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1.
We developed selective monoclonal antibodies and used them for Western and immunocytochemical analyses to determine the tissue and cellular distribution of the human cyclic GMP-stimulated phosphodiesterase (PDE2). Western analysis revealed PDE2A expression in a variety of tissue types, including cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle. Immunocytochemical analysis revealed PDE2A expression in a subset of tissue endothelial cells. PDE2A immunostaining was detected in venous and capillary endothelial cells in cardiac and renal tissue but not in arterial endothelial cells. These results were confirmed by in situ hybridization. PDE2A immunostaining was also absent from luminal endothelial cells of large vessels, such as aorta, pulmonary, and renal arteries, but was present in the endothelial cells of the vasa vasorum. PDE2A immunostaining was detected in the endothelial cells of a variety of microvessels, including those in renal and cardiac interstitial spaces, renal glomerulus, skin, brain, and liver. Although PDE2A was not readily detected in arterial endothelial cells by immunocytochemistry of intact tissue, it was detected at low levels in cultured arterial endothelial cells. These results suggest a possible role for PDE2A in modulating the effects of cyclic nucleotides on fluid and inflammatory cell transit through the endothelial cell barrier.  相似文献   

2.
An experimental kidney infarction model was developed in the mouse to study the uptake of a radiolabeled monoclonal antibody previously shown to bind to degenerating cells in malignant tumors. To determine if this approach is applicable to normal tissue and cell degeneration, kidney infarction was produced by clamping the mouse renal artery for 3 h using surgical procedures. Various groups of mice were injected with 131I-labeled TNT-1 F(ab')2 monoclonal antibody directed against nuclear histone antigens at varying intervals after surgery. Imaging, biodistribution, autoradiography, and histological studies were performed on each group of mice, including sham-operated controls, to quantitate the level of binding and localize the uptake of label in clamped and unclamped (contralateral) kidneys. As additional controls, clamped mice were administered radiolabeled irrelevant monoclonal antibody Lym-1 or mouse albumin. The results showed a marked selective uptake of radiolabeled TNT-1 F(ab')2 in the injured clamped kidney compared with the untreated kidney and other normal organs of the mouse. These studies define a model of normal organ necrosis that may be useful for study of the kinetics of antibody uptake in infarcted tissues.  相似文献   

3.
Green fluorescent protein as a marker in transgenic mice   总被引:8,自引:0,他引:8  
Green fluorescent protein (GFP) found in Aequorea victoria absorbs blue light and emits green fluorescence without exogenous substrates or co-factors. We studied the possibility of using the GFP as a marker in mammals. Transgenic mice were produced using the GFP coding sequence, ligated with the chicken beta-actin promoter. Green fluorescence was observed in muscle, pancreas, kidney, heart and other organs in all the three transgenic mouse lines. Detection of the transgenic mouse was possible by observing a tail or fingers of new born pups under a fluorescent microscope. The marker also enabled us to detect localized expression of the transgene in intact tissues without preliminary steps. It was also demonstrated that the GFP expression could be quantified by measuring the fluorescence in tissue extracts.  相似文献   

4.
Ischemia causes oxygen deprivation, cell injury and related organ dysfunction. Although ischemic injury may be local, it involves many biochemical changes in different cell types. The ability of stem cells to differentiate into different cell lineages provides the possibility of their use in treating a variety of diseases requiring tissue repair or reconstitution, such as stroke, ischemic retinopathy, myocardial infarction, ischemic disorders of the liver, ischemic renal failure, and ischemic limb dysfunction. Several cell types including embryonic stem cells, various progenitor and stem cells of hematopoietic or mesenchymal origin have been used in attempts to reconstitute injured tissue. Xenologous or autologous stem cells may be administered either through the peripheral vascular system or directly by regional injection. The stem cells are then guided to the infarct site by homing signals. Either by cell differentiation or paracrine effects, stem cells or progenitor cells participate in the reconstruction of a favorable microenvironment resulting in neovascularization and tissue regeneration that eventually improve the physiological function of organs with ischemic damage.  相似文献   

5.
Intestine absorption of intact green fluorescent protein (GFP) and its following accumulation in the renal proximal tubule cells after its intragastric administration have been established by confocal microscopy in the rat and frog. Reabsorbed GFP was revealed in the endosomes and lysosomes of the proximal tubule cells by the methods of GFP photooxidation and immunofluorescent microscopy. The GFP intestine absorption rate and GFP accumulation in the kidney were significantly higher in the frog than in the rat. No specific fluorescence was revealed in the liver and colon cells after the GFP intragastric administration. The data obtained indicate the ability of the small intestine in the frog and rat to absorb intact proteins and an important role of the kidney in exogenous protein metabolism.  相似文献   

6.
The pathophysiological sequelae of myocardial infarction include neutrophil infiltration into the infarct zone that contributes to additional damage to viable tissue and removal of cellular debris from necrosed tissue. Reactive chlorinating species produced by myeloperoxidase amplify the oxidant capacity of activated neutrophils. Plasmalogens are a major phospholipid subclass of both endothelial cells and cardiac myocytes. Recent studies have shown that plasmalogens are targeted by neutrophil-derived reactive chlorinating species that lead to the production of alpha-chloro fatty aldehydes. Results herein demonstrate that the alpha-chloro fatty aldehyde 2-chlorohexadecanal (2-ClHDA) accumulates in rat hearts subjected to left anterior descending coronary artery occlusion. Myocardia from rats subjected to surgical infarction had increased 2-ClHDA and neutrophil infiltration levels compared with myocardia from rats subjected to sham surgery. Additionally, infarcted myocardia from rats rendered neutropenic by treatments with an anti-neutrophil antibody had diminished 2-ClHDA and neutrophil infiltration levels compared with infarcted myocardia from normopenic rats; 2-ClHDA was shown to elicit myocardial damage as determined by lactate dehydrogenase release in isolated perfused rat hearts. Additionally, 2-ClHDA treatment of hearts resulted in decreased heart rate and ventricular performance. Taken together, the present results demonstrate a novel neutrophil-dependent myeloperoxidase-based mechanism that results in 2-ClHDA production in response to regional myocardial infarction that may also contribute to cardiac dysfunction.  相似文献   

7.
The appearance of atrial natriuretic peptide (ANP) in the ventricular myocardium was investigated in rat hearts subjected to severe left ventricular infarction. The left coronary artery was ligated for 1, 2, 3, 4 and 6 days and for 3 weeks, and the tissue was prepared for microscopic examination of immunoreactive ANP and for electron microscopy. In the normal and sham-operated hearts, and in hearts subjected to 1 day of coronary ligation, ANP immunoreactivity was restricted to a few ventricular myocytes of the conduction system. Following 2–3 days of coronary ligation, ANP immunoreactivity was detected in the viable myocardium of the lateral border of the infarct and in a few layers of viable cardiac myocytes located in the subendocardial areas of the ischemic left free ventricular wall. Further, during the following days and after 3 weeks of coronary ligation, a gradient of specific labeling was commonly seen across the lateral border area of the infarct. Thus, the strongest immunoreactivities were present in the cardiac myocytes located adjacent to the non-contracting myocardium. Electron microscopic examination of the immunoreactive cardiac myocytes confirmed the presence of electron-dense specific granules within these cells. The present findings suggest that the increased regional production of ANP within the ventricular myocardium is induced by increased mechanical stretch of the cardiac myocytes, and that this might contribute to the increased release of ANP in myocardial infarction.  相似文献   

8.
Mesenchymal stem cells (MSCs) have an ability to migrate in the organism to injured tissue to exert influence on inflammation and reparation in these regions. The aim of this study was to determine the optimal time of MSCs transplantation for myocardial reparation in rat experimental heart failure. The experiments were carried out on inbred line Wistar-Kyoto rats. Myocardial experimental infarction (EI) was induced by ligation of the left descending coronary artery. MSCs were isolated from bone marrow, cultivated in vitro and injected into the tail vein on the day of experimental infarction operation. It was shown that the time of MSCs transplantation exerted an essential influence on angiogenesis in a damaged myocardium, on ventricular dilatation and morphological structure of the scar. The best time for MSCs transplantation was determined within two days before EI, and seven days after EI. As a result, the overload of the border zone of infarct region decreased, and no features of infarction relapse were shown in the border zone.  相似文献   

9.
This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 °C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 µM resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.  相似文献   

10.
Positioned at the boundary between intra- and extravascular compartments, endothelial cells may influence many processes through their production of plasminogen activators (PA). Available data have shown that tissue-type plasminogen activator (t-PA) is the major form produced by human endothelial cells. We have compared the molecular forms of PA produced by human endothelial cells from different microvascular and large vessel sources including two different sites within the circulation of the kidney. Using combined immunoactivity assays specific for u-PA and t-PA activity and antigen, we found that both human renal microvascular and renal artery endothelial cells produced high levels of u-PA antigen (60.48 ng/10(5) cells/24 h and 50.42 ng/10(5) cells/24 h, respectively) and corresponding levels of u-PA activity after activation with plasmin. Activity was not evident before plasmin activation, showing that the u-PA produced is almost exclusively as single chain form U-PA. In contrast, human omental microvascular endothelial cells and human umbilical vein endothelial cells produced exclusively t-PA (8.80 ng/10(5) cells/24 h and 2.17 ng/10(5) cells/24 h, respectively). Neither endothelial cell type from human kidney produced plasminogen activator inhibitor, as determined by reverse fibrin autography and titration assays. Agents including phorbol ester, thrombin, and dexamethasone were shown to regulate the renal endothelial cell production and mRNA expression of both u-PA and t-PA. Among the macro- and microvascular endothelial cells tested, only those from the renal circulation produced high levels of single chain form U-PA, suggesting the vascular bed of origin determines the expression of plasminogen activators.  相似文献   

11.
Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo.   总被引:2,自引:0,他引:2  
Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock. Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli. MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin. Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent. epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain. However, fluorescence mainly accumulated in kidneys. Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules. Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species. We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect. Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.  相似文献   

12.
Angiotensin-converting enzyme (ACE) is expressed in many tissues, including vasculature and renal proximal tubules, and its genetic ablation in mice causes abnormal renal structure and functions, hypotension, and male sterility. To test the hypothesis that specific physiological functions of ACE are mediated by its expression in specific tissues, we generated different mouse strains, each expressing ACE in only one tissue. Here, we report the properties of two such strains of mice that express ACE either in vascular endothelial cells or in renal proximal tubules. Because of the natural cleavage secretion process, both groups also have ACE in the serum. Both groups were as healthy as wild-type mice, having normal kidney structure and fluid homeostasis, though males remained sterile, because they lack ACE expression in sperm. Despite equivalent serum ACE and angiotensin II levels and renal functions, only the group that expressed ACE in vascular endothelial cells had normal blood pressure. Expression of ACE, either in renal proximal tubules or in vasculature, is sufficient for maintaining normal kidney functions. However, for maintaining blood pressure, ACE must be expressed in vascular endothelial cells. These results also demonstrate that ACE-mediated blood pressure maintenance can be dissociated from its role in maintaining renal structure and functions.  相似文献   

13.
Guo Y  Su L  Wu J  Zhang D  Zhang X  Zhang G  Li T  Wang J  Liu C 《Cytotechnology》2012,64(4):391-401
Although green fluorescent protein (GFP) labeling is widely accepted as a tracking method, much remains uncertain regarding the retention of injected GFP-labeled cells implanted in ischemic organs. In this study, we evaluate the effectiveness of GFP for identifying and tracking implanted bone marrow- mesenchymal stem cells (BM-MSCs) and the effect of GFP on the paracrine actions of these cells. MSCs isolated from rat femur marrow were transduced with a recombinant adenovirus carrying GFP. After transplantation of the GFP-labeled BM-MSCs into the infarct zone of rat hearts, the survival, distribution, and migration of the labeled cells were analyzed at 3, 7, 14, and 28 days. To evaluate the effect of GFP on the paracrine actions of BM-MSCs, Western blot analysis was performed to detect the expression of vascular endothelial growth factor (VEGF), b fibroblast growth factor (b FGF), tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinases-2 (MMP-2). GFP was successfully expressed by BM-MSCs in vitro. At 14 days after cell transplantation the GFP-positive cells could not be detected via confocal microscopy. By using a GFP antibody, distinct GFP-positive cells could be seen and quantitative analysis showed that the expression volume of GFP was 6.42 ± 0.92 mm3 after 3 days, 1.24 ± 0.76 mm3 after 7 days, 0.33 ± 0.03 mm3 after 14 days, and 0.09 ± 0.05 mm3 after 28 days. GFP labeling did not adversely affect the paracrine actions of BM-MSCs. GFP labeling could be used to track MSC distribution and their fate for at least 28 days after delivery to rat hearts with myocardial infarction, and this stem cell tracking strategy did not adversely affect the paracrine actions of BM-MSCs.  相似文献   

14.
目的:筛选大鼠急性心梗后的心室重构过程中差异表达的微小RNA(microRNA, miRNA),为miRNA 调控心室重构的机制 研究提供靶标。方法:20 只成年雄性Wistar 大鼠,分组如下:心肌梗死组(MI组,n=10)和假手术组(Sham 组,n=10)。通过结扎大鼠 左冠状动脉前降支构建急性心梗模型建模。4 周后对大鼠进行超声心动图检查和梗死边缘区心肌HE 染色观察心室重构程度。利 用miRNA芯片对心梗边缘区的miRNA进行差异表达检测,采用实时定量PCR验证芯片结果的可靠性。结果:心脏超声显示MI 组大鼠左室重构明显,心梗边缘区心肌HE染色可见细胞间质大量胶原纤维沉积。miRNA 芯片结果显示15 个miRNA在心梗4 周后呈差异表达,其中11 个miRNA(miR-21、miR-23a、miR-125b、miR-132、miR-146b、miR-181b、miR-199a、miR-320、miR-324、 miR-328 和miR-499)表达上调,4 个miRNA(miR-29、miR-30c、miR-133a 和miR-208)表达下调。实时定量PCR 验证结果与芯片结 果一致。结论:这些差异表达的miRNA 可能与心梗后心室重构相关,进一步深入研究特定miRNA 的调控机制有望为基因治疗提 供新靶点。  相似文献   

15.
The mechanism and membrane localization of choroid plexus (CP) organic anion transport were determined in apical (or brush border) membrane vesicles isolated from bovine choroid plexus and in intact CP tissue from cow and rat. Brush border membrane vesicles were enriched in Na(+),K(+)-ATPase (20-fold; an apical marker in CP) and demonstrated specific, sodium-coupled transport of proline, glucose, and glutarate. Vesicular uptake of the anionic herbicide 2, 4-dichlorophenoxyacetic acid (2,4-D) was markedly stimulated by an inward sodium gradient but only in the presence of glutarate, indicating the presence of apical dicarboxylate/organic anion exchange. Consistent with this interpretation, an imposed outward glutarate gradient stimulated 2,4-D uptake in the absence of sodium. Under both conditions, uptake was dramatically slowed and overshoot was abolished by probenecid. Likewise, apical accumulation of 2,4-D by intact bovine choroid plexus tissue in vitro was stimulated by external glutarate in the presence of sodium. Glutarate stimulation was abolished by 5 mM LiCl. Identical findings were obtained using rat CP tissue, which showed both sodium/glutarate-stimulated 2,4-D (tissue/medium (T/M) approximately 8) and p-aminohippurate (T/M = 2) transport. Finally, since the renal exchanger (rROAT1) has been cloned in rat kidney, a rROAT1-green fluorescent protein construct was used to analyze exchanger distribution directly in transiently transfected rat CP. As predicted by the functional studies, the fluorescently tagged transporter was seen in apical but not basolateral membranes of the CP.  相似文献   

16.
Vascular mural cells in healing canine myocardial infarcts.   总被引:4,自引:0,他引:4  
Angiogenesis is a critical process in healing of myocardial infarcts, leading to the formation of highly vascular granulation tissue. However, effective cardiac repair depends on mechanisms that inhibit the angiogenic process after a mature scar is formed, preventing inappropriate expansion of the fibrotic process. Using a canine model of reperfused myocardial infarction, we demonstrated that maturation of the infarct leads to the formation of neovessels, with a thick muscular coat, that demonstrate distinct morphological characteristics. Many of these "neoarterioles" lack a defined internal elastic lamina and demonstrate irregular deposits of extracellular matrix in the media. Vascular mural cells in healing infarcts undergo phenotypic changes, showing minimal expression of desmin during the proliferative phase (1 hr occlusion/7 days reperfusion) but in the mature scar (8 weeks reperfusion) acquire a phenotype similar to that of vascular smooth muscle cells in control areas. Non-muscle myosin heavy chains A and B are induced in infarct endothelial cells and myofibroblasts, respectively, but are not expressed in neovascular mural cells. Recruitment of a muscular coat and formation of neoarterioles in mature scars may inhibit endothelial cell proliferation and vascular sprouting, stabilizing the infarct vasculature.  相似文献   

17.
Beraprost sodium, an orally active prostacyclin analogue, has vasoprotective effects such as vasodilation and antiplatelet activities. We investigated the therapeutic potential of beraprost for myocardial ischemia. Immediately after coronary ligation of Sprague-Dawley rats, beraprost (200 microg/kg/day) or saline was subcutaneously administered for 28 days. Four weeks after coronary ligation, administration of beraprost increased capillary density in ischemic myocardium, decreased infarct size, and improved cardiac function in rats with myocardial infarction. Beraprost markedly increased the number of CD34-positive cells and c-kit-positive cells in plasma. Also, four weeks after coronary ligation of chimeric rats with GFP-expressing bone marrow, bone marrow-derived cells were incorporated into the infarcted region and its border zone. Treatment with beraprost increased the number of GFP/von Willebrand factor-double-positive cells in the ischemic myocardium. These results suggest that beraprost has beneficial effects on ischemic myocardium partly by its ability to enhance neovascularization in ischemic myocardium by mobilizing bone marrow cells.  相似文献   

18.
We previously demonstrated that verapamil inhibits serotonin uptake by bovine pulmonary arterial endothelial cells by a mechanism not involving alterations in calcium fluxes. In this study, we determine whether verapamil inhibition of serotonin uptake occurs in other pulmonary cell types (bovine pulmonary artery smooth muscle cells), in cells from other organs and species (rat epididymal endothelial cells), and in intact organs (isolated rat lungs). We also compare the effects of verapamil with those of nifedipine and diltiazem. At concentrations of 10(-6) M or greater, verapamil is an inhibitor of serotonin uptake by cultured cells and isolated lungs. Nifedipine and diltiazem are weak inhibitors of serotonin uptake by cultured bovine cells only at suprapharmacologic doses and have no effect on serotonin uptake by isolated lungs. Surprisingly, nifedipine stimulates serotonin uptake by rat epididymal endothelial cells. We conclude that inhibition of serotonin uptake by verapamil is a generalized phenomenon, occurring in a variety of cell types, in intact organs, and in different species that does not occur consistently with other calcium channel blockers.  相似文献   

19.
目的:筛选大鼠急性心梗后的心室重构过程中差异表达的微小RNA(microRNA,miRNA),为miRNA调控心室重构的机制研究提供靶标。方法:20只成年雄性Wistar大鼠,分组如下:心肌梗死组(MI组,n=10)和假手术组(Sham组,n=10)。通过结扎大鼠左冠状动脉前降支构建急性心梗模型建模。4周后对大鼠进行超声心动图检查和梗死边缘区心肌HE染色观察心室重构程度。利用miRNA芯片对心梗边缘区的miRNA进行差异表达检测,采用实时定量PCR验证芯片结果的可靠性。结果:心脏超声显示MI组大鼠左室重构明显,心梗边缘区心肌HE染色可见细胞间质大量胶原纤维沉积。miRNA芯片结果显示15个miRNA在心梗4周后呈差异表达,其中11个miRNA(miR-21、miR-23a、miR-125b、miR-132、miR-146b、miR-181b、miR-199a、miR-320、miR-324、miR-328和miR-499)表达上调,4个miRNA(miR-29、miR-30c、miR-133a和miR-208)表达下调。实时定量PCR验证结果与芯片结果一致。结论:这些差异表达的miRNA可能与心梗后心室重构相关,进一步深入研究特定miRNA的调控机制有望为基因治疗提供新靶点。  相似文献   

20.
We examined the cellular distribution of glutathione transferase A4 (GSTA4) in various human tissues by indirect immunoperoxidase using a specific polyclonal antibody raised in rabbit. This enzyme was localized in hepatocytes, bile duct cells, and vascular endothelial cells in liver, upper layers of keratinocytes and sebaceous and sweat glands in skin, proximal convoluted tubules in kidney, epithelial cells of mucosa and muscle cells in colon, muscle cells in heart, and neurons in brain. Staining was increased in pathological situations such as cirrhosis, UV-irradiated skin, and myocardial infarction and was strongly decreased in hepatocellular carcinoma. These results strongly support the view of a close correlation between cellular GSTA4 localization and the formation of reactive oxygen species in the tissues investigated.  相似文献   

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