Effects of aging and HIF-1alpha deficiency on permeability of hippocampal vessels

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.     

Effects of docosahexaenoic acid or arachidonic acid supplementation on the behavior of cardiomyocytes derived from human pluripotent stem cells

Background: Human heart changes its energetic substrates from lactate and glucose to fatty acids during the neonatal period. Noticing the lack of fatty acids in media for the culture of cardiomyocytes derived from human pluripotent stem cells (hiPS-CM), researchers have supplemented mixtures of fatty acids to hiPS-CM and reported the enhancement in the maturation of hiPS-CM. In our previous studies, we separately supplemented two polyunsaturated fatty acids (PUFAs), docosahexaenoic acid (DHA) or arachidonic acid (AA), to rat fetal cardiomyocytes and found that the supplementations upregulated the expressions of mRNAs for cardiomyocyte differentiation, fatty acid metabolism, and cellular adhesion. The enhancement in cellular contractility was attributed to the improvement in intercellular connection rather than a direct enhancement of the contractile force. Methods: This study reports the successive results of the effects of DHA or AA supplementation on hiPS-CM. In addition to the contractile force and mRNA measurements used in the previous study, we further investigated the effect of different cellular aggregations on the contractile force output by means of finite element analysis, measured glucose and fatty acids metabolites, and assessed cTNT and MLC2v expressions through immunofluorecsence evaluation. Results: It showed that the sole supplementation of albumin-conjugated DHA or AA can be taken up by hiPS-CM without other uptake-enhancing factors, and the supplementations may activate the CD36_­ERRγ metabolic pathway. DHA or AA supplementation increased the cellular contractile ratio on collagen gels and AA supplementation stimulated hiPS-CM aggregation to form cellular clusters. The enhancement effect on the hiPS-CM contractile force was modest since the increase in contractile force was not significant. AA supplementation was more effective than DHA supplementation because it significantly upregulated mRNA expressions of P300 and CD36. However, finite element analysis showed that the formation of clusters on a collagen gel attenuated the contractile force exerted by the gel on its surroundings. Conclusion: DHA and AA, as having been supplemented in infant formulas, have no direct and significant enhancement effect on the performance of the hiPS-CM when they were supplemented individually, although they were able to enter the cellular metabolic system. The AA supplementation showed some auxiliary effect on the maturation of hiPS-CM, which is worthy of further investigation under the consideration of membrane composition alteration and remodeling of membrane molecules.     

Regulation of gene expression for neurotransmitters during adaptation to hypoxia in oxygen-sensitive neuroendocrine cells

Reduced oxygen tension (hypoxia) in the environment stimulates oxygen-sensitive cells in the carotid body (CB). Upon exposure to hypoxia, the CB immediately triggers a reflexive physiological response, thereby increasing respiration. Adaptation to hypoxia involves changes in the expression of various CB genes, whose products are involved in the transduction and modulation of the hypoxic signal to the central nervous system (CNS). Genes encoding neurotransmitter-synthesizing enzymes and receptors are particularly important in this regard. The cellular response to hypoxia correlates closely with the release and biosynthesis of catecholamines. The gene expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine biosynthesis, is regulated by hypoxia in the CB and in the oxygen-sensitive cultured PC12 cell line. Recently, genomic microarray studies have identified additional genes regulated by hypoxia. Patterns of gene expression vary, depending on the type of applied hypoxia, e.g., intermittent vs. chronic. Construction of a hypoxia-regulated, CB-specific, subtractive cDNA library will enable us to further characterize regulation of gene expression in the CB.     

Ultrastructural analysis of murine hippocampal neural progenitor cells in culture

Despite the great number of studies devoted to neural stem/progenitor cell biology, the ultrastructural characteristics of these cells in vitro have not been fully studied. To determine the fine structure of hippocampal neural progenitor cells (NPCs) in culture, mouse fetal hippocampi (E18) were extracted, dissected, and cells were expanded as adherent monolayer culture. Electron microscopy revealed that NPCs had an immature phenotype, with a high nuclear/cytoplasmic ratio, small and scant organelles, underdeveloped endoplasmic reticulum, and many free ribosomes and polysomes. Our results may contribute to a better understanding of the fine structure and physiology of hippocampal NPCs in vitro. Microsc. Res. Tech. 78:128–133, 2015. © 2014 Wiley Periodicals, Inc.     

Calcium signalling in cells of oligodendroglial lineage

Intracellular Ca2+ is the key signal that regulates the efficacy of neurotransmitter release and synaptic plasticity in neurons but is also an important second messenger involved in the signal transduction and modulation of gene expression in both excitable and non-excitable cells. Glial cells, including cells of oligodendroglial (OLG) lineage, are capable of responding to extracellular stimuli via changes in the intracellular Ca2+. This review article focuses on the mechanisms of Ca2+ signalling in cells of OLG lineage with the goal of providing the basis for understanding the relevance of receptor- and non-receptor-mediated signalling to oligodendroglial development, myelination, and demyelination. Conclusions to date indicate that cells of OLG lineage exhibit remarkable plasticity with regard to the expression of ion channels and receptors linked to Ca2+ signalling and that perturbation of [Ca2](i) homeostasis contributes to the pathogenesis of demyelinating diseases.     

牛乳腺上皮细胞体外培养研究进展

为研究乳腺上皮细胞增值和分化特性,通过有效的培养方法,实现了牛乳腺上皮细胞的体外培养,并且所获细胞具有正常的生理特性及功能。本文综述了近年来关于牛乳腺上皮细胞体外分离、培养的研究进展。并对牛乳腺上皮细胞在不同培养体系中的生长状态、分化差异;牛乳腺上皮细胞对各种激素和生长因子的应答进行了讨论。     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

Peribronchiolar accumulation of dendritic cells and their close association with CD4(+) T cells in the murine lung hypersensitivity

In order to understand the interaction between dendritic cells (DCs) and helper T (Th) cells in the region exposed to antigens during pulmonary delayed-type hypersensitivity (DTH), which is considered to be mediated by Th1 cells, we immunohistochemically investigated their spatial relationship in the cellular infiltrate. At 24 hours after intratracheal instillation of hapten in sensitized mice, DCs were preferentially accumulated around the bronchioles, whereas macrophages were more abundant around the accompanying arteries. DCs often formed a cluster, in which they were interconnected with each other by projections. Serial section analysis revealed that clustered DCs made a close apposition to Th cells but much less frequently to cytotoxic T cells and B cells. Immunoelectron microscopy demonstrated that lymphocytes extravasated the capillaries in the peribronchiolar interstitium and made conjugation with DCs. In the interstitial tissue, DCs often adhered to the fibroblasts, suggesting the supportive role of the latter cells in DC migration. Eosinophils were also frequent around the arteries, representing the possible involvement of Th2 cytokines. By contrast, in a chronic type of airway inflammation induced by repeated challenges of aerosolized ovalbumin, DCs were densely and diffusely accumulated around the arteries in the same way as macrophages. The present study demonstrated a close association of DCs with Th cells around the bronchioles during pulmonary DTH, suggesting that local interaction between them in the lung may play important roles in the development of this disorder.     

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: Effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.     

Mesenchymal stem cells transplantation attenuates experimentally induced brain injury after neonatal hypoxia by different two routes of administrations

The neonatal hypoxic–ischemic encephalopathy (HIE) is an important cause of neurological morbidity andmortality in neonates. Cell therapy is considered a promising method for treating severe neurological disorders such asthis one. Stem cells have the capacity for self-renewal and differentiation into certain cell lineages. The present study wasaimed to find out the most beneficial route of bone marrow-derived mesenchymal stem cells (BMSCs) administrationfor the attenuation of experimentally induced HIE in neonatal rats. Sixty neonatal rats were divided randomly intofour groups. Group 1: control group. Group 2: rats were exposed to bilateral ligation of cephalic arteries. Group 3: ratswere exposed to bilateral ligation of cephalic arteries and then underwent intravenous (IV) BMSC injection. Group 4:rats were exposed to bilateral ligation of cephalic arteries and then underwent intracerebroventricular (ICV) BMSCinjection. The animals were evaluated by (a) neurobehavioral tests; (b) histopathology, i.e., histological and immunohistochemical studies; and (3) gene expression studies. The BMSC treated groups (3 and 4) showed improvement inneurobehavioral tests, histopathological studies, and gene expression, as compared to non-injected lesioned rats (Group2) with better improvement in Group 4 (ICV injections) than in Group 3 (IV injections).     

Improvement of transfection with reprogramming factors in urinederived cells

Human-induced pluripotent stem cells (iPSCs) are an accessible source of adult-derived, patient-specificpluripotent stem cells for use in basic research, drug discovery, disease modeling, and stem cell therapy. Improving theaccessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application. Therefore,our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells (RECs) usingdifferent extracellular matrices and transfection reagents. In this study, we began by culturing urine-derived cells fromhealthy donors to establish a primary culture of renal epithelial cells, followed by their characterization. Subsequently, wegenerated iPS-like cells by transfecting renal epithelial cells (RECs) with vectors expressing Oct4, Sox2, L-Myc, Lin-28,and Klf4, and we compared the efficacy of different extracellular matrices and transfection reagents. The resultantiPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markersOct3/4, Nanog, Lin28, and Klf4. We concluded that Lipofectamine Stem Cell transfection reagent is more effective thanFuGENE in obtaining iPS-like cells under the conditions tested. Moreover, the three matrices are similar in theirefficiency of obtaining iPS-like cells. This report provides an experimental protocol for obtaining and generating iPS-likecells from urine samples for further cell therapy research on different human diseases.     

Construction of engineered murine embryonic stem cells with conditional knockout of FGFR2 depending on Cre-loxP.

OBJECTIVE: To investigate the functions of Fibroblast Growth Factor Receptor-2 (FGFR2) at different stages of cell differentiation. The engineered murine embryonic stem (ES) cells with conditional knockout of FGFR2 were developed depending on Cre-loxP. METHODS: Cre-loxP system was used in a conditional targeting vector. The competent AM-1 bacteria, which expressed Cre-recombinase, was used to confirm the Cre-mediated deletion of the floxed exons 7 and 8 of FGFR2. The targeting vector was electroporated into the ES cells, and the transfected ES cells were screened with G418 and Ganciclovir. Finally, the ES clones with correct targeting events were identified by Southern Blot and PCR. RESULTS: The targeting vector with conditional knockout of murine FGFR2 was successfully constructed and confirmed by PCR and digestion analysis in bacteria. 86 ES clones were collected by selective culture with G418 and Ganciclovir. Four of the 86 ES clones were found containing the targeting gene sequence in genomic DNA proved by Southern Blot with a 5'-end flank probe. Two of the four ES clones had the correct targeting events that included the insertion of the targeting gene sequence in genomic DNA and were checked by Southern Blot with a 3'-end flanking probe. Finally, the insertion of loxP (loxP3) between exons 8 and 9 in genomic DNA was identified in one of the two ES clones by Southern Blot and PCR. CONCLUSION: FGFR2 conditional knockout depending on Cre-loxP can be successfully used in ES cells.     

Effects of rotational culture on morphology,nitric oxide production and cell cycle of endothelial cells

Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.     

Effects of microwave irradiation and chemical fixation on the localization of perisinusoidal cells in rat liver by gold impregnation

Modified gold impregnation is one of the methods that are used in light microscopical demonstration of hepatic perisinusoidal cells. This method has some disadvantages, such as restriction of fixation time to 16 h, which allows limited time for processing the tissues, especially when dealing with a large amount of material, and a long impregnation time (16–24 h). We investigated the effect of prolonged fixation on the staining of sections, to shorten the time needed for gold impregnation by using microwave irradiation. Liver specimens were fixed in Baker's calcium–formalin for different periods of time. After fixation, frozen sections were impregnated in gold chloride solution either at room temperature or in a microwave oven. The staining quality of the sections which had been impregnated in the microwave oven for a much shorter time were equal to or even superior to the ones impregnated at room temperature. Prolonging the fixation time up to 7 days did not affect the staining results by microwave irradiation, whereas satisfactory results were not obtained from sections stained at room temperature and fixed for more than 3 days. We conclude that microwave irradiation can be used to shorten the impregnation time in gold chloride solution and the duration of fixation can be prolonged up to 3 days in the original method and up to 7 days when microwave irradiation is used during impregnation.     

Effect of Helicobacter pylori eradication on cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression during gastric adaptation to aspirin (ASA) in humans

Gastric adaptation to aspirin is impaired in Helicobacter pylori infection, but the mechanisms underlying this phenomenon are unclear. In this study, we compared gastric mucosal expression of iNOS and COX-2 during 14 days of aspirin ingestion in the same subjects before and 3 months after eradication of H. pylori. Compared to non-infected controls, mucosal expression of COX-2 and iNOS was enhanced before and 3 months after eradication of H. pylori. During aspirin ingestion, mucosal expression of COX-2 remained unchanged before eradication of H. pylori, but increased gradually after successful antimicrobial treatment. Independent of H. pylori status, expression of iNOS increased at the beginning of aspirin intake, but then returned to initial values. We conclude that COX-2 but not iNOS might be involved in gastric adaptation to aspirin in humans and that this mechanism appears to be impaired in H. pylori infection.     

机器人制造单元调度问题研究综述

机器人制造单元在钢铁冶炼、半导体制造和印刷电路板电镀处理等制造行业的应用日益广泛.分析了机器人制造单元调度问题的复杂性,详尽阐述了机器人制造单元调度问题的分类,系统地总结了机器人制造单元调度算法的研究现状,最后指出了该领域研究存在的问题以及未来可能的研究方向.