Enhancing action by sulfated hyaluronan on connexin-26, -32, and -43 gene expressions during the culture of normal human astrocytes

Astrocyte proliferation is strictly controlled during development and in the adult nervous system. In this study, we examined the role of sulfated hyaluronan (SHya) in the proliferation and differentiation of normal human astrocytes (NHAs). Cells were cultured with different concentrations of SHya for 7 days, and the number of viable cells and the presence of neural cell-specific genes were determined to assess their proliferation and development, respectively. With SHya, cell proliferation increased nonsignificantly. Furthermore, remarkable enhancing action by SHya on connexin-26, -32, and -43 gene expressions were observed during the culture of NHAs. It has been suggested that a fraction of NHAs have neural precursor activity that gives rise to astrocytes themselves, oligodendrocytes, and neurons. Our results clearly demonstrated that the expression of specific genes for neural precursor cells, astrocytes, neurons, and oligodendrocytes was significantly increased to 50 mug/mL in SHya-treated cultures when compared with that of the control culture. These findings suggest that SHya plays an important role in the proliferation and differentiation of NHAs and in the production of a novel material for tissue engineering.     

NH+4对星状胶质细胞谷氨酰胺代谢的影响

目的：了解体外培养状态下,ＮＨ４＋对脑星准确无误胶质细胞谷氨酰胺代谢的影响。方法;将不同浓度ＮＨ４ＣＩ加入培养液作用不同的时间,分别收集细胞及上清进行测定。结果：ＮＨ４＾＋作用后,星状胶质细胞谷氨酰胺合成酶活性啬细胞内及上清液内谷氨酰胺含量啬且在一定程度上随着ＮＨ４＾＋氨的浓度的增大而增加。然而,妾ＮＨ４＾＋２作用时间达到一定时限（７２ｈ）后,酶的活性及细胞内谷氨酰胺的量不再增加,甚至有所下降或减     

Heparin structures in FGF-2-dependent morphological transformation of astrocytes

Fibroblast growth factor-2 (FGF-2) participates in the morphological transformation of astrocytes (stellation) during the formation of glial scars in injured brains. In the current study, we used quantitative morphometric analysis to investigate the structural requirements for heparin's enhancement of FGF-2-induced stellation of cultured cortical astrocytes. Native heparin significantly promoted FGF-2-dependent astrocytic stellation, whereas heparin hexasaccharide inhibited FGF-2-dependent stellation. Furthermore, 2-O-, 6-O-, and N-desulfated heparins were unable to promote FGF-2-dependent stellation. The stellation induced by FGF-2 or by a combination of FGF-2 and native heparin was inhibited by SU5402, an FGF receptor inhibitor. These results demonstrate that the length and sulfated position of heparin are important for its enhancement of FGF-2-dependent astrocyte stellation. In addition, our findings show that heparin oligosaccharides are useful for regulating the FGF-2-dependent astrocytic transformation.     

Morphological transformation and proliferation of rat astrocytes as induced by sulfated polysaccharides from the sea cucumber Stichopus japonicus

In this report, we demonstrate that the sulfated polysaccharide, Haishen (HS), which was isolated from the body wall of the sea cucumber Stichopus japonicus can induce morphological transformation and proliferation of astrocytes in vitro when combined with basic fibroblast growth factor 2 (FGF-2). Cell morphology showed no change when induced by HS or FGF-2 alone. However, combinational treatment of HS and FGF-2 promoted transformation of normal astrocyte into a stella morphology (stellation), along with an increase in the expression and rearrangement of glial fibrillary acidic protein (GFAP). Further analysis of HS- and FGF-2-treated cells indicated a reduced percentage of cells in the G0/G1 phase, whereas the cell proliferation index (S phase) was increased. The proportion of 5-bromo-2-deoxyuridine (BrdU)-positive cells increased in response to the combination of HS and FGF-2. With respect to cell cycle signaling, immunoblotting assay demonstrated an accumulation of Cyclin D1. These observations suggest that HS may play a role in astrocyte morphological transformation and proliferation, and this activation requires a synergism with FGF-2.     

Preliminary characterization of an Na,K,Cl co-transport activity in cultured human astrocytes

We have studied the potassium uptake using ⁸⁶Rb⁺ into monolayers of secondary cultures of human astrocytes prepared from cerebral hemispheres of a 4-month-old fetus. With the use of inhibitors we could attribute 30–40% of the ⁸⁶Rb⁺ uptake to an Na⁺,K⁺-ATPase, 50–60% to an anion-cation co-transporter and 10% to potassium leak channels. The anion-cation co-transporter was dependent on the simultaneous presence of both sodium and chloride in the incubation medium and is therefore most likely an Na⁺,K⁺,Cl⁻ co-transporter. This is the first evidence of such an Na⁺,K⁺,Cl⁻ co-transport in human astrocytes.     

Changes in ganglioside composition and morphological features during the development of cultured astrocytes from rat brain

Changes in ganglioside content over a period of days were examined in astrocytes obtained via cell passage from rat cerebral cortex. Thin-layer chromatography revealed that, in the astrocytes, ganglioside GM1 was absent, the predominant ganglioside being GM3. Also, an increased GD3 content in long-term astrocyte cultures was detected. The morphological features of astrocytes were also studied using immunoperoxidase staining. Astroglial features were characterized by high levels of glial fibrillary acidic protein (GFAP) and vimentin, which are the major intermediate-filament proteins present in astrocytes at an early culture stage. In long-term-cultured (greater than 7 months) astrocytes, vimentin and GFAP were increased in process-bearing cells. Ganglioside GD3 recognized by R24 monoclonal antibody was also expressed in these cells. These results suggest that the increase of ganglioside GD3 in long-term-cultured astrocytes may be related to the appearance of multistellate cells showing strong reactivity against GFAP and vimentin during development over a specified period in culture.     

The effect of human eosinophils on cultured human nasal epithelial cell activity and the influence of nedocromil sodium in vitro.

Although there is increasing evidence of a pathogenic role for eosinophils in the airway epithelium, there is little direct evidence which demonstrates that eosinophils influence epithelial cell activity in humans. We have cultured human nasal epithelial cells in vitro and studied the effect of isolated human eosinophils on the ciliary beat frequency (CBF) and cell membrane integrity of these cells after incubation in the absence or presence of 0.1 microM phorbol 12-myristate 13-acetate (PMA) or 0.1 mg/ml opsonized latex beads and the absence or presence of 10(-5) M nedocromil sodium. CBF was monitored by an analogue contrast-enhancement technique, and cell damage was assessed by release of 51Cr from the cells. Cell cultures were also assessed for the percentage of eosinophil cationic protein (ECP) released into the medium at the end of incubation. Neither 0.1 microM PMA, 0.1 mg/ml opsonized latex beads, 10(-5) M nedocromil sodium, nor eosinophils alone altered the CBF of the epithelial cells. PMA-stimulated eosinophils, however, attenuated the CBF significantly, from 10.2 +/- 0.3 to 8.8 +/- 0.4 Hz (P less than 0.05) after 15 h of incubation. Similarly, opsonized latex bead-stimulated eosinophils led to a significant attenuation of CBF from 9.2 +/- 0.3 to 8.4 +/- 0.3 Hz (P less than 0.05), 6.9 +/- 0.5 Hz (P less than 0.001), and 7.5 +/- 0.3 Hz (P less than 0.001) after 2, 15, and 24 h of incubation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

去铁敏对大鼠星型胶质细胞谷氨酸毒性的保护作用

目的:观察去铁敏对谷氨酸所致星型胶质细胞损伤的保护作用。方法:建立原代培养的星型胶质细胞谷氨酸毒性模型,采用免疫组化、Hoechst染色检测细胞形态及核固缩情况。CCK-8法检测细胞活力。生化法检测羟自由基、MDA变化。用显微荧光测量技术监测神经元内钙信号的动态变化。结果:随着谷氨酸浓度的增加细胞损伤也逐渐加重。与对照组相比,去铁敏处理后细胞对0.5,1.0,5.0mmol/L谷氨酸损伤形态保持良好;细胞核固缩率减少,分别为(10.44±5.03)%,(7.93±5.04)%和(11.01±3.73)%(P0.05);细胞活力下降减轻,分别为(94.72±2.20)%(P0.05),(82.49±1.94)%和(67.94±5.55)%(P0.01)。去铁敏处理后羟自由基水平下降,为10.17±1.79(P0.01)。MDA水平下降,为7.36±1.47(P0.01)。去铁敏预处理后钙离子增高的幅度及出现钙离子变化的细胞数较对照有明显减少。结论:去铁敏能减轻谷氨酸导致的星型胶质细胞损伤,能减少谷氨酸导致的星型胶质细胞内钙浓度的升高及自由基水平,这可能是去铁敏保护作用的机理之一。     

Effects of sulfated hyaluronan on keratinocyte differentiation and Wnt and Notch gene expression

Sulfated hyaluronan (SHya), which is composed of a sulfated group and hyaluronan (Hya), has high activity on and biocompatibility with cells. When normal human epidermal keratinocytes (NHEKs) were incubated in dishes coated with SHya, cell proliferation was suppressed in a dose-dependent manner. The expression levels of keratin 1 and loricrin mRNAs, as detected by real-time RT-PCR, were increased significantly. The expressions of Wnt mRNAs, which play important roles in cell proliferation and differentiation, were modulated. Wnt4 and Wnt6 mRNA expressions were increased compared to controls, while expression of Wnt5a was similar to the control and that of Wnt7a mRNA was decreased. In addition, the expression of Notch mRNAs, which play a critical role in keratinocyte differentiation, were affected. Notch3 mRNA was increased significantly, while Notch1 mRNA was decreased compared to controls, and expression of Notch2 was similar to that of control. These results suggested that a SHya-coated scaffold might be useful for regulating cell activity in tissue engineering.     

The effect of endogenous overexpression of hyaluronan synthases on material, morphological, and biochemical properties of uncrosslinked collagen biomaterials

Hyaluronan is an essential component of the native extracellular matrix that has often been added exogenously to biomaterials. The role of endogenously produced hyaluronan on soft tensile tissue mechanics, however, has been largely overlooked. To investigate this aspect of hyaluronan using a cell-mediated approach, cells overexpressing the hyaluronan synthases (has), namely has-1, has-2, has-3 or the empty vector control LXSN, were seeded within collagen gel scaffolds. The resulting engineered tissues were grown under static tension for 6 weeks. Following 6 weeks of culture, the samples were characterized to assess collagen gel contraction, matrix organization, production of hyaluronan, and tissue material properties. The engineered tissues containing cells transfected to overexpress one of the has isozymes had significantly increased retention of hyaluronan within the scaffold; elevated hyaluronan secretion into the culture medium (all but has-2); reduced contraction; reduced collagen density; and significantly altered material properties compared to the LXSN controls. These results indicate that the cell-mediated endogenous overproduction of hyaluronan within biomaterials alters their material, morphological and biochemical characteristics. This investigation, the first to examine the role of endogenously produced hyaluronan in engineered tissue mechanics, suggests that overproduction of hyaluronan in soft connective tissues can transform their biological and biomechanical functionality.     

A morphological study on glutamate-induced swelling of cultured astrocytes: involvement of calcium and chloride ion mechanisms.

Morphological changes in cultured astrocytes exposed to L-glutamate (Glu) were examined light and electron microscopically. The treatment with 0.1 mM Glu for 60 min caused marked swelling of the cells, which was characterized by reduction in staining of cytoplasm with Toluidine blue, disappearance of the cytoplasmic granular ground substances, swollen mitochondrion and nucleus, and dispersed chromatin. The above changes were prevented by the removal of Na+, Ca2+ or Cl- from the incubation medium for Glu treatment. However, the Glu treatment in a Cl(-)-free medium caused conspicuous aggregation of 10 nm filaments.     

人巨细胞病毒感染对体外培养肺成纤维细胞中明胶酶的影响

目的探讨人巨细胞病毒（HCMV）感染对体外培养肺成纤维细胞（HEL）中明胶酶活性的影响。方法体外培养HEL细胞感染HCMV，分为低感染复数（MOI）组及高MOI组，每组重复6例。明胶酶谱法检测HEL细胞中MMP-2及MMP-9的明胶酶活性，用半定量RT-PCR检测各组HEL细胞中MMP-9及TIMP-1的转录水平。结果在低MOI组及高MOI组HEL细胞中MMP-9及MMP-2活性均增强（P〈0．05），高MOI组MMP-9及MMP-2活性较低MOI组显著增加（P〈0．05）。进一步检查MMP-9及TIMP-1的mRNA水平发现，正常对照组HEL细胞中MMP-9及TIMP-1的mRNA处于一个较低的水平，HCMV感染使HEL细胞中MMP-9及TIMP-1的mRNA水平均明显升高（P〈0．05），低MOI组和高MOI组差异无统计学意义（P〉0．05）。在低MOI组及高MOI组，HEL细胞MMP-9／TIMP-1的比值和正常对照组相比明显升高（P〈0．05），表明MMP-9升高更为显著。高MOI组和低MOI组中MMP-9／TIMP-1的比值差异无统计学意义（P〉0．05）。结论HCMV感染可以造成MMP-9和TIMP-1转录和MMP-9／TIMP-1的失衡，同时造成MMP-9及MMP-2明胶酶活性增强，导致肺泡结构的破坏和肺纤维化的发生，这在CMV肺炎的发病机制中起着重要的作用。     

Functional expression of CXCR3 in cultured mouse and human astrocytes and microglia

The aging brain is characterized by selective neurochemical changes involving several neural populations. A deficit in the cholinergic system of the basal forebrain is thought to contribute to the development of cognitive symptoms of dementia. Attempts to prevent age-associated cholinergic vulnerability and deterioration therefore represent a crucial point for pharmacotherapy in the elderly. In this paper we provide evidence for the protective effect of nicergoline (Sermion) on the degeneration of cholinergic neurons induced by nerve growth factor deprivation. Nerve growth factor deprivation was induced by colchicine administration in rats 13 and 18 months old. Colchicine induces a rapid and substantial down-regulation of choline acetyltransferase messenger RNA level in the basal forebrain in untreated adult, middle-aged and old rats. Colchicine failed to cause these effects in old rats treated for 120 days with nicergoline 10 mg/kg/day, orally. Moreover, a concomitant increase of both nerve growth factor and brain-derived neurotrophic factor content was measured in the basal forebrain of old, nicergoline-treated rats. Additionally, the level of messenger RNA for the brain isoform of nitric oxide synthase in neurons of the basal forebrain was also increased in these animals.Based on the present findings, nicergoline proved to be an effective drug for preventing neuronal vulnerability due to experimentally induced nerve growth factor deprivation.     

Induction of human leukocyte antigen-A,B,C and -DR on cultured human oligodendrocytes and astrocytes by human gamma-interferon

gamma-Interferon (IFN-gamma) is known to induce expression of major histocompatibility complex (MHC) class II antigens on murine astrocytes and MHC class I antigens on murine oligodendrocytes. We studied whether the human IFN-gamma could induce the expression of Human Leukocyte Antigen (HLA)-A, B, C and -DR antigens on cultured human glia from autopsied brain white matter tissue. HLA-A, B, C antigens were induced on both human astrocytes and oligodendrocytes, whereas HLA-DR antigens were induced only on some astrocytes. From these results, it is suggested that IFN-gamma affects the expression of MHC class I and class II antigens on astrocytes and oligodendrocytes derived from human brain. The relationship between the induction of MHC class I and class II antigens by IFN-gamma and the pathogenesis of multiple sclerosis is discussed.     

Lack of effect of LTB4 on human natural killer cell activity and T lymphocyte transformation

Human natural killer (NK) cell activity was not significantly affected by leukotriene (LT) B4 over a wide concentration range (10(-6)-10(-14) M), whether added directly to the NK cell assay or after preincubation of effector cells for 2 h with the drug before addition of Cr labelled K562 target cells. In addition, LTB4 did not affect the kinetics of NK cell mediated cytotoxicity. Addition of LTB4 (10(-6)-10(-10) M) to concanavalin A (Con A) or phytohaemagglutinin (PHA)-stimulated human peripheral blood mononuclear cells (PBMC) had no significant effect on proliferation measured by tritiated thymidine incorporation, using both optimal and sub-optimal mitogen concentrations. Whilst it is clear that LTB4 is an important mediator of inflammation involving polymorphonuclear (PMN) leukocytes in vivo, it does not affect NK cell or T cell function in vitro.     

体外血小板活化因子对神经元和星形胶质细胞的作用

目的：研究血小板活化因子（PAF）对神经元活力及星形胶质细胞胶质纤维酸性蛋白（GFAP）表达的影响。 方法： 分别取BALB/c胎鼠和新生小鼠大脑皮层，纯化培养神经元和星形胶质细胞，设正常对照组和实验组，实验组中分别加入4、8和16 μmol/L的PAF并分别作用4 h、24 h和72 h,用MTT法和免疫组织化学方法测定神经元活力和星形胶质细胞表达GFAP的平均灰度值。 结果： PAF作用后，神经元活力降低；星形胶质细胞数量减少，但存活细胞GFAP表达增加。两者均呈浓度依赖关系。 结论： PAF不仅直接作用于神经元，且可通过作用于星形胶质细胞间接影响神经元的存活。     

The SV40 small t antigen is essential for the morphological transformation of human fibroblasts

The morphological transformation of human fibroblasts as measured in an assay for dense focus formation required, besides the SV40 large T antigen, an intact SV40 small t antigen. Using a G418-resistant colony formation assay it also was found that expression of the SV40 large T antigen only is not sufficient for the morphological transformation of human fibroblasts. Therefore it is concluded that the SV40 small t antigen is essential for the morphological transformation of human fibroblasts.     

Proliferation and morphological differentiation of neurblastoma cells in cultured under the effect of avermectins

The effect of natural avermectin complex (Aversectin C) and Abamectin on the processes of proliferation and morphological differentiation of the neural cells was studied using N1E-115 murine neuroblastoma cells (clone C-1300) as a model. Aversectin C in concentrations 10(-7)-10(-8) was shown to induce morphological differentiation of cultured nervous cells. Treatment with Abamectin resulted in the changes of proliferation pattern of the cells. Morphological differentiation of the cultured nervous cells treated with Aversectin C was associated with electrophysiological one.     

Transferrin receptors on the plasma membrane of cultured rat astrocytes

It is generally accepted that transferrin receptor is located in the endothelial cells of the brain, but its existence in other brain cell types is less established. In this study, a [¹²⁵I]transferrin binding assay was used to determine whether there is transferrin receptor on the membrane of cultured rat cortical astrocytes (type 1) in vitro. The results demonstrated that cortical astrocytes (type 1) in suspension attracted [¹²⁵I]transferrin with a saturable and specific binding. Scatchard and Hill plot analysis showed that the dissociation constant (K_D) of the binding was about 3.5×10^–8 M and the number of receptors was about 7.1×10⁴/cell. The Hill coefficient was 0.99, approaching 1, indicating the absence of cooperativity. The receptor was specific both for rat and human transferrin. The binding of rat [¹²⁵I]transferrin could be competitively and specifically inhibited by unlabeled iron-saturated rat and human transferrin, and no difference was found between interaction of rat or human transferrin with this receptor. The interaction of duck or camel transferrin with this receptor was found to be very weak. This study provides evidence for the presence of transferrin receptor on the plasma membrane of cultured rat brain astrocytes. Received: 25 May 1999 / Accepted: 18 August 1999