脑挫伤大鼠GFAP、ApoE、S-100、炎症因子时序性表达及代谢组学研究

发布时间：2019-01-11 15:06

【摘要】：目的:本实验拟采用大鼠颅脑挫伤模型,应用免疫组化和酶联免疫法,结合计算机图像分析技术,对脑组织胶质细胞的阳性标志物GFAP的形态、数目、分布和炎性因子血清水平和蛋白含量的表达进行研究,检测大鼠脑挫伤后在不同时间段挫伤灶及其周围GFAP、ApoE、S-100蛋白和炎性因子的时序性表达情况及基于1H-NMR代谢组学技术研究脑损伤血清生物标志物。方法:选取健康雄性SD大鼠60只,随机分成正常对照组(n=10)、假性损伤组(n=10)和脑损伤组(n=40)。损伤组在0.5h、1d、3d、7d时相点,各10只。1)建立大鼠局灶性脑挫伤模型,运用光学显微镜观察脑组织形态学的改变,利用免疫组织化学染色法、分子生物学技术和酶联免疫法对三组大鼠不同时间组内脑匀浆中的GFAP、S-100和ApoE检测。2)酶联免疫法对三组大鼠不同时间组内脑匀浆中的炎症因子进行检测。3)基于1H-NMR代谢组学方法确定脑挫伤大鼠血浆生物标志物,并作统计学分析。结果:1)正常对照组及假性脑损伤组大鼠大脑皮质浅层少数神经元或星形胶质细胞内出现淡棕色颗粒,GFAP呈弱阳性表达。脑挫伤大鼠0.5h组,在皮质挫伤灶周围的出现黄棕色颗粒;1d~3d组上述部位的阳性细胞逐渐增加,7d组挫伤灶及其周围大量神经元及星型胶质细胞胞浆内出现深棕色颗粒,阳性表达。正常对照组和假性损伤组可见GFAP蛋白和mRNA表达未见明显改变;脑挫伤各亚组GFAP蛋白和mRNA均增高,随挫伤时间延长GFAP蛋白和mRNA表达明显增强;2)与正常对照组比较,假性脑损伤组大鼠大脑组织TNF-α、IL-6、MMP-9匀浆含量未见明显变化;脑挫伤大鼠0.5h、1d、3d、7d TNF-α、IL-6、MMP-9含量均明显增高,差异有统计学意义(P0.05)。3)Cr、Lac、Cho、Glu含量增高,NAA峰值下降。结论:1)Feeney氏自由落体撞击法可制作稳定的局灶性脑挫伤动物模型。2)大鼠脑挫伤后GFAP、S-100、ApoE、炎症因子在挫伤灶的表达有一定的时间规律性,在第7d达到高峰,故可以联合用来较准确地推断脑挫伤形成的时间。3)1H-NMR能评价脑损伤严重程度的指征。Lac、Cr、Cho含量明显升高,NAA明显降低,提示脑挫伤大鼠脑组织神经元受损,缺血缺氧,能量代谢异常。
[Abstract]:Objective: to investigate the morphology and number of GFAP, a positive marker of glial cells in brain tissue, using immunohistochemical and enzyme-linked immunosorbent assay (IHC) and computer image analysis (image analysis) in a rat model of brain contusion. The distribution and the expression of serum and protein of inflammatory factors were studied to detect the GFAP,ApoE, of contusion focus and its surrounding area in different time after brain contusion in rats. The temporal expression of S-100 protein and inflammatory factors and the study of serum biomarkers of brain injury based on 1H-NMR metabolomics. Methods: sixty healthy male SD rats were randomly divided into normal control group (n = 10), sham injury group (n = 10) and brain injury group (n = 40). In the injury group, the focal brain contusion model was established at 0. 5 h, 1 d, 3 d and 7 d, 10 rats each). The morphologic changes of brain tissue were observed by optical microscope, and immunohistochemical staining method was used. Molecular biological techniques and enzyme-linked immunosorbent assay for GFAP, in brain homogenate of rats in different time groups S-100 and ApoE assays. 2) the levels of inflammatory factors in brain homogenate of three groups were detected by enzyme-linked immunosorbent assay (Elisa). 3) the plasma biomarkers of brain contusion rats were determined and statistically analyzed based on 1H-NMR metabolic method. Results: 1) the light brown granules appeared in the superficial few neurons or astrocytes of the cerebral cortex in the normal control group and the sham brain injury group, and the expression of GFAP was weakly positive. In the 0.5 h group, the yellowish brown granules appeared around the contusion of cortex. In the 1d~3d group, the positive cells increased gradually, and dark brown granules were found in the cytoplasm of the contusion focus and its surrounding neurons and astrocytes on the 7th day. The expression of GFAP protein and mRNA in normal control group and pseudoinjury group were not significantly changed, GFAP protein and mRNA were increased in all subgroups of brain contusion, and the expression of GFAP protein and mRNA were significantly increased with the extension of contusion time. 2) compared with the control group, the contents of TNF- 伪 and IL-6,MMP-9 in the brain tissue of the rats with pseudo-brain injury did not change significantly. The content of TNF- 伪 and IL-6,MMP-9 in brain contusion rats increased significantly at 0. 5 h, 1 d, 3 d and 7 d, and the difference was statistically significant (P0.05). The content of Cr,Lac,Cho,Glu increased and the peak value of NAA decreased. Conclusion: 1) Feeney's free fall impingement method can make a stable animal model of focal brain contusion. 2) the expression of GFAP,S-100,ApoE, inflammatory factors in the contusion area of rats has a certain time regularity and reaches its peak on the 7th day. Therefore, it can be used to infer the time of formation of brain contusion more accurately. 3) 1H-NMR can be used to evaluate the indication of the severity of brain contusion. The content of Lac,Cr,Cho is obviously increased and NAA is obviously decreased, suggesting that the neurons of brain tissue are damaged in brain contusion rats. Ischemia and hypoxia, abnormal energy metabolism.
【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R651.15

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