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dc.contributor.advisor이순남-
dc.contributor.author최희정-
dc.creator최희정-
dc.date.accessioned2016-08-26T03:08:50Z-
dc.date.available2016-08-26T03:08:50Z-
dc.date.issued2001-
dc.identifier.otherOAK-000000001018-
dc.identifier.urihttps://dspace.ewha.ac.kr/handle/2015.oak/205731-
dc.identifier.urihttp://dcollection.ewha.ac.kr/jsp/common/DcLoOrgPer.jsp?sItemId=000000001018-
dc.description.abstractInterleukin (IL)-18 is a pro-inflammatory cytokine which functions as an interferon (IFN)-γ inducing factor and contributes to the generation of the T helper type 1 (Th1) response. IL-18 also increases human immunodeficiency virus type 1 (HIV-1) production in a chronically infected monocytic cell line and in a lymphocytic cell line. The IL-18 effect on HIV-1 production for primary cells was investigated using p24 enzyme-linked immunosorbent assay (ELISA) and IFN-γ was measured by electrochemiluminescence (ECL) in peripheral blood mononuclear cells (PBMC) cultures. In contrast to the results obtained in cell lines, IL-18 added immediately after infection of PBMC with monocyte (M)-tropic HIV-1 inhibited p24 antigen (Ag) production by a maximum of 72% (p < 0.001) after 4 days of culture. In PBMC infected with T lymphocyte (T)-tropic HIV-1, an inhibition of 57% (p < 0.01) was observed. IFN-γ levels in PBMC cultures were increased in cells infected with M- or T-tropic virus and then exposed to IL-18, indicating an inverse relationship between HIV-1 production and IFN-g levels (r = -0.96, p = 0.008 in M-tropic virus), which suggests that IFN-γ may function as an anti-viral agent in these cultures. Pre-incubation of PBMC with IL-18 during the 2 days prior to infection with M-tropic HIV-1 also inhibited p24 production by 64% (p < 0.01) in 4 day-culture conducted in the absence of additional exogenous IL-18. However, compared to the degree of IL-18 inhibition observed after 4 days of exposure, there was no additional IL-18 inhibitory effect during days 5-13. These results demonstrate that IL-18 inhibits HIV-1 production in infected PBMC, and suggest that inhibition occurs through intermediate IFN-γ production. Furthermore, a predominant inhibitory effect of IL-18 appears to be present during the early rather than the late stages of PBMC infection. This effect on early infection is further supported by reduced formation of HIV-1 2 long terminal repeat (LTR) in IL-18-exposed PBMC.; 인터루킨(Interleukin, IL)-18은 IFN-γ유도인자이며 T helper type 1 면역반응을 일으키는데 기여하는 전구 염증형 cytokine이다. IL-18은 또한 만성적으로 인간면역 부전 바이러스 제 1형 (HIV-1)에 감염되어 있는 단핵구 세포와 림프구세포에서는 그 바이러스 생성을 증가시킨다. 본 연구는 말초 혈액 단핵구 세포에서 IL-18이 HIV-1 생성에 미치는 효과를 알아보고자 시행되었다. 건강한 공여자로부터 얻은 말초 혈액 단핵구 세포를 분리하여 HIV-1으로 감염시켜 4일간 배양 후, 세포를 분해시킨 뒤 HIV-1 생성은 p24 ELISA로, IFN-γ에 대해서는 electrochemiluminescence로 그 배양액을 검사하였다. 말초 혈액 단핵구 세포를 단핵구향성 바이러스로 감염시킨 뒤 IL-18에 노출된 경우, 바이러스 단백질 항원인 p24 생성이 72%까지 감소하였다 (p < 0.001). T세포향성 바이러스로 감염시킨 말초 혈액 단핵구 세포에서는 IL-18은 p24 생성을 57%까지 감소시켰다 (p < 0.01). 세포배양액에서 측정된 IFN-γ는 IL-18에 노출된 단핵구향성 바이러스와 T세포향성 바이러스로 감염시킨 말초 혈액 단핵구 세포모두에서 증가되었다. 특히, 단핵구향성 바이러스로 감염시킨 세포에서는 바이러스 생성과 IFN-γ정도 사이에 역상관관계가 있어 (r = -0.96, p = 0.008), 이는 IL-18의 효과가 IFN-γ를 매개로 한다는 것을 시사한다. T세포향성 바이러스로 감염시킨 말초 혈액 단핵구 세포에서도 p24와 IFN-γ의 생성사이에 역상관관계를 보였으나, 통계적으로 유의하지 않았다 (r = -0.69, p = 0.19). PBMC를 IL-18으로 2일간 처치한 후 세척하고, 단핵구향성 바이러스로 세포를 감염시킨뒤, 더 이상 IL-18을 처치하지 않고 4일간 배양한 뒤에 측정한 p24도 생성이 64%까지 감소하였다. 하지만, 동일한 바이러스로 세포를 감염시킨 뒤 IL-18을 처치하여 13일간 계속 배양한 말초 혈액 단핵구 세포 배양에서는, 4일간 IL-18을 처치한 말초 혈액 단핵구 세포에서 얻은 p24 억제정도와 비교하여 더 이상의 IL-18 억제효과는 관찰되지 않았다. IL-18 5 nM을 처치하였을 때, p24 억제정도는 배양 4, 7, 10, 그리고 13일째 각각 58, 46, 52, 52%이었다. 또한, 중합 효소 연쇄 반응을 사용하여, IL-18을 처리한 HIV-1에 감염된 말초 혈액 단핵구 세포에서 HIV-1 2LTR 형성이 감소되었다. 본 연구에서 IL-18은 HIV-1을 감염시킨 PBMC에서 바이러스를 억제하였으며 이러한 억제효과는 IFN-γ를 매개로 일어난다는 것을 시사한다. 더욱이, IL-18의 억제효과는 PBMC의 바이러스 감염시기 초기에 우선적으로 일어나는 것을 보여준다.-
dc.description.tableofcontentsLIST OF FIGURES ----------------------------------------------------- ⅶ LIST OF TABLES ------------------------------------------------------ ⅷ ACKNOWLEDGMENTS ----------------------------------------------------- ⅸ ABSTRACT OF DISSERTATION -------------------------------------------- ⅹ I. INTRODUCTION ----------------------------------------------------- 1 1.1 Literature Review ---------------------------------------------- 1 1.1.1 Effects of Cytokine on Pathogenesis of HIV-1 ---------------- 1 1.1.2 Review of IL-18 --------------------------------------------- 4 1.1.3 Implications of IL-18 for HIV-1 ----------------------------- 6 1.1.4 HIV-1 Life Cycle -------------------------------------------- 9 1.2 Research Objectives -------------------------------------------- 13 II. MATERIALS AND METHODS ------------------------------------------- 15 2.1 Reagents ------------------------------------------------------- 15 2.2 Methods -------------------------------------------------------- 16 2.2.1 Isolation of PBMC ------------------------------------------- 16 2.2.2 Infection of PBMC with HIV-1 -------------------------------- 16 2.2.3 Enzyme-Linked Immunosorbent Assay (ELISA) for HIV-1 p24 Antigen ------------------------------------------------------------- 19 2.2.4 Measurement of IFN-g ---------------------------------------- 20 2.2.5 Polymerase Chain Reaction (PCR) for Detection of HIV-1-Specific DNA ------------------------------------------ 21 2.2.6 Cell Viability Assay ---------------------------------------- 22 2.2.7 Statistical Analysis ---------------------------------------- 23 III. RESULTS -------------------------------------------------------- 24 3.1 Exogenous IL-18 Inhibits p24 Ag Levels but Induces IFN-g Production in PBMC Infected with M-tropic HIV-1 --------------------------- 24 3.2 Exogenous IL-18 Inhibits p24 Ag Levels but Induces IFN-g Production in PBMC Infected with T-tropic HIV-1 --------------------------- 29 3.3 Comparison of HIV-1 Production and IFN-g Synthesis in PBMC Infected with M- or T-tropic HIV-1 --------------------- 32 3.4 Pre-treatment with IL-18 2 days prior to HIV-1 Infection Inhibits p24 Production in PBMC ----------------------------------------- 35 3.5 Effect of Prolonged Exposure to IL-18 on p24 Ag and IFN-g Production in Infected PBMC Cultures -------------------------------------- 38 3.6 Inefficient Nuclear Translocation Induced by IL-18 contributes to Restricted Replication of HIV-1 -------------------------------- 41 IV. DISCUSSION ------------------------------------------------------ 43 V. CONCLUSIONS ------------------------------------------------------ 51 APPENDIX ------------------------------------------------------------ 54 A.1 Expansion and Quantification of HIV-1 -------------------------- 54 A.1.1 Amplification of Virus -------------------------------------- 54 A.1.2 Titration of HIV-1 Stock ------------------------------------ 55 REFERENCES ---------------------------------------------------------- 58 ABSTRACT IN KOREAN -------------------------------------------------- 73-
dc.formatapplication/pdf-
dc.format.extent811134 bytes-
dc.languageeng-
dc.publisher이화여자대학교 대학원-
dc.titleThe effect of Interleukin-18 on human immunodeficiency virus type 1 production in peripheral blood mononuclear cells-
dc.typeDoctoral Thesis-
dc.identifier.thesisdegreeDoctor-
dc.identifier.major대학원 의학과-
dc.date.awarded2001. 2-
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