幽門螺旋桿菌的感染與許多上消化道的疾病相關，像是慢性胃
炎、消化性潰瘍、黏膜相關淋巴組織淋巴癌 （MALT lymphoma）以
及胃癌。感染幽門螺旋桿菌患者若無以抗生素加以治療，將轉變為慢
性、持續性感染 ; 而上述疾病的發生則主要起因於該病原菌的慢性
感染。並且該菌所分泌的熱休克蛋白60 已被證實為一黏附分子可連
結幽門螺旋桿菌以及人類胃上皮細胞，進而造成胃部疾病。
對於幽門螺旋桿菌熱休克蛋白60 在免疫調節功能上，較多的研
究是指出此蛋白可引發前發炎激素（pro-inflammatory cytokine）例
如：干擾素-g （IFN-g）、腫瘤壞死因子-a（TNF-a）、白介素6、8
（IL-6、8）而引起發炎反應。特別是在氨基酸序列300 到435 的位
置可引發大量IL-8 的表現。在另一方面，近年來也有研究指出不同
物種的熱休克蛋白60 似乎在人體免疫系統可以抵禦病原菌的慢性感
染。例如人類與日本血吸蟲的熱休克蛋白60 可引發調節型T 細胞
（regulatory T cells）的增生達到免疫抑制的效果。然而，幽門螺旋桿
菌熱休克蛋白60 對於免疫調節的機制依然不清楚。在本篇實驗中，
我們利用人類周邊血液單核球細胞（ human peripheral blood
mononuclear cells, PBMCs）作為研究系統，探討幽門螺旋桿菌熱休
克蛋白60 之N 端蛋白對其增生所造成之影響。研究結果顯示，幽門
螺旋桿菌熱休克蛋白60 之N 端位置於101-200 和1-200 之蛋白能夠
降低PBMCs 之增生以及引發TGF-b之產生。根據上述研究結果，幽
門螺旋桿菌熱休克蛋白60 可藉由接近N 端的101-200 之處引發
TGF-b並且達到免疫抑制的效果。

Helicobacter pylori can lead to variety of upper gastrointestinal
disorders, such as chronic gastritis, peptic ulcer disease, gastric
mucosa-associated lymphoid tissue (MALT) lymphoma and gastric
cancer. Without treatment, H. pylori would become chronic infection in
almost all of those patients. The expression of heat shock protein 60 by H.
pylori (HpHSP60) has been shown as an adhesion molecule that interacts
with host gastric epithelial cells.
For immunoregulation, many literatures showed that HpHSP60 can
induce the secretions of pro-inflammatory cytokines, such as
IFN?{g, TNF-a, IL-?{6, IL-8 and cause inflammation. Particularly, the
sequence of HpHSP60 from 300 to 435 can induce dramatically IL-8
expression. Interestedly, researchers found that HSP60 in different
species seems to be related to the regulation of immune responses in
chronic infection disease. Literatures indicated that HSP60 in human and
S. japonicum can induce regulatory T cell (Treg) expression and suppress
immunity. However, the feature of HpHSP60 for immunoregulation still
remains unknown. In this study, the N-terminal domains of HpHSP60
were constructed and measured their activities on immune response. The
results showed that the treatment with the sequence 101-200
(HpHSP60101-200) or 1-200 (HpHSP601-200) of HpHSP60 to human
peripheral blood mononuclear cells (PBMCs) decreased the proliferation
rate. Furthermore, HpHSP60101-200 and HpHSP601-200 could increase
TGF-b secretion from THP-1 cells. Taken together, the sequence of
HpHSP60 from 101 to 200 could induce the expression of TGF-b and
which may have contributions to their immune suppressive activity.

中文摘要……………………………………………………………….i
Abstract……………………………………………………………… iii
Contents……………………………………………………………….vi
Introduction…………………………………………….......................1
Materials and methods……………………………………………….5
Preparation of recombinant HpHSP60………………………………...5
The coupling of HpHSP60 and FITC………………………………….6
The binding of proteins and cells……………………………………...7
Cloning and expression and purification of recombinant protein……..7
IL-8 secretion from cells……………………………………………….8
Peripheral blood mononuclear cells (PBMCs) isolation………………9
PBMC proliferation assay……………………………………………10
Statisticalanalysis…………………………………………………….11
Results………………………………………………………………..12
The binding activities of HpHSP60 to cells………………………….12
The N-terminal sequence of HpHSP60 for cell binding activity……..13
The functions of HpHSP60……………………………………….13-15
Discussion……………………………………………………………16
Figure and Legend…………………………………………………..20
References…………………………………………………………...49

Algood, H. M. &Cover, T. L. (2006). Helicobacter pylori persistence: an
overview of interactions between H. pylori and host immune
defenses. Clin Microbiol Rev 19(4): 597-613.
Bulut, Y., Faure, E., Thomas, L., Karahashi, H., Michelsen, K. S., Equils,
O., Morrison, S. G., Morrison, R. P. &Arditi, M. (2002).
Chlamydial Heat Shock Protein 60 Activates Macrophages and
Endothelial Cells Through Toll-Like Receptor 4 and MD2 in a
MyD88-Dependent Pathway. J. Immunol. 168: 1435-1440.
Bulut, Y., Shimada, K., Wong, M. H., Chen, S., Gray, P., Alsabeh, R.,
Doherty, T. M., Crother, T. R. &Arditi, M. (2009). Chlamydial
Heat Shock Protein 60 Induces Acute Pulmonary Inflammation in
Mice via the Toll-Like Receptor 4- and MyD88-Dependent
Pathway INFECTION AND IMMUNITY 77: 2683–2690.
Ellis, J. (1992). Cytosolic chaperonin confirmed. NATURE 358: 191-192.
Francisco J. Quintana, P. C., Felix Mor, and Irun R. Cohen (2003). DNA
Fragments of the Human 60-kDa Heat Shock Protein (HSP60)
Vaccinate Against Adjuvant Arthritis: Identification of a
Regulatory HSP60 Peptide. The Journal of Immunology 171:
3533-3541.
Friedland, J. S., Shattock, R., Remick, D. G. &Griffin, G. E. (1993).
Mycobacterial 65-kD heat shock protein induces release of
proinflammatory cytokines from human monocytic cells. Clin Exp
Immunol 91(1): 58-62.
Galdiero, M., de l'Ero, G. C. &Marcatili, A. (1997). Cytokine and
adhesion molecule expression in human monocytes and endothelial
cells stimulated with bacterial heat shock proteins. Infect. Immun.
65: 699-707.
Huesca, M., Borgia, S., Hoffman, P. &Lingwood, C. A. (1996). Acidic
pH changes receptor binding specificity of Helicobacter pylori: a
binary adhesion model in which surface heat shock (stress) proteins
mediate sulfatide recognition in gastric colonization. Infect Immun
64(7): 2643-2648.
Jang, T. J. (2010). The
numberofFoxp3-positiveregulatoryTcellsisincreased in
Helicobacter pylori gastritisandgastriccancer. Pathology–Research
and Practice 206: 34-38.
Karin, N. (2010). The multiple faces of CXCL12 (SDF-1 ) in the
regulation of immunity during health and disease. Journal of
Leukocyte Biology 88: 1-11.
Kol, A., Bourcier, T., Lichtman, A. H. &Libby, P. (1999). Chlamydial
and human heat shock protein 60s activate human vascular
endothelium, smooth muscle cells, and macrophages. J. Clin.
Invest. 103: 571–577.
Lin, C. S., He, P. J., Hsu, W. T., Wu, M. S., Wu, C. J., Shen, H. W.,
Hwang, C. H., Lai, Y. K., Tsai, N. M. &Liao, K. W. (2010a).
Helicobacter pylori-derived Heat shock protein 60 enhances
angiogenesis via a CXCR2-mediated signaling pathway. Biochem
Biophys Res Commun 397(2): 283-289.
Lin, C. S., He, P. J., Tsai, N. M., Li, C. H., Yang, S. C., Hsu, W. T., Wu,
M. S., Wu, C. J., Cheng, T. L. &Liao, K. W. (2010b). A potential
role for Helicobacter pylori heat shock protein 60 in gastric
tumorigenesis. Biochem Biophys Res Commun 392(2): 183-189.
Lin, C. Y., Huang, Y. S., Li, C. H., Hsieh, Y. T., Tsai, N. M., He, P. J.,
Hsu, W. T., Yeh, Y. C., Chiang, F. H., Wu, M. S., Chang, C. C.
&Liao, K. W. (2009a). Characterizing the polymeric status of
Helicobacter pylori heat shock protein 60. Biochem Biophys Res
Commun 388(2): 283-289.
Lin, C. Y., Huang, Y. S., Li, C. H., Hsieh, Y. T., Tsai, N. M., He, P. J.,
Hsu, W. T., Yeh, Y. C., Chiang, F. H., Wu, M. S., Chang, C. C.
&Liao, K. W. (2009b). Characterizing the polymeric status of
Helicobacter pylori heat shock protein 60. Biochemical and
Biophysical Research Communications 388: 283–289.
Lin, S. N., Ayada, K., Zhao, Y., Yokota, K., Takenaka, R., Okada, H.,
Kan, R., Hayashi, S., Mizuno, M., Hirai, Y., Yoshihito Fujinami, Y.
&Oguma, K. (2005). Helicobacter pylori heat-shock protein 60
induces production of the pro-inflammatory cytokine IL8 in
monocytic cells. Journal of Medical Microbiology 54: 225-233.
Macchia, G., Massone, A., Burroni, D., Covacci, A., Censini, S.
&Rappuoli, R. (1993). The Hsp60 protein of Helicobacter pylori:
structure and immune response in patients with gastroduodenal
diseases. Moiecuiar Microbiology 3: 645-652.
Mahabaleshwar, H., Boldajipour, B. &Raz, E. (2008). The role of
CXCR7 in regulating primordial germ cell migration. Cell
Adhesion & Migration 2: 69-70.
Matsuura, E., Kobayashi, K., Matsunami, Y., Shen, L., Quan, N.,
Makarova, M., Suchkov, S. V., Ayada, K., Oguma, K. &Lopez, L.
R. (2009). Autoimmunity, infectious immunity, and atherosclerosis.
J Clin Immunol 29(6): 714-721.
Quinn, T. J., Taylor, S., Wohlford-Lenane, C. L. &Schwartz, D. A.
(2000). IL-10 reduces grain dust-induced airway inflammation and
airway hyperreactivity. J Appl Physiol 88(1): 173-179.
Takenaka, R., Yokota, K., Ayada, K., Mizuno, M., Zhao, Y., Fujinami, Y.,
Lin, S. N., Toyokawa, T., Okada, H., Shiratori, Y. &Oguma, K.
(2004). Helicobacter pylori heat-shock protein 60 induces
inflammatory responses through the Toll-like receptor-triggered
pathway in cultured human gastric epithelial cells. Microbiology
150: 3913–3922.
Toms, C. &Powrie, F. (2001). Control of intestinal inflammation by
regulatory T cells. Microbes Infect 3(11): 929-935.
Vabulas, R. M., Ahmad-Nejad, P., da Costa, C., Miethke, T., Kirschning,
C. J., Ha¨cker, H. &Wagner, H. (2001). Endocytosed HSP60s Use
Toll-like Receptor 2 (TLR2) and TLR4 to Activate the
Toll/Interleukin-1 Receptor Signaling Pathway in Innate Immune
Cells. THE JOURNAL OF BIOLOGICAL CHEMISTRY 276:
31332–31339.
Wang, X., Zhou, S., Chi, Y., Wen, X., Hoellwarth, J., He, L., Liu, F., Wu,
C., Dhesi, S., Zhao, J., Hu, W. &Su, C. (2009). CD4+CD25+ Treg
induction by an HSP60-derived peptide SJMHE1 from
Schistosoma japonicum is TLR2 dependent. Eur. J. Immunol. 39:
3052–3065.
Wieten, L., Broere, F., Zee, R. V. D., Koerkamp, E. K., Wagenaar, J.
&Eden, W. V. (2007). Cell stress induced HSP are targets of
regulatory T cells: A role for HSP inducing compounds as
anti-inflammatory immuno-modulators? FEBS Letters 581:
3716-3722.
Yoshimura, A., Wakabayashi, A. &Mori, T. (2010). Cellular and
molecular basis for the regulation of inflammation by TGF-beta. J.
Biochem. 147: 781-792.
Zanin-Zhorov, A., Cahalon, L., Tal, G., Margalit, R., Lider, O. &Cohen, I.
R. (2006). Heat shock protein 60 enhances CD4+CD25+ regulatory
T cell function via innate TLR2 signaling. The Journal of Clinical
Investigation 116: 2022–2032.
Zhao, Y., Yokota, K., Ayada, K., Yamamoto, Y., Okada, T., Shen, L.
&Oguma, K. (2007).Helicobacter pylori heat-shock protein 60
induces interleukin-8 via a Toll-like receptor (TLR)2 and
mitogen-activated protein (MAP) kinase pathway in human
monocytes. In Journal of Medical Microbiology, Vol. 56, 154–164.