登革病毒是RNA病毒的一種，它被歸類到黃病毒科，黃病毒屬中，共有四種血清型。人感染登革病毒可能會發展成如登革出血熱或登革休克症等的致命性併發症。本論文利用兩種免疫細胞：T細胞與單核球細胞研究登革二型病毒感染以後不同免疫細胞所表現出的各種差異。分析的重點為細胞的成長、死亡。我們使用一組登革二型病毒特異性的核酸序列為引子於聚合酵素連鎖反應中證實病毒可以在所有研究的細胞中複製，並且用隙縫點墨法來定量病毒RNA的在細胞內的量，以及免疫螢光染色觀察病毒蛋白的表現。我們發現，登革病毒感染吸附單核球及T細胞株的能力類似，但是在單核球內的複製能力較T細胞差。受登革二型病毒感染後，Molt-4和Jurkat兩株T細胞株的生長受壓制；反之在單核球細胞株中，U-937細胞株的生長速率加快，而HL-60細胞株的生長則不受病毒感染所影響。另一方面，以death-ELISA and Merocyanine 540 binding assay來分析細胞的計畫性死亡，結果顯示Molt-4和Jurkat兩株T細胞株在受登革二型病毒感染後48小時，細胞計畫性死亡速度增加，然而在U-937 和HL-60兩株單細胞株卻不受影響。在死亡相關基因表現的研究上，發現Bcl-2的 mRNA量並不因病毒感染而有太大改變。但是T細胞內Fas-L及Fas的mRNA則受病毒感染而有增加。綜合以上結果，雖然這些細胞皆可受登革二型病毒感染，但是不同類的細胞反應則各有不同。

Infection with dengue virus, an RNA virus classified to flaviviridae, may develop life-threatening complications such as dengue hemorrhagic fever and dengue shock syndrome. A variety of human mononuclear cell lines can be infected with dengue virus in vitro. In this study we compared the responses of T cells and monocytes after dengue-2 virus infection to determine their possible roles in the pathogenesis. Cell growth and death were analyzed. Reverse transcription-polymerase chain reaction analysis using primers specific to dengue viral RNA confirmed the replication of dengue-2 virus in cells. Viral RNA and viral E protein was analyzed by Slot blotting and immunostaining, respectively. We found that lesser viral RNA and viral protein were detected in monocytes than that in T cells. Futhermore, dengue-2 virus infection suppressed the growth of Molt-4 and Jurkat cells, while enhanced it of U-937 cells. The growth of HL-60 cells was not affected by dengue-2 virus infection. An increase in apoptosis, determined by death-ELISA and Merocyanine 540 binding assay, in Molt-4 and Jurkat cells was observed after 48-hr virus infection. Dengue virus did not enhance apoptosis in HL-60 and U-937 cells. Moreover, we found that there was more fas-L and fas mRNA, but not bcl-2, in virus infected cells. In conclusion, different cells might have distinct responses to dengue-2 virus infection.

中文摘要 I
英文摘要 III
誌謝 IV
目錄 V
圖目錄 VII
符號及縮寫 VIII
主文
I、前言 1
II、材料 6
A、 細胞株 6
B、登革二型病毒 6
C、培養基及相關藥品 6
D、試劑 7
E、器材 8
III、方法 11
A、T細胞株 (Molt-4＆Jurkat cells)與單細胞株
(HL-60＆U-937 cells)培養的狀況 11
B、細胞感染登革熱二型病毒後的成長情況 13
C、反轉錄酵素-聚合酵素連鎖反應 15
D、免疫螢光染色法 19
E、隙縫點墨法 21
F、細胞計畫性死亡的測定 24
IV、結果 30
一、 登革二型病毒在細胞中的感染與複製情形 30
二、 登革二型病毒蛋白在細胞中的表現 32
三、 登革二型病毒對細胞生長所造成的影響 33
四、 登革二型病毒的感染可造成T細胞株進行細胞凋亡 34
五、 登革二型病毒造成T細胞株進行細胞凋亡中的相關性
基因表現的偵測 35
V、討論 36
VI、參考文獻 42
圖 49
實驗流程 69
作者經歷 70
圖目錄
Fig. 1 DEN-2 virus RNA deteted by RT-PCR. 49
Fig. 2 DEN-2 virus RNA deteted by nest PCR. 50
Fig. 3 Slot blotting analysis of DEN-2 viral negative
stranded RNA. 51
Fig. 4 Immunofluorecent staining of DEN-2 viral E protein.
52
Fig. 5 Immunofluorecent staining of DEN-2 viral E protein.
53
Fig. 6 Percentage of DEN-2 viral E protein positive cells in
immunofluorecent staining. 54
Fig. 7a Representative histogram of Jurkat cells at 96 hr-
postinfection. 55
Fig. 7b Immunofluorecent staining of DEN-2 viral E protein.
56
Fig. 8a Representative histogram of U-937 cells at 96 hr-
postinfection. 57
Fig. 8b Immunofluorecent staining of DEN-2 viral E protein.
58
Fig. 9 Effect of DEN-2 virus infection on T cell lines growth. 59
Fig. 10 Effect of DEN-2 virus infection on monocytic cell
lines growth. 60
Fig. 11 A representative histogram of MC540 stainingof Jurkat
cell after DEN-2 virus infection. 61
Fig. 12 Preapoptosis detection in Molt-4 cells. 62
Fig. 13 Preapoptosis detection in Jurkat cells. 63
Fig. 14 Preapoptosis detection in HL-60 cells. 64
Fig. 15 Preapoptosis detection in U-937 cells. 65
Fig. 16 Apoptosis detection by death-ELISA assay. 66
Fig. 17 RT-PCR for determining mRNA expression of death-
related genes. 67
Fig. 18 RT-PCR for determining mRNA expression of death-
related genes. 68

1. Scott, B. H., 1988. Pathogenesis of Dengue: Challenges to
Molecular Biology, Science 239:476-481.
2. Halstead, S. B., 1980. Immunological parameters of togavirus
disease syndromes, p. 107-173. In R. W. Schlesinger (ed.),
The togaviruses. Academic Press, Inc., New York.
3. Sabin, A. B., 1959. Dengue, p. 361-373. In T. Rivers and F.
Horsfall (ed.), Viral and rickettsial infection of man. J.
B. Lippincott Co., Philadelphia.
4. World Health Organization. 1975. Technical guides for
diagnosis, treatment, surveillance, prevention and control
of dengue hemorrhagic fever. World Health Organization,
Geneva.
5. Monath, T. P., 1985. Flaviviruses, p. 955-1004. In B. W.
Fields, D. M. Knipa, R. M. Chanock, J. L. Malnick, B.
Roizman, and R. E. Shope (ed.), Virology. Raven Press, New
York.
6. Sangkawibha, N., Rojanasuphot, S., Ahandrik, S.,
Viriyapongse, S., Jatanasen, S., Salitul, V.,
Phanthumachinda, B., Halstead, S. B., 1984. Risk factors in
dengue shock syndrome：A prospective epidemiologic study in
Rayong, Thailand. Am. J. Epidemiol. 120:653-669
7. Guzman, M. G., Kouri, G. P., Bravo, J., Soler, M., Vazquez,
S., Morier, L., 1990. Dengue hemorrhagic fever in Cuba,
1981: A retrospective seroepidemiologic study. Am. J. Trop.
Med. Hyg. 42:179-184.
8. Halstead, S. B., 1981. The Alexander D. Langmuir Lewcture.
The pathogenesis of dengue: Molecular epidemiology in
infectious disease. Am. J. Epidemiol. 114:632-648.
9. Kliks, S. C., Nimmanitya, S., Nisalak, A., Burke, D. S.,
1988. Evidence that maternal dengue antibodies are important
in the development of dengue hemorrhagic fever in infants.
Am. J. Trop. Med. Hyg. 38:411-419.
10.Halstead, S. B., 1977. Dengue viruses and mononuclear
phagocytes. I. Infection enhancement by non-neutralizing
antibody. J Exp. Med. 146:201-217.
11.Kliks, S. C., Nisalak, A., Brandt, W. E., Wahl, L., Burke,
D. S., 1989. Antibody dependent enhancement of dengue virus
growth in human monocytes as a risk factor for dengue
hemorrhagic fever. Am. J Trop. Med. Hyg. 40:444-451.
12.Morens, D. M., Marchette, N. J., Chu, M. C., Halstead, S.
B., 1991. Growth of dengue type 2 virus isolates in human
peripheral blood leukocytes correlates with severe and mild
dengue disease. Am. J. Trop. Med. Hyg. 45: 644-651.
13.Rosen, L., 1977. The emperor's new clothes revisited, or
reflections on the pathogenesis of dengue hemorrhagic fever.
Am. J. Trop. Med. Hyg. 26:337-343.
14.Shaio, M. F., Chang, F. Y., Hou, S. C., 1992. Complement
pathoway activity in serum from patients with classical
dengue fever. Trans. Roy. Soc. Trop. Med. Hyg. 86:672-675.
15.Shaio, M. F., 1994. Influence of dengue virus infection on
the up take and the respiratory burst activity of human
monocytes. Immunol. Infect. Dis. 4:69-75.
16.Chang, D. M., Shaio, M. F., 1994. Production of interleukin-
1 (IL-1) and IL-1 inhibitor by human monocytes exposed to
dengue virus. J. Infect. Dis. 170:811-817.
17.Shaio, M. F., Cheng. S. N., Yuh, Y. S., Yang. K. D., 1995.
Cytotoxic factors released by dengue virus-infected human
blood monocytes. J. Med. Virol. 46:216-223.
18.Yang, K. D., Lee, C. S., Hwang, K. P., Chu, M. L., Shaio, M.
F., 1995. A model to study cytokine profiles in primary and
hetrologously secondary dengue-2 virus infections. Acta.
Virol. 39:19-21.
19.Yang, K. D., Lee, C. S., Shaio, M. F., 1995. A higher
production of platelet activating factor in ex vivo
heterologously secondary dengue-2 virus infections. Acta.
Micro. Immunol. Hungarica. 42:403-407.
20.Yang, K. D., Wang, C. L., Shaio, M. F., 1995. Production of
cytokines and platelet activating factor in secondary dengue
virus infection. J. Infect. Dis. 172:604-605.
21.Kurane, I., B. L. Innis, A. Nisalak, C. Hoke, S. Nimmanitya,
A. Meager, and F. A. Ennis. 1989. Human responses to dengue
virus antigens. Proliferative responses and interferon gamma
production. J. Clin. Invest. 83:506-513.
22.Bukowski, J. F, I. Kurane, C. J. Lai, M. Bray, B. Falgout,
and F. A. Ennis. 1989. Dengue virus-specific cross-reactive
CD8+ human cytotoxic T lymphocytes. J. Virol. 63:5086-5091.
23.Kurane, I., A. Meager, and F. A. Ennis. 1989. Dengue virus-
specific human T cell clones. Serotype cross-reactive
proliferation, interferon gamma production, and cytotoxic
activity. J. Exp. Med. 170:763-775.
24.Konty, U., I. Kurane, and F. A. Ennis. 1988. Gamma
interferon of human monocytic cells. J. Virol. 62:3928-3933.
25.Bukowski, J. F., Kurane, I., Lai, C. J., Bray, M., Falgout,
B., Ennis, F. A., 1989. Dengue virus-specific cross-reactive
CD8+ human cytotoxic T lymphocytes. J. Virol. 12:5086-91.
26.Schlesinger, R. W. 1977. Dengue viruses. Springer-Verlag,
New York.
27.Despres, P., Flamand, M., Ceccaldi, P. E., Deubel, V., 1996.
Human isolates of dengue type 1 virus induce apoptosis in
mouse neuroblastoma cells. J. Virol. 70:4090-4096.
28.Marianneau, P., Cardona, A., Edelman, L., Deubel, V.,
Despres, P., 1997. Dengue virus replication in human
hepatoma cells activates NF-B which in turn induces
apoptotic cell death. J. Virol. 71:3244-3249.
29.Despres, P., Frenkiel, M. P., Ceccaldi, P. E., Duarte, Dos.,
Santos, C., Deubel, V., 1998. Apoptosis in the mouse
central nervous system in response to infection with mouse-
neurovirulent dengue viruses. J. Virol. 72:823-829.
30.Monath, T. P., 1985. Flaviviruses. In：Fields BN, et al.
(eds) Virology. Raven Press, New York, p 955.
31.Halstead, S. B., O'Rourke, E. J., Allison, A. C., 1977.
Dengue virus and mononuclear phagocytes. II. Identity of
blood and tissue leukocytes supporting in vitro infection.
J. Exp. Med. 146:218-229.
32.Kurane, I., Kontny, U., Janus, J., Ennis, F. A., 1990.
Dengue-2 virus infection of human mononuclear cell lines and
establishment of persistent infections. Arch. Viro. 110:91-
101.
33.Halstead S. B., 1979. In vivo enhancement of dengue virus
infection in rhesus monkeys by passively transferred
antibody. J. Infec. Dis. 140:527-33.
34.Marchette, N. J., Halstead, S. B., Chow, J. S., 1976.
Replication of dengue viruses in cultures of peripheral
blood leukocytes from dengue-immune rhesus monkeys. J.
Infec. Dis. 133:274-282.
35.Marchette, N. J., 1974. Immunopathogenesis of dengue
infection in the rhesus monkey. Transplant. Proce. 6:197-201.
36.La Russa, V. F., Innis, B. L., 1995. Mechanisms of dengue
virus-induced bone marrow suppression. Bailli. Clin. Haema.
8:249-270.
37.Murgue, B., Cassar, O., Guigon, M., Chungue, E., 1997.
Dengue virus inhibits human hematopoietic progenitor growth
in vitro. Journal of Infectious Diseases. 175:1497-1501.
38.Rothwell, S. W., Putnak, R., La Russa, V.F., 1996. Dengue-2
virus infection of human bone marrow: characterization of
dengue-2 antigen-positive stromal cells. Am. J. Trop. Med.
Hyg. 54:503-510.
39.Collins, K. L., Mary, and Abelardo Lopez Rivas, 1993. The
control of apoptosis in mammalian cells. TIBS 18:307-309.
40.Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J.,
Bratton, D. L., Henson, P. M., 1992. Exposure of
phosphatidylserine on the surface of apoptotic lymphocytes
triggers specific recognition and removal by macrophages. J.
Immuno. 148:2207-2216.
41.Mower, D. A. Jr., Peckham, D. W., Illera,V. A., Fishbaugh,
J. K., Stunz, L. L., Ashman, R. F., 1994. Decreased
membrane phospholipid packing and decreased cell size
proceed DNA cleavage in mature mouse B cell apoptosis. J.
Immuno. 152:4832-4842.
42.Duke, R. C., Cohen, J. J., 1986. IL-2 addiction: withdrawal
of growth factor activates a suicide program in dependent T
cells. Lympho. Resear. 5:289-99.
43.Siebenlist, U., Franzoso, G., Brown, K., 1994. Structure,
regulation and function of NF-B. Annu. Rev. Cell Biol.
10:405-455.
44.Thanos, D., Maniatis, T., 1995. NF-B：a lesson in family
values. Cell 80:529-532.
45.Lin, K. I., Lee, S. H., Narayanan, R., Baraban, J. M.,
Hardwick, J. M., Ratan, R. R., 1995. Thiol agents and Bcl-2
identify an alphavirus-induced apoptotic pathway that
requires activation of the transcription factor NF-B. J.
Cell Biol. 131:1149-1161.
46.Cleveland, L., John, and James, N., Ihle, 1995. Contenders
in FasL/TNF death signaling. Cell 81: 479-182.
47.Halstead, S. B., 1990. Global epidemiology of dengue
hemorrhagic fever. Southeast Asia J. Trop. Med. Public.
Health. 21:636.
48.Morens, D. M., Halstead, S. B., Repik, P. M., Putvatana, R.,
Raybourne, N., 1985. Simplified plaque reduction
neutralization assay for dengue viruses by semimicro methods
in BHK-21 cells: comparison of the BHK suspension test with
standard plaque reduction neutralization. J. Clin.
Microbiol. 22:250-254
49.Sambrook, J., 1989. Molecular cloning.
50.Liu, H. S., Tzeng, H. C., Chang, T. Y., Chen, C. C., 1994.
Using a radioactive and non-radioactive exchangeable
labeling system for laboratory diagnosis. Kaohsiung. J. Med.
Sci. 10:165-169.
51.Wengler, G., Wengler, G., Gross, H. J., 1978. Studies on
virus-specific nucleic acids synthesized in vertebrate and
mosquito cells infected with Flavivirus. Virology 89:423-437.
52.Guirakhoo, F., A. R. Hunt, J. G. Lewis, and J. T. Roehring.
1993. Selection and partial characterization of dengue 2
virus mutants that induce fusion at elevated pH. Virology
194:219-223
53.Kawano, H., V. Rostapshov, and C-J. Lai. 1993. Genetic
determinents of dengue type 4 virus neurovirulence to mice.
J. Virol. 67:6567-6575.
54.O'Sullivan, M. A., Killen, H. M., 1994. The differentiation
state of monocytic cells affects their susceptibility
infection and the effects of infection by dengue virus.
75:2387-2392.
55.Tsujimoto, Y., Croce, C. M., 1986. Analysis of the
structure, transcripts, and protein products of bcl-2, the
gene involved in human follicular lymphoma. 83:5214-5218.
56.Ivanov VN. Lee RK. Podack ER. Malek TR. 1997. Regulation
of Fas-dependent activation-induced T cell apoptosis by cAMP
signaling: a potential role for transcription factor NF-
kappa B. Oncogene. 14:2455-2464.