The expression and effect of LINGO-1 during the differentiation of spinal cord derived neural stem cells

doi:10.3969/j.issn.2095-4344.2017.33.023

Abstract

Abstract:

BACKGROUND: It is of great significance to explore the expression and effect of LINGO-1 in the differentiation of spinal cord derived neural stem cells (SpNSCs) for regulating neural stem cell differentiation and repairing spinal cord injury.
OBJECTIVE: To investigate the expression features and biological effects of LINGO-1 in the differentiation of SpNSCs.
METHODS: SpNSCs were isolated from the rat spinal cord and cultured in vitro. The expression characteristics of LINGO-1 was observed through double immunofluorescence staining of LINGO-1 and Nestin (neural stem cells), β-Tubulin III (neurons), GFAP (astrocytes) and O₄ (oligodendrocyte) at 0-5 days of differentiation. SpNSCs isolated from the rat spinal cord were cultured in vitro and divided into siRNA group and control group. The siRNA group was transfected with LINGO-1 shRNA lentiviral vector to down-regulate the expression of LINGO-1, and the control group was transfected with Scramble-shRNA lentiviral vector. The growth of neurites was detected by immunofluorescence staining at 5 days after transfection.
RESULTS AND CONCLUSION: The SpNSCs could differentiate into neurons, astrocytes and oligodendrocytes. LINGO-1 was expressed in SpNSCs, neurons and oligodendrocytes, but not in astrocytes. The neurite length of the siRNA group was significantly longer than that of the control group (P < 0.05). In summary, the SpNSCs have the potential of multi-directional differentiation, and LINGO-1 has a negative effect on the neurite growth.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Spinal Cord, Neural Stem Cells, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Xu Gang, Zhao Chen-guang, Sun Wei, Ju Fen, Yuan Hua, Mou Xiang. The expression and effect of LINGO-1 during the differentiation of spinal cord derived neural stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(33): 5394-5399.

Figures/Tables 2

2.1 脊髓神经干细胞多向分化潜能脊髓神经干细胞培养2周后可见中等大小的圆形细胞克隆球形成，将克隆球接种到载玻片后第1天即见到有细胞分化出现，随诱导分化时间延长Nestin表达减弱，β-Tubulin Ⅲ阳性细胞(神经元细胞)出现并逐渐增多(图1A-C)，神经元成龛状分化生长，即在广泛星形胶质细胞背景下，神经元呈圆形向外放射状生长；GFAP阳性细胞(星形胶质细胞)在种植到载玻片上第1天即可被检测到，第3天可见明显增多(图1D-F)，整个视野下星形胶质细胞散在较均匀分布，数量较多且密集；O4阳性细胞(少突胶质细胞)在分化后第2天就开始出现，第5天数量明显增多(图1G-I)。 2.2 LINGO-1在脊髓神经干细胞分化过程中的表达特征为了进一步探索LINGO-1在脊髓神经干细胞分化过程中的表达特征，运用LINGO-1抗体及Nestin抗体(脊髓神经干细胞)、β-Tubulin Ⅲ抗体(神经元)、GFAP抗体(星形胶质细胞)、O4抗体(少突胶质细胞)进行免疫荧光双染色观察。实验结果表明(95±2.1)%细胞在分化第0天时表达Nestin，并且这些细胞(100±0)%表达LINGO-1(图2A-D)。至分化第5天时进行免疫荧光染色，结果显示(82.0±1.5)%神经元(图2E-H)、(0±0)%星形胶质细胞(图2I-L)及(98.0±1.9)%少突胶质细胞(图2M-P)表达LINGO-1。 2.3 LINGO-1干扰序列的选择为了筛选检测设计的干扰片序列，对设计方案进行RNA干扰实验，然后进行Real-time PCR检测，发现pRNAi-Lingo1可使LINGO-1的RNA表达下降至对照组的30%(图3)。 2.4 LINGO-1对分化形成神经元突起长度的影响在分化第5天应用β-Tubulin Ⅲ抗体对神经元进行免疫荧光染色。结果提示LINGO-1干扰组的神经元突出长度(图4E-H)明显长于对照组神经元突出长度(图4A-D)，两者突出长度分别为(37.1±2.5) μm及(54.8±3.9) μm，差异有显著性意义(P < 0.05)，见图5。"

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