解剖学报

解剖学报 ›› 2022, Vol. 53 ›› Issue (6): 689-697.doi: 10.16098/j.issn.0529-1356.2022.06.001

• 神经生物学 •    下一篇

蛋白酪氨酸激酶7和受体酪氨酸激酶样孤儿受体2在hSOD1-G93A突变肌萎缩侧索硬化症转基因小鼠脑干中的表达

孟凡迪1,2 管英俊1,2* 赵振涵1,2 陈燕春1,2 刘金梦1,2 王雪枚1,2 张皓云2 周风华2    

  1. 1.潍坊医学院组织学与胚胎学教研室; 2.神经疾病与再生修复重点实验室，山东 潍坊261053
  • 收稿日期:2021-12-06 修回日期:2022-02-20 出版日期:2022-12-06 发布日期:2022-12-06
  • 通讯作者: 管英俊 E-mail:guanyj@wfmc.edu.cn
  • 基金资助:
    山东省高校科技发展项目重点项目;山东省高等学校青创科技支持计划;山东省自然科学基金项目;山东省自然科学基金项目;国家自然科学基金项目

Expression of protein tyrosine kinase 7 and receptor tyrosine kinase-like orphan receptor 2 in the brainstem of amyotrophic lateral sclerosis transgenic mice with hSOD1-G93A mutation

MENG  Fan-di1,2  GUAN  Ying-jun1,2*  ZHAO  Zhen-han1,2  CHEN  Yan-chun1,2  LIU  Jin-meng1,2  WANG  Xue-mei1,2 ZHANG  Hao-yun2  ZHOU  Feng-hua2#br#

#br#
  

  1. 1.Histology and Embryology Department; 2.Laboratory of Neurologic Disorders and Regenerative Repair, Weifang Medical University, Shandong Weifang261053, China
  • Received:2021-12-06 Revised:2022-02-20 Online:2022-12-06 Published:2022-12-06
  • Contact: GUAN Ying-jun E-mail:guanyj@wfmc.edu.cn

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摘要：

目的 探讨脑干中蛋白酪氨酸激酶7（PTK7）和受体酪氨酸激酶样孤儿受体2（ROR2）的表达变化与肌萎缩侧索硬化症（ALS）发病的关系。  方法 选取44只人超氧化物歧化酶1（hSOD1）-G93A突变型ALS转基因小鼠，以同等数量的同窝野生型小鼠作为对照，于出生后70 d、95 d、108 d和122 d分离脑干，尼氏染色法观察模型小鼠脑干舌下神经核（12N）及面神经核（7N）神经元形态变化，RT-PCR、Western blotting技术分别检测模型小鼠脑干中PTK7和ROR2 mRNA及蛋白表达变化，免疫荧光双标技术观察12N及7N中PTK7和ROR2的细胞定位及分布特点。  结果 尼氏染色结果显示，ALS转基因小鼠脑干12N及7N可见神经元尼氏体明显减少，神经元空泡样变性，胞体萎缩，核体积变小。RT-PCR结果发现，与同窝野生型小鼠相比，ALS转基因小鼠脑干ROR2和PTK7 mRNA在70 d、95 d、108 d和122 d升高。Western blotting结果显示，与同窝野生型小鼠相比，ALS转基因小鼠脑干中PTK7在70 d、95 d、108 d和122 d表达升高，ROR2在70 d、95 d、108 d表达升高，122 d表达降低。免疫荧光结果显示，在ALS转基因小鼠和野生型小鼠脑干12N及7N均可检测到ROR2/神经元核抗原（NeuN）和PTK7/NeuN双阳性细胞，ROR2/胶质纤维酸性蛋白（GFAP）和PTK7/GFAP双阳性细胞，提示ROR2和PTK7在神经元和星形胶质细胞中共表达。  结论 PTK7和ROR2在ALS转基因小鼠脑干中表达异常与ALS发病密切相关。

关键词: 肌萎缩侧索硬化症, 脑干, 蛋白酪氨酸激酶7, 受体酪氨酸激酶样孤儿受体2, 免疫荧光, 转基因小鼠

Abstract:

Objective To investigate the relationship between changes in protein tyrosine kinase 7 (PTK7) and receptor tyrosine kinase-like orphan receptor 2 (ROR2) expression in the brainstem and the pathogenesis of amyotrophic lateral sclerosis (ALS).   Methods Forty-four human superoxide dismutase 1(hSOD1)-G93A mutant ALS transgenic mice were selected, and an equal number of wild-type littermates was used as control. The brainstems were isolated at day 70, day 95, day 108 and day 122 after birth, and the morphology of hypoglossal nucleus (12N) and nucleus of facial nerve (7N) neurons in the brainstem of the model mice were observed by Nissl staining. The mRNA and protein expression of PTK7 and ROR2 were detected by RT-PCR and Western blotting respectively, and the cellular localization and distribution of PTK7 and ROR2 in 12N and 7N were observed by immunofluorescence double-labeling technique.   Results The result  of Nissl staining showed that Nissl bodies in the neurons reduced distinctly with vacuolar degeneration of neurons, cell body atrophy and nuclear volume reduction in the 12N and 7N brainstems of ALS transgenic mice. RT-PCR result  indicated that ROR2 and PTK7 mRNA level in the brainstem of ALS transgenic mice were up-regulated at day 70, day 95, day 108 and day 122 compared with wild-type littermates. Western blotting result  showed that PTK7 protein was up-regulated at day 70,  day 95, day 108 and day 122, ROR2 protein was up-regulated at day 70, day 95, day 108, and down-regulated at day 122 in the brainstem of ALS transgenic mice compared with wild-type littermates. Immunofluorescence result  showed that ROR2/neuronal nuclei (NeuN)and PTK7/NeuN double positive cells, ROR2/glial fibrillary acidic protein (GFAP) and PTK7/GFAP double positive cells were observed in the 12N and 7N of the brainstem of ALS transgenic mice and wild-type mice, suggesting that ROR2 an  PTK7 were expressed both in neurons and astrocytes.   Conclusion PTK7 and ROR2 are abnormally expressed in the brainstem of ALS transgenic mice, which is closely related to the pathogenesis of ALS.

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