詹東榮臺灣大學：獸醫學研究所吳欣穎Wu, Hsin-YingHsin-YingWu2007-11-282018-07-092007-11-282018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/60000安非他命和苯二氮平類藥物在台灣是兩種常被濫用而導致成癮的藥物，本論文探討單獨或合併投予甲基安非他命（MA）和二氮平（DZ）對小鼠抗原專一性免疫反應之免疫毒性，並且進一步探討這二種藥物抑制抗體生成之作用機制。小鼠連續5日接受MA和DZ給藥以模擬藥物成癮，接著以alum吸附之卵白蛋白(ovalbumin; OVA)免疫，實驗結果顯示每日1次投與MA 和DZ（5 mg/kg）連續5日，不論是單獨或是合併投予MA和DZ，皆具有顯著抑制OVA免疫小鼠血清中抗體生成的作用，受到藥物抑制的OVA專一性抗體包括 IgM、IgG1和IgG2a。分離接受MA和DZ給藥的OVA兩次重複免疫小鼠之脾臟細胞，發現其IL-4和IFN-γ表現量在給藥各組明顯較對照組為低。此外，MA 和DZ會抑制抗原專一性脾臟細胞的增生反應且增加其凋亡，MA 和DZ也會降低T-bet的表現，並且增加SOCS3的表現。綜合上述，本研究結果指出MA和DZ對小鼠的抗原專一性免疫反應具有相當顯著的免疫毒性，包括抑制血清中抗體生成和抑制脾臟細胞表現細胞激素，其作用機制可能係經由抑制抗原專一性脾臟細胞的增生反應並且增加其凋亡所造成。Amphetamines and benzodiazepines are commonly abused drugs in Taiwan. The objective of this thesis was to investigate the effects of methamphetamine (MA) and diazepam (DZ), either alone or in combination, on the antigen-specific antibody production in ovalbumin (OVA)-sensitized mice and to study the underlying mechanisms responsible for MA-and DZ-mediated effects on antibody production. BALB/c mice were daily administered with MA and DZ for 5 consecutive days to mimic the status of drug addiction, followed by sensitization with aluminum potassium sulfate-absorbed ovalbumin (OVA/alum). The results demonstrated that repeated dosage of MA and DZ (5 mg/kg), either alone or in combination, significantly attenuated the serum levels of OVA-specific IgM, IgG1 and IgG2a. In accordant with the effect on antibody production, the steady state mRNA expression and protein secretion of IL-4 and IFN-γ by splenocytes isolated from drug-treated and OVA/alum-sensitized mice were attenuated as compared to the vehicle-treated control. In addition, MA and DZ attenuated the proliferation of splenocytes stimulated with OVA or the T-cell mitogen concanavalin A, and increased the percentage of apoptotic splenocytes. MA and DZ moderately decreased expression of T-bet and increased expression of SOCS3. In conclusion, the present studies demonstrated that both MA and DZ administration exhibit marked immunosuppressive effects in OVA/alum-sensitized mice. MA- and DZ-mediated suppression of antigen-specific antibody production is correlated with down regulation of cytokine expression. One of the potential mechanisms of MA- and DZ-mediated immunosuppression is via the inhibition of antigen-specific splenocyte proliferation and the enhancement of antigen-specific splenocyte apoptosis.致 謝 i 中文摘要 ii Abstract iii Contents v Figures vii Tables viii Abbreviations ix 1. Introduction 1 1.1. Drug abuse, microbial infections and immunomodulation 1 1.2. Methamphetamine 1 1.3. Immunomodulation by MA 4 1.4. Benzodiazepines 5 1.5. Immunomodulation by BZDs 7 1.6. Antigen specific immune responses 9 1.7. Objective of the thesis 10 2. Materials and methods 12 2.1. Chemicals and reagents 12 2.2. Animals 13 2.3. The formula of the vehicle for DZ 13 2.4. Administration and sensitization protocol 16 2.4. Isolation of the serum 16 2.5. Measurement of OVA-specific IgM, IgG1 and IgG2a by ELISA 16 2.6. Preparation of splenocytes 17 2.7. Cytokines production by OVA-stimulated splenocytes 18 2.8. IFN-γ and IL-4 measurement 19 2.9. Reverse transcription- polymerase chain reactions (RT-PCR) 19 2.10. Flow cytometric analysis of splenocyte cellularity 22 2.11. MTT assay 23 2.12. Bromodeoxyuridine (BrdU) incorporation and flow cytometric analysis 23 2.13. Flow cytometric analysis of the cell cycle 24 2.14. Statistical analysis 25 3. Results 26 3.1. Suppression of antibody production by MA and DZ in OVA/alum-sensitized mice 26 3.2. Inhibition of MA and DZ on IL-4 and IFN-γ protein secretion by splenocytes isolated from OVA/alum-sensitized mice 27 3.3. Effect of MA and DZ on the cellularity of splenocytes isolated from OVA/alum-sensitized mice 29 3.4. Inhibition of Con A- or OVA-induced splenocyte proliferation by MA and DZ in OVA/alum-sensitized mice 29 3.5. MA and DZ increased the percentage of apoptotic splenocytes stimulated with OVA 30 3.6. Inhibition of IL-4, IFN-γ, T-bet and increase of SOCS3 mRNA expression by MA and DZ 31 4. Discussion 44 5. References 52610901 bytesapplication/pdfen-US甲基安非他命二氮平抗原專一性免疫反應免疫球蛋白細胞激素卵白蛋白methamphetaminediazepamantigen-specific immune responsesimmunoglobulincytokineovalbumin[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/60000/1/ntu-95-R92629005-1.pdf