An in vitro cell culture system to study the influence of external pneumatic compression on endothelial function

PURPOSE: External pneumatic compression (EPC) is an effective means of prophylaxis against deep venous thrombosis. However, its mechanism remains poorly understood. Understanding of the biological consequences of EPC is an important goal for optimizing performance of the EPC-generating device and providing guidance for clinical use. We present a new in vitro cell culture system (Venous Flow Simulator) that simulates blood flow and vessel collapse conditions during EPC, and we examine the influence of these factors on endothelial cell (EC) fibrinolytic activity and vasomotor function. METHODS: An in vitro cell culture system was designed to replicate the hemodynamic shear stress and vessel wall strain associated with induced blood flow during different modes of EPC. Human umbilical vein endothelial cells were cultured in the system and subjected to intermittent flow, vessel collapse, or a combination of the two. The biologic response was assessed through changes in EC morphology and the expression of fibrinolytic factors tissue plasminogen activator, plasminogen activator inhibitor type 1, profibrinolytic receptor (annexin II), and vasomotor factors endothelial nitric oxide synthase and endothelin-1. RESULTS: The cells remained attached and viable after being subjected to intermittent pulsatile flow (F) and tube compression (C). In F and F + C, cells aligned in the direction of flow after 6 hours. Northern blot analysis of messenger RNA shows that there is an upregulation of tissue plasminogen activator expression (1.95 +/- 0.19 in F and 2.45 +/- 0.46 in FC) and endothelial nitric oxide synthase expression (2.08 +/- 0.25 in F and 2.11 +/- 0.21 in FC). Plasminogen activator inhibitor type 1, annexin II, and endothelin 1 show no significant change under any experimental conditions. The results also show that pulsatile flow, more than vessel compression, influences EC morphology and function. CONCLUSION: Effects on ECs of intermittent flow and vessel collapse, either individually or simultaneously, were simulated with an in vitro system of new design. Initial results show that intermittent flow associated with EPC upregulates EC fibrinolytic potential and influences factors altering vasomotor tone. The system will facilitate future studies of EC function during EPC.     

In vitro effects of hyperbaric oxygen on sickle cell morphology.

STUDY OBJECTIVE: To determine in vitro whether hyperbaric oxygen has any effect on the morphology of sickle cells. DESIGN: Prospective, in vitro, study, with each patient sample serving as its own control. SETTING: University medical center. PATIENTS: 10 children known to be homozygous for hemoglobin S. INTERVENTIONS: Blood samples were obtained from 10 children during routine visits to the University sickle cell clinic. Blood samples were exposed to room air to achieve maximal sickling. Each sample was divided into control and study aliquots, and the study portions placed in a research hyperbaric chamber with 100% oxygen at 3 atmospheres absolute pressure for 15 min. Then smears were prepared from all samples at regular intervals and examined by technicians in the sickle cell clinic who were blinded as to the details of this study. MEASUREMENTS: Percentages of normal cells, sickle cells and sickle forms were reported. Data were interpreted using t-tests. MAIN RESULTS: Hyperbaric oxygen appeared to have no effect on sickle cell morphology. Percentages of each cell type were unaffected by hyperbaric oxygen exposure. CONCLUSIONS: Hyperbaric oxygen appears to have no effect on the morphology of sickle cells in vitro. Other mechanisms may account for the beneficial clinical effects of hyperbaric oxygen in sickle cell crisis, although in vivo studies are warranted.     

三磷酸腺苷对体外培养大鼠雪旺细胞的作用

目的观察三磷酸腺苷（ATP）对体外培养的大鼠雪旺细胞的影响，探讨ATP促进周围神经再生的作用机制。方法用SD乳鼠坐骨神经双酶消化后的剩余组织块培养雪旺细胞，G-418纯化后培养24h，分成5组继续培养10d：ATP组（按不同浓度分4组）和正常组。相差显微镜下观察计数，绘制各自的增殖曲线。在24和72h对各组细胞进行流式细胞分析。结果0．1mmol ATP组雪旺细胞的增殖不明显；1、10mmol组24h和72h后处于S期的雪旺细胞明显增多；100mmol组雪旺细胞数则明显减少；1、10、100mmol组和对照组的倍增时间分别为6．4、5．6、10．2、8．0d，差异有统计学意义（P〈0．05）。结论ATP能显著地促进雪旺细胞的增殖从而促进外周神经再生，但其作用具有浓度依赖性，高浓度ATP反而抑制雪旺细胞的增殖。     

In vitro effects of bleomycin on a carcinoma of the oesophagus cell line.

A dose of 10 microgram/ml bleomycin inhibited the growth of oesophageal carcinoma cells and non-squamous cells in vitro. Drug concentration, density of cells and the time of exposure to the drug were important variables. The oesophagus cancer cells increased in volume by 2,5 +/- 0,2% per hour until the onset of lysis at 96 hours. The effects of the drug were irreversible even when the exposure time was only 24 hours. A certain proportion of the cell population was resistant to bleomycin and these cells remained resistant when the drug was removed for 48 hours and then replaced. No drug inactivation or uptake could be detected with the use of microbiological assay after 4 days of incubation with 3 cell types. There is poor correlation between in vitro and in vivo results.     

Effect of cannabinoids on human Sertoli cell function in vitro

Exposure to marijuana adversely affects spermatogenesis in humans and rodents. Since the Sertoli cell interfaces between changes in the serum hormonal milieu and the adluminal compartment and acts in various ways to support the germinal epithelium, this adverse marijuana mediated effect might be transmitted locally in the testes of humans by interference with Sertoli cell function. This report describes the effect of three active marijuana components: delta-9-tetrahydrocannabinol (THC), cannabidiol (CD) and cannabinol (CN) on various human Sertoli cell markers: transferrin, protein secretion and gamma glutamyl transpeptidase activity utilizing cultured human Sertoli cells. THC, CD, and CN tested in concentrations up to 200 ng/ml did not change transferrin secretion by these human Sertoli cells. Transferrin represents 1-4% of the total newly synthesized proteins of human Sertoli cells in culture. Even when the Sertoli cells were incubated for 30 days with THC there was no effect on transferrin secretion. The presence or absence of follicle-stimulating hormone, testosterone (T) or Fe3+ in the culture media did not modify the effect of THC. THC, CD, and CN also did not alter total protein secretion by Sertoli cells nor affect the level of gamma glutamyl transpeptidase activity.     

Direct effects of ethane dimethanesulphonate on epididymal function in adult rats. An in vitro demonstration.

It was recently demonstrated that the Leydig cell toxicant ethane dimethanesulphonate (EDS) produces multiple effects on the epididymis after a single in vivo exposure. To determine whether any of the perturbations were mediated by a direct action of the compound, we used a novel system for the coculture of epididymal epithelial cells and sperm from the caput epididymidis. This system maintains the morphologic integrity and cell polarity of the epididymal epithelial cells before and during coculture, and the sperm recovered after coculture have intact plasma and acrosomal membranes. In addition, several functions required for epididymal sperm maturation are expressed, including the secretion of protein by the epididymal epithelium, the association of secreted protein with the plasma membrane of cocultured sperm, and the acquisition of progressive motility by cocultured sperm. In vitro exposure of epididymal epithelial cells and sperm to EDS results in a significant decline in protein secretion by the epithelial cells during coculture, and in particular, a dose-dependent decline in a 36- to 38-kd protein (PI 4.0 to 4.5) and a 34- to 36-kd protein (PI 4.5 to 5.0). Moreover, these and other proteins are not recovered from the sperm membrane of cocultured sperm after EDS treatment. Finally, EDS results in a dose-dependent decline in the percentage of both motile and progressively motile sperm recovered after coculture compared with that of sperm from untreated cocultures. These effects on sperm motility were not observed when sperm were pretreated with EDS and subsequently cocultured with untreated epithelial cells. We conclude that EDS alters epididymal sperm maturation by acting directly on the epididymal epithelium to mediate changes in sperm membrane protein, and that this may subsequently alter the development of the progressive motility of sperm.     

The effects of immunosuppressive agents on the function of human hepatocytes in vitro

Calcineurin inhibitors (tacrolimus) and steroids continue to be an important component of hepatocyte transplantation protocols, despite reports of hepatotoxicity and inhibitory effects of steroids on cell proliferation. The aim of the study was to investigate whether isolated human hepatocytes were more vulnerable to the toxicity of these agents and also to investigate their effects on hepatocyte VEGF secretion, a vascular permeability factor suggested to be involved in the cell engraftment process. Human hepatocytes were isolated from donor livers/segments rejected or unused for orthotopic liver transplantation using a collagenase perfusion technique. Hepatocytes were plated for cell function tests and to determine VEGF production. Tacrolimus (0-50 ng/ml) and methylprednisolone (0-500 ng/ml) were added to the culture media and cells incubated for 24 h. Cell metabolic activity was assessed using the MTT assay, cell number using the SRB assay, and cell attachment from hepatocyte total protein content and protein synthesis using [14C]leucine incorporation. VEGF in culture supernatants was measured by ELISA. Tacrolimus and methylprednisolone had no statistically significant inhibitory effects on metabolic activity or protein synthesis compared to controls at all concentrations of the agents tested when added after plating. There were also no significant effects on cell attachment when tacrolimus or methylprednisolone was added at the time of cell plating. There were no differences in the responses obtained when either fresh or cryopreserved hepatocytes were used. The amount of VEGF secreted by untreated hepatocytes was highly variable (0-1400 pg/10(6) cells/24 h). VEGF levels in the culture supernatant from hepatocytes isolated from < or = 20-year-old donors (687 +/- 59 pg/10(6) cells/24 h) was significantly greater than from older donors (61 +/- 7 pg/10(6) cells/24 h; p = 0.003). Tacrolimus and methylprednisolone did not significantly affect VEGF secretion by hepatocytes. Tacrolimus and methylprednisolone did not have detrimental effects on the metabolic function of human hepatocytes, cell attachment, or VEGF secretion after cell isolation.     

沙利度胺对前列腺癌PC3细胞的体外作用及机制研究

目的 研究沙利度胺对激素非依赖性前列腺癌(AIPC)细胞株PC-3体外生长的抑制作用及其可能的机制.方法 将不同浓度的沙利度胺作用于AIPC细胞株PC-3,采用CCK-8法检测沙利度胺对PC-3细胞的增殖抑制作用;流式细胞仪检测凋亡率;通过RT-PCR法检测沙利度胺处理前后PC-3细胞中低氧诱导因子-1(HIF-1α)和血管内皮生长因子(VEGF) mRNA的表达水平;Western blot检测沙利度胺作用后HIF-1α、VEGF、Bcl-2、Bax蛋白的表达水平.结果 不同浓度的沙利度胺作用于PC-3细胞后,细胞增殖水平明显下降(P＜0.05),且沙利度胺对PC-3细胞增殖的抑制作用呈浓度-时间依赖性.流式细胞仪检测结果表明不同浓度的沙利度胺诱导PC-3细胞发生凋亡,和对照组相比差异有统计学意义(P＜0.05).随着沙利度胺浓度的增加,PC-3细胞中HIF-1 α、VEGF mRNA的表达逐渐下调;HIF-1α、VEGF和Bcl-2蛋白的含量逐渐下降,而Bax蛋白的含量逐渐增加.结论 沙利度胺可能通过诱导AIPC细胞的凋亡和抑制肿瘤血管的生成而发挥双重抗肿瘤生长的作用.     

The effects of bupivacaine and pipecoloxylidide on platelet function in vitro

The influence of bupivacaine and its major metabolite, pipecoloxylidide, on human platelet function was studied in vitro. Significant inhibition of ADP and collagen-induced platelet aggregation occurred only with concentrations of bupivacaine above 10 micrograms.ml-1. This concentration (10-25 micrograms.ml-1) is much higher than would be expected in routine clinical use of bupivacaine for epidural analgesia. The inhibition of platelet aggregation was associated with a significant decrease in beta-thromboglobulin secretion. In contrast, pipecoloxylidide had no effect on platelet aggregation or the beta-thromboglobulin release. We conclude that the previously reported 30-min time-lag between the maximal plasma concentration of bupivacaine and the inhibition of platelet aggregation is unlikely to be due to a metabolism of bupivacaine to pipecoloxylidide.     

Direct effects of phosphate on vascular cell function

Elevated serum phosphate has clinically been associated with vascular stiffness and cardiovascular mortality. Mechanistic studies over the past decade regarding local effects of phosphate on the vessel wall have provided insight into various pathways that culminate in vascular calcification. Smooth muscle cell phenotype change and apoptosis play prominent roles. The sodium-phosphate cotransporter PiT-1 is required for the osteochondrogenic differentiation of smooth muscle cells in vitro. Less is known about phosphate-driven valve interstitial cell calcification and elastin degradation. In this article, we review the current knowledge about phosphate-induced changes in the vascular wall.     

Effects of different anaesthetic agents on immune cell function in vitro

BACKGROUND AND OBJECTIVE: Anaesthesia may affect the regulatory balance of postoperative immune response. The aim of this study was to investigate the effects of different volatile and non-volatile anaesthetic agents and particularly of clinically used agent combinations on the proliferation capacity and cytokine production of immune cells. METHODS: Peripheral blood mononuclear cells from healthy donors were PHA-activated in the presence or absence of various concentrations of thiopental, propofol, fentanyl, sufentanil, sevoflurane, nitrous oxide and combinations of these anaesthetics. Cell proliferation was assessed by tritiated thymidine uptake. Interleukin-2 production and release of the soluble IL-2 receptor were determined by enzyme immunoassays and used as measures of lymphocyte activation. RESULTS: Thiopental inhibited cell proliferation in a dose dependent manner (P < 0.001) and reduced sIL-2R release (2090-970 microg mL(-1); P < 0.05). Propofol reduced sIL-2R release at the high concentration of 10 microg mL(-1) (2220 pg mL(-1) 1780 microg mL(-1); p < 0.05). Fentanyl and sufentanil did not compensate for or enhance the inhibitory effects of thiopental. Nitrous oxide, but not sevoflurane, reduced the proliferation of human peripheral blood mononuclear cells (P < 0.05). In combinations with thiopental or nitrous oxide, sevoflurane compensated the inhibitory effects of these two agents. Fentanyl, sufentanil, sevoflurane and nitrous oxide did not affect PHA-induced IL-2 and sIL-2 receptor release by human peripheral blood mononuclear cells. CONCLUSION: Thiopental and nitrous oxide have immunosuppressive activity. In contrast, sevoflurane may have a beneficial effect by alleviating the immunosuppressive effects of both substances.     

Influence of rat testicular macrophages on Leydig cell function in vitro

The influence of co-cultures of rat testicular macrophages and Leydig cells (LC) on LC morphology and steroidogenesis was investigated with and without macrophage stimulation by a bacterial lipopolysaccharide (LPS). LC showed an elongated form in the presence of stimulated testicular macrophages. In the presence of non-stimulated testicular macrophages a significant inhibition of testosterone production was observed (decrease of 33%) from 48 h in co-culture while an increase of 16% was obtained at the same culture time, after stimulation of macrophages by LPS. When LC were treated with testicular macrophage-conditioned media (MCM) obtained from LPS-treated macrophages, they became fusiform and there was stimulation (78%) of steroid production. After human FSH stimulation (1-1000 mIU ml-1), MCM from testicular macrophages was no more effective in enhancing testosterone production by LC than was media from untreated LC. Similar experiments with LPS were conducted with macrophages of peritoneal origin. Peritoneal macrophages stimulated or not by LPS in co-cultures with LC or peritoneal MCM did not significantly modify testosterone production. However, these cells were able to modify LC morphology when LPS-MCM was added to LC-culture medium. The present results suggest strongly that testicular macrophage-LC interactions could be important in the control of LC steroidogenesis.     

体外肾系膜细胞对成骨细胞增殖及功能的影响

目的:从生长因子角度研究肾小球系膜细胞对成骨细胞增殖及功能的影响和机制。方法:应用体外细胞培养的方法,将成骨细胞分为正常血清组和系膜细胞组,分别予10%正常血清培养液及20%系膜细胞培养上清液。MTT法分别检测培养24、48、72、120 h后两组成骨细胞增殖情况;在培养72及144 h后检测上清液骨钙素(BGP)及骨桥蛋白(OPN)浓度;在培养144 h后检测培养液胰岛素样生长因子-α(IGF-α)、转化生长因子-β(TGF-β)的浓度。结果:在培养的各个时间点,系膜细胞组的成骨细胞增殖均明显高于正常血清组(P<0.05)。在培养72、144 h后,系膜细胞组BGP及OPN的浓度分别高于正常血清组11.3%、16.4%和55.0%、39.6%。在培养144 h后,系膜细胞组的IGF-α、TGF-β浓度比正常血清组分别增高10.1%和47.7%。结论:肾小球系膜细胞能直接作用于成骨细胞,促进成骨细胞的增殖及功能。这种作用可能是通过促进成骨细胞分泌TGF-β来实现的。     

Differential effects of glucocorticoids on human osteoblastic cell metabolism in vitro

Clinical observations suggest that the onset and severity of glucocorticoid (GC) induced osteoporosis is dependent on the duration of the GC treatment and the applied GC compound. To test whether these in vivo observations are reflected by different in vitro effects of various synthetic GCs on human bone cell metabolism we isolated human osteoblast-like cells (HOC) from bone biopsies of healthy (no clinical symptoms of arthritis or arthrosis) adults who underwent selective orthopedic surgery. HOC were identified as bone cells by 1,25-vitamin D₃-stimulated increase of specific alkaline phosphatase (ALP) activity, secretion of osteocalcin and type-I procollagen peptide, and the ability to form mineral in vitro. We investigated the effects of dexamethasone (dexa), methylprednisolone (mpred), prednisolone (pred), and deflazacort (defla) on DNA-synthesis, ALP, and osteocalcin (OC)- and type-I procollagen peptide secretion of HOC in vitro. In summary, (1) GC exposure stimulates DNA synthesis after 6–12-hour treatment periods; (2) dex and mpred strongly inhibit DNA (48-hour treatment) and collagen synthesis but stimulate ALP, whereas pred and defla exhibit smaller effects on DNA synthesis, ALP, and collagen production; and (3) all tested glucocorticoids inhibit OC secretion by HOC in vitro. Thus, the effect of GC on DNA synthesis of HOC varies with the duration of GC exposure, and dex and mpred more potently affect HOC metabolism in vitro than pred and defla.This work was presented in part at the 23rd European Symposium on Calcified Tissues in Heidelberg 1993.     

Inhibitory effects of intravenous anesthetics on mast cell function

Mast cells play a protective role in the inflammation and auto-tissue injury. The impairment of mast cell function may influence defense against infection. We investigated the effect of four IV anesthetics (thiopental, midazolam, ketamine, and propofol) on the chemotaxis and exocytosis of mast cells. Canine mast cell chemotaxis was measured by the Boyden's blindwell chamber technique using 100 microg/mL of substance P as a stimulator. We measured mast cell exocytosis by measuring released histamine from mast cells using substance P or gamma-monomeric IgG-mediated crosslinking as a stimulator. Thiopental, midazolam, and propofol exerted a dose-dependent inhibitory effect on mast cell chemotaxis. Ketamine, midazolam, and propofol had a dose-dependent inhibitory effect on mast cell exocytosis. In conclusion, midazolam and propofol inhibited both chemotaxis and exocytosis of mast cells, while thiopental only inhibited chemotaxis, and ketamine only inhibited exocytosis. IMPLICATIONS: Mast cells play an important role in the antibacterial host-defense mechanism. Thiopental, midazolam, and propofol exerted a dose-dependent inhibitory effect on mast cell chemotaxis. Ketamine, midazolam, and propofol had a dose-dependent inhibitory effect on mast cell exocytosis. The impairment of mast cell function by IV anesthetics may influence the defense against infection.