本文關(guān)鍵詞： 下咽癌 多藥耐藥 ABCB1 ABCG2 JNK　出處：《山東大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：下咽癌是頭頸部鱗狀細胞癌中惡性程度較高的腫瘤之一,由于發(fā)病位置隱蔽,發(fā)現(xiàn)時多數(shù)已為中晚期,患者的5年生存率僅為30%-50%,傳統(tǒng)的手術(shù)方法由于不能保留喉器官,導(dǎo)致患者的生存質(zhì)量下降.以器官保留為目的的放化療并不能提高患者生存期,5年生存率仍處于40%-60%.近年來人們提倡手術(shù)聯(lián)合放化療以提高患者生存率和生活質(zhì)量,臨床上比較常用的化療藥物包括順鉑(cisplatin DDP),5-氟尿嘧啶(5-fluorouracil5-FU),多柔比星(doxorabicin Dox),和長春新堿(vincristine VCR)等。但是,隨著化療藥物的應(yīng)用,腫瘤細胞逐漸出現(xiàn)了多藥耐藥性(multidrug resistance, MDR),從而降低了治療效果。紫杉醇作為新一代化療藥物在晚期或復(fù)發(fā)下咽癌中被廣泛應(yīng)用并取得較好的療效,研究表明其臨床試驗療效高于其他傳統(tǒng)化療藥物。盡管如此,腫瘤細胞對紫杉醇產(chǎn)生的耐藥性仍然是其發(fā)揮作用的最大障礙。為進一步了解下咽癌細胞對紫杉醇的耐藥機制,我們通過用紫杉醇誘導(dǎo)下咽癌細胞株FaDu的方法建立其多藥耐藥細胞株FaDu/T.通過比較兩種細胞株間多藥耐藥蛋白ABCB1和ABCG2表達變化及JNK信號轉(zhuǎn)導(dǎo)途徑在這種變化中的作用,進一步揭示了下咽癌的多藥耐藥及調(diào)控機制,并為臨床治療下咽癌提供理論支持。 目的 1.檢測FaDu/T細胞的多藥耐藥性。 2.檢測多藥耐藥蛋白ABCB1,ABCG2分別在FaDu和FaDu/T細胞系中的不同表達。 3.探討JNK信號轉(zhuǎn)導(dǎo)途徑在ABCB1及ABCG2變化表達中的作用,為進-步逆轉(zhuǎn)下咽癌細胞的多藥耐藥性奠定理論基礎(chǔ)。 材料與方法 以人下咽癌細胞株FaDu為親本細胞,采用濃度梯度遞增法成功建立下咽癌細胞株FaDu的耐紫杉醇細胞株FaDu/T,紫杉醇的起始用藥濃度為FaDu的IC25：5×10-9mol/L,截止實驗時耐藥濃度為200×10-9mol/L,整個過程持續(xù)約14個月。四甲基偶氮唑藍法(Methyl-Thiazolyl-Tetrazolium MTT)分別檢測FaDu和FaDu/T對DDP,5-FU, Dox,和VCR的多藥耐藥性,IC50軟件分別計算出其半數(shù)致死率；多藥耐藥基因及蛋白ABCB1及ABCG2表達變化情況分別通過反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR), Western blotting和激光共聚焦檢測；FaDu/T細胞中茴香霉素(Anisomycin)的加入前后不同時間JNK信號轉(zhuǎn)導(dǎo)通路相關(guān)蛋白的表達變化通過Western blotting檢測。 結(jié)果 1. FaDu細胞的耐紫杉醇細胞株FaDu/T成功建立,FaDu/T細胞可以在含有紫杉醇的培養(yǎng)基中正常生長。 2.耐藥細胞株FaDu/T比FaDu細胞具有更強的多藥耐藥性。 3.與FaDu細胞相比,FaDu/T細胞株中多藥耐藥基因和蛋白ABCB1的表達增加但ABCG2表達下降。 4.紫杉醇初始作用于FaDu細胞后JNK信號轉(zhuǎn)導(dǎo)通路被激活,但是在FaDu/T細胞中JNK信號轉(zhuǎn)導(dǎo)通路呈失活狀態(tài),MAPK信號轉(zhuǎn)導(dǎo)通路的激活劑茴香霉素(Anisomycin)則可以使FaDu/T細胞的JNK信號通路重新激活。 5.耐藥細胞株FaDu/T中加入Anisomycin時ABCB1表達下調(diào),ABCG2的表達升高,但是在預(yù)先加入JNK特異性抑制劑SP600125的FaDu/T細胞株中加入Anisomycin時,ABCB1和ABCG2的表達無明顯變化,提示JNK信號通路在下咽癌的多藥耐藥過程中具有重要的調(diào)控作用。 結(jié)論 1.多藥耐藥蛋白ABCB1在FaDu/T細胞的高表達是其產(chǎn)生多藥耐藥的重要原因。 2.紫杉醇通過JNK信號轉(zhuǎn)導(dǎo)通路影響多藥耐藥蛋白ABCB1和ABCG2表達 3.各種耐藥蛋白交替變化表達從而對不同的化療藥物產(chǎn)生耐藥性是導(dǎo)致臨床腫瘤化療失敗的重要原因。
[Abstract]:Hypopharyngeal cancer is one of malignant squamous cell carcinoma of head and neck cancer incidence higher, due to the hidden location, the majority found already in the late stage, with a 5 year survival rate is only 30%-50%, the traditional surgical method due to the preservation of laryngeal organs, leading to the survival quality of the patients decreased. With the purpose of organ retention. Chemotherapy does not improve survival rate, 5 year survival rate is still in the 40%-60%. in recent years, people advocate surgery combined with radiotherapy and chemotherapy to improve survival and quality of life of patients, the clinical commonly used chemotherapeutic drugs including cisplatin (cisplatin DDP), 5- (5-fluorouracil5-FU), fluorouracil, doxorubicin and vincristine (doxorabicin Dox). (vincristine VCR). However, with the application of chemotherapy, tumor cells appeared multidrug resistance (multidrug resistance, MDR), thereby reducing the therapeutic effect of paclitaxel as a new generation. Chemotherapy pharyngeal cancer is widely used and has good efficacy in advanced or recurrent, studies show that the clinical curative effect than other traditional chemotherapy drugs. However, drug resistance of tumor cells to paclitaxel remains the biggest obstacle to play a role. The pharyngeal resistance mechanism of cancer cells to paclitaxel for further we know, through the method of paclitaxel induced hypopharyngeal carcinoma cell line FaDu in the establishment of multidrug resistance cell line FaDu/T. by comparing the two kinds of cell lines of multidrug resistance protein ABCB1 and ABCG2 expression and JNK signal transduction pathway in this kind of change in the role, further reveals the multidrug resistance and regulation mechanism pharyngeal cancer, and provide theoretical support for the clinical treatment of hypopharyngeal carcinoma.
objective
1. the multidrug resistance of FaDu/T cells was detected.
2. the multidrug resistance protein ABCB1 and ABCG2 were detected in different expressions in FaDu and FaDu/T cell lines.
3. to explore the role of JNK signal transduction pathway in the expression of ABCB1 and ABCG2, and to lay a theoretical basis for reversing the multidrug resistance of hypopharyngeal cancer cells.
Materials and methods
In human hypopharyngeal carcinoma cell line FaDu as the parental cells, the method of increasing successful establishment of hypopharyngeal carcinoma cell line FaDu in paclitaxel resistant cell line FaDu/T by concentration gradient, the initial drug concentration of paclitaxel for FaDu IC25:5 * 10-9mol/L, the cut-off resistance concentration is 200 * 10-9mol/L, the whole process lasted about 14 months. Four methyl thiazolyl tetrazolium assay (Methyl-Thiazolyl-Tetrazolium MTT) and FaDu/T FaDu detection of DDP, 5-FU, Dox, multidrug resistance and VCR, IC50 software to calculate the half lethal rate; multidrug resistance gene and protein expression of ABCG2 and ABCB1 respectively by reverse transcription polymerase chain reaction (RT-PCR), Western blotting and laser confocal detection; FaDu/T cells of anisomycin (Anisomycin) expression changes before and after the addition of different time of JNK signal transduction pathway related proteins by Western blotting test.
Result
The taxol resistant cell line FaDu/T of 1. FaDu cells was successfully established, and FaDu/T cells could grow normally in the medium containing paclitaxel.
The 2. drug resistant cell line, FaDu/T, has a stronger multidrug resistance than the FaDu cell.
3. compared with FaDu cells, the expression of multidrug resistance gene and protein ABCB1 in FaDu/T cell lines increased, but the expression of ABCG2 decreased.
4. after paclitaxel initial action on FaDu cells, JNK signal transduction pathway was activated, but JNK signaling pathway was inactivated in FaDu/T cells. The activation of MAPK signal transduction pathway, anisomycin (Anisomycin), could activate JNK signaling pathway in FaDu/T cells.
Adding 5. Anisomycin resistant cell line FaDu/T ABCB1 expression, the expression of ABCG2 increased, but the addition of Anisomycin in FaDu/T cells and pretreatment with JNK specific inhibitor of SP600125, no significant changes in the expression of ABCB1 and ABCG2, has an important role in the regulation of JNK signaling pathway in hypopharyngeal cancer multidrug resistance in the process of.
conclusion
The high expression of multidrug resistant protein ABCB1 ABCB1 in FaDu/T cells is an important cause of multidrug resistance.
2. paclitaxel affects the expression of multidrug resistant protein ABCB1 and ABCG2 through JNK signal transduction pathway
3. the alternately altered expression of various drug resistant proteins, thus producing resistance to different chemotherapeutic drugs, is an important reason for the failure of clinical tumor chemotherapy.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R739.63

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相關(guān)期刊論文 前1條

1 張宗敏,唐平章,徐震綱,李慶宏,胡郁華,徐國鎮(zhèn),高黎,屠規(guī)益;下咽鱗癌不同治療方案的臨床分析[J];中華腫瘤雜志;2005年01期

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本文編號：1529798