Astrocyte- and Endothelial-Targeted CCL2 Conditional Knockout Mice: Critical Tools for Studying the Pathogenesis of Neuroinflammation
Astrocyte- and Endothelial-Targeted CCL2 Conditional Knockout Mice: Critical Tools for Studying the Pathogenesis of Neuroinflammation
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DOI：
10.1007/s12031-009-9197-4
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发表时间：
2009-09-01
影响因子：
3.1
通讯作者：
Pachter, Joel S.
中科院分区：
文献类型：
--
作者：
Ge, Shujun;Murugesan, Nivetha;Pachter, Joel S.
尽管C-C趋化因子配体2（CCL 2）在中枢神经系统（CNS）中的表达与许多神经炎性病症相关，但该趋化因子的关键细胞来源（其负责疾病过程以及相关的致病机制）仍未得到解决。由于抗CCL 2治疗剂在治疗神经炎性疾病中的潜力可能取决于有效的药物递送至CNS中CCL 2作用的来源和/或靶标，因此强调神经炎症期间CCL 2作用过程的工具是必要的。为了满足这一需求，我们使用Cre/loxP和FLP-FRT重组系统开发了前两种细胞条件性CCL 2敲除小鼠-分别将CCL 2基因消除靶向星形胶质细胞和内皮细胞，这两种细胞被认为在神经炎症性疾病中发挥关键但未定义的作用。具体地，将含有floxed CCL 2等位基因的小鼠与GFAP-Cre或Tie 2-Cre转基因小鼠杂交，以分别产生具有CCL 2缺陷型星形胶质细胞（星形胶质细胞KO）或内皮细胞（内皮KO）的小鼠。聚合酶链反应，逆转录聚合酶链反应/定量逆转录聚合酶链反应，和酶联免疫吸附试验的CCL 2基因，RNA和蛋白质，分别从培养的星形胶质细胞和脑微血管内皮细胞（BMEC）建立的效率和特异性的CCL 2基因缺失和CCL 2空表型在这些CNS细胞。在体内环境中也证实了CCL 2的有效细胞条件性敲除，其中通过免疫引导的激光捕获显微切割从经历急性脂多糖介导的内毒素血症的小鼠脑中的原位位置取回星形胶质细胞和BMEC以诱导CCL 2基因表达。体内分析进一步揭示了BMEC和星形胶质细胞之间关于星形胶质细胞CCL 2表达的调节的明显串扰。星形胶质细胞KO和内皮细胞KO小鼠的使用应证明在阐述神经炎性疾病的致病机制和优化治疗方面至关重要。
While the expression of the C-C chemokine ligand 2 (CCL2) in the central nervous system (CNS) is associated with numerous neuroinflammatory conditions, the critical cellular sources of this chemokine, which is responsible for disease processes-as well as associated pathogenic mechanisms, remain unresolved. As the potential for anti-CCL2 therapeutics in treating neuroinflammatory disease is likely to be contingent upon effective drug delivery to the source(s) and/or target(s) of CCL2 action in the CNS, tools to highlight the course of CCL2 action during neuroinflammation are imperative. In response to this need, we used the Cre/loxP and FLP-FRT recombination system to develop the first two, cell-conditional CCL2 knockout mice-separately targeting CCL2 gene elimination to astrocytes and endothelial cells, both of which have been considered to play crucial though undefined roles in neuroinflammatory disease. Specifically, mice containing a floxed CCL2 allele were intercrossed with GFAP-Cre or Tie2-Cre transgenic mice to generate mice with CCL2-deficient astrocytes (astrocyte KO) or endothelial cells (endothelial KO), respectively. Polymerase chain reaction, reverse transcription polymerase chain reaction/quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay of CCL2 gene, RNA, and protein, respectively, from cultured astrocytes and brain microvascular endothelial cells (BMEC) established the efficiency and specificity of the CCL2 gene deletions and a CCL2 null phenotype in these CNS cells. Effective cell-conditional knockout of CCL2 was also confirmed in an in vivo setting, wherein astrocytes and BMEC were retrieved by immune-guided laser capture microdissection from their in situ positions in the brains of mice experiencing acute, lipopolysaccharide-mediated endotoxemia to induce CCL2 gene expression. In vivo analysis further revealed apparent cross-talk between BMEC and astrocytes regarding the regulation of astrocyte CCL2 expression. Use of astrocyte KO and endothelial KO mice should prove critical in elaborating the pathogenic mechanisms of and optimizing the treatments for neuroinflammatory disease.