檢測細胞色素1A1及1B1在人類肺腺癌組織及戴奧辛與NNK在小鼠共同誘發的肺腺瘤的表現情形

中山醫學大學機構典藏 CSMUIR > 醫學院 > 醫學分子毒理學研究所 > 博碩士論文 > Item 310902500/382

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Title:	檢測細胞色素1A1及1B1在人類肺腺癌組織及戴奧辛與NNK在小鼠共同誘發的肺腺瘤的表現情形 Study of the expression of Cytochrome P4501A1 and 1B1 in human lung adenocarcinomas and TCDD/NNK- induced lung adenomas in A/J mice by immunohistochemistry
Authors:	蔡宛霖 Wan-Lin Tsai
Contributors:	中山醫學大學：醫學分子毒理學研究所許國堂林嬪嬪
Keywords:	戴奧辛細胞色素1A1 細胞色素1B1 人類肺腺癌肺腺瘤 2,3,7,8-tetrachlorodibenzo-p-dioxin 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone Adenocarcinoma Aryl hydrocarbon receptor Cytochrmoe P450 1A1 Cytochrmoe P450 1B1 Immunohistochemistry
Date:	2006/07/19
Issue Date:	2009-12-22T08:19:48Z (UTC)
Abstract:	流行病學或動物實驗證實暴露環境毒物(TCDD為代表)或香菸煙霧中的有害物質(4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone，NNK為代表)會增加罹患肺癌的機率。戴奧辛(TCDD)為多環芳香烴受器(AhR)的配基，當細胞暴露到TCDD之後，活化AhR進而促使細胞色素P450 1A1/ P450 1B1(CYP1A1/CYP1B1)表現，CYP1A1/CYP1B1酵素會代謝活化一些致癌物。香菸濃縮物(CSC)具有多種致癌物，NNK是在CSC中的致癌物之一，NNK的代謝物會形成DNA adduct，經錯誤的DNA修復會使DNA發生突變，因而產生致癌性。本實驗室之前使用quantitative real-time RT-PCR方法去偵測人類淋巴球CYP1A1基因表現，發現抽菸者比不抽菸者較常表現CYP1A1 mRNA；而使用免疫染色法檢測人類肺癌組織中CYP1B1蛋白，發現CYP1B1蛋白的過度表現主要發生於肺腺癌，其他肺癌組織形態包括鱗狀上皮癌相對較少發生。另外，有研究報告指出cyclooxygenase-2 (COX-2) 蛋白在肺腺癌的表現明顯高於鱗狀上皮癌。因此，本研究的目的有二：(1)檢測CYP1A1以及CYP1B1在人類肺腺癌組織的表現與抽菸行為的相關性。收集107例人類肺腺癌組織採用免疫染色，分層檢視抽菸行為或性別，AhR對於CYP1A1、CYP1B1或COX-2表現的影響。(2)探討雌性A/J小鼠暴露於TCDD、NNK、CSC或暴露於其中兩種毒物，飼養24週後，觀察小鼠誘發肺腫瘤的情形以及對AhR/CYP1A1/CYP1B1表現的影響。實驗結果顯示，人類肺腺癌組織中AhR、CYP1A1或CYP1B1的表現與性別或抽菸行為無關，但是COX-2較常在女性肺癌組織中表現。當肺腺癌病人有抽菸習慣且癌組織AhR過度表現時，會使得CYP1A1表現增加；但是，使用多變項logistic regression同時檢定性別、抽菸行為與AhR過度表現時，顯示AhR過度表現是最重要的因子，也證實CYP1A1與AhR表現呈正相關。CYP1B1的過度表現僅與AhR過度表現有關，而與性別/抽菸行為無關。COX-2的表現無論與性別、抽菸習慣與AhR表現都無關。動物實驗結果顯示A/J mice連續暴露TCDD 4週，然後繼續飼養20週之後，不會產生肺腫瘤；單獨暴露於單一劑量CSC或2 mg NNK，或共同暴露TCDD + CSC或TCDD + 1 mg NNK，飼養24週後，都會產生肺腫瘤：其中單獨處理1 mg NNK的小鼠有10%產生肺腫瘤；25 mg CSC有60%；TCDD + CSC或TCDD + 1 mg NNK組，皆為40%；而在2 mg NNK為100%。因此，由動物實驗結果推測 TCDD具有促進NNK誘發肺腫瘤形成的能力。同時，觀察A/J mice肺小支氣管上皮與肺腫瘤細胞的AhR、CYP1A1與CYP1B1表現。值得注意的發現是CYP1B1過度表現會發生在控制組的自發性肺腺癌(12.7%)及NNK+TCDD實驗組的肺腺瘤(15.3%)。A/J mice飼養24週，發現單獨暴露TCDD或TCDD + 1 mg NNK實驗組小支氣管上皮的CYP1B1表現率都高於控制組。單獨暴露TCDD (37.5%)或TCDD + NNK (24.7%) 4週實驗組的CYP1A1會高於控制組(0.2%)；飼養24週A/J mice中，所有組別的肺小支氣管上皮或腫瘤都沒有表現CYP1A1。由以上的動物實驗觀察發現CYP1A1蛋白的表現與CYP1B1不同，推論CYP1A1蛋白的誘發(inducibility)或穩定性與CYP1B1蛋白不同。CCSP是Clara cell的標記。在A/J mice 4週實驗組中，TCDD或NNK各實驗組Clara cell的數目(26.6%-34.3%)與控制組(34.2%)比較，並沒有明顯的差異；反觀A/J mice 24週實驗組中，除了TCDD+NNK (1 mg)實驗組外，其他各實驗組Clara cell的數目與控制組(55.9%)比較明顯下降11.7%-41.4%，顯示暴露NNK、TCDD、CSC或TCDD+CSC皆會傷害Clara cell，暗示Clara cell對這些環境毒物比其他呼吸道上皮細胞敏感。 Epidemiologic or animal studies showed that exposure to environmental toxicants (such as TCDD and 4-(N-methyl-N-nitrosamino)- 1-(3-pyridyl)-1-butanone, NNK) increased the risk of human lung cancer. Dioxin (TCDD) is one of aryl hydrocarbon receptor (AhR) ligands. TCDD activates AhR to up-regulate gene expression of cytochrome P450 1A1/P450 1B1 (CYP1A1/CYP1B1), leading to the formation of carcinogenic metabolites. Cigarette smoke condensate (CSC) contains many carcinogens including NNK. Our earlier studies demonstrated that induction of CYP1A1 mRNA in human leukocytes was more common in smokers than in nonsmokers. In addition, CYP1B1over-expression was more commonly detected in human adenocarcinoma cells than in other tumor types including squamous cell carcinoma. Some researchers have reported that cyclooxygenase-2 (COX-2) over-expressed in human lung adenocarcinoma but not in squamous cell carcinoma. Thus, the aims of this study were (1) to examine the relationship between expression of AhR, CYP1A1, CYP1B1 or COX-2 in human lung adenocarcinomas and smoking behavior using immunohistochemistry; (2) to examine the formation of lung tumor and the CYP1A1/CYP1B1 expression in A/J mice exposed to TCDD, NNK or CSC, as well as co-exposure to TCDD+NNK or TCDD+CSC after 4- or 24-weeks. Our results showed no association between patients’ smoking behavior, gender, and expression of AhR, CYP1A1 or CYP1B1 in lung adenocarcinomas. AhR overexpression positively associated with CYP1A1/CYP1B1 but not COX-2 expression in human lung adenocarcinomas adjusted for patients’ gender and smoking behavior using a logistic regression testing. Our present animal studies showed lung tumor formation in A/J mice exposed to 1 mg or 2 mg NNK, CSC, TCDD + 1 mg NNK or TCDD + CSC, but not in mice exposed to TCDD alone 24-weeks later. The percentage of the lung tumors was 10% in mice with 1 mg NNK treatment, 60% in 25 mg CSC/ kg treatment, 40% in co-exposure to TCDD+NNK or TCDD+CSC, and 100% in 2 mg NNK. Thus, These results suggested that TCDD promoted the formation of lung tumor in A/J mice. CYP1A1 protein was not detected in tumors or bronchiolar epithelia at 24th week. But CYP1B1 protein was more commonly detected in bronchiolar epithelia of TCDD or NNK + TCDD exposed groups. At 4th week, CYP1A1 protein was detected in TCDD and TCDD+NNK exposed groups. These results indicated that both CYP1A1 and CYP1B1 protein was inducible by TCDD in A/J mice. But CYP1A1 induction was less stable than CYP1B1 induction. CCSP is a biomarker for respiratory Clara cells. At 4th week, the percentages of Clara cells over bronchiolar epithelial cells were similar between groups. At 24th week, the percentages of Clara cells were reduced in mice exposed to TCDD, NNK, CSC, or TCDD+CSC exposure, but not in mice exposed to TCDD+1 mg NNK. Thus, these findings suggested that TCDD, NNK, CSC, or TCDD+CSC exposure injuried Clara cells to lose CCSP protein in mice lung.
URI:	http://140.128.138.153:8080/handle/310902500/382
Appears in Collections:	[醫學分子毒理學研究所] 博碩士論文

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