發(fā)布時(shí)間：2019-01-11 15:06

【摘要】：目的:本實(shí)驗(yàn)擬采用大鼠顱腦挫傷模型,應(yīng)用免疫組化和酶聯(lián)免疫法,結(jié)合計(jì)算機(jī)圖像分析技術(shù),對(duì)腦組織膠質(zhì)細(xì)胞的陽(yáng)性標(biāo)志物GFAP的形態(tài)、數(shù)目、分布和炎性因子血清水平和蛋白含量的表達(dá)進(jìn)行研究,檢測(cè)大鼠腦挫傷后在不同時(shí)間段挫傷灶及其周圍GFAP、ApoE、S-100蛋白和炎性因子的時(shí)序性表達(dá)情況及基于1H-NMR代謝組學(xué)技術(shù)研究腦損傷血清生物標(biāo)志物。方法:選取健康雄性SD大鼠60只,隨機(jī)分成正常對(duì)照組(n=10)、假性損傷組(n=10)和腦損傷組(n=40)。損傷組在0.5h、1d、3d、7d時(shí)相點(diǎn),各10只。1)建立大鼠局灶性腦挫傷模型,運(yùn)用光學(xué)顯微鏡觀察腦組織形態(tài)學(xué)的改變,利用免疫組織化學(xué)染色法、分子生物學(xué)技術(shù)和酶聯(lián)免疫法對(duì)三組大鼠不同時(shí)間組內(nèi)腦勻漿中的GFAP、S-100和ApoE檢測(cè)。2)酶聯(lián)免疫法對(duì)三組大鼠不同時(shí)間組內(nèi)腦勻漿中的炎癥因子進(jìn)行檢測(cè)。3)基于1H-NMR代謝組學(xué)方法確定腦挫傷大鼠血漿生物標(biāo)志物,并作統(tǒng)計(jì)學(xué)分析。結(jié)果:1)正常對(duì)照組及假性腦損傷組大鼠大腦皮質(zhì)淺層少數(shù)神經(jīng)元或星形膠質(zhì)細(xì)胞內(nèi)出現(xiàn)淡棕色顆粒,GFAP呈弱陽(yáng)性表達(dá)。腦挫傷大鼠0.5h組,在皮質(zhì)挫傷灶周圍的出現(xiàn)黃棕色顆粒;1d~3d組上述部位的陽(yáng)性細(xì)胞逐漸增加,7d組挫傷灶及其周圍大量神經(jīng)元及星型膠質(zhì)細(xì)胞胞漿內(nèi)出現(xiàn)深棕色顆粒,陽(yáng)性表達(dá)。正常對(duì)照組和假性損傷組可見(jiàn)GFAP蛋白和mRNA表達(dá)未見(jiàn)明顯改變;腦挫傷各亞組GFAP蛋白和mRNA均增高,隨挫傷時(shí)間延長(zhǎng)GFAP蛋白和mRNA表達(dá)明顯增強(qiáng);2)與正常對(duì)照組比較,假性腦損傷組大鼠大腦組織TNF-α、IL-6、MMP-9勻漿含量未見(jiàn)明顯變化;腦挫傷大鼠0.5h、1d、3d、7d TNF-α、IL-6、MMP-9含量均明顯增高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。3)Cr、Lac、Cho、Glu含量增高,NAA峰值下降。結(jié)論:1)Feeney氏自由落體撞擊法可制作穩(wěn)定的局灶性腦挫傷動(dòng)物模型。2)大鼠腦挫傷后GFAP、S-100、ApoE、炎癥因子在挫傷灶的表達(dá)有一定的時(shí)間規(guī)律性,在第7d達(dá)到高峰,故可以聯(lián)合用來(lái)較準(zhǔn)確地推斷腦挫傷形成的時(shí)間。3)1H-NMR能評(píng)價(jià)腦損傷嚴(yán)重程度的指征。Lac、Cr、Cho含量明顯升高,NAA明顯降低,提示腦挫傷大鼠腦組織神經(jīng)元受損,缺血缺氧,能量代謝異常。
[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- 偽 and IL-6,MMP-9 in the brain tissue of the rats with pseudo-brain injury did not change significantly. The content of TNF- 偽 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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R651.15

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