臺灣博碩士論文加值系統

人類腸胃道中未受控制的腸道免疫便會造成慢性IBD，就像Crohn disease與潰瘍性結腸炎 (UC)。IBD是一群慢性的疾病，它會導致大腸或小腸中的發炎與潰瘍。在之前研究中發現，介白素十重組蛋白與經基因改造細菌所分泌介白素十可以改善由糊精硫酸鈉 (DSS)所引發的老鼠結腸炎。近來，人類介白素十九被界定為介白素十的家族之ㄧ。它會影響T細胞成熟並改變Th1/Th2平衡而偏向Th2。再者，介白素十九是介白素十的轉錄活化因子，且在介白素十九刺激的周邊血單核球細胞中會引發高量的介白素十上升。在本篇研究中，我們利用減毒豬霍亂沙門氏桿菌攜帶表現老鼠介白素十九的質體以減緩DSS所引發的老鼠急性結腸炎，並減緩其致死率與減輕其許多臨床上的發炎指標。我們也發現在介白素十九所治療的結腸炎老鼠中，在早期便可以誘發介白素十的上升而達到預防DSS所引發的老鼠結腸炎。在未來利用減毒豬霍亂沙門氏桿菌攜帶表現老鼠介白素十九的治療將提供ㄧ個新的臨床應用的方法來治療人類IBD.

Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn’s disease and ulcerative colitis. IBD is a group of chronic disorder that causes inflammation or ulceration in the small and/or large intestines. In previous studies, recombinant interleukin (IL)-10 and genetically modified bacteria secreting IL-10 ameliorated the dextran sulfate sodium (DSS)-induced colitis of IBD model. Recently, human IL-19 has been identified as a member of the IL-10 cytokine family. It influences the maturation of T cells and alters the balance of Th1/Th2 cells in favor of Th2. Furthermore, IL-19 is a transcriptional activator of IL-10 and IL-10 is strongly induced in IL-19-stimulated peripheral blood mononuclear cells. In this study, we investigated whether the expression of murine IL-19 gene was beneficial in ameliorating IBD. Our results showed that the use of the attenuated Salmonella choleraesuis carrying murine IL-19 expressing plasmid for IBD gene therapy reduced the mortality and many clinical markers in DSS- induced acute colitis as compared with those untreated counterparts. We also found that IL-10 was induced in IL-19 treated colitis mice and it prevented the progression at the early stage of DSS-induced colitis. The S. choleraesuis encoding IL-19 for the treatment of IBD provides a new strategy in future clinical application.

Abstract I
Chinese abstract III
Contents VI
Figure contents VIII
Abbreviations X
Introduction 1
Inflammatory bowel disease (IBD) 1
Current treatments and interleukin (IL)-10 gene therapy of IBD 3
Interleukin (IL)-19 5
Bacteria as the gene delivery vehicle 6
IL-19 gene delivery by attenuated Salmonella choleraesuis and treatment of IBD 7
Material and Methods 8
Isolation of murine IL-19 cDNA 8
Integrated plasmids and luciferase gene-abeled bacteria 8
Southern blot analysis 9
Plasmids and bacteria 10
Cell lines and mice 11
Assays of gene transfer in vitro and in vivo 12
Immunohistochemistry 13
Isolation of spleen cells 14
Induction of colitis and challenge with the DSS induced colitis mice 14
TNF-α, IL-6, IL-1β, and IL-10 concentration in the colonic tissues 15
Histological observations and scoring 15
Statics 16
Results 17
Generation of the attenuated S. choleraesuis expressing the luciferase gene 17
S. choleraesuis transfers functional genes in vitro and in vivo 18
Murine IL-19 induced IL-10 production in RAW264.7 and mouse splenocytes 19
S.C./pLJD-CMV-mIL19 mediated IL-19 expression induced IL-10 production in RAW264.7 and mouse splenocytes 20
S.C./pLJD-CMV-mIL19 treatment induced IL-10 production in the DSS induced colitis mice 22
S.C./pLJD-CMV-mIL19 treatment reduced the mortality and body weight loss in DSS induced acute colitis mice 23
S.C./pLJD-CMV-mIL19 diminished the colonic markers in DSS induced acute colitis mice 24
S.C./pLJD-CMV-mIL19 treatment decreased intestinal inflammation in DSS induced acute colitis mice 25
S.C./pLJD-CMV-mIL19 treatment decreased colon immune cell infiltration in C57BL/6 mice with DSS colitis 26
S.C./pLJD-CMV-mIL19 treatment decreased colonic pro-inflammatory cytokine production 27
Discussion 29
Reference 35
Figure 1. Diagram representative of construction of the integrated plasmid. 41
Figure 2. Homologous recombination in Salmonella choleraesuis. 42
Figure 3. Mutant types of attenuated S. choleraesuis with the luciferase gene were confirmed by Southern blot analysis. 43
Figure 4. In vivo monitor the spatial and temporal changes in the movement of attenuated S. choleraesuis with the luciferase gene (S.C.-Luc)...44
Fig 5. S.C/pLJD-CMV-mIL19 transfers functional gene in vitro and in vivio.. 45
Fig 6. Murine IL-19 induced IL-10 production in RAW 264.7 and mice spleen cells. 46
Fig 7. S.C./pLJD-CMV-mIL19 transferred IL-19 to Caco-2 cells and induced IL-10 production in mouse splenocytes and RAW264.7.. 47
Fig 8. The schedule of gene therapy in DSS induced colitis mice. 48
Fig 9. S.C./pLJD-CMV-mIL19-treated mice induced IL-10 production in DSS induced colitis mice. 49
Fig 10. The effects of S.C./pLJD-CMV-mIL19 treatment on the various manifestations of acute DSS colitis in C57BL/6 mice.. 50
Fig 11. The effect of S.C./pLJD-CMV-mIL19 treatment on the clinical markers of acute DSS colitis in C57BL/6 mice. 51
Fig 12. The effect of S.C./pLJD-CMV-mIL19 treatment on the histological appearances of acute DSS colitis in C57BL/6 mice colons.. 53
Fig 13. The effect of S.C./pLJD-CMV-mIL19 treatment on the histological manifestations of acute DSS colitis in C57BL/6 mice.. 55
Fig 14. The overall cytokine secretion of S.C./pLJD-CMV-mIL19 treatment in C57BL/6 mice induced with acute DSS colitis.. 56

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