臺灣博碩士論文加值系統

香菸已知是導致慢性阻塞性肺部疾病 (Chronic obstructive pulmonary disease, COPD) 的主要因素之一，其形成過程會透過增加細胞內活性氧族群 (Reactive oxygen species, ROS)、氧化壓力及持續性慢性肺發炎。相關文獻指出香菸與肺部發炎及活化活性氧族群的調控機制有關，然而香菸如何透過活性氧族群活化下游一連串的訊息傳遞，引發持續性慢性發炎，進而導致阻塞性肺部疾病的病理機制仍尚未完全明瞭。瞬態電壓感受器陽離子通道錨蛋白類第1型 (Transient receptor potential ankyrin 1, TRPA1) 與瞬態電壓感受器陽離子通道M類第8型 (Transient receptor potential melastatin 8, TRPM8) 皆屬於一種對溫度具有敏感性且非特異性穿透細胞膜的陽離子通道。當TRPA1與TRPM8活化時其通透的離子主要為鈣離子，目前研究此兩通道大多表現於感覺神經元。於肺部感覺神經元上，愈來愈多的證據顯示TRPA1可以被許多種氧化物包括香菸所活化，而TPRM8則會受到冷溫度、薄荷與氧化物所調控活化。然而，肺部非神經細胞上TRPA1與TRPM8的表現及功能，與香菸是否會透過活化此兩通道進而引發肺發炎反應及其後續相關的調控機轉仍待進一步釐清。
本論文第一部分的研究，主要探討TRPA1於香菸引發肺部發炎所扮演的角色及其相關的調控機轉。我們分別利用香菸暴露小鼠，以及香菸萃取物刺激人類支氣管上皮細胞 (Human bronchial epithelial cells, HBECs) 來進行實驗。在動物實驗結果顯示，肺部組織有TRPA1蛋白的表現；香菸暴露Wildtype小鼠四週後，可明顯提升TRPA1蛋白表現量；此外，也會增加支氣管肺泡灌流液中或者是肺部的巨噬細胞炎症蛋白-2 (Macrophage inflammatory protein-2, MIP-2)，以及肺部氧化壓力與白血球數量增加等發炎現象。然而此發炎現象，在TRPA1-/- 小鼠組別則明顯被抑制。接著進一步在細胞實驗發現，隨著香菸萃取物漸增的濃度及時間刺激下，TRPA1的蛋白表現量也有明顯上升；此外也證實香菸萃取物本身內含的活性氧族群能活化TRPA1引起鈣離子流入細胞內。接著預處理TRPA1抑制劑或以小干擾核醣核酸引發TRPA1的基因靜默可有效減少香煙萃取物引起的IL-8。重要的是，預先給予細胞處理各種相關抑制劑可以減少鈣離子流入細胞內、活性氧族群、NADPH oxidase活性、有絲分裂活化蛋白質激酶 (Mitogen-activated protein kinases, MAPKs) 中的胞外信號調節激酶 (Extracellular-signal-regulated kinases, ERK) 與c-Jun N端蛋白質激酶 (c-Jun N-terminal kinases, JNK) 磷酸化，以及轉錄因子nuclear factor-kappa B (NF-kappaB) 核內表現量。以上結果可證實，香菸會透過其本身的活性氧族群活化TRPA1，引發鈣離子流入細胞內，活化NADPH oxidase並造成細胞內活性氧族群增加而促使下游訊息傳遞，進而導致肺發炎反應。
本論文第二部分的研究，主要探討TRPM8 於正規香菸或薄荷香菸誘發肺部發炎中所扮演的角色，並進一步研究此兩種香菸所造成發炎反應之差異程度與其相關的調控機轉。我們分別利用正規香菸或薄荷香菸暴露小鼠，以及兩者的香菸萃取物分別刺激人類支氣管上皮細胞 (HBECs) 來進行實驗。在動物實驗結果顯示，肺部組織有TRPM8蛋白的表現；正規香菸與薄荷香菸分別暴露Wildtype小鼠一週後，可明顯發現薄荷香菸組別的老鼠相較於正規香菸組別提升較多的TRPM8蛋白表現量；而以上此現象也於支氣管肺泡灌流液中或者是肺部的巨噬細胞炎症蛋白-2 (MIP-2)，以及白血球數量有相似的結果。然而上述的發炎現象，在兩種不同香菸的小鼠組別給予TRPM8抑制劑後則皆明顯被抑制。接著在細胞實驗發現，薄荷香菸萃取物相較於正規香菸萃取物，會誘發較高的IL-8蛋白表現量。而無論是正規香菸或薄荷香菸，其本身內含的活性氧族群皆能活化TRPM8，此外也證實相較於正規香菸，薄荷香菸能造成雙重刺激活化TRPM8而引起較大量的鈣離子流入細胞內。接著分別利用預處理TRPM8抑制劑或以小干擾核醣核酸引發TRPM8的基因靜默可有效減少兩者香菸萃取物引起的IL-8；另外利用剃除人類支氣管上皮細胞中TRPM8基因表現，或將TRPM8基因轉送入人類胚胎腎細胞293 (Human Embryonic Kidney Cells 293 cells, HEK 293 cells) 再給予TRPM8抑制劑也皆可有效抑制兩者香菸萃取物所引起的鈣離子與IL-8增加的現象。預先給予細胞處理各種相關抑制劑可以分別減少兩者香菸所引發鈣離子流入細胞內、活性氧族群、有絲分裂活化蛋白質激酶中的胞外信號調節激酶與c-Jun N端蛋白質激酶磷酸化，以及轉錄因子nuclear factor-kappa B (NF-kappaB) 核內表現量。最後，分別用薄荷、正規香菸或薄荷結合正規香菸給予細胞刺激，相較於薄荷或正規香菸組別，薄荷結合正規香菸組別會引發較高的鈣離子流入細胞內以及IL-8表現量增加。以上這些結果可證實無論是正規香菸或薄荷香菸中的活性氧族群皆會活化TRPM8，造成鈣離子流入調控細胞內活性氧族群增加，進而引發下游一連串的訊息傳遞，最後導致發炎反應；重要的是，相較於正規香菸，在薄荷香菸中由於額外添加的薄荷成分會與香菸本身內含的活性氧族群達到雙重刺激效果，因而促使TRPM8活化加劇，導致更嚴重的發炎反應生成。
綜合兩者研究主題結果，無論是在動物或細胞上，皆可得知正規香菸內含的活性氧族群會透過活化TRPA1與TRPM8，引發鈣離子流入後再進一步調控細胞內活性氧族群增加，接著透過下游訊息傳遞導致肺發炎反應生成。此外，在薄荷香菸的研究可發現香菸內含的薄荷成分會與香菸本身內含的活性氧族群達到雙重刺激效果，因而促使TRPM8活化加劇，引發更大量的鈣離子流入，並導致下游發炎相關的訊息傳遞以及發炎反應增強。經由我們的研究不僅可發現，薄荷香菸確實會比正規香菸引發更嚴重的肺發炎反應，另外也可得知TRPA1與TRPM8於持續性慢性肺發炎進而形成COPD疾病的病理機轉中扮演相當重要的角色。

Cigarette smoke (CS) is known to the major factor that contribute to the development and progression of chronic obstructive pulmonary disease (COPD), which results from the direct damage cells by CS via reactive oxygen species (ROS)-related pathways causes oxidative stress and persistent lung inflammation. However, the pathogenetic mechanisms and the role of increased ROS in the CS-induced regulation of lung inflammation are still largely unknown. Transient receptor potential ankyrin 1 (TRPA1) and transient receptor potential melastatin 8 (TRPM8), are nonselective cation channels sensitive to temperature and chemical stimuli, belong to TRP superfamily. TRPA1 and TRPM8 are mainly permeable to Ca2+, and they are predominately expressed in nociceptive sensory neurons. Increasing evidence suggests that in lung sensory neurons, TRPA1 are activated by many oxidants including CS and TRPM8 are activated by cold temperature, menthol and oxidants. However, the expression and function of TRPA1 and TRPM8 in non-neuronal cells in the lungs are fully unknown.
Our first study investigated the novel role of TRPA1 in CS-induced lung inflammation and its underlying mechanisms. To achieve this goal, wildtype and TRPA1 knockout mice or culture human bronchial epithelial cells (HBECs) were exposed to CS or cigarette smoke extract (CSE). In vivo, we showed that TRPA1 expressed in lung. Besides, mice were exposed to chronic CS for 4 weeks promoted expression of MIP-2 (an IL-8 homolog) in bronchoalveolar lavage fluid and lungs, caused oxidative stress, as well as lung inflammation, all of which were reduced in TRPA1 knockout mice. In vitro, our results found that CSE dose- and time-dependently increased the production of TRPA1. Moreover, CSE-induced extracellular ROS activated TRPA1 to increase the intracellular level of Ca2+ leading to NADPH oxidase activation and intracellular ROS production, respectively. Pretreatment with TRPA1 inhibitors or knockdown of TRPA1 by small interfering RNA prevented CSE-induced IL-8 expression. Importantly, pretreatment with various pharmacological inhibitors suppressed the increased Ca2+ intracellular levels, attenuated the intracellular ROS production, inhibited the activity of NADPH oxidase, reduced phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), as well as prevented the activation of nuclear factor-kappa B (NF-kappaB) in HBECs exposed to CSE. These results suggest that CS-induced extracellular ROS caused TRPA1 activation leading to increase intracellular Ca2+ levels. The Ca2+ levels increase sequentially resulted in NADPH oxidase activation, intracellular ROS production and downstream signaling activation leading to lung inflammation.
Our second study investigated the role of TRPM8 in Non-menthol-cigarette smoke- (Non-M-CS) or menthol-cigarette smoke (M-CS)-induced lung inflammation and the mechanisms for the different effects between these two types of CS. To achieve the purpose, wildtype mice were exposed to Non-M-CS or M-CS, and HBECs were exposed to Non-M-CSE or M-CSE. In vivo, Mice were exposed to Non-M-CS or M-CS for a week. Our results showed that TRPM8 expressed in lung, and M-CS group caused higher TRPM8 expression than Non-M-CS in lung. Besides, compared with the group of Non-M-CS exposure, M-CS group promoted higher expression of MIP-2 (an IL-8 homolog) in bronchoalveolar lavage fluid and lungs, as well as severe lung inflammation, all of which were reduced after administration of TRPM8 antagonist. In vitro, compared to Non-M-CSE, M-CSE induced higher IL-8 expression. Non-M-CSE- or M-CSE-induced the similar extracellular ROS levels, however, M-CSE activated TRPM8 to increase the higher intracellular level of Ca2+. Pretreatment with TRPM8 inhibitors, knockdown of TRPM8 by small interfering RNA prevented IL-8 induction to both CSE type. In addition, knockout of TRPM8 in HBECs or pretreatment with TRPM8 inhibitor to HEK293 cells transfected with hTRPM8 also found that M-CSE promoted greater Ca2+ levels and IL-8 induction than Non-M-CSE. Moreover, exposed to both CSE types, the increased intracellular Ca2+ levels, intracellular ROS production, phosphorylation of ERK and JNK and the activation of NF-kappaB were all attenuated by pretreatment with various pharmacological inhibitors. Last, a combination of Non-M-CSE and menthol induced more elevations of intracellular Ca2+ and IL-8 induction than Non-M-CSE alone. These results suggest that both CS type induced the similar extracellular ROS and then sequentially caused TRPM8 activation, increase intracellular Ca2+ levels, intracellular ROS production and downstream signaling activation leading to lung inflammation. Importantly, compared with Non-M-CSE, M-CSE caused greater inflammatory responses. This phenomenon may be caused a double hit which resulted from the ROS produced by CS and menthol in M-CS.
In conclusion, Non-M-CS-induced extracellular ROS activates TRPA1 and TRPM8, which cause intracellular Ca2+ levels increase. The elevations of intracellular Ca2+ levels induce intracellular ROS production and then activate MAPK/NF-kappaB signaling leading to lung inflammation. In addition, menthol in M-CS and ROS produced by CS may cause a double hit that activates TRPM8 to induce greater intracellular Ca2+ levels, MAPK/NF-kappaB signaling and inflammatory responses. Our findings may provide novel information that M-CS causes more severe lung inflammation than Non-M-CS. Moreover, TRPA1 and TRPM8 play a vital role in the development and progression of COPD disease.

目錄
目錄 --------------------------------------- i
圖目錄 --------------------------------------vi
縮寫對照表 ---------------------------------ix
中文摘要 -----------------------------------xi
英文摘要 -----------------------------------xiv
壹、文獻回顧及研究目的 ------------------------1
一、香菸的起源 -------------------------------2
1.香菸製程 --------------------------------2
2.香菸的總類 -------------------------------3
3.香菸的毒性 -------------------------------3
二、呼吸道上皮細胞 -------------------------5
三、香菸造成肺部損傷及呼吸道的發炎反應 --------6
四、瞬態電壓感受器陽離子通道 (Transient Receptor
Potential channels, TRP channels) ---------7
五、瞬態電壓感受器陽離子通道錨蛋白類第一型 (Transient
Receptor Potential Ankyrin 1, TRPA1) -------8
(1)TRPA1結構與配基 --------------------------8
(2)TRPA1分佈與功能 --------------------------9
(3)調控TRPA1通道 -------------------------9
六、瞬態電壓感受器陽離子通道M類第八型 (Transient
Receptor Potential Melastatin 8, TRPM8) ---------11
(1)TRPM8結構與配基 --------------------11
(2)TRPM8分佈與功能 -----------------------12
(3)調控TRPM8通道 --------------------------13
七、研究目的 ---------------------------14
貳、材料與方法 --------------------------17
一、儀器設備 -------------------------18
二、實驗材料與試劑 ---------------------19
香菸萃取物之製備 (Preparation of cigarette smoke extract)-19
細胞培養 (Cell culture) --------------------19
逆轉錄聚合酶鏈式反應 (Reverse transcriptase PCR, RT-PCR) ---19
西方墨漬法 (Western blot) -----------------20
NADPH oxidase 活性測定 (NADPH oxidase activity assay) --21
細胞內外ROS螢光測量 -------------------------21
組織免疫分析法 (Immunohistochemistry) ------------21
聚合酶酵素分析法 (ELISA assay) -------------21
細胞內鈣離子螢光的測量 (Fluo-8 calcium assay kit) -----21
三、實驗用溶液配方 --------------------21
四、實驗方法 ----------------------25
1.動物實驗 -----------------------25
2.動物的麻醉及手術 ----------------------25
3.支氣管肺泡灌洗液中，發炎細胞之總細胞計數，及分類細胞
計數 ------------------------------26
4.肺泡-微血管通透度的測定 -------------------27
5.組織免疫分析法 (Immunohistochemistry) -----------27
6.En face組織染色 ----------------------28
7.蘇木紫與伊紅染色 (Hematoxylin & Eosin staining; H&E
staining) -------------------------29
8.香煙萃取物的製備 (Preparation of cigarette smoke extract)-30
9.細胞培養 (Cell culture) --------------30
10.西方墨漬法 (Western blotting) -------------31
11.酵素結合免疫吸附法 (Enzyme-linked immunosobent
assay, ELISA) -----------------------36
12.細胞內鈣離子螢光的測定 (Screen QuestTM Fluo-8 medium
removal calcium assay kit) ------------------37
13.細胞內外ROS的測定 (Determination of intracellular ROS) --38
14.支氣管肺泡灌流液中ROS的測定 (Determination of ROS in
BALF) --------------------------39
15.細胞內NADPH oxidase活性測定 (NADPH oxidase activity
assay) -----------------------------40
16.小干擾核醣核酸引發基因靜默 (Small interfering RNA,
siRNA-mediated gene silencing) -----------------41
17.CRISPR/Cas9 系統調控剔除基因 (CRISPR/Cas9-mediated
gene knockout) ----------------------42
18.數據統計分析 ----------------------43
參、實驗結果 -------------------------44
一、第一部分研究：TRPA1於香菸引發肺部發炎所扮演的角色及
其相關的調控機轉 ------------------------45
1.TRPA1在小鼠肺組織的表現情形 -----------------45
2.香菸 (CS)對小鼠肺組織裡TRPA1表現量之影響 ----------45
3.TRPA1對香菸 (CS)造成小鼠氧化壓力產生之影響 ---------45
4.TRPA1對於香菸 (CS)導致小鼠肺組織產生的損傷之影響 ----46
5.TRPA1對於暴露香菸 (CS)下對小鼠呼吸道產生發炎之影響 ---46
6.TRPA1對於香菸 (CS)導致小鼠呼吸道發炎激素增加之影響 ---47
7.香菸萃取物 (CSE)對於支氣管上皮細胞內TRPA1表現的濃度及
時間點之影響 ---------------------------------47
8.TRPA1抑制劑 (HC)對於香菸萃取物 (CSE)調控細胞內白血球介
素-8 (IL-8)之影響 ----------------------------------48
9.將TRPA1基因靜默對於香菸萃取物 (CSE)誘導支氣管上皮細胞
IL-8表現之影響 ------------------------------------48
10.香菸萃取物 (CSE)刺激細胞對於活化TRPA1時間點之影響 ----49
11.TRPA1與ROS對於香菸萃取物 (CSE)調控鈣離子流入細胞內之
影響 --------------------------------------49
12.TRPA1對香菸萃取物 (CSE)於不同時間點調控細胞內外氧化性
族群之影響-----------------------------50
13.TRPA1對於香菸萃取物 (CSE)調控細胞內NADPH oxidase活性
之影響 ------------------------------50
14.TRPA1與ROS對於香菸萃取物 (CSE)調控細胞內ERK和JNK磷酸
化之影響 ----------------------------51
15.TRPA1與ROS對於香菸萃取物 (CSE)調控細胞內轉錄因子
NF-kappaB p65進入細胞核內之影響 --------------------51
二、第二部分研究：TRPM8於正規香菸或薄荷香菸誘發肺部發炎
中所扮演的角色及相關調控機轉 -------------------------52
1.TRPM8在小鼠肺組織中的表現情形 ---------------------52
2.正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)對小鼠肺組織裡
TRPM8表現量之影響 ---------------------------------52
3.TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)導致
小鼠肺組織產生的損傷之影響 --------------------------53
4.TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)對小
鼠呼吸道產生發炎之影響 ------------------------------53
5.TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)導致
小鼠呼吸道發炎激素增加之影響 ------------------------54
6.不同濃度的正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物
(M-CSE)對於支氣管上皮細胞內IL-8表現之影響 -----------55
7.探討TRPM8是否參與在對於正規香菸萃取物 (Non-M-CSE)或薄
荷香菸萃取物 (M-CSE)調控支氣管上皮細胞內IL-8表現之影響 ---55
8.TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃
取物 (M-CSE)調控支氣管上皮細胞內IL-8表現之影響 ---------56
9.TRPM8對正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物
(M-CSE)調控支氣管上皮細胞內外氧化性族群之影響 ------------57
10.TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷香菸
萃取物 (M-CSE)調控細胞內ERK、JNK磷酸化及轉錄因子
NF-kappaB p65進入細胞核內之影響 ----------------------57
11.探討正規香菸萃取物 (Non-M-CSE)結合薄荷醇 (Menthol)對
於細胞活化TRPM8與誘導發炎蛋白IL-8之影響 ----------------58
12.於人類胚胎腎臟細胞 (HEK293)送入人類TRPM8 (hTRPM8)質體
來探討正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物 (M-CSE)
對於活化TRPM8與發炎蛋白IL-8分泌之影響 ------------------59
13.正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物 (M-CSE)對於
調控支氣管上皮細胞氧化壓力相關因子之影響 ---------------59
肆、討論 -------------------------------------------61
伍、附圖 -------------------------------------------69
陸、參考文獻 -------------------------------113
柒、附件----------------------------------134

圖目錄
圖一、TRPA1在小鼠肺組織中的表現量 -----------------71
圖二、香菸 (CS)對小鼠肺組織裡TRPA1表現量之影響 ---------72
圖三、TRPA1對香菸 (CS)造成小鼠氧化壓力產生之影響 ---------73
圖四、TRPA1對於香菸 (CS)導致小鼠肺組織產生的損傷之影響 ----74
圖五、TRPA1對於暴露香菸 (CS)下對小鼠呼吸道產生發炎之影響 ---75
圖六、TRPA1對於香菸 (CS)導致小鼠呼吸道或肺組織發炎激素增加
之影響 ----------------------------------------------76
圖七、香菸萃取物 (CSE)對於支氣管上皮細胞內TRPA1表現的濃度及
時間點之影響 ------------------------------------------77
圖八、TRPA1抑制劑 (HC)對於香菸萃取物 (CSE)調控支氣管上皮細胞
內IL-8之影響 -----------------------------------------78
圖九、將TRPA1基因靜默對於香菸萃取物 (CSE)誘導支氣管上皮細胞
IL-8表現之影響 ----------------------------------------79
圖十、香菸萃取物 (CSE)刺激支氣管上皮細胞對於活化TRPA1時間點
之影響 -----------------------------------------------80
圖十一、TRPA1與ROS對於香菸萃取物 (CSE)調控支氣管上皮細胞內
鈣離子之影響 -------------------------------------------81
圖十二、TRPA1對香菸萃取物 (CSE)於不同時間點調控支氣管上皮細
胞內外氧化性族群之影響 ----------------------------------82
圖十三、TRPA1對於香菸萃取物 (CSE)調控支氣管上皮細胞內
NADPH oxidase活性之影響 -------------------------------83
圖十四、TRPA1與ROS對於香菸萃取物 (CSE)調控支氣管上皮細胞
內ERK和JNK磷酸化之影響 --------------------------------84
圖十五、TRPA1與ROS對於香菸萃取物 (CSE)調控支氣管上皮細胞內
轉錄因子NF-kappaB p65進入細胞核內之影響 ----------------85
圖十六、香菸 (CS)與TRPA1關係路徑圖 ---------------------86
圖十七、TRPM8在小鼠肺組織中的表現情形 -------------------88
圖十八、正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)對小鼠肺組織裡
TRPM8表現量之影響 ------------------------------------89
圖十九、TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸
(M-CS)導致小鼠肺組織產生的損傷之影響 -------------------90
圖二十、TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)
對小鼠呼吸道產生發炎之影響 -----------------------------91
圖二十一、TRPM8對於暴露正規香菸 (Non-M-CS)或薄荷香菸 (M-CS)
導致小鼠呼吸道發炎激素增加之影響 ------------------------92
圖二十二、不同濃度的正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃
取物 (M-CSE)對於支氣管上皮細胞內IL-8表現之影響 ----------93
圖二十三、TRPM8抑制劑 (AMTB)對於正規香菸萃取物 (Non-M-CSE)
或薄荷香菸萃取物 (M-CSE)調控支氣管上皮細胞內IL-8之影響 ---94
圖二十四、將TRPM8基因靜默對於正規香菸萃取物 (Non-M-CSE)或
薄荷香菸萃取物 (M-CSE)誘導支氣管上皮細胞IL-8表現之影響 ----95
圖二十五、將TRPM8基因剔除對於正規香菸萃取物 (Non-M-CSE)或
薄荷香菸萃取物 (M-CSE)誘導支氣管上皮細胞IL-8表現之影響 ----96
圖二十六、正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物 (M-CSE)
刺激支氣管上皮細胞對於活化TRPM8時間點之影響 --------------97
圖二十七、將TRPM8基因剔除對於正規香菸萃取物 (Non-M-CSE)或
薄荷香菸萃取物 (M-CSE)調控支氣管上皮細胞內鈣離子之影響 ----98
圖二十八、TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷
香菸萃取物 (M-CSE)調控支氣管上皮細胞內鈣離子之影響 --------99
圖二十九、TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷
香菸萃取物 (M-CSE)調控支氣管上皮細胞內IL-8之影響 --------100
圖三十、TRPM8對正規香菸萃取物 (Non-M-CSE)或薄荷香菸萃取物
(M-CSE)於不同時間點調控支氣管上皮細胞內外氧化性族群之影響 ------101
圖三十一、TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷
香菸萃取物 (M-CSE)調控細胞內ERK和JNK磷酸化之影響 --------102
圖三十二、TRPM8與ROS對於正規香菸萃取物 (Non-M-CSE)或薄荷
香菸萃取物 (M-CSE)調控細胞內轉錄因子 NF-kappaB p65進入細胞核內之影響 ------------------103
圖三十三、不同濃度的薄荷醇 (Menthol)對於支氣管上皮細胞活化
TRPM8時間點之影響 ---------------------------104
圖三十四、將正規香菸萃取物 (Non-M-CSE)與薄荷醇 (Menthol)合
併處理對於活化支氣管上皮TRPM8時間點之影響 ----------------105
圖三十五、TRPM8對於合併處理正規香菸萃取物 (Non-M-CSE)與薄
荷醇 (Menthol)調控細胞內IL-8之影響 --------------------106
圖三十六、將人類TRPM8 (hTRPM8)質體轉送入人類胚胎腎臟細胞
(HEK 293)表達 -----------------------------107
圖三十七、探討在表現人類TRPM8 (hTRPM8)的人類胚胎腎臟細胞
(HEK 293)中正規香菸萃取物 (Non-M-CSE)與薄荷香菸萃取物
(M-CSE)對於活化TRPM8時間點之影響 ------------------108
圖三十八、在表現人類TRPM8 (hTRPM8)的人類胚胎腎臟細胞
(HEK 293)中探討TRPM8對於正規香菸萃取物 (Non-M-CSE)或
薄荷香菸萃取物 (M-CSE)調控細胞內鈣離子之影響 -------------109
圖三十九、在表現人類TRPM8 (hTRPM8)的人類胚胎腎臟細胞
(HEK 293)中探討TRPM8對於正規香菸萃取物 (Non-M-CSE)或
薄荷香菸萃取物 (M-CSE)調控IL-8分泌之影響 ----------110
圖四十、與氧化壓力相關調控因子對於正規香菸萃取物 (Non-M-CSE)
或薄荷香菸萃取物 (M-CSE)刺激支氣管上皮細胞內IL-8表現之影響 --111
圖四十一、TRPM8與正規香菸(Non-M-CS)或薄荷香菸(M-CS)關係
路徑圖 -------------------------------------------112

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