论文总字数：24242字

摘 要

肠道内寄生菌群之间的关系和平衡对于维持肠道健康有着十分重要的作用。加氏乳杆菌作为一种益生菌，在动物的肠道、人体阴道内都可以发现它的存在，并且占据菌群的主导地位。一些研究已经指出了加氏乳杆菌对肠道的调节作用。然而对于肠炎的影响以及潜在的免疫调节效应还未有人涉及。本实验通过在肠道内人工定植加氏乳杆菌和建立斑马鱼肠炎模型，来探讨其在肠炎的免疫调节方面具有的功效。首先，我们通过实验证实了加氏乳杆菌可以定植于斑马鱼肠道内，而后利用TNBS诱导肠炎，测试了有加氏乳杆菌和无加氏乳杆菌定植情况下的斑马鱼存活率。以及诱导肠炎后，对特定组分的斑马鱼进行解剖取出肠道做RT-PCR分析和检测斑马鱼肠道内SOD、MDA、CAT、GSH、Caspase-3和Caspase-9含量来探讨加氏乳杆菌的影响。结果表明，加氏乳杆菌可能对TNF-α和IL-1β具有调节作用，同时与氧化应激和细胞自噬也有一定关联。

关键词：加氏乳杆菌 斑马鱼 肠炎 免疫调节

Effects of Lactobacillus Gasseri on Regulation of Innate Immune Responses in Colitis

Abstract

The interaction and balance between intestinal microbiota play a critical role in the maintenance of bowel health. Lactobacillus gasseri, as one of the probiotics which has been found to predominate in some animal intestines and human vagina, is discovered to regulate intestinal situations in various ways. However, little information is found on modulation of inflammatory bowel disease by L.gasseri. This experiment practiced factitious intestinal colonization of L.gasseri in zebrafish and setting colitis models in order to investigate potential effects of L.gasseri in immune responses. First, it’s been proved that L.gasseri is capable of colonizing zebrafish’s intestine and then we induced intestinal inflammation to testify the survival rate of zebrafish with or without L.gasseri colonization by using TNBS. Also, after 6 hours of TNBS injection, certain groups of zebrafish were dissected for the RT-PCR test of intestines and the detection of SOD, MDA, CAT, GSH, Caspase-3 and Caspase-9 for the purpose of probing possible effects of L.gasseri. The results showed that L.gasseri might have regulatory functions on TNF-αand IL-1β, and also connected with oxidative stress and autophagy.

Keyword: Lactobacillus gasseri; Zebrafish; IBD; Immune Regulation

目录

摘要.....................................................Ⅰ

Abstract.................................................Ⅱ

一 文献综述...............................................1

• 1. 益生菌对肠炎的免疫调节的机制和认识.....................1

1.1.1 益生菌对肠炎的保护.............................. 1

1.1.2 斑马鱼肠炎的益生菌调节.......................... 1

1.1.3 益生菌调节肠炎的特定免疫........................ 1

1.1.4信号通路在肠免疫调节的论述....................... 1

1.1.5 氧化应激、细胞自噬和凋亡的论述...................2

• 1. 加氏乳杆菌及其免疫调节的机制.......................... 3

1.2.1加氏乳杆菌的研究及其免疫调节功能论述............. 3

1.3研究目的与意义.........................................4

二 材料与方法.............................................5

2.1 实验材料与仪器........................................ 5

2.2 实验方法与步骤........................................ 6

2.2.1 斑马鱼的养殖................................... 6

2.2.2 加氏乳杆菌的活化和接种......................... 7

2.2.3 加氏乳杆菌的离心和染色......................... 7

2.2.4 斑马鱼肠炎诱导方法............................. 7

2.2.5 加氏乳杆菌在斑马鱼肠道内的定植实验............. 8

2.2.6 加氏乳杆菌定植对诱导肠炎的斑马鱼存活率实验..... 8

2.2.7 加氏乳杆菌定植对肠炎的RT-PCR检测...............9

2.2.8肠道的氧化应激与细胞自噬指标检测................ 9

三 实验结果..............................................11

3.1 加氏乳杆菌在肠道内的定植结果......................... 11

3.2 加氏乳杆菌定植对诱导肠炎的斑马鱼存活率实验........... 11

3.3 加氏乳杆菌定植对肠炎的RT-PCR检测结果................12

3.4 氧化应激和细胞细胞自噬指标检测结果................... 14

四 结论与展望

4.1 实验结论............................................. 20

4.2 课题展望............................................. 20

参考文献.................................................22

致谢.....................................................27

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