Effect of formyl peptide receptor on the differentiation of neural stem/progenitor cells into neurons

doi:10.3969/j.issn.2095-4344.0419

Abstract

Abstract:

BACKGROUND: Previous studies have observed the expression of formyl peptide receptors (FPRs) in neural stem/progenitor cells (NSPCs) and confirmed that FPRs can promote the migration of NSPCs and induce them to differentiate into neurons. FPRs ligands are present in damaged tissues, but the binding of different ligands with FPRs may lead to different and even opposite biological effects.
OBJECTIVE: To investigate the effect on the differentiation of NSPCs into neurons after the binding of the ligands produced following spinal cord injury with FPRs.
METHODS: Immunofluorescence staining, western blot and flow cytometry were used to analyze the expression of FPRs in NSPCs. Immunofluorescent staining with confocal microscope detection was used to analyze the effect of homogenates of the spinal cord on the differentiation of FPR1⁺ or FPR2⁺ NSPCs into neurons.
RESULTS AND CONCLUSION: Some of NSPCs expressed FPR1 and FPR2, not only on the cell membrane, but also in the cytoplasm. The expression level of FPR1 was obviously lower than that of FPR2. The homogenate group for FPR1⁺ or FPR2⁺ NSPCs could produce more β-III tubulin-positive cells and fewer GFAP-positive astrocytes, and the effects could be blocked by FPR1 or FPR2 inhibitor Boc2 or WRW4. These experimental findings show that the spinal cord homogenate can induce FPR1 or FPR2 positive NSPCs to differentiate into neurons and inhibit their differentiation to astrocytes, and moreover, this effect is specific.

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: Neural Stem Cells, Receptors, Formyl Peptide, Spinal Cord Injuries, Cell Differentiation, Tissue Engineering

CLC Number:

R394.2

Zhang Liang, Cheng Hui, Leng Ming-hao, Pan Jian-hai, He Xiang-chun, Zhang Yi-ming, Lu Shan, Chen Hua. Effect of formyl peptide receptor on the differentiation of neural stem/progenitor cells into neurons[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(1): 107-112.

Figures/Tables 1

2.1 神经干/祖细胞的培养及鉴定来源于胎龄12.5 d的胎鼠大脑皮质的细胞，在含有2% B27、20 μg/L表皮生长因子和20 μg/L碱性成纤维细胞生长因子的DMEM/F12培养基中培养7 d后，在倒置显微镜下观察呈典型的神经球(图1A)。为了进一步鉴定神经球细胞具有干细胞特征，选用两个能够标记神经干/祖细胞特性的分子进行免疫荧光染色：SOX2和Nestin。共聚焦显微镜观察结果显示：几乎所有的细胞胞核SOX2表达阳性，同时大部分细胞胞质Nestin染色阳性(图1B)。神经干/祖细胞另外一个特征就是能够分化成为类神经元、星形胶质细胞和少突胶质细胞。因此对神经球细胞的分化能力也进行了鉴定。分别用β-Ⅲ tubulin、GFAP和Olig2来标记神经元、星形胶质细胞和少突胶质细胞。共聚焦显微镜观察结果显示：神经球细胞分化后大部分细胞GFAP表达阳性，少部分细胞β-Ⅲ tubulin和Olig2表达阳性，说明培养的神经球细胞具有向类神经元、星形胶质细胞和少突胶质细胞分化的能力，同时说明神经干/祖细胞大部分分化成星形胶质细胞，少部分分化为类神经元和少突胶质细胞(图1C-E)。上述实验结果充分说明在体外成功建立了神经干/祖细胞的培养体系，为进一步实验打下基础。 2.2 FPR1和FPR2在神经干/祖细胞中的表达免疫荧光染色共聚焦检测结果显示：部分细胞表达FPR1和FPR2，同时二者主要表达在细胞膜和细胞质中；FPR2的免疫荧光强度明显高于FPR1(图2A)。为了进一步证实这种结果，又进行了Western blotting实验，结果提示：神经干/祖细胞表达FPR1和FPR2，同时FPR2的表达量要比FPR1多(图2B)。 2.3 流式分析FPR1和FPR2在神经干/祖细胞中的表达为了进一步观察神经干/祖细胞中FPR1和FPR2的表达水平，又进行了流式细胞仪检测，结果提示：神经球细胞中，大约有45.8%的细胞表达FPR1，大约有45.3%的细胞表达FPR2(图3)。 2.4 大鼠脊髓损伤后组织匀浆液通过FPR1或FPR2导向神经干/祖细胞向类神经元分化为了观察脊髓损伤后产生的FPR1和FPR2配体是否能够通过神经干/祖细胞表达的FPR1和FPR2受体对神经干/祖细胞分化产生影响，实验首先提取了大鼠脊髓损伤后2 d的匀浆液，又用流式细胞仪分选出FPR1或FPR2阳性的神经干/祖细胞。共聚焦显微镜观察结果显示：与对照组相比，匀浆液能够使FPR1或FPR2阳性神经干/祖细胞分化后产生的β-Ⅲ tubulin阳性神经元比例升高，这种作用能够被FPR1或FPR2阻断剂Boc2或WRW4阻断。同时，与对照组相比，匀浆液能够使FPR1或FPR2阳性神经干/祖细胞分化后产生的GFAP阳性星形胶质细胞比例降低，这种作用能够被FPR1或FPR2阻断剂Boc2或WRW4阻断(图4A-D)。这些实验结果说明匀浆液能够导向FPR1或FPR2阳性神经干/祖细胞向类神经元分化，抑制其向星形胶质细胞分化，同时这种作用具有特异性。"

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