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吸煙是引起骨質疏鬆症的因素之一。臨床研究清楚表明吸煙者的骨密度降低，但其他干擾因素可能掩蓋了吸煙對骨頭的不良效果。使用動物模型用以研究吸煙和骨質疏鬆症之間是否有直接的因果關係與它潛在的機制是有必要的。為此，我們使用年輕和雌激素耗盡的小鼠作被動吸煙模型以及小鼠成骨細胞和破骨細胞株來作研究。, 年輕的Balb/c小鼠暴露於2%或4% (v/v)的香煙煙霧中，代表中度和重度吸煙的人。骨代謝生物標誌物明顯增加，4%吸煙組在14週後股骨的微觀結構4%顯著下降，這相等於人類吸煙12年。此外，雌性Balb/c小鼠進行4%吸煙和/或卵巢切除術(OVX)。吸煙+OVX組增加血清中骨轉換指標水平。4%吸煙組的股骨生長板較薄。μ-CT數據進一步表明，相對骨體積(BV / TV)和結構模型指數(SMI)在吸煙組有顯著影響，而且在吸煙+ OVX組上有更大程度的影響。, 在細胞研究中使用氯仿(CSE)和乙醇的香煙提取物(ESE)。CSE抑制小鼠細胞株RAW 264.7形成破骨細胞，並刺激小鼠成骨細胞株的分化和功能。這個與體內研究矛盾的結果暗示直接從煙霧中提取的化學成分並不是引起骨質疏鬆的元兇。影響骨代謝的很可能是其他從煙霧中生成的活性代謝物和一些吸煙引起的內源性激素物質。在吸煙引起的骨質流失中，這些代謝物或內源性物質可能是非常重要的。, 有見及此，4%吸煙小鼠的血清用以研究其對成骨細胞和破骨細胞活動的影響。吸煙小鼠血清顯著降低在成骨細胞中鹼性磷酸酶(ALP)活性和鈣沉積，一些成骨細胞標記基因和蛋白表達均下降，而且 Wnt/β-catenin信號通路下調。此外，吸煙小鼠血清顯著增加形成破骨細胞的數量，組織蛋白酶K的基因和蛋白表達增加，在NF-κB和p-38 MAPK信號傳導途徑的信號分子表達增加。, 總而言之，大量吸煙可能影響年輕小鼠和雌激素耗竭小鼠的骨代謝和微結構，通過類似的行動機制，人類也可能有同樣的骨疾病風險。這項研究揭示了吸煙導致的骨質疏鬆症在青少年和絶經後婦女的發病機制。這也給我們線索如何預防和治療與吸煙有關的骨骼疾病。這項研究還傳達了一個明確的信息：在年輕時應開始應控制吸煙。, Cigarette smoking is one of the risk factors for osteoporosis. Clinical studies clearly showed that smokers have lower bone mineral density, but other confounding factors could mask the adverse actions of smoking on bones. Animal models are warranted to study the direct causal relationship between cigarette smoking and osteoporosis, and also the underlying mechanisms. In this regard, we used a mouse passive smoking model in both young and estrogen depleted mice, and the mouse osteoblast and osteoclast cell lines., Young Balb/c mice were exposed to 2 or 4% (v/v) of cigarette smoke, similar to moderate or heavy smoking respectively in humans. Biomarkers for bone turnover were increased and bone microstructure of femur was significantly deteriorated after 4% smoking for 14 weeks which is similar to cigarette smoking for 12 years in humans. Furthermore, the effects of heavy smoking on ovariectomized mice were also investigated. Female Balb/c mice were subjected to 4% cigarette smoking and/or ovariectomy (OVX). Cigarette smoking together with OVX further increased the levels of bone turnover markers in serum. Femur growth plate was thinner in the 4% smoking group when compared to those in the SHAM- and OVX-operated groups. Micro-CT data further indicated that the relative bone volume (BV/TV) and structural model index (SMI) were significantly affected in the smoking groups, and to a greater extent in the 4% smoking + OVX group., Chloroform (CSE) and ethanol smoke extracts (ESE) were used in cell studies. CSE suppressed the formation of osteoclasts, and stimulated the differentiation and function of mouse osteoblasts. These findings are contradictory to those found in in vivo study implying that chemical components directly extracted from cigarette smoke are not the culprits in causing bone disorder in animals. It is likely that other active metabolites from cigarette smoke and some endogenous hormonal substances released by cigarette smoking could affect bone metabolism. These active metabolites together with the endogenous bone hormones are perhaps crucial in smoking-induced bone loss in the body., In view of this hypothesis, sera from 4% smoking mice were used to investigate their effects on osteoblast and osteoclast activities. It was found that the alkaline phosphatase (ALP) activity and calcium deposition in osteoblast were reduced significantly by the sera from smoking mice. Gene and protein expressions of some osteoblast markers were also decreased. The downregulation of Wnt/β-catenin signaling pathway was observed after the treatment with the serum obtained from the 4% smoking group. Moreover, the number of osteoclasts being formed was increased significantly by the smoking mouse serum. Cathepsin K gene and protein expressions were also induced. The increased expressions of the signaling molecules including NF-κB and p-38 MAPK were also observed., In conclusion, heavy cigarette smoking could deteriorate bone metabolism and microstructures in young female and also estrogen depleted mice. The same kind of risk in bone disease may also apply to humans through similar mechanisms of action. This study sheds light in understanding the pathogenesis of smoking-induced bone disorders in teenagers and also postmenopausal women. It also gives us clues how to prevent and treat smoking related bone diseases. This study also conveys a clear message that cigarette smoking control should be started in young ages., Detailed summary in vernacular field only., Chan, Lok Yi Ruby., Thesis (Ph.D.) Chinese University of Hong Kong, 2014., Includes bibliographical references (leaves 182-207)., s also in Chinese., http://library.cuhk.edu.hk/record=b6115668, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)

肺癌是一個世界性的健康難題。大量研究證據顯示，煙草及其致癌物NNK對環氧酶（COX）-2及其下游產物具有促進效應。血栓素（TxA）2是COX-2的關鍵性下游產物之一，該論文闡述了TxA2在NNK導致的肺癌增長中的可能作用。, 我們發現相对于非吸烟者，吸煙者肺癌組織表达更高水平的TxA2合酶（TxAS）。NNK可以刺激培養的肺癌細胞TxA2合成。用TxAS抑制劑和TxA2受體（TP）拮抗劑分別阻抑TxA2的合成與功能可以引起細胞凋亡，從而有效抑制NNK導致的細胞增殖效應。在TxA2合成受抑制的情況下，TP激動劑U46619幾乎可以重建NNK效應，說明TP在NNK效應中的重要作用。研究還顯示，激活的TP可以通過PI3K/Akt和ERK通路進一步激活CREB，從而參與NNK對肺癌細胞的促生長效應。, 緊接著，我們的研究顯示TP 可以調節NNK對COX-2 和TxA2的誘導，而且發現NNK刺激的TxA2合成主要依賴於COX-2活性。COX-2和TxA2功能抑製劑對NNK的促細胞生長作用具有相似的抑制效用。考慮到TP是TxA2的功能受體，該資料說明TP在NNK處理的肺癌細胞中傳遞了上游因子COX-2的促腫瘤作用。在使用COX-2小干擾RNA（siRNA）抑制NNK作用的情況下，TP激動劑U46619幾乎可以恢復NNK的效應證實了TP的傳遞者角色。研究還發現 TPα而不是TPβ在培養的肺癌細胞系中廣泛表達，並且過表達TPα具有促進腫瘤生長的作用。在用NNK處理細胞的條件下，TPα還具有促COX-2表達和TxA2生成的作用。, 我們的研究進一步發現，在吸煙者肺癌組織中TPα表達增高，這與TxAS的表達相似。与此结果相一致，在經NNK處理的A/J小鼠肺癌組織中，TxAS和TP表達水準也是明顯上升的。在細胞培養實驗中，NNK能夠提高TxAS蛋白和信使RNA（mRNA）的表達水準。但是，在TP的兩個亞型TPα和TPβ中， NNK僅能促進TPα的蛋白表達，對它們的mRNA均無影響。NNK對TxAS的促表達作用是核轉錄因數(NF)-κB依賴性的。其他的幾個關鍵轉錄因數，諸如特異性蛋白(SP)-1，CREB和活化受體 （PPAR）γ均未參與NNK對TxAS和TPα的表達促進作用。進一步的，轉錄後機理被證實參與了NNK對TPα的作用。TPα而不是TPβ經鑒別在NNK的促NF-κB 激活 和 促TxAS 表達效應中起正向調節作用。, 總之， 我們的研究說明TxA2相關通路在NNK的促肺癌細胞生長效應中起正向調節作用。我們的研究揭示了TPα的自我激活環路。通過該環路，TxA2，或者說TxAS和TPα參與了NNK的肺癌促生長效應。因此，我們的研究為肺癌的防治了提供了一個新的方向，即靶向TxAS和TPα是一種可能有效的策略。, Lung cancer concerns a world-wide health problem. There is considerable evidence of that tobacco smoke and its carcinogen 4-methylnitrosamino-1-3-pyridyl-1-butanone (NNK) have the potential effects on the production of cyclooxygenase (COX)-2 and its downstream products in tumor cells. This thesis is constructed to describe the study focused on the role of thromboxane A2 (TxA2), one of the key downstream products of COX-2, in NNK-induced lung tumor growth., We found that as compared to non-smokers, lung cancer tissues obtained from smokers tended to express more TxA2 synthase (TxAS). Moreover, NNK could stimulate TxA2 synthesis in lung cancer cells. Blockade of TxA2 synthesis and action by TxAS inhibitor and TxA2 receptor (TP) antagonist completely blocked NNK-promoted cell proliferation via inducing apoptosis. Moreover, TP agonist U46619 reconstituted a near full proliferative response to NNK when TxAS was inhibited, affirming the role of TP in NNK-induced cell growth. Furthermore, we revealed that the activated TP may then activate CREB through PI3K/Akt and ERK pathways, thereby contributing to the NNK-induced lung cancer cell growth., We subsequently showed that TP could modulate the induction of COX-2 and TxA2 by NNK. The synthesis of TxA2 stimulated by NNK was found to be mainly dependent on COX-2 activity. Intriguingly, there are similar inhibitory effects on NNK-induced cell growth between pharmacological inhibition of COX-2 and the blockade of TxA2 synthesis and action. Because TP is the natural receptor of TxA2, these results suggest that TP may function as a mediator for the tumor-promoting effects of COX-2 upon NNK treatment, which was confirmed by the data showing that U46619 almost restored NNK effects in the presence of COX-2-siRNA. Importantly, TPα, but not TPβ was found to be widely expressed in lung cancer cells and be able to promote tumor growth, COX-2 expression and TxA2 synthesis upon NNK treatment., We further demonstrated that in lung tumor tissues obtained from smoker, TPα protein was increased, which was similar to the change in TxAS protein. The increased levels of TxAS and TP proteins were also found in lung cancer tissues of A/J mice treated with NNK. In cell culture experiments, NNK could increase TxAS at both protein and mRNA levels. However, TPα rather than TPβ was increased by NNK at protein but not mRNA level. NNK-stimulated TxAS expression was dependent on nuclear factor (NF)-κB signaling. Other key transcriptional factors, such as specificity protein(SP)-1, CREB and peroxisome proliferator-activated receptor-gamma (PPARγ), were not involved in NNK-induced TxAS and TPα expression. Further experiments revealed that post-transcriptional mechanisms were responsible for NNK-induced TPα expression. TPα rather than TPβ was finally identified to have a positive role in NNK-induced NF-κB activation and TxAS expression., Taken together, our study suggests that TxA2 pathway has a positive role in NNK-induced lung cancer cell growth. An auto-positive feedback loop of TPα activation to facilitate lung tumor growth in the presence of NNK is delineated by these results. Therefore, targeting TxAS or/and TPα may represent a promising strategy for prevention and treatment of lung cancer., Detailed summary in vernacular field only., Huang, Runyue., Thesis (Ph.D.)--Chinese University of Hong Kong, 2012., Includes bibliographical references (leaves 119-146)., Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web., also in Chinese., p.I, 摘要, Publications, Acknowledgement, Abbreviations, Table of contents, Chapter Chapter 1 --- General introduction--Tobacco smoking, COX-2 pathway and cancer, Chapter 1.1 --- Abstract --- p.1, Chapter 1.2 --- Introduction --- p.2, Chapter 1.3 --- Cyclooxygenase and prostanoids --- p.5, Chapter 1.4 --- The effects of tobacco smoking on COX-2 pathway, and the related pathologies --- p.8, Chapter 1.4.1 --- Smoking, PGE2, inflammation and immunosupression --- p.8, Chapter 1.4.2 --- Smoking, TxA2, platelet activation, cell contraction and angiogenesis --- p.11, Chapter 1.4.3 --- Smoking and PGI2 --- p.16, Chapter 1.5 --- The role of cyclooxygenase-2 pathway in the progression of tobacco smoke-related cancers --- p.19, Chapter 1.5.1 --- Lung cancer --- p.19, Chapter 1.5.2 --- Gastrointestinal cancer --- p.23, Chapter 1.5.3 --- Bladder cancer --- p.24, Chapter 1.5.4 --- Head and neck squamous cell carcinoma --- p.25, Chapter 1.5.5 --- The signaling mechanisms underlying the induction of COX-2 by smoking in tumors --- p.26, Chapter 1.6 --- Summary, future directions and key questions --- p.28, Chapter Chapter 2 --- NNK induces lung cancer cell growth by stimulating TxA2 and its receptor, Chapter 2.1 --- Abstract --- p.32, Chapter 2.2 --- Introduction --- p.33, Chapter 2.3 --- Materials and Methods --- p.35, Chapter 2.3.1 --- Cell lines and cell culture --- p.35, Chapter 2.3.2 --- Chemicals and drug treatment --- p.35, Chapter 2.3.3 --- Thromboxane B2 EIA assay --- p.36, Chapter 2.3.4 --- MTT assay --- p.36, Chapter 2.3.5 --- BrdU cell proliferation assay --- p.37, Chapter 2.3.6 --- Flow cytometry for analysis of apoptosis --- p.37, Chapter 2.3.7 --- Transfection of cells with CREB siRNA --- p.38, Chapter 2.3.8 --- Western blot analysis and antibodies --- p.38, Chapter 2.3.9 --- Statistical analysis --- p.39, Chapter 2.4 --- Results --- p.41, Chapter 2.4.1 --- High expression of TxAS in lung cancer tissues of smoker --- p.41, Chapter 2.4.2 --- NNK stimulated TxA2 synthesis in lung cancer cells --- p.43, Chapter 2.4.3 --- Blockade of TxA2 synthesis and action prevented NNK-induced cell growth --- p.44, Chapter 2.4.4 --- TxA2 mimetic U46619 reconstituted NNK-enhanced cell proliferation under TxA2-inhibited condition --- p.47, Chapter 2.4.5 --- Blockade of TxA2 synthesis or action induced the apoptosis of the NNK-exposed cells --- p.47, Chapter 2.4.6 --- CREB is accountable for the key role of TxA2 in NNK-enhanced cell proliferation --- p.49, Chapter 2.4.7 --- PI3K/Akt and ERK rather than JNK and p38 pathways were mediated by TxA2 in the NNK-exposed cells --- p.52, Chapter 2.4.8 --- CREB is located downstream of the PI3K/Akt and ERK pathways in NNK-treated cells --- p.53, Chapter 2.5 --- Discussion --- p.55, Chapter Chapter 3 --- The positive role of TPα in the induction of COX-2, TxA2 and cell growth by NNK in human lung cancer cells, Chapter 3.1 --- Abstract --- p.62, Chapter 3.2 --- Introduction --- p.63, Chapter 3.3 --- Materials and methods --- p.65, Chapter 3.3.1 --- Cell culture and chemicals --- p.65, Chapter 3.3.2 --- Transient transfections --- p.66, Chapter 3.3.3 --- TxB2 measurement --- p.66, Chapter 3.3.4 --- Cell growth detection --- p.67, Chapter 3.3.5 --- Analysis of apoptosis --- p.67, Chapter 3.3.6 --- Western blot analysis and antibodies --- p.67, Chapter 3.3.7 --- Statistical analysis --- p.68, Chapter 3.4 --- Results --- p.70, Chapter 3.4.1 --- Examination of TP as the modulator for induction of COX-2 and TxA2 by NNK --- p.70, Chapter 3.4.2 --- The TxA2 generated in cells treated with NNK is mainly dependent on COX-2 activity --- p.72, Chapter 3.4.3 --- Examination of TP as the key mediator for the tumor-promoting effect of COX-2 --- p.72, Chapter 3.4.4 --- The expression and action of α and β isoforms of TP in human lung cancer cells --- p.77, Chapter 3.4.5 --- the identification of positive role of TPα in NNK-induced COX-2, TxA2 and cell growth in lung cancer cells --- p.79, Chapter 3.5 --- Discussion --- p.81, Chapter Chapter 4 --- TP-α facilitates lung tumor growth through an autoregulatory feedback mechanism, Chapter 4.1 --- Abstract --- p.88, Chapter 4.2 --- Introduction --- p.89, Chapter 4.3 --- Materials and methods --- p.91, Chapter 4.3.1 --- Human lung tissue and immunohistochemical analysis --- p.91, Chapter 4.3.2 --- Animal treatment --- p.91, Chapter 4.3.3 --- Cell culture and chemicals --- p.92, Chapter 4.3.4 --- Transient transfection --- p.93, Chapter 4.3.5 --- Real-time PCR --- p.93, Chapter 4.3.6 --- Western blot analysis and antibodies --- p.94, Chapter 4.3.7 --- Statistical analysis --- p.95, Chapter 4.4 --- Results --- p.96, Chapter 4.4.1 --- The effects of smoking on the expression of TP in human lung cancer tissue --- p.96, Chapter 4.4.2 --- The effects of NNK on the expression of TxAS and TP in lung tissues of A/J mice --- p.98, Chapter 4.4.3 --- The effects of NNK on the expression of TxAS and TPα in lung cancer cells --- p.99, Chapter 4.4.4 --- Identification of the roles of NF-κB, CREB and SP1 in NNK-induced TxAS and TPα expression --- p.101, Chapter 4.4.5 --- The negative role of PPARγ in NNK-induced TxAS and TPα expression --- p.104, Chapter 4.4.6 --- NNK-induced TPα expression via post-transcriptional mechanism --- p.105, Chapter 4.4.7 --- Examination of TPα auto-activation mechanism in lung cancer cells stimulated with NNK --- p.106, Chapter 4.5 --- Discussion --- p.109, Chapter Chapter 5 --- Conclusion and future works, Chapter 5.1 --- Conclusion --- p.114, Chapter 5.2 --- Future works --- p.115, Chapter 5.2.1 --- The possible role of miR-34c in the auto-regulatory loop of TxAS expression or TPα activation --- p.116, Chapter 5.2.2 --- The possible role of FOXO3a in the auto-regulatory loop of TxAS expression or TPα activation --- p.116, References --- p.119, http://library.cuhk.edu.hk/record=b5549645, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)