期刊介绍
期刊导读
- 10/21世茂的地标式拿地 这次覆盖了三线城市肇庆
- 10/21SKP要去武汉了?喜茶开了首家宠物主题店、美国
- 10/20融创隐溪晓院 | “锋·隐 大师的小院”产品发布
- 10/19国家电子商务新经济平台成立发布会暨湖南省首
- 10/17虹口?区商务委走访宝华商业广场项目
Inhibition of neurite outgrowth using comme(4)
Figure 5 Linear dose effects of MAG-Fc on neurite outgrowth from neuro-2a cells.(A) Neuro-2a cells treated with a series of MAG-Fc concentrations from 5–40 nM for 24–72 hours showed dose-dependent inhibition of neurite growth based on anti-βIII tubulin (red) immunofluorescence staining (Alexa Fluorescence-594 labeling). Scale bar: 50 μm. (B) The total neurite length of neuro-2a cells was decreased at 24 hours by 5–40 nM MAG treatment; the decrease in neurite length also appeared to be dose-dependent, although there was no significant difference with incubation time. (C) Adding MAGFc to neuro-2a cells also decreased the percentage of cells with drop in the percentage was clearly observed at 48 and 72 hours of MAG treatment. Data are expressed as the mean ± SE (one-way analysis of variance followed by t-test). *P <0.05, **P < 0.01, ***P < 0.001, vs. 0 nM group. MAG: Myelin-associated glycoprotein; h: hours.
Similar to the altered activation of RhoA, levels of phosphorylated ROCKII (p-ROCKII) were also increased after treatment with MAG-Fc for 5, 10 and 20 minutes. More-over, p-ROCK levels were also increased after 40 minutes of MAG treatment, as observed for RhoA, although no significant difference was found (Figure 3). In addition, the immuno fluorescence of p-ROCKII also increased in the cytoplasm after MAG-Fc treatment compared with addition to the increase in p-ROCK immunofluorescence, the staining was also translocated from deep within the cytoplasm toward the cytoplasmic area adjacent to the membrane. Some stronger p-ROCK immunopositive foci were found at random locations adjacent to the membrane(Figure 4). In summary, treatment of neuro-2a cells with exogenous MAG activated RhoA and the phosphorylation of its downstream signaling protein, ROCKII.
Effect of exogenous MAG-Fc on neuritogenesis in neuro-2a cells
Because MAG-specific receptors were present in neuro-2a cells, their functional effect on neurite outgrowth was investigated. All neurites were visualized by the immuno fluorescence of βIII tubulin. Linear concentrations of MAG-Fc (5, 10, 20 and 40 nM) were added to the medium, and cells were incubated for 24, 48, or 72 hours. The neurite lengths were measured,revealing that neurite outgrowth was inhibited by MAG-Fc treatment. The inhibition of neurite growth also exhibited a linear pattern. The maximum inhibition of neurite growth appeared after 24 hours of treatment with 40 nM MAG-Fc (37.63± 1.08 μm), which was 20% shorter than the neurites of the control group (47.03 ± 1.30 μm). Moreover, further incubation with MAG-Fc for 48 hours or 72 hours did not induce any further inhibition on neurite outgrowth (Figure 5B).
MAG-Fc treatment also affects neuritogenesis because the number of neurites on neuro-2a cells was decreased upon MAG-Fc treatment. This decrease was seen at all time points of MAG-Fc incubation (Figure 5C). The percentage of cells with neurites began to reduce after treatment for 24 hours with 5 nM and 10 nM MAG-Fc, but a statistically significant decrease was not observed until 24 hours of treatment with 20 or 40 nM MAG-Fc (P < 0.05). Furthermore, at the final time points of MAG-Fc incubation (48 and 72 hours), a percentage decrease in cells with neurites was observed for all MAG-Fc concentrations (Figure 5C).
All of these functional effects of MAG-Fc on neurite outgrowth indicate that neuro-2a cells are susceptible to endocytosis of MAG-Fc was mediated through the same specific receptors as endogenous MAG.
Discussion
The neuro-2a cell line was originally derived from mouse neuroblastoma cells from a spontaneous tumor in the A strain of white mice (Klebe and Ruddle, 1969; LePage et al., 2005). The cells are a type of brain tumor cell, but they carry many inherent morphological and physiological properties that resemble neural stem cells in neuronal development. However, neuronal processes appeared in only a few cultured neuro-2a cells under regular medium supplemented with 10% fetal bovine serum. Moreover, no study has shown that these cells contain the necessary machinery for MAG-Fc interaction, such as a membrane specific receptor for MAG and the related intracellular signaling proteins. Neuro-2a cells act differently to primary cerebellar neurons when used in in vitro cell models(Nieder?st et al., 2002). Because of several features under culture conditions, such as the ability to replicate, monolayer growth, homogeneous cellular constituents, and minimal interference with others cells, neuro-2a cells are versatile and more economical than primary neuronal cultures. Therefore,many researchers have chosen neuro-2a cells as a substitute for primary neuron cultures. There are hundreds of publications regarding the use of these cells for different purposes,such as studies of neuronal survival, cytotoxicity, mechanisms of neuronal metabolic dysfunctions, neuronal differentiation,and ligand-receptor interactions (Eom et al., 2015; Nicolas et al., 2015; Wesley et al., 2015; Grimm et al., 2016). However,whether the cell line is susceptible to myelin-associated nerve growth inhibitors has not yet been verified.
文章来源:《新商务周刊》 网址: http://www.xswzkzzs.cn/qikandaodu/2020/1021/526.html