脑血管内皮细胞敲除Prmt5导致脑血管损伤及星形胶质细胞活化*

目的: 研究蛋白质精氨酸甲基转移酶5(protein arginine methyltransferase 5,Prmt5)在小鼠脑血管发育、稳态维持中的功能,并考察脑血管内皮细胞特异性敲除Prmt5后对中枢神经系统的影响。方法: 利用脑血管内皮细胞特异性表达SP-A-Cre转基因小鼠和Prmt5条件基因打靶小鼠交配,构建脑血管内皮细胞特异性Prmt5敲除小鼠。利用H-E染色、免疫荧光染色、激光散斑成像、Sulfo-NHS-Biotin染料灌注等方法评价脑血管内皮细胞特异性Prmt5敲除小鼠脑血管结构、脑血流量、血脑屏障渗透性等;利用实时定量PCR进一步检测补体C1q(complement C1q,C1q)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-1β(interleukin 1β,IL-1β)等细胞因子的表达水平。通过免疫荧光、Western blot等检测胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)、S100钙结合蛋白β(S100 calcium-binding protein β protein,S100β)和补体C3(complement C3,C3)的表达,检测小鼠皮层、丘脑和小脑中星形胶质细胞活化水平。结果: 脑血管内皮细胞特异性敲除Prmt5导致血管损伤, C1q、TNF-α和IL-1β等炎症因子表达水平上调,活化星形胶质细胞比例明显增加。结论: 脑血管内皮细胞中Prmt5在小鼠脑血管稳态维持中发挥了重要功能。

Deletion of Prmt5 in Cerebral Endothelial Cells Leads to Cerebrovascular Disease and Astrocyte Activation

Objective: To study the role of protein arginine methyltransferase 5 (Prmt5) in cerebral vascular development and homeostasis maintenance in mice, and to investigate the effect of Prmt5 specific knockout on the central nervous system. Methods: We crossed Prmt5^fl/fl mice with SP-A Cre transgenic mice that express Cre recombinase in cerebrovascular endothelial to generate cerebrovascular endothelial cell-specific Prmt5 knockout mice. H-E staining and immunostaining were performed to observe the vascular structures of control and Prmt5^fl/fl mutant mice. Laser speckle contrast imaging was used to detect cerebral blood flow in control and mutant mice. Sulfo-NHS-Biotin was intraperitoneally injected into control and mutant mice to examine the blood brain barrier(BBB) integrity. The expression levels of astrocyte glial fibrillary acidic protein (GFAP), S100 calcium-binding protein β (S100β), complement C3 (C3), C1q, tumor necrosis factor alpha(TNF-α) and Interleukin-1 beta(IL-1β) were detected by immunofluorescence and Western blot to evaluate the activation level of astrocytes in cortex, thalamus and cerebellum of knockout mice and control mice. Furthermore, activators of astrocytes, such as C1q, TNF-α, IL-1β and other cytokines, were also detected by real-time PCR. Results: We found that cerebrovascular endothelial cell-specific Prmt5 knockout mice exhibited aberrant cerebrovascular structure, and increased the number of reactive astrocytes. The expression levels of TNF-α and IL-1β in the whole brain, as well as the C1q, TNF-α and IL-1β, were all increased in Prmt5^fl/fl mutant mice. Conclusion: Prmt5 plays an essential role in the maintenance of cerebrovascular homeostasis, suggesting that it might act as a potential therapeutic target for cerebrovascular diseases.