摘要
背景:褪黑素主要是由松果体分泌的一种神经内分泌激素,能够促进许旺细胞增殖并促进周围神经损伤后再生。目的:探究褪黑素对许旺细胞迁移活性的影响及其相关分子机制。方法:体外培养大鼠许旺细胞RSC96细胞株并鉴定;CCK-8检测褪黑素对许旺细胞增殖活性的影响;采用Transwell细胞迁移小室,观察0,100 nmol/L,1μmol/L,10μmol/L褪黑素作用20 h对许旺细胞迁移活性的影响;采用qRT-PCR和Western-blot检测褪黑素受体1,2在许旺细胞上的表达情况;通过Transwell细胞迁移实验,观察褪黑素受体拮抗剂luzindole或典型Wnt/β-catenin信号通路抑制剂IWR-1对褪黑素促进许旺细胞迁移活性的影响。Western-blot检测褪黑素对许旺细胞中Wnt/β-catenin信号通路活性的调控作用。结果与结论:①贴壁培养的RSC96细胞具有许旺细胞的典型形态,细胞呈纺锤状或三角形,细胞两极伸出细长的突起;许旺细胞标志物S-100免疫荧光染色阳性;②相较于空白对照组,1,10μmol/L褪黑素能够显著促进许旺细胞迁移,对其产生化学性吸引作用,其中1μmol/L褪黑素作用最显著;③许旺细胞主要表达褪黑素受体1;Transwell细胞迁移实验表明,和单纯褪黑素处理组比较,luzindole和褪黑素共同干预组迁移的细胞数无显著性改变,而IWR-1和褪黑素共同干预组许旺细胞迁移的细胞数显著低于单纯褪黑素处理组(P<0.05);④qRT-PCR和Western-blot结果表明,相较于空白对照组,褪黑素组许旺细胞中P-Lrp6,LEF-1和细胞核β-catenin蛋白表达水平显著上调(P<0.05);⑤单纯褪黑素组和IWR-1+褪黑素组许旺细胞中LEF-1和核β-catenin蛋白表达量均显著高于空白对照组和单纯IWR-1组(P<0.05),表明褪黑素在一定程度上拮抗IWR-1对Wnt/β-catenin信号通路活性的抑制作用;⑥上述数据证实,褪黑素能够通过激活Wnt/β-catenin信号通路促进许旺细胞迁移。
BACKGROUND:Melatonin,a neuroendocrine hormone,is mainly secreted by the pineal gland,which can promote the proliferation of Schwann cells and the regeneration of peripheral nerve after injury.OBJECTIVE:To investigate the role of melatonin in the migratory activity of Schwann cells together with the molecular mechanism involved.METHODS:The rodent RSC96 Schwann cell line was cultured in vitro followed by identification.The CCK-8 assay was performed to detect the effect of melatonin on Schwann cells proliferation.The Transwell chamber was adopted to observe the efficacy of melatonin with a series of concentrations,which is 0,100 nmol/L,1μmol/L and 10μmol/L,on the migratory activity of Schwann cells following 20 hours.The expression of melatonin receptor 1 and 2 in Schwann cells was assessed using qRT-PCR and western blot assay.Transwell cell migration system was conducted to determine the influence of melatonin receptor antagonist luzindole or inhibitor IWR-1 of the canonical Wnt/β-catenin signaling on the enhanced migratory activity exerted by melatonin.Western blot assay was performed to investigate the regulatory effect of melatonin on the activity of Wnt/β-catenin signal cascades.RESULTS AND CONCLUSION:(1)The cultured RSC96 cells showed the typical morphology of Schwann cells,showing a spindle-like or triangular shape with thin and long processes on the two cell poles.The Schwann cells marker S-100 immunofluorescence staining was positive.(2)Compared with the blank control group,1μmol/L and 10μmol/L melatonin could promote Schwann cell migration dramatically compared to the control group,and displayed a chemoattractive effect,with 1μmol/L showing the most significant effect.(3)Melatonin receptor 1 was the dominating receptor of melatonin expressed in Schwann cells.The Transwell assay results displayed that the number of migratory cells that melatonin induced was not significantly changed with luzindole added,as compared with treatment with melatonin alone,showing no statistical significance.The number of Schwann cells treated with IWR-1 and melatonin was significantly lower than those treated with melatonin(P<0.05).(4)Results of qRT-PCR and western blot assay showed that compared with the blank control group,the expression levels of P-Lrp6,LEF-1 and nuclearβ-catenin protein in Schwann cells of melatonin group were significantly increased(P<0.05).(5)The expression levels of LEF-1 and nuclearβ-catenin protein in Schwann cells of melatonin group and IWR-1+melatonin group were significantly higher than those of blank control group and IWR-1 group(P<0.05),indicating that melatonin can antagonize the inhibition of IWR-1 on Wnt/β-catenin signaling pathway activity to some extent.(6)The above data confirm that melatonin can promote Schwann cell migration by activating Wnt/β-catenin signaling pathway.
作者
吕建伟
马剑雄
马信龙
Lü Jianwei;Ma Jianxiong;Ma Xinlong(Department of Orthopedics,Tianjin Hospital,Tianjin 300211,China;Orthopedics Research Institute,Tianjin Hospital,Tianjin 300050,China)
出处
《中国组织工程研究》
CAS
北大核心
2020年第31期5030-5037,共8页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81572154),项目负责人:马信龙,项目参与者:吕建伟
天津市自然科学基金(17JCQNJC10900),项目参与者:马剑雄。
关键词
褪黑素
许旺细胞
因子
通路
细胞迁移
周围神经再生
蛋白
实验
melatonin
Schwann cell
factor
pathway
cell migration
peripheral nerve regeneration
protein
experiment
作者简介
吕建伟,男,1988年生,河北省内丘县人,汉族,2016年天津医科大学毕业,硕士,医师,主要从事骨科研究;通讯作者:马信龙,硕士,教授,天津市天津医院骨科,天津市300211。
