詹東榮Jan, Tong-Rong臺灣大學:獸醫學研究所李繼雅Lee, Chi-YaChi-YaLee2010-05-042018-07-092010-05-042018-07-092008U0001-2007200814094800http://ntur.lib.ntu.edu.tw//handle/246246/178896藥物濫用為全球性的問題，其中大麻類名列前矛。大麻二酚 (cannabidiol；CBD) 為一天然大麻生物鹼，對於大麻受體cannabinoid receptor 1 (CB1) 及CB2親合力均低，因此不具中樞活性，關於CBD的研究近年來著重於引起細胞凋亡的抗腫瘤活性，包括人類神經癌、血癌及乳房癌等，但對神經膠細胞 (glial cell) 及單核球 (monocyte) 等初代細胞，現有的證據指出CBD並不會誘發其顯著的凋亡，而且此類報告對於CBD與免疫反應之關聯性探討甚少。本實驗室先前研究結果顯示，給予小鼠腹腔注射CBD後，再以雞卵白蛋白免疫，其抗原專一性的抗體生成，包括IgM、IgG1和IgG2a，以及T細胞製造細胞激素包含IL-2、IL-4和IFN-γ，皆呈現出顯著的下降趨勢，由此結果可以得知以CBD處理小鼠會抑制其體液性免疫反應，推論其可能的作用機轉，CBD在小鼠所引起的免疫功能下降可能是由於其造成細胞凋亡所致。胞凋亡 (apoptosis) 為細胞程序性死亡，主要包含內源性 (intrinsic pathway) 及外源性 (extrinsic pathway) 兩路徑。在CBD引起細胞凋亡的機制方面，則是一直沒有確切定論。近期有些研究指出CBD所引起的細胞凋亡可能與ROS (reactive oxygen species) 造成的氧化性傷害有關。本論文探討CBD於初代及癌化免疫細胞所引起之細胞凋亡及其機制，實驗結果顯示CBD於初代胸腺細胞 (thymocyte)、單核球及癌化之胸腺瘤細胞 (EL-4 thymoma) 均會引起細胞凋亡，且在胸腺瘤及胸腺細胞給予CBD處理後，皆可見到ROS的產生，伴隨細胞內抗氧化劑glutathione (GSH) 下降。進一步探討其機制則發現以CBD處理三種細胞皆可活化凋亡蛋白酶caspase-8和-9，且於胸腺瘤及胸腺細胞引起粒腺體膜電位的下降。此外，給予抗氧化劑 (N-acetyl-L-cysteine；NAC) 則能有效地以不同比例回復不同濃度CBD在胸腺瘤及胸腺細胞引起之凋亡及GSH下降現象。綜合上述，本研究結果指出CBD於初代及癌化免疫細胞皆會引起凋亡，其作用機制可能與CBD處理後所產生ROS造成之氧化性傷害及粒腺體膜電位下降有關。Drug abuse is a global problem, and marijuana is one of the most commonly abused drugs. CBD is the major non-psychoactive phytocannabinoids contained in marijuana, which lacks significant binding affinity to CB1 and CB2 receptors. Many reports showed that CBD exerted a growth inhibitory effect on various cancer cells, such as glioma, leukemia and breast carcinoma, whereas CBD did not affect their primary counterparts, such as glia cells and monocytes. These results suggest that CBD may be used as a potential anti-cancer agent. However, little information pertaining to the immunomodulatory effects of CBD is available. Previous studies from our laboratory have demonstrated that CBD exhibited a broad spectrum of immunosupressive effects on humoral responses in ovalbumin-sensitized BALB/c mice, including the production of antigen-specific IgM, IgG1 and IgG2a. In addition, the production of cytokines, including IL-2, IL-4 and IFN-致謝 i文摘要 iibstract ivontents viigures ixhapter 1. Introduction 1 1.1 Marijuana and cannabinoid background 1 1.2 Cannabinoid receptors 2 1.3 Cannabinoid agonists 3 1.4 Cannabinoid actions 4 1.5 Immunomodulation by cannabinoids 7 1.6 Apoptotic effects of cannabinoids 9 1.7 Objective of the study 11hapter 2. Materials and methods 14 2.1 Chemicals and reagents 14 2.2 Cell lines 15 2.3 Animals 15 2.4 Culture of thymocytes 16 2.5 Isolation of peripheral blood mononuclear cells (PBMC) 16 2.6 Cell cycle analysis 17 2.7 Terminal dUTP nick-end labeling (TUNEL) assay 18 2.8 Hoechst staining of apoptotic nuclei 18 2.9 Measurement of intracellular reactive oxygen species (ROS) 19 2.10 Detection of intracellular thiols using flow cytometry 20 2.11 Analysis of mitochondrial membrane potential 21 2.12 Detection of caspases activation 21 2.13 Statistical analysis 22hapter 3 Results 23 3.1 CBD induced apoptosis in primary and transformed immune cells. 23 3.2 CBD caused oxidative stress in thymocytes and EL-4 cells. 25 3.3 CBD induced glutathione (GSH) diminishment in thymocytes and EL-4 cells 25 3.4 N-acetyl-L-cysteine (NAC) attenuated CBD-triggered oxidative stress and apoptosis in thymocytes and EL-4 cells. 26 3.5 CBD caused caspase-8 and -9 activation in thymocytes and EL-4 cells. 27 3.6 CBD induced dissipation of mitochondrial membrane potential in thymocytes and EL-4 cells. 27 3.7 CBD induced apoptosis in primary monocytes. 28hapter 4 Discussion 44eferences 50application/pdf658698 bytesapplication/pdfen-US大麻二酚凋亡胸腺細胞胸腺瘤細胞單核球氧化性傷害cannabidiolapoptosisthymocytesEL-4monocytesoxidative stress[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/178896/1/ntu-97-R95629006-1.pdf