Increased stemness and migration of human mesenchymal stem cells in hypoxia is associated with altered integrin expression

Human mesenchymal stem cells (hMSCs) are regularly cultured and characterised under normoxic (21% O(2)) conditions, although the physiological oxygen tension in the stem cell niche is known to be as low as 1-2%. Oxygen itself is an important signalling molecule, but the distinct impact on various stem cell characteristics is still unclear. Therefore, the aim of this study was to evaluate the influence of oxygen concentration on the hMSC subpopulation composition, cell morphology and migration on different surfaces (polystyrene, collagen I, fibronectin, laminin) as well as on the expression of integrin receptors. Bone marrow-derived hMSCs were cultured either in normoxic (21% O(2)) or hypoxic (2% O(2)) conditions. The hMSC subpopulations were assessed by aspect ratio and cell area. Hypoxia promoted a more homogeneous cell population with a significantly higher fraction of rapidly self-renewing cells which are believed to be the true stem cells. Under hypoxic conditions hMSC volume and height were significantly decreased on all surfaces as measured by white light confocal microscopy. Furthermore, low oxygen tension led to a significant increase in cell velocity and Euclidian distance on all matrixes, which was evaluated by time-lapse microscopy. With regard to cell-matrix contacts, expression of several integrin subunits was evaluated by semi-quantitative RT-PCR. Increased expression of the subunits α(1), α(3), α(5,) α(6), α(11), α(v), β(1) and β(3) was observed in hypoxic conditions, while α(2) was higher expressed in normoxic cultured hMSCs. Taken together, our results indicate that hypoxic conditions promote stemness and migration of hMSC along with altering their integrin expression.     

Effect of hypoxia on equine mesenchymal stem cells derived from bone marrow and adipose tissue

ABSTRACT: BACKGROUND: Mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (AT-MSCs) are being applied to equine cell therapy. The physiological environment in which MSCs reside is hypoxic and does not resemble the oxygen level typically used in in vitro culture (20% O2). This work compares the growth kinetics, viability, cell cycle, phenotype and expression of pluripotency markers in both equine BM-MSCs and AT-MSCs at 5% and 20% O2. RESULTS: At the conclusion of culture, fewer BM-MSCs were obtained in hypoxia than in normoxia as a result of significantly reduced cell division. Hypoxic AT-MSCs proliferated less than normoxic AT-MSCs because of a significantly higher presence of non-viable cells during culture. Flow cytometry analysis revealed that the immunophenotype of both MSCs was maintained in both oxygen conditions. Gene expression analysis using RT-qPCR showed that statistically significant differences were only found for CD49d in BM-MSCs and CD44 in AT-MSCs. Similar gene expression patterns were observed at both 5% and 20% O2 for the remaining surface markers. Equine MSCs expressed the embryonic markers NANOG, OCT4 and SOX2 in both oxygen conditions. Additionally, hypoxic cells tended to display higher expression, which might indicate that hypoxia retains equine MSCs in an undifferentiated state. CONCLUSIONS: Hypoxia attenuates the proliferative capacity of equine MSCs, but does not affect the phenotype and seems to keep them more undifferentiated than normoxic MSCs.     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

Oxygen tension differentially influences osteogenic differentiation of human adipose stem cells in 2D and 3D cultures

Skeletal defects commonly suffer from poor oxygen microenvironments resulting from compromised vascularization associated with injury or disease. Adipose stem cells (ASCs) represent a promising cell population for stimulating skeletal repair by differentiating toward the osteogenic lineage or by secreting trophic factors. However, the osteogenic or trophic response of ASCs to reduced oxygen microenvironments is poorly understood. Moreover, a direct comparison between 2D and 3D response of ASCs to hypoxia is lacking. Thus, we characterized the osteogenic and angiogenic potential of human ASCs under hypoxic (1%), normoxic (5%), and atmospheric (21%) oxygen tensions in both 2D and 3D over 4 weeks in culture. We detected greatest alkaline phosphatase activity and extracellular calcium deposition in cells cultured in both 2D and 3D under 21% oxygen, and reductions in enzyme activity corresponded to reductions in oxygen tension. ASCs cultured in 1% oxygen secreted more vascular endothelial growth factor (VEGF) over the 4‐week period than cells cultured in other conditions, with cells cultured in 2D secreting VEGF in a more sustained manner than those in 3D. Expression of osteogenic markers revealed temporal changes under different oxygen conditions with peak expression occurring earlier in 3D. In addition, the increase of most osteogenic markers was significantly higher in 2D compared to 3D cultures at 1% and 5% oxygen. These results suggest that oxygen, in conjunction with dimensionality, affects the timing of the differentiation program in ASCs. These findings offer new insights for the use of ASCs in bone repair while emphasizing the importance of the culture microenvironment. J. Cell. Biochem. 110: 87–96, 2010. © 2010 Wiley‐Liss, Inc.     

Moderate Hypoxia Influences Potassium Outward Currents in Adipose-Derived Stem Cells

Moderate hypoxic preconditioning of adipose-derived stem cells (ASCs) enhances properties such as proliferation and secretion of growth factors, representing a valuable strategy to increase the efficiency of cell-based therapies. In a wide variety of cells potassium (K⁺) channels are key elements involved in the cellular responses to hypoxia, suggesting that ASCs cultured under low oxygen conditions may display altered electrophysiological properties. Here, the effects of moderate hypoxic culture on proliferation, whole-cell currents, and ion channel expression were investigated using human ASCs cultured at 5% and 20% oxygen. Although cell proliferation was greatly enhanced, the dose-dependent growth inhibition by the K⁺ channel blocker tetraethylammonium (TEA) was not significantly affected by hypoxia. Under both normoxic and hypoxic conditions, ASCs displayed outward K⁺ currents composed by Ca²⁺-activated, delayed rectifier, and transient components. Hypoxic culture reduced the slope of the current-voltage curves and caused a negative shift in the voltage activation threshold of the whole-cell currents. However, the TEA-mediated shift of voltage activation threshold was not affected by hypoxia. Semiquantitative real-time RT-PCR revealed that expression of genes encoding for various ion channels subunits related to oxygen sensing and proliferation remained unchanged after hypoxic culture. In conclusion, outward currents are influenced by moderate hypoxia in ASCs through a mechanism that is not likely the result of modulation of TEA-sensitive K⁺ channels.     

Mesenchymal stem cells cultured under hypoxia escape from senescence via down-regulation of p16 and extracellular signal regulated kinase

Hypoxia has been considered to affect the properties of tissue stem cells including mesenchymal stem cells (MSCs). Effects of long periods of exposure to hypoxia on human MSCs, however, have not been clearly demonstrated. MSCs cultured under normoxic conditions (20% pO₂) ceased to proliferate after 15-25 population doublings, while MSCs cultured under hypoxic conditions (1% pO₂) retained the ability to proliferate with an additional 8-20 population doublings. Most of the MSCs cultured under normoxic conditions were in a senescent state after 100 days, while few senescent cells were found in the hypoxic culture, which was associated with a down-regulation of p16 gene expression. MSCs cultured for 100 days under hypoxic conditions were superior to those cultured under normoxic conditions in the ability to differentiate into the chondro- and adipogenic, but not osteogenic, lineage. Among the molecules related to mitogen-activated protein kinase (MAPK) signaling pathways, extracellular signal regulated kinase (ERK) was significantly down-regulated by hypoxia, which helped to inhibit the up-regulation of p16 gene expression. Therefore, the hypoxic culture retained MSCs in an undifferentiated and senescence-free state through the down-regulation of p16 and ERK.     

Low-oxygen and knock-out serum maintain stemness in human retinal progenitor cells

Using stem and progenitor cells to treat retinal disorders holds great promise. Using defined culture conditions to maintain the desires phenotype is of utmost clinical importance. We cultured human retinal progenitor cells (hRPCs) in different conditions: such as normoxia (20% oxygen), and hypoxia (5% oxygen) with and without knock-out serum replacement (KOSR) to evaluate its effect on these cells. KOSR is known nutrient supplement often used to replace bovine serum for culturing embryonic or pluripotent stem cells, especially those destined for clinical applications. The purpose of this study was to identify the impact of different environmental and chemical cues to determine if this alters the fate of these cells. Our results indicate that cells cultured with or without KOSR do not show significant differences in viability, but that the oxygen tension can significantly change their viability (higher in hypoxia than normoxia). However, cells with KOSR in hypoxia condition expressed significantly higher stemness markers such as C-myc and Oct4 (31.20% and 13.44% respectively) in comparison to hRPCs cultured in KOSR at normoxia (12.07% and 4.05%). Furthermore, levels of markers for retinal commitment such as rhodopsin were significantly lower in the KOSR supplemented cells in hypoxia culture compared to normoxia. KOSR is known to improve proliferation and maintain stemness of embryonic cells and our experiments suggest that hRPCs maintain their proliferation and stemness characteristics in hypoxia with KOSR supplement. Normoxia, however, results in mature cell marker expression, suggesting a profound effect of oxygen tension on these cells.

     

Effects of oxygen and culture system on in vitro propagation and redifferentiation of osteoarthritic human articular chondrocytes

Regenerative medicine-based approaches for the repair of damaged cartilage rely on the ability to propagate cells while promoting their chondrogenic potential. Thus, conditions for cell expansion should be optimized through careful environmental control. Appropriate oxygen tension and cell expansion substrates and controllable bioreactor systems are probably critical for expansion and subsequent tissue formation during chondrogenic differentiation. We therefore evaluated the effects of oxygen and microcarrier culture on the expansion and subsequent differentiation of human osteoarthritic chondrocytes. Freshly isolated chondrocytes were expanded on tissue culture plastic or CultiSpher-G microcarriers under hypoxic or normoxic conditions (5% or 20% oxygen partial pressure, respectively) followed by cell phenotype analysis with flow cytometry. Cells were redifferentiated in micromass pellet cultures over 4 weeks, under either hypoxia or normoxia. Chondrocytes cultured on tissue culture plastic proliferated faster, expressed higher levels of cell surface markers CD44 and CD105 and demonstrated stronger staining for proteoglycans and collagen type II in pellet cultures compared with microcarrier-cultivated cells. Pellet wet weight, glycosaminoglycan content and expression of chondrogenic genes were significantly increased in cells differentiated under hypoxia. Hypoxia-inducible factor-3α mRNA was up-regulated in these cultures in response to low oxygen tension. These data confirm the beneficial influence of reduced oxygen on ex vivo chondrogenesis. However, hypoxia during cell expansion and microcarrier bioreactor culture does not enhance intrinsic chondrogenic potential. Further improvements in cell culture conditions are therefore required before chondrocytes from osteoarthritic and aged patients can become a useful cell source for cartilage regeneration.     

Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.     

Autocrine fibroblast growth factor 2‐mediated interactions between human mesenchymal stem cells and the extracellular matrix under varying oxygen tension

Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor‐2 (FGF‐2). The decellularized ECM matrices derived from hMSC culture under both hypoxic (e.g., 2% O₂) and the standard culture (e.g., 20% O₂) conditions have different binding capacities to the cell‐secreted and exogenenous FGF‐2. The reduced hMSC proliferation in the presence of FGF‐2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF‐2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi‐potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. 114: 716–727, 2013. © 2012 Wiley Periodicals, Inc.     

The biosynthetic pathway of pyrimidine (deoxy)nucleotides: A sensor of oxygen tension necessary for maintaining cell proliferation?

Culture experiments on Ehrlich ascites tumor cells revealed that a low oxygen tension (about 20% in normoxic atmosphere) induced an increase in the length of the growth cycle. The relative growth of aerobic control cells after transfer to the second in vitro passage was 145% within 24 h, and reduced to 50% at 1% O2 and about 30% at 0.1% O2. The increase in protein and DNA content of these hypoxic cultures was equally impaired. Also, the cell cycle traverse as analyzed by flow cytometry was affected predominantly at the G1/early S stage. Uptake of labeled thymidine into acid-insoluble material of hypoxic cells was below that of controls whereas incorporation of uridine exceeded that of normoxic controls. Supplementation of cells cultured under 0.1 and 1% O2 with 0.1 mM uridine or 0.1 mM deoxycytidine + 0.01 mM deoxyadenosine and deoxyguanosine improved all growth parameters; deoxynucleosides were more effective than uridine in cells under 0.1% O2 whereas in cells cultured under 1% O2 similar effects of both were observed. This points to an insufficient supply of nucleic acid precursors even under moderate limitations of oxygen tension and not only under strict hypoxia. Whereas a 12-h cultivation time at 0.1% O2 hardly impaired cell growth after reoxygenation, a cultivation time of 24 h considerably reduced the cellular capability to recover. This was alleviated by addition of (deoxy)nucleosides from the beginning of hypoxic culture. The results are interpreted as supporting the concept that the biosynthetic pathway of pyrimidine (deoxy)nucleotides--because of two oxygen-dependent enzymes, dihydroorotate dehydrogenase and ribonucleotide reductase--is a potential transducer of environmental limitations in oxygen tension to the proliferative capacity of cells.     

Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.     

Hypoxia promotes satellite cell self-renewal and enhances the efficiency of myoblast transplantation

Microenvironmental oxygen (O(2)) regulates stem cell activity, and a hypoxic niche with low oxygen levels has been reported in multiple stem cell types. Satellite cells are muscle-resident stem cells that maintain the homeostasis and mediate the regeneration of skeletal muscles. We demonstrate here that hypoxic culture conditions favor the quiescence of satellite cell-derived primary myoblasts by upregulating Pax7, a key regulator of satellite cell self-renewal, and downregulating MyoD and myogenin. During myoblast division, hypoxia promotes asymmetric self-renewal divisions and inhibits asymmetric differentiation divisions without affecting the overall rate of proliferation. Mechanistic studies reveal that hypoxia activates the Notch signaling pathway, which subsequently represses the expression of miR-1 and miR-206 through canonical Hes/Hey proteins, leading to increased levels of Pax7. More importantly, hypoxia conditioning enhances the efficiency of myoblast transplantation and the self-renewal of implanted cells. Given the robust effects of hypoxia on maintaining the quiescence and promoting the self-renewal of cultured myoblasts, we predict that oxygen levels in the satellite cell niche play a central role in precisely balancing quiescence versus activation, and self-renewal versus differentiation, in muscle stem cells in vivo.     

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm × 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O₂) or hypoxic (5% O₂) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10–20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (±25°; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro. This work was supported by the Swiss National Science Foundation (grant no. 3200B0-104083).     

Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.     

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rγ^null (NOG) mice. Hypoxic culture (1% O₂) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34⁺CD38⁻ cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.     

Hypoxia protects articular chondrocytes from thapsigargin-induced apoptosis

The present study investigates the effect of low oxygen concentrations on thapsigargin-induced apoptosis and reactive oxygen species (ROS)-related signaling in articular chondrocytes. Chondrocytes were obtained from normal canine knee cartilage and were treated with different concentrations of thapsigargin for 24 h under normoxic (21% oxygen tension) or hypoxic (1% oxygen tension) conditions. The cells treated with thapsigargin under normoxic conditions showed a dose-dependent induction of apoptosis. However, the cellular changes and apoptotic events that occurred following thapsigargin treatment, were completely inhibited by hypoxia, including loss of mitochondrial transmembrane potential (MTP), ROS generation and JNK phosphorylation. Moreover, the cells exposed to hypoxic conditions showed increased expression of the anti-apoptotic proteins xIAP-2 and Bcl-2. We demonstrate that hypoxia inhibited thapsigargin-induced apoptosis in chondrocytes by regulating ROS-related signaling and the expression of anti-apoptotic proteins. We propose that maintaining hypoxic conditions in articular cartilage may be required for the prevention of chondrocyte and cartilage diseases such as arthritis.     

缺氧对新生小牛肺血管平滑肌细胞增殖的影响及764—3的抑制作用

本研究应用细胞培养、~3H—TdR参入技术,探讨缺氧对新生小牛肺血管平滑肌细胞(PASMC)DNA合成和细胞增殖的影响及746—3和川芎嗪抗细胞增殖效果。结果表明:缺氧的肺血管内皮细胞(PAEC)培养液可以明显增加PASMC中~3H—TdR的参入;缺氧24h亦可直接刺激PASMC,使其中~3H—TdR的参入显著增加(P<0.001);在缺氧的PASMC培养基中加入764—3,~3H—TdR参入值与单纯缺氧组相比显著下降,并使细胞数减少36.3％,764—3还可以显著抑制常氧PASMC的增殖;川芎嗪的作用较小。实验结果提示,缺氧不仅可以刺激内皮细胞(EC)产生促分裂素使PASMC增殖,而且还可以直接刺激PASMC的增殖,764—3可抑制缺氧及血清刺激的PASMC的增殖。