Analysis of mouse UNC93A gene expression in innate immune response = 선천면역반응에서 마우스 UNC93A 유전자의 발현분석

다국어 초록 (Multilingual Abstract)

Thesis Abstract
The function of UNC93B1 is related with deliverance of nucleotide-sensing Toll-like receptors (TLR) including TLR9 to endolysosomes. However, there is no report concerning the function of UNC93A, which is one of UNC-93 superfamily. To figure out mouse UNC93B1 and UNC93A expression pattern, CpG-ODNs, liposome-encapsulated CpG-ODN (Lipoplex(O)), LipoplexGC(O) were injected into mouse peritoneal cavity. UNC93A mRNA expression was increased on time-dependent by Lipoplex(O) immunization, but UNC93B1 was not changed in expression level. To evaluate whether the expression of mUNC93A is involved in the TLR9, TLR9 knock out (TLR-/-) mice were injected i.p with Lipoplex(O) or LipoplexGC(O), and the peritoneal cells were harvested after 12 h injection. The expression of mUNC93A was not detected in TLR9-/- mice. Further, CpG-ODNs, Lipoplex(O), LipoplexGC(O),and several cytokines were stimulated to peritoneal cells in vitro. However, UNC93A expression was not induced by several stimulations. To identify cellular localization of UNC93A, UNC93A stably expressing HEK 293 cells were established and analyzed by confocal microscopy. The hUNC93A and mUNC93A proteins were detected in the cytoplasm. These results suggest that mouse UNC93A in BALB/c mice are closely associated with TLR9 signaling with Lipoplex(O) stimulation. Further investigations of function on UNC93A in the regulation of CpG-DNA-mediated immune response may support the applicational diversity of CpG-DNA immunotherapeutics.

목차 (Table of Contents)

• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 4
• 1. ODNs 4
• 2. Preparation of CpG-ODN encapsulated in liposome complex 4
• 3. Mice and ODN treatment 5
• 4. Preparation of peritoneal cells, splenocytes and bone marrow cells 5
• 5. Stimulation of primary cells with cytokines 6
• 6. RNA isolation and reverse-transcriptional PCR analysis 6
• 7. Cell lines 7
• 8. Construction of recombinant hUNC93A 7
• 9. Construction of recombinant mUNC93A 8
• 10. Construction of UNC93A expressing stable cell line 8
• 11. Western blotting 9P
• 12. Indirect immunofluorescence and confocal microscopyp 9
• III. RESULTS 11
• 1. TLR9-dependent expression of UNC93A by Lipoplex(O) stimulation in BALB/c peritoneal cells 11
• 2. Expression of UNC93A in human total tissues 12
• 3. Effect of in vitro stimulation with ODNs or Lipoplex(O) in cultured BALB/c peritoneal cells 12
• 4. Effect of cytokines on UNC93A expression in BALB/c peritoneal cells, splenocytes, and bone marrow cells 12
• 5. Overexpression of UNC93A in HEK 293 cells 13
• 6. Localization of UNC93A in HEK 293 cells 14
• IV. DISCUSSION 15
• V. REFERENCES 17
• VI. FIGURES 22
• VII. ABSTRACT i
• VIII. ABSTRACT IN KOREA ii