Bcl-2硫代反义核酸(G3139)OPC纯化方法

目的 建立Bcl-2硫代反义核酸(G3139)OPC纯化方法。方法 将G3139从合成柱上解离并去保护，用OPC柱分离纯化化学合成的G3139。紫外分光光度计测OD值，20％PAGE鉴定其纯度。结果 经OPC柱纯化后G3139制备量可达mg水平，回收率为66％，电泳结果显示为一条亮带。结论 Bcl-2硫代反义核酸OPC纯化法是一种高效、快速、简便的方法。     

A role for the MAPK/ERK pathway in oligodendroglial differentiation in vitro: stage specific effects on cell branching

The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway is important for both long-term survival and timing of the progression of oligodendrocyte differentiation. Oligodendroglial cells treated with MEK inhibitor were distinguished by using stage specific markers: NG2 proteoglycan, A2B5, 2′3′nucleotide-cyclic 3′phosphodiesterase (CNPase) and myelin basic protein (MBP), and classified according to their morphology into different developmental stages. Treatment significantly increased the number of cells with more immature morphologies and decreased the number of mature cells. Furthermore, it increased the number of rounded cells that could not be classified into any of the oligodendroglial developmental stages. The strongest effects were usually observed shortly after treatment. Rounded cells were CNPase/MBP positive and they were not stained by anti-NG2 or A2B5, indicating that they were mature cells unable either to extend and/or to maintain their processes. These data showed an effect of the MAPK/ERK pathway on oligodendroglial branching, with possible consequences for the formation of the myelin sheath.     

NG2 cells differentiate into astrocytes in cerebellar slices

NG2-glia are an abundant population of glial cells that have been considered by many to be oligodendrocyte progenitor cells (OPCs). However, growing evidence suggests that NG2-glia may also be capable of differentiating into astrocytes and neurons under certain conditions. Here, we have examined NG2-glia in cerebellar slices, using transgenic mice in which the astroglial marker glial specific protein (GFAP) drives expression of the reporter gene enhanced green fluorescent protein (EGFP). Immunolabelling for NG2 shows that NG2-glia and GFAP-EGFP astroglia are separate populations in most areas of the brain, although a substantial population of NG2-glia in the pons also express the GFAP-EGFP reporter. In the cerebellum, NG2-glia did not express EGFP, either at postnatal day (P)12 or P29–30. We developed an organotypic culture of P12 cerebellar slices that maintain cytoarchitectural integrity of Purkinje neurons and Bergmann glia. In these cultures, BrdU labelling indicates that the majority of NG2-glia enter the cell cycle within 2 days in vitro (DIV), suggesting that NG2-glia undergo a ‘reactive’ response in cerebellar cultures. After 2 DIV NG2-glia began to express the astroglial reporter EGFP and in some cases the respective GFAP protein. However, NG2-glia did not acquire phenotypic markers of neural stem cells or neurons. The results suggest that NG2-glia are not lineage restricted OPCs and are a potential source of astrocytes in the cerebellum.     

Diosgenin promotes oligodendrocyte progenitor cell differentiation through estrogen receptor-mediated ERK1/2 activation to accelerate remyelination

Differentiation of oligodendrocyte progenitor cells (OPCs) into mature oligodendrocytes is a prerequisite for remyelination after demyelination, and impairment of this process is suggested to be a major reason for remyelination failure. Diosgenin, a plant-derived steroid, has been implicated for therapeutic use in many diseases, but little is known about its effect on the central nervous system. In this study, using a purified rat OPC culture model, we show that diosgenin significantly and specifically promotes OPC differentiation without affecting the viability, proliferation, or migration of OPC. Interestingly, the effect of diosgenin can be blocked by estrogen receptor (ER) antagonist ICI 182780 but not by glucocorticoid and progesterone receptor antagonist RU38486, nor by mineralocorticoid receptor antagonist spirolactone. Moreover, it is revealed that both ER-alpha and ER-beta are expressed in OPC, and diosgenin can activate the extracellular signal-regulated kinase 1/2 (ERK1/2) in OPC via ER. The pro-differentiation effect of diosgenin can also be obstructed by the ERK inhibitor PD98059. Furthermore, in the cuprizone-induced demyelination model, it is demonstrated that diosgenin administration significantly accelerates/enhances remyelination as detected by Luxol fast blue stain, MBP immunohistochemistry and real time RT-PCR. Diosgenin also increases the number of mature oligodendrocytes in the corpus callosum while it does not affect the number of OPCs. Taking together, our results suggest that diosgenin promotes the differentiation of OPC into mature oligodendrocyte through an ER-mediated ERK1/2 activation pathway to accelerate remyelination, which implicates a novel therapeutic usage of this steroidal natural product in demyelinating diseases such as multiple sclerosis (MS).     

Effects of isozyme-selective phosphodiesterase inhibitors on rat aorta and human platelets: smooth muscle tone,platelet aggregation and cAMP levels

The inhibitors of the cGMP-inhibited, low-K_m cAMP phosphodiesterase—milrinone and OPC 3911-and an inhibitor of a non-cGMP-inhibited low-K_m cAMP phosphodiesterase—rolipram—were used to evaluate the functional importance of the two cAMP phosphodiesterase activities in vascular smooth muscle and in platelets. Vinpocetine, an inhibitor of a calcium-calmodulin-dependent phosphodiesterase was also studied. OPC 3911 and milrinone relaxed the contracted rat aorta, inhibited ADP-induced platelet aggregation and also enhanced isoprenaline-induced relaxation as well as the antiaggregatory effects of adenosine. In platelets, OPC 3911 and milrinone increased cAMP levels, but in the rat aorta the increase was significant only for milrinone (OPC 3911 P= 0.062). In both tissues OPC 3911 and milrinone enhanced the increase in cAMP caused by activators of adenylate cyclase (isoprenaline/adenosine). Rolipram had no effects on aggregation or cAMP levels in platelets and no overadditive effects in combination with adenosine. Rolipram had little effect on relaxation and cAMP levels, did not alter isoprenaline-induced relaxation of guanfacin-contracted rat aorta, but showed synergistic effects with isoprenaline in raising cAMP levels. In PGF_2α-contracted aorta rolipram enhanced relaxation caused by isoprenaline. Vinpocetine had a relaxant effect without affecting cAMP levels, but had no effect on platelets. These results support the concept that the cGMP-inhibited phosphodiesterase is an important modulator of vascular smooth muscle tone and platelet function. The role of the non-cGMP-inhibited phosphodiesterase in these tissues is less obvious.     

The inotropic mechanism of the phosphodiesterase inhibitor OPC 3911 alone and in combination with rolipram,studied in papillary muscles of ferret and guinea–pig and isolated myocytes of guinea–pig ventricular muscle

OPC 3911 is a potent inhibitor and PDE III is a specific inhibitor in cardiac muscle. The effects of the drug alone and in combination with the non–inotropic PDE IV inhibitor rolipram were analysed using hearts from guinea–pigs and ferrets. OPC 3911 had an EC₅₀ value of 0.1μM. At 0.1 fiM peak force was increased by 50.7 ± 7.6% (n = 6, P< 0.001), time to peak tension (TPT) reduced by 18,7±5.6% (n = 6, P < 0.05). Time to half relaxation (THR) was prolonged by 19±4.2% (n= 6, P < 0.001). After addition of rolipram (30 μM) there was a potentiation of peak force at all concentrations of OPC 3911. At 0.1 μM OPC rolipram increased peak force by 82.8±8.9% (n= 6, P < 0.001), reduced TPT by 73 ± 6% (n = 6, P < 0.005) and increased THR by 27±5% (P<0.01). OPC 3911 shortened action potential duration (APD) at 50% repolarization by 5.3 ±2.5% (n= 6, P < 0.05). Addition of rolipram prolonged APD by 3.7 ± 2.5% (n = 6, P < 0.05). Second inward current (I_8l) was increased at 3μM OPC 3911 by 46 ± 6% (n = 6, P< 0.05). The combination of OPC 3911 and rolipram intensified the I_8l to 101 ± 5% (n= 3). Rolipram slowed the rate of restitution and the onset of restitution was prolonged. Relative maximum post–extrasystolic potentiation was reduced in the presence of OPC 3911 from 67 ± 5% to 45 ±6%. Adding rolipram caused potentiation of 55 ± 6%. OPC 3911 increased the recirculation fraction of activator calcium from 0.36 to 0.42 (n = 10, P < 0.05). After addition of rolipram the recirculation fraction was 0.41 ± 0.04 (n =10, P < 0.05). The results suggest that rolipram exerts its potentiating effect on OPC 3911 via an increased I_sl.     

Live imaging of remyelination in the adult mouse corpus callosum

Oligodendrocyte precursor cells (OPCs) retain the capacity to remyelinate axons in the corpus callosum (CC) upon demyelination. However, the dynamics of OPC activation, mode of cell division, migration, and differentiation on a single-cell level remain poorly understood due to the lack of longitudinal observations of individual cells within the injured brain. After inducing focal demyelination with lysophosphatidylcholin in the CC of adult mice, we used two-photon microscopy to follow for up to 2 mo OPCs and their differentiating progeny, genetically labeled through conditional recombination driven by the regulatory elements of the gene Achaete-scute homolog 1. OPCs underwent several rounds of symmetric and asymmetric cell divisions, producing a subset of daughter cells that differentiates into myelinating oligodendrocytes. While OPCs continue to proliferate, differentiation into myelinating oligodendrocytes declines with time, and death of OPC-derived daughter cells increases. Thus, chronic in vivo imaging delineates the cellular principles leading to remyelination in the adult brain, providing a framework for the development of strategies to enhance endogenous brain repair in acute and chronic demyelinating disease.

In the central nervous system (CNS), oligodendrocytes form myelin sheaths around axons, allowing for the rapid transduction of electrical impulses and providing metabolic support (1, 2). Myelin remodeling occurs throughout life, is regulated by neuronal activity, and has been associated with learning and circuit plasticity in mice and humans (3–10). However, a number of neurological diseases, multiple sclerosis (MS) among others, leads to impaired oligodendrocyte function and an eventual loss of myelin sheaths, causing demyelination (1, 11). Demyelination is associated with progressive axonal degeneration and neurological decline (2). Whereas substantial progress has been made over the last decade to reduce myelin loss in acute phases of demyelinating diseases such as MS, regenerative approaches to enhance remyelination remain scant (1, 12, 13). In rodents, remyelination is achieved via activation of oligodendrocyte precursor cells (OPCs) that are capable of generating new functional oligodendrocytes (1, 14–17). Snapshot-based genetic lineage tracing experiments showed that OPCs expand within areas of damage through a combination of migration and proliferation in response to demyelinating injury (1, 18). Recruited OPCs differentiate into oligodendrocytes and myelin sheaths are eventually restored within lesions. Remyelination also occurs in humans in the context of demyelinating diseases, in which differentiated oligodendrocytes possibly also contribute to myelin repair (4, 19, 20). However, myelin recovery in humans often remains incomplete. Thus, new therapeutic approaches to enhance myelin repair are needed (1).Understanding OPC dynamics on a single-cell level could facilitate the development of remyelination-promoting strategies with the aim to prevent disease progression or to ameliorate symptoms. Recently, OPC kinetics, oligodendrocyte differentiation, and myelin formation in the healthy and injured adult mouse brain have begun to be studied in superficial layers of the cortex using in vivo two-photon imaging (7, 9, 21–23). However, myelination and remyelination in gray and white matter follows different dynamics (24). The clonal behavior of OPCs during remyelination in gray and white matter structures remains largely unknown (1). For example, the mode of cell division, self-renewal potential, and successive rounds of division of individual OPCs is unclear. We here used in vivo two-photon imaging to analyze OPC behavior and subsequent remyelination in the corpus callosum (CC), a white matter area connecting the two brain hemispheres that is commonly affected in demyelinating disease (11). Combining lysophosphatidylcholin (LPC, also called lysolecithin)–induced demyelination and repeated in vivo imaging (25, 26), we characterize the cellular principles of OPC behavior upon demyelination, allowing for insights that may guide future strategies to enhance regenerative brain repair.     

The applicability of new TNM classification for humanpapilloma virus-related oropharyngeal cancer in the 8th edition of the AJCC/UICC TNM staging system in Japan: A single-centre study

Objective

The purpose of this study is to validate the applicability of new TNM classification for human papillomavirus (HPV)-related oropharyngeal cancer (OPC) in the 8th edition of the American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) TNM staging system in Japan.