학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2014. 2. 윤철희.In the respiratory system, chemokines, cytokines and antimicrobial peptides (AMPs) play important roles for regulation of immune response. AMPs can protect the foreign molecules and chemokines can recruit the leukocytes that can remove the pathogen. Cytokines can enhance the effect of AMPs and chemokines and activate various immune cells.
The mucosal adjuvant has been studied by many researchers. Cholera toxin (CT), lipopolysaccharide (LPS) and Bacillus subtilis spore were, also, examined for the development of mucosal adjuvant. However, the effects of these adjuvants have not been understood completely through the administration of intranasal route in the lung and bronchoalveolar lavage fluid (BALF).
In the present study, the effect of the mucosal adjuvants, CT, B. subtilis spore and LPS, was examined by the analysis on the expression of effector molecules and migration of leukocytes in the lung. The adjuvants, CT, B. subtilis spore or LPS, were administered through the intranasal route to mice maintained at SPF facility. Then lung, BALF and serum were taken from the mice at 6, 12, 24 and 72 hr and analyzed mRNA and protein levels of effector molecules. Furthermore, the migration of cells into BALF was examined through analysis of the surface marker using flow cytometer.
The results showed that mRNA of CCL2, CCL4 and CXCL10 were induced by spore or LPS in the lung. However, there are no differences on the mRNA expression of AMPs between the adjuvants. Spore and LPS promoted the expression and secretion of CCL2 and CCL4 proteins in the lung and BALF. In analysis of pro-inflammatory cytokines, spore and LPS dramatically induced the secretion of IL-1β, IL-6 and TNF-α in the lung and BALF. CT induced the increase of IL-6 in the serum. In analysis of surface marker, spore highly promoted the migration of Gr-1+CD11b+ cells at 6 hr and F4/80+CD11b+Gr-1- cells at 72 hr. LPS, also, induced the migration of Gr-1+CD11b+ cells at 12 and 24 hr. In conclusion, B. subtilis spore can induce the chemokine and cytokine expression in the lung and BALF and enhance the migration of neutrophils into BALF.I. Introduction 1
II. Review of Literature 3
1 Immune modulation in the lung 3
1.1 Recruitment of leukocytes in respiratory tract 3
1.2 Mucosal immunization via intranasal administration 3
2 Effector molecules in the lung 4
2.1 Chemokines 4
2.2 Anti-microbial peptides 5
2.3 Cytokines 7
3 The effect of mucosal adjuvant in lung 8
3.1 Cholera toxin 8
3.2 Lipopolysaccharide 9
3.3 Bacillus subtilis spore 10
III. Materials and Methods 12
1. Reagents 12
2. Preparation and isolation of Bacillus subtilis spore 12
3. Approval for animal study 12
4. Isolation of serum, lung and bronchoalveolar lavage fluid (BALF) 13
5. Isolation of total lysate from lung 13
6. Isolation of total RNA from lung 13
7. Reverse transcription polymerase chain reaction 14
8. Real time - polymerase chain reaction 15
9. Analysis of surface marker 16
10. Measurement of chemokines and cytokines production 16
IV. Results and discussion 17
1. The mRNA of chemokines, CXCL10, CCL2 and CCL4, was remarkably increased in the whole lung of mouse treated with B. subtilis spore or LPS during the early phase of immune response 17
2. B. subtilis spore and LPS enhanced the secretion of CCL2 and CCL4 proteins into BALF during the early immune response. 20
3. CCL2 and CCL4 expression were induced by administration of B. subtilis spore and LPS in the lung 21
4. B. subtilis spore and LPS induced IL-1β expression in the lung 23
5. B. subtilis spore and LPS induced IL-6 and TNF-α secretion into BALF 24
6. B. subtilis spore and LPS promoted the recruitment of Gr-1+CD11b+ cells into BALF in early phase 27
V. Literature Cited 30
VI. Summary in Korean 41Maste
