β-1，4-半乳糖基转移酶-Ⅰ在大鼠中枢神经系统炎症反应中的表达及意义

发布时间：2018-01-04 22:09

本文关键词：β-1，4-半乳糖基转移酶-Ⅰ在大鼠中枢神经系统炎症反应中的表达及意义　出处：《南通大学》2009年硕士论文　论文类型：学位论文

【摘要】：目的:观察β-1,4-半乳糖基转移酶-I(β-1,4-galactosyltransferase-I,β-1,4-GalT-I)及其催化合成的Galβ1-4GlcNAc糖链在大鼠脊髓内注射内毒素(Lipopolysaccharide,LPS)致炎模型及大鼠大脑炓质内注射LPS致帕金森病(PD)模型中的表达情况及细胞定位,进一步明确炎症刺激小胶质细胞β-1,4-GalT-I表达上调的意义,并探讨其在炎症激活小胶质细胞中的生物学作用。方法:(1)应用SD大鼠脊髓内注射LPS建立脊髓炎症模型,利用实时荧光定量PCR(Real Time-PCR)检测术后脊髓组织中β-1,4-GalT-I在mRNA水平的表达变化;免疫荧光检测β-1,4-GalT-I及Galβ1-4GlcNAc糖链在脊髓内注射LPS后的定位和细胞分布;利用免疫共沉淀检测LPS对E-选择素与β-1,4-半乳糖基化作用的影响;PCR检测LPS对小胶质细胞β-1,4-GalT-I表达的影响;应用ELISA检测了脊髓内注射LPS后TNF-α释放情况;PCR检测TNF-α对小胶质细胞β-1,4-GalT-I表达的影响。(2)应用SD大鼠大脑炓质内注射LPS建立大鼠PD模型,分别利用Real Time-PCR、Western-blot、Lectin-blot、免疫荧光双标法等方法观察β-1,4-GalT-I及其催化合成的Galβ1-4GlcNAc糖链在术后炓质中的表达情况及细胞定位。(3)应用胚胎SD大鼠中脑神经元-胶质细胞共培养建立体外PD模型,利用Western-blot观察了LPS对共培养细胞β-1,4-GalT-I蛋白表达的影响;应用LPS或β-1,4-GalT-I抗体处理共培养细胞,免疫荧光和ELISA检测共培养细胞中小胶质细胞活化和吞噬作用。结果:(1)大鼠脊髓内注射LPS诱导脊髓中β-1,4-GalT-I mRNA表达上调。β-1,4-GalT-I mRNA的表达水平呈时间依赖性关系,在术后6-8h表达到达高峰;β-1,4-GalT-I蛋白水平和Galβ1-4GlcNAc糖链在术后24h达到高峰,主要是注射侧灰质和白质表达增多,且这些分子主要定位于活化的小胶质细胞、中性粒细胞、巨噬细胞。术后荧光双标检测到E-选择素与Galβ1-4GlcNAc糖链共定位,运用免疫共沉淀进一步证实脊髓炎症过程中E-选择素与β-1,4-半乳糖基化作用增强。并且在脊髓炎症中检测到TNF-α表达增加。PCR显示LPS或TNF-α刺激体外培养的小胶质细胞,β-1,4-GalT-I mRNA的表达水平呈现剂量依赖性和时间依赖性关系。 (2)大鼠大脑炓质内注射LPS后,β-1,4-GalT-I mRNA表达出现上调趋势,在术后8h达到高峰,之后表达逐渐下降到正常水平。Western-blot显示β-1,4-GalT-I蛋白在炓质内注射LPS后2h即表达增加,持续至术后3 dβ-1,4-GalT-I蛋白表达仍较高, 3 d后表达降低;Galβ1-4GlcNAc糖链表达亦随之升高,在术后12-24 hβ-1,4-半乳糖基化作用增强尤为明显。此外,在大鼠PD病理过程中,β-1,4-GalT-I和Galβ1-4GlcNAc糖链主要定位于活化的小胶质细胞。 (3)LPS诱导共培养细胞中β-1,4-GalT-I蛋白表达增加,作用8-18 h表达达到高峰,然后逐渐下降到正常水平。免疫荧光显示LPS诱导共培养细胞中小胶质细活化和吞噬功能增强,这一作用可以被β-1,4-GalT-I抗体减弱。ELISA结果表明β-1,4-GalT-I抗体影响LPS导致的TNF-α的释放。结论:(1)β-1,4-GalT-I及其催化合成的Galβ1-4GlcNAc糖链可能参与LPS所致的中枢神经系统炎症反应,尤其可能在炎症细胞向炎症部位迁移这一过程中发挥重要作用。 (2)在中枢神经系统炎症反应过程中,TNF-α可能参与β-1,4-GalT-I的表达变化。 (3)在中枢神经系统炎症反应过程中,β-1,4-GalT-I可能参与介导小胶质细胞的激活,从而在神经退变性疾病发病过程中发挥重要作用。
[Abstract]:Objective: To observe the -1,4- beta galactosyltransferase -I (beta -1,4-galactosyltransferase-I and beta -1,4-GalT-I) injection and catalytic synthesis of Gal beta 1-4GlcNAc sugar chain in the rat spinal cord induced by endotoxin (Lipopolysaccharide, LPS) LPS brain Liao Intraplasmic injection and inflammatory model of rats induced by Parkinson disease (PD) expression and cell localization model the further inflammation of microglia stimulated the expression of -1,4-GalT-I beta, and investigate its biological role in microglia activation in inflammation.
Methods: (1) the application of SD in the rat spinal cord with LPS to establish spinal cord inflammation model, using real-time fluorescence quantitative PCR (Real Time-PCR) expression of beta -1,4-GalT-I detection in spinal cord tissue after operation at the mRNA level; immunofluorescence detection of beta -1,4-GalT-I and Gal beta 1-4GlcNAc sugar chain positioning injection in the spinal cord after LPS and cell distribution co precipitation; detection of LPS effects and beta -1,4- galactosylation of E- by immune; PCR detection effect of LPS on the expression of microglia in beta -1,4-GalT-I; ELISA was used to detect the TNF- alpha release in the spinal cord after LPS injection; effect of PCR detection of TNF- alpha on the expression of microglia (beta -1,4-GalT-I. 2) application of SD rat brain Liao Intraplasmic injection LPS rats PD model, respectively, by Real Time-PCR, Western-blot, Lectin-blot, immunofluorescence method to observe the beta -1,4-GalT-I and its catalytic synthesis of Gal beta 1-4GlcNAc sugar After Liao chain in the expression and cellular localization. (3) the application of SD rat embryonic midbrain neurons and glial cells were cultured in vitro PD model, the effects of LPS co cultured cells beta -1,4-GalT-I protein expression by Western-blot; application of LPS or beta -1,4-GalT-I antibody treated cells co culture, immunofluorescence and detection of ELISA cells Co cultured microglia activation and phagocytosis.
Results: (1) upregulation of beta -1,4-GalT-I and mRNA expression in spinal cord in the spinal cord of rats induced by injection of LPS. The expression level of -1,4-GalT-I beta mRNA was time dependent relationship, reached the peak expression at 6-8h after operation; beta -1,4-GalT-I and Gal beta 1-4GlcNAc protein level in the sugar chain reached the peak at 24h after the injection side, is mainly gray and white the increased expression of these molecules and matter, mainly located in activated microglial cells, neutrophils and macrophages. After fluorescence double staining to E- selectin and Gal beta 1-4GlcNAc sugar chain co localization, further confirmed using immune E- selection of spinal cord inflammation process enhancement and beta -1,4- galactosylation Co precipitation. And in the spinal cord inflammation was detected in TNF- LPS or.PCR showed increased expression of alpha TNF- alpha stimulation in cultured microglia, the expression level of mRNA beta -1,4-GalT-I showed a dose-dependent and time-dependent manner.
(2) rat brain Liao Intraplasmic injection after LPS, the expression of -1,4-GalT-I beta mRNA appears upward trend, reaching a peak after 8h, then the expression decreased gradually to the normal level of.Western-blot showed beta -1,4-GalT-I protein in Liao Intraplasmic injection after LPS 2H expression increased to 3 d after the operation of beta -1,4-GalT-I protein expression is still high 3, the expression of D decreased; the expression of Gal beta 1-4GlcNAc sugar chain is also increasing, in the postoperative effect of 12-24 h -1,4- beta galactosyl enhanced obviously. In addition, the pathological process of PD rats, beta -1,4-GalT-I and Gal beta 1-4GlcNAc sugar chain located mainly in activated microglial cells.
(3) LPS induced by co culture increased -1,4-GalT-I protein expression of beta cells, 8-18 expression of H reached the peak, then gradually decreased to normal level. Immunofluorescence showed that LPS induced cell co culture of microglial activation and fine phagocytosis, and this effect can be reduced.ELISA beta -1,4-GalT-I antibody -1,4-GalT-I antibody showed that beta effect due to LPS the TNF- alpha release.
Conclusion: (1) beta -1,4-GalT-I and its Gal beta 1-4GlcNAc sugar chain may be involved in LPS induced inflammatory response in the central nervous system, especially in the process of migration of inflammatory cells to inflammatory sites.
(2) during the inflammatory response of the central nervous system, TNF- alpha may be involved in the changes in the expression of beta -1,4-GalT-I.
(3) in the inflammatory reaction of central nervous system, beta -1,4-GalT-I may participate in the activation of microglia, and play an important role in the pathogenesis of neurodegenerative diseases.

【学位授予单位】：南通大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

【共引文献】