臺灣博碩士論文加值系統

TXA又稱氨甲環酸，是一種合成的離氨酸(lysine)類似物，根據研究顯示，可抑制纖維蛋白溶解，促進血栓的穩定性，產生止血作用，並可減輕發炎反應。臨床上廣泛應用於治療各種出血性疾病和手術時異常出血。同時，TXA可以抑制肝斑中的色素沉著，黑色素細胞的活性，具有美白效果，為衛生署核可外用美白成分。
乾癬(Psoriasis)是一種慢性免疫介導的炎症性皮膚病，其成因為免疫系統異常，使角質細胞過度增殖及異常的發育，導致皮膚呈現紅斑及脫屑，因此又稱為銀屑病。研究顯示，IL-17在乾癬患者的血清和皮膚損傷中過度表現，所以它可作為刺激劑刺激細胞在體外模擬乾癬(psoriasis)的模式。
在本篇論文中，選用TXA作為預先處理的保護劑，以探討TXA對IL-17 (500ng/mL)誘導人類角質細胞(HaCaT)的發炎與增生的表現是否有抑制作用。因此本研究的目的有三：第一探討TXA對IL-17(500ng/mL)誘導的人類角質細胞(HaCaT) 發炎反應是否有抑制作用。研究顯示，以500ng/mL IL-17處理人類角質細胞後，會促使NF-κB 路徑的蛋白(IKK???}??)磷酸化，以及NF-κB下游細胞因子IL-8的表現。但以TXA預先處理人類角質細胞2小時後，則會減少IL-17(500ng/mL)導致的IKK???}?畛C酸化、IκB的降解和NF-κB入核表現和IL-8的分泌量。
第二由於角蛋白17（Keratin17，K17）是一種肌上皮角蛋白，在乾癬損傷表皮中過度表達，促使角質細胞過度增殖。因此，K17被認為是乾癬的標誌，參與乾癬的致病機制。而先前研究指出，IL-17透過促進乾癬表皮中Nrf2的轉錄入核，上調角蛋白17 (K17)的表達。因此，探討TXA對IL-17(500ng/mL)誘導的人類角質細胞增生反應(proliferation )是否有抑制作用。研究顯示，以500ng/mL IL-17處理人類角質細胞後，會促使Nrf2增加。但以TXA預先處理人類角質細胞2小時後，則會減少Nrf2入核表現，並降低K17的表現量。
第三探討TXA是否能改善Imiquimod 誘導之乾癬小鼠動物實驗模式。結果發現，TXA可以改善BALB/C品系之小鼠乾癬病徵，包括背部及耳朵之皮膚紅腫、皮屑和增厚及減少脾臟腫大的現象。綜合以上結果，In vitro模式中，TXA透過NF-κB 和Nrf2路徑，抑制IL-17誘導人類角質細胞(HaCaT)的發炎與增生的表現，而在小鼠動物模式中，TXA能改善Imiquimod 誘導的類似乾癬病變。

TXA, also known as tranexamic acid, is a synthetic derivative of lysine that inhibits fibrinolysis, promotes thrombus stability and reduces inflammation. It is widely used to treat various bleeding disorders and abnormal bleeding during surgery. TXA can also inhibit pigmentation in Melasma and is a topical whitening ingredient according to Ministry Of Health.
Psoriasis is a chronic inflammatory immnue disease. It is caused by abnormal immune system, which causes hyperproliferation and abnormal development of keratinocytes, resulted in scaly papulosquamous plaque. Studies have shown that IL-17 is overexpressed in serum and skin lesions in psoriatic patients but not in healthy epidermis.
In this study, we investigated whether TXA can inhibits the inflammation and proliferation induced by IL-17 (500 ng/mL) in HaCaT cells. Studies have shown that treatment of HaCaT cells with 500 ng/mL IL-17 promotes phosphorylation of IKK???}?? and expression of IL-8. However, pretreat HaCaT cells with TXA for 2 hours reduced IL-17 induced IKK ???}???nphosphorylation, IκB degradation, NF-κB nuclear translocation and IL-8 secretion.
Secondly, Keratin 17 (K17) is a hyperproliferation-associated keratin, is overexpressed in psoriatic epidermis but bot found in normal epidermis. Therefore, K17 is considered to be a hallmark of psoriasis and involved in the pathogenesis of psoriasis. Previous studies have indicated that IL-17 upregulates the expression of K17 by promoting Nrf2 nuclear translocation . Our studies shown that, pretreat with TXA reduced Nrf2 nuclear translocation and decreased K17 expression in IL-17 induced HaCaT cells.
Finally, topical application of TXA can improve Imiquimod-induced psoriasiform dermatits in BALB/C mice.Treatment with TXA improve BALB/C mice skin inflammation and the thickness of right ear and attenuated of spleen mass.
Taken together, In vitro study, TXA inhibits IL-17-induced inflammation and proliferation through NF-κB and Nrf2 pathways in HaCaT cells, whereas in animal models, TXA improves Imiquimod-induced psoriasis-like inflammation in BALB/C mice.
 

中文摘要 IX
Abstrate XI
縮寫表 XII
第壹章、前言 1
第貳章、文獻探討 3
第一節 乾癬(Psoriasis) 4
一、乾癬的介紹 4
二、乾癬的種類和臨床症狀 4
三、乾癬的發病原因及風險因子 5
四、乾癬的免疫發病機制 6
五、乾癬目前的治療方式 10
第二節 IL-17 (interleukin-17) 13
一、IL-17 A (interleukin-17)與乾癬的關係 13
二、IL-17 訊息傳遞路徑 15
第三節 Nrf2與乾癬的關係 16
一、Nrf2 16
二、角蛋白 (Keratin 17) 18
第四節 角質細胞(Keratinocytes)與乾癬的關係 19
第五節 傳明酸Tranexamic acid (TXA) 22
第六節Imiquimod (IMQ) 25
一、Imiquimod 25
二、Imiquimod與乾癬之間的關聯 25
第參章、研究動機與實驗架構圖 26
一、研究動機與假説 27
二、實驗設計流程圖 28
第肆章、實驗材料與方法 30
第一節.實驗材料 31
第二節 實驗儀器 34
第三節 實驗方法 36
一、細胞培養 36
二、細胞存活率試驗 41
三、細胞總蛋白質萃取 43
四、細胞核與細胞質蛋白質萃取 46
五、蛋白質定量 49
六、西方墨點法 51
七、細胞激素(cytokine)之測定—使用R&D system 59
八、乾癬動物模式 62
九、乾癬病變指數評分表(PASI) 65
十、脾臟細胞萃取 66
十一、流式細胞儀分析 68
十二、動物組織切片蘇木精?{伊紅?vHematoxylin and Eosin Stain)染色法 70
十三、統計分析 71
第伍章、實驗結果與圖表 72
第一節 TXA對IL-17誘導的HaCaT細胞的發炎反應之影響 73
一、TXA及IL-17對HaCaT細胞之細胞存活率影響 73
二、IL-17對於HaCaT細胞NF-κB 路徑相關發炎蛋白之影響 73
三、TXA 對於IL-17誘導的HaCaT細胞NF-κB 路徑相關發炎蛋白之影響 74
四、TXA 對於IL-17誘導的HaCaT細胞發炎轉錄因子NF-κB入核表現之影響 74
五、IL-17對於HaCaT細胞細胞激素IL-8分泌之影響 75
六、TXA 對IL-17誘導HaCaT細胞細胞激素IL-8分泌之影響 75
七、TXA 對IL-17誘導HaCaT細胞ACT1之影響 76
第二節 TXA對IL-17誘導的HaCaT細胞的增生反應Nrf2/Keratin 17 路徑之影響 85
一、TXA 對IL-17誘導HaCaT細胞Keratin 17 蛋白之表現 85
二、IL-17誘導HaCaT細胞Nrf2蛋白質表現的影響 85
三、TXA 對IL-17誘導HaCaT細胞Nrf2入核表現 86
第三節、評估TXA對於IMQ誘導乾癬小鼠模式的影響 90
一、TXA對於IMQ 誘導乾癬樣小鼠之體重的影響 90
二、TXA不會誘導小鼠背部皮膚產生類似乾癬的病徵 (紅腫、增厚、皮屑) 90
三、TXA改善IMQ誘導小鼠背部皮膚產生類似乾癬的病徵 (紅腫、增厚、脫屑) 91
四、TXA抑制IMQ誘導小鼠右耳增厚的現象 91
五、TXA能改善IMQ誘導小鼠之脾臟腫大 92
六、TXA對IMQ誘導小鼠脾臟的CD4+細胞表現IL-17A之影響 92
七、TXA對IMQ誘導小鼠脾臟的CD4+細胞表現IFN-?蚺尬v響 92
八、TXA處理不會引起小鼠右耳皮膚增厚(H&E) 93
九、TXA能抑制IMQ誘導小鼠右耳皮膚增厚(H&E) 93
十、TXA 能抑制IMQ誘導小鼠右耳皮膚之過度角化、增厚和發炎 93
第陸章、討論 105
第柒章、結論 110
第捌章、參考文獻 113

1.Meephansan, J., et al., Pathogenic Role of Cytokines and Effect of Their Inhibition in Psoriasis, in An Interdisciplinary Approach to Psoriasis. 2017, InTech.
2.Greb, J.E., et al., Psoriasis. Nature Reviews Disease Primers, 2016. 2: p. 16082.
3.鄭亘佑, Imiquimod 誘導類似乾癬皮膚發炎之小鼠模式的發展與抗生素 Azithromycin 改善乾癬之評估. 2014.
4.Nickoloff, B.J. and F.O. Nestle, Recent insights into the immunopathogenesis of psoriasis provide new therapeutic opportunities. The Journal of clinical investigation, 2004. 113(12): p. 1664-1675.
5.Di Cesare, A., P. Di Meglio, and F.O. Nestle, The IL-23/Th17 axis in the immunopathogenesis of psoriasis. Journal of Investigative Dermatology, 2009. 129(6): p. 1339-1350.
6.Yamaguchi, Y., et al., Decreased expression of caveolin-1 contributes to the pathogenesis of psoriasiform dermatitis in mice. Journal of Investigative Dermatology, 2015. 135(11): p. 2764-2774.
7.Perera, G.K., P. Di Meglio, and F.O. Nestle, Psoriasis. Annu Rev Pathol, 2012. 7: p. 385-422.
8.Chiricozzi, A., et al., Scanning the immunopathogenesis of psoriasis. International journal of molecular sciences, 2018. 19(1): p. 179.
9.Beringer, A., M. Noack, and P. Miossec, IL-17 in Chronic Inflammation: From Discovery to Targeting. Trends in Molecular Medicine, 2016. 22(3): p. 230-241.
10.Beringer, A., M. Noack, and P. Miossec, IL-17 in Chronic Inflammation: From Discovery to Targeting. Trends Mol Med, 2016. 22(3): p. 230-241.
11.Mitsuishi, Y., et al., Nrf2 Redirects Glucose and Glutamine into Anabolic Pathways in Metabolic Reprogramming. Cancer Cell, 2012. 22(1): p. 66-79.
12.Yang, L., et al., Nrf2 promotes keratinocyte proliferation in psoriasis through up-regulation of keratin 6, keratin 16, and keratin 17. Journal of Investigative Dermatology, 2017. 137(10): p. 2168-2176.
13.Jiang, C.K., et al., Disease-activated transcription factor: allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17. Mol Cell Biol, 1994. 14(7): p. 4759-69.
14.Shen, Z., et al., HLA DR B1*04, *07-restricted epitopes on Keratin 17 for autoreactive T cells in psoriasis. Journal of Dermatological Science, 2005. 38(1): p. 25-39.
15.Shi, X., et al., IL-17A upregulates keratin 17 expression in keratinocytes through STAT1- and STAT3-dependent mechanisms. J Invest Dermatol, 2011. 131(12): p. 2401-8.
16.Collaborators, C.-. The importance of early treatment with tranexamic acid in bleeding trauma patients: an exploratory analysis of the CRASH-2 randomised controlled trial. The Lancet, 2011. 377(9771): p. 1096-1101. e2.
17.Lethaby, A., C. Farquhar, and I. Cooke, Antifibrinolytics for heavy menstrual bleeding. The Cochrane database of systematic reviews, 2000(4): p. CD000249-CD000249.
18.Shakur, H., et al., The WOMAN Trial (World Maternal Antifibrinolytic Trial): tranexamic acid for the treatment of postpartum haemorrhage: an international randomised, double blind placebo controlled trial. Trials, 2010. 11(1): p. 40.
19.Bala, H.R., et al., Oral Tranexamic Acid for the Treatment of Melasma: A Review. Dermatologic Surgery, 2018. 44(6): p. 814-825.
20.Peng, Z., et al., Intraluminal tranexamic acid inhibits intestinal sheddases and mitigates gut and lung injury and inflammation in a rodent model of hemorrhagic shock. The journal of trauma and acute care surgery, 2016. 81(2): p. 358.
21.van der Fits, L., et al., Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis. J Immunol, 2009. 182(9): p. 5836-45.
22.Liu, T., et al., NF-κB signaling in inflammation. Signal Transduction And Targeted Therapy, 2017. 2: p. 17023.
23.Xiong, H., et al., Glycyrrhizin ameliorates imiquimod-induced psoriasis-like skin lesions in BALB/c mice and inhibits TNF-alpha-induced ICAM-1 expression via NF-kappaB/MAPK in HaCaT cells. Cell Physiol Biochem, 2015. 35(4): p. 1335-46.
24.Han, L., et al., Formula PSORI-CM01 inhibits the inflammatory cytokine and chemokine release in keratinocytes via NF-kappaB expression. Int Immunopharmacol, 2017. 44: p. 226-233.
25.Zhang, S., et al., Epigallocatechin-3-gallate (EGCG) inhibits imiquimod-induced psoriasis-like inflammation of BALB/c mice. BMC Complement Altern Med, 2016. 16(1): p. 334.