[보고서]묘소병과 세균성 혈관종증을 일으키는 Batonella hensella에서 BadA의 병인인자로서의 역할 조사

이희우

묘소병과 세균성 혈관종증을 일으키는 Batonella hensella에서 BadA의 병인인자로서의 역할 조사
The role of BadA as a pathogenic factor in Batonella hensella pathogenesis including cat scratch disease and bacillary angiomatosis 원문보기

보고서 정보
주관연구기관	서울대학교 수의과대학 Seoul National University
연구책임자	이희우
보고서유형	최종보고서
발행국가	대한민국
언어	한국어
발행년월	2011-06
과제시작연도	2010
주관부처	교육과학기술부
사업 관리 기관	한국연구재단
등록번호	TRKO201200002698
과제고유번호	1345121696
사업명	일반연구자지원
DB 구축일자	2013-05-20
키워드	혈관내피세포.염증반응.균력인자.병원성인자.세균 부착능.균막형성능.Bartonella henselae.Bartonella adhesion A.endothelial cells.inflammatory response.virulence.pathogenic factor.BadA-/- mutant.bacterial adhesion.biofilm formation.

초록 ▼

- 재조합 Bartonella adhesion A 단백질이 사람의 혈관내피세포에서 염증반응과 혈관생성과 관련한 세포활성에 미치는 효과를 분석
- BadA에 대한 특이적 다클론 항체를 제작하여 B. henselae의 효과적인 검출법을 개발.
- B. henselae 감염에 중요한 균력인자로서의 BadA의 역할을 규명
- 마우스에서 $<TEX>$BadA^{-/-}$</TEX>$ - 재조합 Bartonella adhesion A 단백질이 사람의 혈관내피세포에서 염증반응과 혈관생성과 관련한 세포활성에 미치는 효과를 분석
- BadA에 대한 특이적 다클론 항체를 제작하여 B. henselae의 효과적인 검출법을 개발.
- B. henselae 감염에 중요한 균력인자로서의 BadA의 역할을 규명
- 마우스에서 $<TEX>$BadA^{-/-}$</TEX>$ mutant 또는 wild strain을 감염시켜 조직학적 및 병리학적으로 비교 관찰하고 염증관련 사이토카인들의 발현을 비교 분석함으로서 병원성인자로서의 BadA의 역할을 규명

Abstract ▼

- To investigate the effects of the recombinant Bartonella adhesion A (BadA) on inflammatory response and angiogenesis-related cell activity in Human umblical vein vein endothelial cells (HUVECs)
- To develop the effective B. henselae detection tool using a specific BadA polyclonal antibody
- To evaluate the role of BadA as virulence factor involved in B. henselae infection
- To investigate the role of BadA as a pathogenic factor through the analysis of histology, histopathology, and inflammatory response-related cytokines expression in $<TEX>$BadA^{-/-}$</TEX>$ mutant- or wild strain- infected mice

목차 Contents

일반연구자지원사업 최종보고서 ... 1
목차 ... 3
I. 연구 계획 요약문 ... 4
1. 국문 요약문 ... 4
II. 연구 결과 요약문 ... 5
한글요약문 ... 5
SUMMARY ... 6
III. 연구내용 및 결과 ... 7
1. 연구 개발 과제의 개요 ... 7
2. 국내외 기술 개발 현황 ... 10
3. 연구 수행 내용 및 결과 ... 11
4. 목표 달성도 및 관련 분야에의 기여도 ... 36
5. 연구 결과의 활용 계획 ... 39
6. 연구 과정에서 수집한 해외 과학기술 정보 ... 40
7. 주관연구책임자(공동연구원) 대표적 연구 실적 ... 41
8. 참고 문헌 ... 42
9. 연구 성과 ... 43
10. 기타사항(중요 연구 변경사항 등) ... 44
[별첨1] 대표연구성과 ... 45
자체평가 의견서 ... 50

표/그림 (25)

표 B. heselae infection. Nature Reviews Microbiology 3, 621-631
표 Primer sequeces used in this study
표 Primer sequences used in this study
표 Primer sequence used in this study
표 Crystal violet staining of B. henselae cultured on blood agar plate.
표 The influence on cytokine production in PBMCs isolated from human after in vitro stimulation of B. henselae. (X: unstimulated well, Bh: B. henselae-stimulated PBMCs)
표 Construct of BadA-pET28a expression vector and purification of recombinant BadA protein. M: size marker, NC: Negative control(E. coli strain BL21/DE3)
표 Cytotoxicity assay of HUVECs treated with recombinant BadA protein for 1, 2 and 3 days.
표 Proliferation assay of HUVECs treated with recombinant BadA protein for 1, 2 and 3 days.
표 RT-PCR analysis of proinflammatory response-related adhesion molecules (E-selectin, ICAM-1 and VCAM-1) in HUVECs treated with BadA protein for 6, 12, 24 and 48h. The density of each band in each lane was quantitated by scanning densitometry and then expressed as mean + SD. Data are expressed as ratios of E-selectin, ICAM-1 and VCAM-1 mRNA normalized to GAPDH mRNA.
표 RT-PCR analysis of proinflammatory response-related cytokines (MCP-1, IL-6 and IL-8) in HUVECs treated with BadA protein for 6, 12, 24 and 48h. The density of each band in each lane was quantitated by scanning densitometry and then expressed as mean + SD. Data are expressed as ratios of MCP-1, IL-6 and IL-8 mRNA normalized to GAPDH mRNA.
표 ELISA analysis of IL-6 and IL-8 in HUVECs treated with BadA protein for 12, 24 and 48h. Data are expressed as mean + SD. *p<0.001 compared with the corresponding control value, as determined by student t-test.
표 Adhesion assay of HUVECs to U937 monocytes after treatment BadA protein for a day. Data are expressed as mean + SD. *p<0.001 compared with the corresponding control value, as determined by student t-test.
표 BadA gene PCR-based detection of B. henselae in canine and feline blood samples. Agarose gel (1.5%) demonstrating amplified products from template genomic DNA derived from animal blood is shown. M: DNA size marker (100 bp), lane 1: B. henselae ATCC 49882 (positive control), lane 2: negative control, lane 3, 4: canine positive blood samples, lane 5, 6: feline positive blood samples.
표 Prevalence of B. henselae infection in blood samples of cats and dogs
표 Western blot of BadA using polyclonal BadA antibody. Lane 1: 10 ng of purified recombinant BadA protein, Lane 2: 20 ng of purified recombinant BadA protein.
표 Comparative RT-PCR analysis of pro-inflammatory response-related adhesion molecules (E-selectin, VCAM-1 and ICAM-1) in HUVECs infected B. henselae. A) RT-PCR products of human E-selectin, VCAM-1 and ICAM-1 from after 2, 4, 8, 12 and 24h incubation in HUVECs infected B. henselae. B)The amounts of E-selectin, VCAM-1 and ICAM-1 mRNA quantities were analyzed in triplicate, normalized against GAPDH as acontrol gene. Values were expressed as a relative mRNA expression. Data are shown as mean +SD. *P<0.05 and **P <0.01 compared with the corresponding control value as determined by Student’s t-test.
표 Comparative RT-PCR analysis of pro-inflammatory response-related cytokines (IL-6, IL-8 and MCP-1) in HUVECs infected B. henselae. A) RT-PCR products of human IL-6, IL-8 and MCP-1 from after 2, 4, 8, 12 and 24h incubation in HUVECs infected B. henselae. B) The amounts of IL-6, IL-8 and MCP-1 mRNA quantities were analyzed in triplicate, normalized against GAPDH as a control gene. Values were expressed as a relative mRNA expression. Data are shown as mean +SD. *P <0.05 and **P <0.01 compared with the corresponding control value as determined by Student’s t-test.
표 ELISA of pro-inflammatory cytokines (IL-6, IL-8 and MCP-1) in HUVECs infected B. henselae. For determination of secreted pro-inflammatory cytokine levels, HUVECs were infected, and supernatants (triplicates) were analyzed 24 hours after infection by ELISA. (A) IL-6, (B) IL-8 and (C) MCP-1 were significantly increased in HUVECs infected B. henselae. Data are shown as mean+SD. ** P < 0.01 compared with the corresponding control value as determined by Student’s t-test.
표 Adherence of BadA-/- mutant to HUVECs. The number of bacteria adherent to HUVECs was determined by counting. Data are expressed as the mean +SD. * P < 0.01 compared with the corresponding control value as determined by Student’s t-test.
표 Invasion assay of BadA-/- mutant to HUVECs. The number of bacteria inserted into HUVECs was determined by counting. Data are expressed as the mean +SD. * P < 0.01 compared with the corresponding control value as determined by Student’s t-test.
표 FACS analysis of HUVECs adherent BadA protein. HUVECs adherent BadA were analyzed by FACS with rat anti-BadA polyclonal antibody.
표 Biofilm formation assay of BadA-/- mutant on polystyrene surface. (A) Biofilms stained with gentian violet 0.1% for 30 min. (B) The gentian violet-stained biofilms were solubilised with ethanol 95% for 5 min, and biofilm cell- associated dye was measured at OD570 using an ELISA reader. Data are expressed as the mean +SD. * P < 0.01 compared with the corresponding control value as determined by Student’s t-test. C: wild strain B. henselae , M: BadA-/- mutant.
표 In vivo imaging of wild strain B. henselae or BadA-/- mutant colonization in mouse spleen and liver. Spleen and liver sections were observed after green flourescent labeling B. henselae or BadA-/- mutant infection by intraperitoneal for 2 weeks with confocal fluorescent microscopy. Nucleus of tissue cells were counterstained with Hochest 33422 (blue) and the bacteria (green) were fluorescence stained. Scale bars indicate 10 um in figures (x400).
표 Histological analysis in liver tissue of mice infected with wild strain B. henselae or BadA-/-mutant. Scale bars indicate 5 um (H&E, MT stain) and 10 um (TB stain).

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연합인증

묘소병과 세균성 혈관종증을 일으키는 Batonella hensella에서 BadA의 병인인자로서의 역할 조사
The role of BadA as a pathogenic factor in Batonella hensella pathogenesis including cat scratch disease and bacillary angiomatosis 원문보기