. [Ca2+](i) was measured in single cells by ratio imaging of Fura-2 fluorescence after various periods of cold hypoxia (ischaemia phase) in different preservation solutions [UW, HTK, EC and Krebs-Henseleit buffer (KH)] and following warm normoxic reperfusion (re-oxygenation phase) with KH. Cell survival was measured simultaneously by trypan-blue exclusion. Cell survival decreased, depending on preservation solution and preservation time. The partially tremendous [Ca2+](i) change under cold hypoxia did not correlate with the change in cell survival. For example, UW-stored cells showed a [Ca2+](i) loss from 280 nM to 56 nM, compared with KH-stored cells with a [Ca2+](i) increase of up to 445 nM. Our results indicate that [Ca2+](i) plays only a minor role in the pathomechanisms of hypoxic and re-oxygenation hepatocellular injury.     

HIF activation protects from acute kidney injury

The contribution of hypoxia to cisplatin-induced renal tubular injury is controversial. Because the hypoxia-inducible factor (HIF) pathway is a master regulator of adaptation to hypoxia, we measured the effects of cisplatin on HIF accumulation in vitro and in vivo, and tested whether hypoxic preconditioning is protective against cisplatin-induced injury. We found that cisplatin did not stabilize HIF-1alpha protein in vitro or in vivo under normoxic conditions. However, hypoxic preconditioning of cisplatin-treated proximal tubular cells in culture reduced apoptosis in an HIF-1alpha-dependent fashion and increased cell proliferation as measured by BrdU incorporation. In vivo, rats preconditioned with carbon monoxide before cisplatin administration had significantly better renal function than rats kept in normoxic conditions throughout. Moreover, the histomorphological extent of renal damage and tubular apoptosis was reduced by the preconditional treatment. Therefore, development of pharmacologic agents to induce renal HIF might provide a new approach to ameliorate cisplatin-induced nephrotoxicity.     

Intracellular mechanism of mitochondrial adenosine triphosphate-sensitive potassium channel activation with isoflurane

The precise mechanism of isoflurane and mitochondrial adenosine triphosphate-sensitive potassium channel (mitoK(ATP)) interaction is still unclear, although the mitoK(ATP) is involved in isoflurane-induced preconditioning. We examined the role of various intracellular signaling systems in mitoK(ATP) activation with isoflurane. Mitochondrial flavoprotein fluorescence (MFF) was measured to quantify mitoK(ATP) activity in guinea pig cardiomyocytes. To confirm isoflurane-induced MFF, cells were exposed to Tyrode's solution containing either isoflurane (1.0 +/- 0.1 mM) or diazoxide and then both drugs together (n = 10 each). In other studies, the following drugs were each added during isoflurane administration: adenosine or the adenosine receptor antagonist 8-(p-sulfophenyl)-theophylline (SPT); the protein kinase C (PKC) activators phorbol-12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu); the PKC inhibitors polymyxin B and staurosporine; the tyrosine kinase inhibitor lavendustin A; or the mitogen-activated protein kinase inhibitor SB203580 (n = 10 each). Isoflurane potentiated MFF induced by diazoxide (100 micro M), and diazoxide also increased isoflurane-induced MFF. PMA (0.2 micro M), PDBu (1 micro M), and adenosine (100 micro M) induced MFF. However, SPT (100 micro M), polymyxin B (50 micro M), staurosporine (200 nM), lavendustin A (0.5 micro M), and SB203580 (10 micro M) all failed to inhibit the effect of isoflurane. Our results show that isoflurane, adenosine, and PKC activate mitoK(ATP). However, our data do not support an action of isoflurane through pathways involving adenosine, PKC, tyrosine kinase, or mitogen-activated protein kinase. These results suggest that isoflurane may directly activate mitoK(ATP). IMPLICATIONS: Our results show that isoflurane activates mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, but not through pathways involving adenosine, protein kinase C, tyrosine kinase, or p38 mitogen-activated protein kinase. Isoflurane may directly activate mitoK(ATP) channels.     

Haemodynamic and metabolic changes induced by repeated episodes of hypoxia in pigs

Background : Repeated hypoxia and surgical trauma trigger a potent neuroendocrine response and their association is thought to play a pivotal role in the pathogenesis of multi-organ dysfunction. We investigated the cardiovascular and metabolic responses to repeated acute hypoxia in anaesthetised and surgically instrumented pigs. Methods : Under ketamine-midazolam anaesthesia, 15 pigs were surgically instrumented for measurements of cardiac output, vascular pressures and organ blood flows. Lactate production and O₂ uptake were determined in the brain, liver, kidney and intestine. Ten animals were subjected to two 12-min periods of ventilatory hypoxia (FIO₂=7%) followed by re-oxygenation and 5 animals underwent 120–min normoxic ventilation (Control group). Results : Both hypoxic challenges produced a comparable release of catecholamines that was associated with increased cardiac output and redistribution of blood flow away from the intestinal and renal areas towards the brain and the liver; O² uptake was markedly reduced in the intestine (– 56 ± 10%, P < 0.05) and least affected in the brain and the kidney (– 19±12% and — 23±21%, respectively). During the second hypoxic test, lethal cardiovascular depression occurred in 5 animals; these non-survivors demonstrated impaired hyperdynamic response and incomplete recovery of intestinal O₂ uptake during the first hypoxia/reoxygenation test. In the Control group, normoxic ventilation was not associated with significant haemodynamic and metabolic changes. Conclusion : Intraoperative hypoxia causes marked heterogeneity in organ blood flow and metabolism. The inability to develop a hyperdynamic cardiovascular response during a first hypoxic event, as well as a persistent intestinal O² debt following re-oxygenation, predict the occurrence of death during the second hypoxic insult.     

Hypoxia regulates PDGF-B interactions between glomerular capillary endothelial and mesangial cells

BACKGROUND: Platelet-derived growth factor (PDGF)-B regulates mesangial cell and vessel development during embryogenesis, and contributes to the pathogenesis of adult renal and vascular diseases. Endothelial cell PDGF-B exerts paracrine effects on mesangial cells, but its regulation is not well defined. We examined the impact of hypoxia on PDGF-B-mediated interactions between glomerular endothelial and mesangial cells, a condition of potential relevance in developing, and diseased adult, kidneys. METHODS: Glomerular endothelial or mesangial cells were subjected to hypoxia and responses compared to normoxic cells. Endothelial PDGF-B was studied by Northern and Western analysis. Mesangial proliferative responses to PDGF-B were assessed by (3)H-thymidine incorporation, and migration by a modified Boyden chamber assay. Hypoxia-induced changes in receptor specific binding capacity were studied by saturation binding assays. RESULTS: Hypoxia stimulated increases in endothelial PDGF-B mRNA and protein. In normoxic mesangial cells, PDGF-B stimulated dose-dependent proliferation, but the proliferative response of hypoxic cells was two to three times greater. Exogenous PDGF-B also caused prompter migration in hypoxic mesangial cells. Mesangial cells were treated with endothelial cell-conditioned medium. More cells migrated when hypoxic cells were stimulated with hypoxic conditioned medium, than when normoxic cells were stimulated with normoxic conditioned medium. Preincubating conditioned medium with PDGF-B neutralizing antibody greatly decreased the chemoattractant activity. Binding studies demonstrated increased specific binding capacity in hypoxic cells. CONCLUSION: Hypoxia enhances PDGF-B paracrine interactions between glomerular endothelial and mesangial cells. These hypoxia-regulated interactions may be important during glomerulogenesis in fetal life and during the pathogenesis of adult glomerular disease.     

Reoxygenation results in cell death of human alveolar epithelial cells.

BACKGROUND: The functional response of isolated alveolar epithelial cells (AECs) to ischemia/reperfusion injury (I/R) is incompletely understood. Using a cell culture model, we investigated the tolerance of human type II alveolar cells (ATII) to hypoxia and subsequent reoxygenation. METHODS: Cell cultures of A549 cells (human lung adenocarcinoma) and primary ATII were incubated in 95% N(2)/5% CO(2) saturated medium at 37 degrees C for 48 hours or 72 hours. The hypoxic medium was subsequently exchanged to normoxic medium at 37 degrees C. Lactate dehydrogenase (LDH) release and mitochondrial viability, as assessed by WST-1 metabolism, were determined during both hypoxia and reoxygenation. A549 cells and ATII maintained under normoxic conditions served as controls. RESULTS: Before reoxygenation, after 48 or 72 hours of hypoxia, WST-1 metabolism in A549 cells was significantly reduced (p < 0.05), but LDH release remained low in both cell types. Reoxygenation after 48 h of hypoxia was associated with recovery of WST-1 metabolism and an only minimal increase in LDH release. Reoxygenation after 72 hours of hypoxia, in contrast, induced marked injury in both A549 cells and primary ATII as indicated by significantly reduced WST-1 metabolism and a dramatic increase of LDH release compared with normoxic controls (p < 0.05). CONCLUSIONS: Viability of alveolar cell lines and primary ATII is maintained during hypoxia for up to 72 hours. Reoxygenation after 72 hours of hypoxia results in rapid development of injury and cell death in both cell types.     

缺氧对人脐静脉内皮细胞株增殖与活力的影响

目的了解缺氧对人脐静脉内皮细胞增殖与活力的影响。方法将体外培养的人脐静脉内皮细胞株 EA.hy926分为常氧组和缺氧组。常氧组常规培养,缺氧组细胞充入混合气体(含体积分数94%N_2、5%CO_2、1%O_2)后置于37℃缺氧培养1、3、6、12 h。提取两组细胞总蛋白,用蛋白质印迹法检测血管内皮生长因子(VEGF)和增殖细胞核抗原(PCNA)的表达水平;以噻唑蓝法检测细胞活力,并用流式细胞仪检测细胞周期。结果与常氧组比较,缺氧组细胞培养1 h VEGF 表达增高,6 h 达高峰,12 h 降低但仍明显高于常氧组;培养3 h 时细胞 PCNA 蛋白表达开始升高,6 h 达峰值并持续至12 h。培养1、3 h,缺氧组细胞活力较常氧组明显增高(P<0.05),6 h 开始下降,至12 h 时低于常氧组但差异无统计学意义(P>0.05)。缺氧组细胞培养1、3、6 h,G_0/G_1期细胞均较常氧组少,S 期和 G_2/M 期细胞增多,增殖指数(PI)各为(43±9)%、(39±11)%、(40±11)%,均高于常氧组(32±9)%,其中3、6 h 时差异有统计学意义(P<0.05);12 h 时 PI 为(27±4)%,降至常氧组水平以下(P<0.05)。结论缺氧早期可促进人脐静脉内皮细胞增殖,但随着缺氧时间的延长细胞增殖将受抑制。