CD47敲除对小鼠颅脑创伤后血管生成的影响及其分子机制

Effect of CD47 knockout on angiogenesis after traumatic brain injury in mice and its molecular mechanism

目的:研究整合素相关蛋白(IAP)CD47敲除对小鼠颅脑创伤(TBI)后血管生成的影响,阐明其可能的分子机制。方法:体内实验,采用液压颅脑打击仪制备成年C57BL/6小鼠TBI模型,将小鼠随机分为假手术组和TBI组。TBI后24 h取小鼠脑组织测定脑组织含水量,采用伊文思蓝(EB)注射法检测小鼠血脑屏障通透性(即EB水平),HE染色观察小鼠脑组织病理形态表现,免疫荧光染色法检测小鼠脑组织中血管内皮细胞生长因子受体1(VEGFR1)和血小板-内皮细胞黏附分子CD31蛋白表达水平。体外实验,选用CD47敲除同时内源性表达绿色荧光蛋白(GFP)的C57BL/6小鼠(CD47KO-GFP组)和野生型C57BL/6小鼠(野生型组),采用液压颅脑打击仪分别制备TBI模型,分离脑组织中微血管内皮细胞,并混合培养,荧光显微镜下观察TBI后小鼠脑组织中微血管内皮细胞的再生和血管生成能力,采用免疫荧光染色法检测小鼠损伤部位脑组织微血管内皮细胞中核因子κB(NF-κB)的活化情况。结果:与假手术组比较,TBI组小鼠脑组织含水量和EB水平明显增加(P<0.05或P<0.01)。HE染色,与假手术组比较,TBI组小鼠脑组织结构无法辨认,出血点明显。免疫荧光染色,TBI组小鼠脑组织中VEGFR1和CD31蛋白表达水平明显升高(P<0.01)。荧光显微镜下观察,与野生型组比较,CD47KO-GFP组小鼠脑组织中参与血管生成的微血管内皮细胞百分率明显增加(P<0.05)。免疫荧光检测,与野生型组比较,CD47KO-GFP组小鼠脑组织微血管内皮细胞中NF-κB活化状态明显降低。结论:TBI后小鼠血脑屏障通透性明显增加,损伤脑组织的血管生成增强,CD47敲除有利于TBI后血管新生。

Objective:To study the effect of integrin-associated protein(IAP)CD47 knockout on the angiogenesis after traumatic brain injury(TBI)in the mice,and to clarify its possible molecular mechanism.Methods:In the in vivo experiment,the TBI models of adult C57BL/6 mice were prepared by hydraulic craniocerebral percussion instrument,and the experimental mice were randomly divided into sham operation group and TBI group.The brain tissue was collected 24 h after TBI for the detection of water content in brain tissue;Evans blue(EB)injection was used to detect the blood brain barrier permeability(EB level)and HE staining was used to observe the pathomorphology of brain tissue of the mice.Immunofluorescence staining method was performed to detect the expression levels of vascular endothelial growth factor receptor 1(VEGFR1)and platelet-endothelial cell adhesion molecule CD31 proteins in brain tissue of the mice.In the in vitro experiment,the C57BL16 mice with CD47 knockout and endogenous expression of green fluorescent protein(GFP)labeled(CD47KO-GFP group)and wild type(WT)mice(WT group)were selected.The TBI models were prepared with hydraulic craniocerebral percussion.The brain microvascular endothelial cells were extracted and mixedly cultured.The regeneration and tube-forming capacities of cerebral microvascular endothelial cells after TBI were observed by fluorescence microscope;immunofluorescence staining method was used to detect the activation of nuclear factor-κB(NF-κB)in the cerebral microvascular endothelial cells in injured area of the mice.Results:Compared with sham operation group,the water content in brain tissue and EB level of the mice in TBI group were significantly increased(P<0.05).The HE staining results showed that compared with sham operation group,the brain tissue structure of the mice in TBI group was unrecognizable and the bleeding spots were obvious.The immunofluorescence staining results showed that compared with sham operation group,the expression levels of VEGFR1 and CD31 proteins in brain tissue of the mice in TBI group were significantly increased(P<0.01).The fluorescence microscope results showed that compared with WT group,the number of brain microvascular endothelial cells participating in angiogensis of the mice in CD47KO-GFP group was significantly increased(P<0.05).The immunofluorescence staining results showed that compared with WT group,the activation of NF-κB in the cerebral microvascular endothelial cells in CD47KO-GFP group was decreased significantly.Conclusion:After TBI,the blood brain barrier permeability and angiogenesis of injured brain tissue are increased significantly,and CD47 knockout can promote the angiogenesis.