【作者】 刘玉如；

【导师】 佘锐萍；

【作者基本信息】 中国农业大学 ， 基础兽医学， 2005， 博士

【摘要】 为探讨在兔圆小囊肠道黏膜免疫防御反应中神经肽分子SP、VIP的功能作用及其对免疫相关细胞的调节作用,本研究采用组织培养、免疫组化、原位分子杂交、免疫电镜细胞化学、组织微阵列和图像分析等技术,对巴氏杆菌和大肠杆菌感染后早期的实验兔圆小囊组织,和受到巴氏杆菌和大肠杆菌等抗原刺激后早期的离体圆小囊组织中P物质(SP)及其SPmRNA、血管活性肠肽(VIP)、突触素(SYP)三种脑肠肽的动态表达和CD25、CD68、溶菌酶和抗菌肽等免疫相关因子的动态表达进行了系统地定性、定位和定量观察。对SP、VIP作用受抑后和施加外源性SP后圆小囊组织中免疫反应相关因子和淋巴细胞的活化分子的动态变化进行了系统的定量观察。结果如下: 家兔感染巴氏杆菌、大肠杆菌后,圆小囊各部位中SP和VIP的表达量均出现了明显的变化,相应部位的淋巴细胞活化分子、巨噬细胞及DNES细胞的数量也呈现出显著的变化。相关性分析结果表明,圆小囊圆顶部位的SP表达量与淋巴细胞表达CD25和巨噬细胞数量呈显著的正相关性。溶菌酶和抗菌肽的表达也出现了一定的升高。 离体圆小囊受巴氏杆菌、大肠杆菌刺激后,圆小囊各个部位SP的表达量均明显升高,VIP表达量则出现了明显下降。同时,圆小囊内各个部位淋巴细胞的活化分子表达量、CD68和SYP表达量、溶菌酶和抗菌肽表达量也都出现了显著升高。在滤泡区和圆顶部位SP的表达量与CD25、CD68、SYP表达量呈显著的正相关性。 原位分子杂交观察结果表明活体感染巴氏杆菌、大肠杆菌后,圆小囊组织中SPmRNA的表达不明显;而离体圆小囊受巴氏杆菌、大肠杆菌刺激后,圆小囊中SPmRNA的表达量明显升高。这表明在脱离机体整体调控后SP在圆小囊的自主调节中起作用。 采用抗SP抗体使SP作用受到抑制后,圆小囊组织中淋巴细胞活化分子、DNES细胞表达SYP和巨噬细胞表达CD68分子的量均出现一定程度的减少;而当VIP作用受到抑制后,圆小囊组织中淋巴细胞活化分子、DNES细胞表达SYP和巨噬细胞表达CD68分子的量均出现一定程度的增多。外源性SP可以在一定程度上促进圆小囊组织CD25、CD68的表达。 免疫电镜细胞化学观察发现在兔圆小囊内除了DNES等细胞内有SP和VIP的分布以外,在细胞间隙和淋巴细胞、巨噬细胞等的胞浆膜上也常见SP和VIP免疫电镜阳性反应信号。这些观察结果提供了SP、VIP和DNES细胞通过细胞信号转导途径调节局部黏膜免疫的形态学依据。 上述结果表明圆小囊中的神经肽分子SP、VIP对局部黏膜免疫有一定的调节作用。SP可促进组织中CD25、CD68分子表达,及DNES细胞SYP的表达,而VIP的作用在以上方面则具有一定的反向调节效应。提示在兔肠道黏膜免疫防御反应中SP和VIP这两种神经肽可能是正负调节因子,对于维持肠道黏膜免疫的平衡起一定的调节作用。 本研究结果进一步揭示了圆小囊参与肠道黏膜免疫的反应特点,初步揭示了圆小囊在细菌感染早期肠道黏膜免疫反应过程中神经肽SP、VIP的调节作用机制,为丰富免疫-神经-内分泌网络学说提供了新的有价值的资料,为生产实践中更好地防治肠源性细菌感染提供了新的理论依据。更多还原

【Abstract】 To investigate the mechanism of substance P (SP) and vasoactive intestinal peptide (VIP) in the mediation of intestinal mucosal immunity and the interplaying of SP, VIP with immune related cells, the distribution and expression of SP, SPmRNA, VIP, CD25, CD68, Lysozyme and anti-bacterial peptide(ABP) in the Sacculus Rotundus(SR) of rabbits were investigated using tissue culture methods, immunohistochemistry, in situ hybridization, immune electron cytochemistry, tissue micro-arraying and image analysis during the early stage of the rabbits were infected with Pasteurella and E. coli. and the cultural tissues of SR were stimulated by the same pathegons. The results were showed as followed:1. The expression of SP, VIP in some subfields of sacculus rotundus altered markedly after the rabbits were inoculated with Pasteurella and E.coli. At the same time, the numbers of activated lymphocytes, macrophages, DNES cells and the expression of Lysozyme also up-regulated in some subfields of sacculus rotundus. There were correlated relations between SP expression and the numbers of macrophges, DNES cells, CD25-expressing lymphocytes in some extent.2. When it was stimulated by the Pasteurella and E. coli in vitro, the tissue of SR up-regulated expression of SP greatly and down-regulated the expression of VIP markedly. Meantime, the expression of SYP, CD25, CD68, lysozyme, and ABP increased markedly. There’s a close correlationship between the SP, VIP expression and CD25, CD68, SYP expression.3. The SP mRNA was up-regulated markedly in in-vitro experiments and no such results were found in the in-vivo experiments.4. When the cultural fliud were added into the antibodies of SP, the activation and the expression of CD68, SYP were inhibited somehow. Meanwhile, the antibodies of VIP enhanced the activation of lymphocytes and the expression of CD68, SYP in sacculus rotundus. The added SP can upregulate expression of CD25, CD68 and downregulate SYP to some extent in the tissue of SR.5. The ultrastructures of the SP, VIP secretory cells showed there were several kinds of cells with different morphology which could express these neuropeptides in sacculus rotundus. Some intercellular immunoreactive products and cells with immunoreactive membrane (including lymphocytes, macrophages, etc.) were found in the SR, which might favour the transduction of these two peptides.The results suggested that SP, VIP may play an important role in the regulation of intestinal mucosal immunity by mediating of the activation of lymphocytes, the macrophages and DNES cells. VIP displayed downregulating effect in these reactions somehow. SP and VIP may be a couple moleculars with reversed function in the regulation of local intestinal mucosal immunity.The characteristics of immune reaction of SR and the mechanism of SP, VIP in SR which involved in the regulation of the intestinal mucosal immunity were discussed in this study, which richer the datafor the immnuo-neuro-endocrine network theory and provided some new knowledge for reference of making new strategy to control the disease of intestinal bacterial infection.更多还原