解剖学报

解剖学报 ›› 2019, Vol. 50 ›› Issue (4): 405-410.doi: 10.16098/j.issn.0529-1356.2019.04.001

• 神经生物学 •    下一篇

大鼠脑缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A的表达变化

吴燕平 秦新月* 张融融 黄思源 张磊   

  1. 重庆医科大学附属第一医院神经内科，重庆市神经病学重点实验室，重庆 400016
  • 收稿日期:2018-09-04 修回日期:2018-10-16 出版日期:2019-08-06 发布日期:2019-08-06
  • 通讯作者: 秦新月 E-mail:qinxinyue2018@sina.com
  • 基金资助:
    国家自然科学基金面上项目

Expression of repulsive guidance molecule A in polarized microglia after rat focal cerebral ischemia/reperfusion injury

WU Yan-ping QIN Xin-yue* ZHANG Rong-rong HUANG Si-yuan ZHANG Lei   

  1. Department of Neurology，the First Affiliated Hospital of Chongqing Medical University，Chongqing Key Laboratory of Neurology，Chongqing 400016，China
  • Received:2018-09-04 Revised:2018-10-16 Online:2019-08-06 Published:2019-08-06
  • Contact: QIN Xin-yue E-mail:qinxinyue2018@sina.com
  • Supported by:
    General Program of National Natural Science Foundation of China

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摘要：

目的 探讨大鼠大脑中动脉缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A（RGMa）的表达变化。 方法 取雄性SD大鼠30只，随机分成对照组，缺血再灌注损伤7 d、14 d模型组（I/R），采用线栓法制作大鼠大脑中动脉缺血再灌注损伤模型(tMCAO)；Real-time PCR检测M1、M2型小胶质细胞标记分子白细胞介素-1β（IL-1β）、诱导性一氧化氮合酶（iNOS）、精氨酸酶1（Arg-1）及CD206 mRNA的表达；免疫荧光检测缺血区小胶质细胞RGMa的表达；体外培养小胶质细胞，分别用M1或M2型小胶质细胞的诱导物脂多糖（LPS）或IL-4诱导24 h后，利用Real-time PCR检测M1、M2型小胶质细胞标记分子IL-1β、iNOS、Arg-1、CD206 mRNA的表达；Western blotting检测M1、M2型小胶质细胞上RGMa的表达。 结果 缺血再灌注损伤后7 d、14 d，M1、M2型小胶质细胞的标记分子表达增加。缺血后7 d、14 d激活的小胶质细胞上RGMa大量表达。RGMa在体外培养的LPS诱导极化的M1型小胶质细胞和IL-4诱导极化的M2型小胶质细胞上表达均显著增加。结论 大鼠大脑中动脉缺血再灌注损伤后，RGMa在缺血区M1型和M2型极化的小胶质细胞上均大量表达，RGMa可能在小胶质细胞激活极化过程中发挥重要作用。RGMa可能是缺血性脑卒中治疗的新靶点。

关键词: 排斥性导向分子A, 脑缺血, 再灌注损伤, 小胶质细胞极化, 免疫印迹法, 实时定量聚合酶链反应, 大鼠

Abstract:

Objective To investigate the expression of repulsive guidance molecule A (RGMa) in polarized microglia after rat focal cerebral ischemia/reperfusion injury. Methods Adult male Sprague-Dawley (SD) rats were randomly divided into control group, 7 days cerebral ischemia/reperfusion injury group (I/R), and 14 days I/R group. The transient middle cerebral occlusion (tMCAO) model was induced by ligation with nylon monofilament. Real-time PCR was used to test the mRNA expression of M1 and M2 microglia marker interleukin-1β(IL-1β), inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and CD206 in ipsilateral cortex at day 7 and day 14.Double immunofluorescence staining was performed to investigate the co-expression of RGMa and Iba1 in microglia. The microglia was incubated with lipopolysaccharides (LPS)or IL-4 in vitro. The mRNA expression of M1 and M2 microglia marker (IL-1β, iNOS, Arg-1, CD206) was tested by Real-time PCR. Western blotting was used to assess the proteins level of RGMa in M1 and M2 microglia. Results M1 and M2 microglia marker mRNA level were increased in ipsilateral cortex at day 7 and day 14. RGMa was strongly expressed in reactive microglia at day 7 and day 14. LPS or IL-4 treatment polarized microglia to M1 or M2 in vitro. The expression of RGMa protein in M1 and M2 microglia was increased. Conclusion RGMa was strongly expressed in reactive M1 and M2 microglia after ischemia/reperfusion injury. RGMa may play an important role in the process of microglia activation and polarization. RGMa may be a novel therapeutic target for stroke.

Key words: Repulsive guidance molecule A, Brain ischemia, Reperfusion injury, Microglia polarization, Western blotting, Real-time PCR, Rat