【摘要】：背景與目的:甲狀腺癌已成為常見(jiàn)的、多發(fā)性疾病。其中甲狀腺乳頭狀癌是發(fā)病率最高的一種,占所有類型甲狀腺癌的85-90%。BRAFV600E基因突變是BRAF基因最常見(jiàn)的一種突變,可改變癌細(xì)胞生物學(xué)狀態(tài),加速了惡性腫瘤的進(jìn)展期,在甲狀腺癌、結(jié)腸癌以及黑色素瘤等惡性腫瘤中發(fā)生率較高,是目前新型的腫瘤標(biāo)志物,提示臨床預(yù)后較差。而BRAFV600E基因突變?cè)诩谞钕偃轭^狀癌中發(fā)生率最高,在其它類型的甲狀腺癌也已發(fā)現(xiàn),如甲狀腺未分化癌。雖然目前臨床上治療甲狀腺乳頭狀癌的主要方式為手術(shù)切除,內(nèi)分泌治療和放射性碘治療,然而對(duì)于侵襲性甲狀腺癌,放射性碘治療耐受的患者,這些傳統(tǒng)治療方式的預(yù)后效果仍然不理想。雖然針對(duì)BRAFV600E基因突變的靶向藥物在臨床前期和臨床治療中已經(jīng)應(yīng)用,然而經(jīng)過(guò)臨床觀察BRAFV600E抑制劑例如PLX4032(vemurafenib)在治療BRAFV600E基因突變的黑色素瘤時(shí)展示了強(qiáng)效的抗腫瘤特性,而對(duì)BRAFV600E基因突變的甲狀腺癌的治療中,效果一般。近年來(lái)研究發(fā)現(xiàn)BRAFV600E抑制劑可激活BRAFV600E基因突變的結(jié)腸癌細(xì)胞中EGFR磷酸化高表達(dá),從而導(dǎo)致其對(duì)BRAF抑制劑產(chǎn)生耐藥性,目前已證實(shí)EGFR抑制劑和BRAF抑制劑聯(lián)合應(yīng)用治療可產(chǎn)生很好的協(xié)同作用。然而由于EGFR抑制劑和BRAF抑制劑價(jià)格昂貴,以及其自身毒副作用,極大的限制其在臨床應(yīng)用的可行性,因此尋找新型的治療甲狀腺癌細(xì)胞的藥物代替BRAFV600E抑制劑,研究其生物學(xué)活性和抗腫瘤的機(jī)制,為其在今后臨床應(yīng)用打下基礎(chǔ)。方法:為了尋找能抑制甲狀腺癌細(xì)胞活性的小分子化合物,我們采用高通量藥物篩選方法,將2種含有BRAFV600E基因突變的人甲狀腺癌細(xì)胞株8505C和KTC-1作為篩選對(duì)象,而將另外1種非甲狀腺癌并具有BRAFV600E基因突變的人黑色素瘤細(xì)胞株Malme-3M做為參照。篩選5種藥庫(kù)共包含了約5200種小分子化合物,對(duì)篩選出的候選藥物藥物秋水仙堿進(jìn)行重新定位及其機(jī)理的研究。通過(guò)Alamar Blue染色法檢測(cè)細(xì)胞活力,細(xì)胞計(jì)數(shù)方法檢測(cè)其細(xì)胞增殖能力,通過(guò)流式細(xì)胞術(shù)測(cè)定細(xì)胞周期確定藥物的生物學(xué)特性;通過(guò)westernblot方法和Annexin V-FITC與PI雙染色結(jié)合流式細(xì)胞術(shù)測(cè)定細(xì)胞凋亡;通過(guò)Westernblot方法和建立穩(wěn)定秋水仙堿耐藥細(xì)胞克隆的方法尋找其誘導(dǎo)凋亡的機(jī)制。建立小鼠甲狀腺癌細(xì)胞(8505C和WRO)接種模型,確定秋水仙堿在體內(nèi)動(dòng)物試驗(yàn)的有效性,以及其在小鼠體內(nèi)有無(wú)毒副作用。結(jié)果:高通量藥物篩選成功發(fā)現(xiàn)了74個(gè)候選藥物,將候選藥物之一AV-412與針對(duì)Malme-3M的藥物PLX4032進(jìn)行驗(yàn)證試驗(yàn),結(jié)果與高通量篩選實(shí)驗(yàn)相符,證實(shí)該系統(tǒng)的可靠性及可信性。秋水仙堿作為候選藥物之一作為本次課題主要研究對(duì)象。通過(guò)Alamar Blue染色實(shí)驗(yàn)和細(xì)胞計(jì)數(shù)實(shí)驗(yàn),秋水仙堿可明顯抑制BRAFV600E(8505C、KTC-1)和BRAFWT(WRO、TPC-1)甲狀腺癌細(xì)胞活力和細(xì)胞增殖能力,流式細(xì)胞術(shù)測(cè)定發(fā)現(xiàn)秋水仙堿可誘導(dǎo)8505C和WRO細(xì)胞阻滯在G2/M期,而進(jìn)入G1期細(xì)胞明顯減少。Annexin V-FITC與PI雙染檢測(cè)秋水仙堿誘導(dǎo)8505C和WRO細(xì)胞凋亡,與濃度和時(shí)間相關(guān)。蛋白免疫印跡試驗(yàn)表明,在8505C細(xì)胞中,秋水仙堿以濃度依賴和時(shí)間依賴的方式誘導(dǎo)PARP切割形成凋亡片段,并同時(shí)激活A(yù)KT,MEK/ERK,P38和JNK/c-Jun通路。然而通過(guò)應(yīng)用選擇性抑制劑U0126和SP600125分別對(duì)MEK1/2和JNK通路進(jìn)行抑制,干擾了秋水仙堿誘導(dǎo)凋亡的能力,導(dǎo)致其細(xì)胞活力增高,同時(shí)激活Caspase3和誘導(dǎo)PARP形成切割片段的能力明顯降低,凋亡細(xì)胞數(shù)顯著減少。相反,SB203580和LY294002分別抑制p38 MAPK和AKT通路后,對(duì)秋水仙堿誘導(dǎo)細(xì)胞凋亡作用無(wú)影響。在WRO細(xì)胞中,秋水仙堿以濃度依賴和時(shí)間依賴的方式誘導(dǎo)PARP切割形成凋亡片段,MEK,P38和JNK/c-Jun的磷酸化水平明顯上調(diào)。AKT磷酸化在秋水仙堿處理24小時(shí)表達(dá)下調(diào),而在48和72小時(shí)無(wú)明顯變化,ERK也無(wú)明顯變化,通過(guò)應(yīng)用選擇性抑制劑U0126和SP600125分別對(duì)MEK1/2和JNK進(jìn)行抑制,干擾了秋水仙堿誘導(dǎo)凋亡的能力,導(dǎo)致其細(xì)胞活力增高,Caspase 3和PARP形成切割片段的能力明顯降低,凋亡細(xì)胞數(shù)量顯著減少。SB203580抑制p38 MAPK通路后,只輕度干擾了秋水仙堿誘導(dǎo)凋亡的能力。而LY294002抑制AKT通路后,對(duì)秋水仙堿誘導(dǎo)細(xì)胞凋亡作用無(wú)明顯影響。通過(guò)建立8505C秋水仙堿耐藥細(xì)胞株和蛋白免疫印跡檢測(cè)表明,MEK1/2,ERK1/2和JNK的磷酸化水平在親代細(xì)胞中表達(dá)明顯增高,而在耐藥細(xì)胞R2和R4中幾乎未見(jiàn)激活。不同的是p38的磷酸化在耐藥細(xì)胞株中表達(dá)仍增高,與親代細(xì)胞相似。通過(guò)建立小鼠移植瘤8505C和WRO模型,每日注射不同劑量的秋水仙堿,共2周,發(fā)現(xiàn)治療組的腫瘤體積和腫瘤重量較對(duì)照組明顯降低,并具有濃度和時(shí)間依賴性;通過(guò)免疫組化PHH3染色發(fā)現(xiàn)經(jīng)秋水仙堿治療后,有絲分裂的細(xì)胞數(shù)較對(duì)照組相比明顯減少,具有濃度依賴性。通過(guò)TUNEL染色發(fā)現(xiàn)治療組的腫瘤凋亡細(xì)胞與對(duì)照組相比明顯增多,具有濃度依賴性。同時(shí),對(duì)小鼠重要臟器進(jìn)行HE染色鏡下觀察,秋水仙堿治療組與對(duì)照組均無(wú)組織損傷和毒性作用;體重監(jiān)測(cè)也表明秋水仙堿在小鼠動(dòng)物模型試驗(yàn)的安全性及耐受性。結(jié)論:1、高通量藥物篩選作為一種有效方法對(duì)藥物的生物學(xué)活性進(jìn)行重新定性提供了新的平臺(tái)。高通量篩選方法成功篩選出了熱點(diǎn)藥物秋水仙堿,并為甲狀腺癌藥物治療的選擇提供了新的思路和方法。2、秋水仙堿不僅能對(duì)具有BRAFV600E基因突變的甲狀腺癌有明顯的抑制作用,而且對(duì)其他BRAF野生型的甲狀腺癌同樣有強(qiáng)效的抗腫瘤的功能。3、秋水仙堿通過(guò)對(duì)細(xì)胞周期G2/M阻滯和誘導(dǎo)凋亡抑制了多種甲狀腺癌細(xì)胞的生長(zhǎng),具有濃度和時(shí)間相關(guān)性。4、秋水仙堿通過(guò)激活MEK/ERK和JNK/c-Jun磷酸化途徑從而誘導(dǎo)細(xì)胞凋亡,p38的磷酸化表達(dá)僅部分的導(dǎo)致了WRO細(xì)胞的凋亡,而在8505C細(xì)胞中無(wú)明顯的作用。AKT磷酸化表達(dá)并未參與細(xì)胞凋亡的過(guò)程。5、秋水仙堿在抑制甲狀腺癌8505C和WRO小鼠移植瘤的生長(zhǎng)展現(xiàn)了良好的抗腫瘤效果,而且對(duì)動(dòng)物模型無(wú)明顯的毒副作用。同時(shí)秋水仙堿作為一種已知常見(jiàn)的廣泛治療痛風(fēng)的藥物,值得在其他臨床領(lǐng)域例如甲狀腺癌中進(jìn)一步關(guān)注和研發(fā)。
[Abstract]:BACKGROUND & OBJECTIVE: Thyroid carcinoma has become a common and multiple disease. Papillary thyroid carcinoma is the most common type of thyroid cancer, accounting for 85-90% of all types of thyroid cancer. Colorectal cancer, melanoma and other malignant tumors have a high incidence and are new tumor markers, suggesting a poor clinical prognosis. BRAFV600E mutation has the highest incidence in papillary thyroid cancer and has been found in other types of thyroid cancer, such as undifferentiated thyroid cancer. Although the current clinical treatment of papillary thyroid cancer. Surgical resection, endocrine therapy, and radioiodine therapy are the main modalities of carcinomas. However, the prognosis of these traditional therapies is still unsatisfactory for patients with invasive thyroid cancer who are tolerant to radioiodine therapy. BRAFV600E inhibitors such as PLX4032 (vemurafenib) have shown potent antitumor properties in the treatment of melanoma with BRAFV600E gene mutation, while BRAFV600E gene mutation has been shown to be effective in thyroid cancer. Recent studies have shown that BRAFV600E inhibitors can activate BRAFV600E gene mutation in colorectal cancer cells. High phosphorylation of EGFR leads to resistance to BRAF inhibitors. It has been proved that EGFR inhibitors combined with BRAF inhibitors can produce a good synergistic effect. However, the high price of EGFR inhibitors and BRAF inhibitors, as well as their own toxic and side effects, greatly limit the feasibility of clinical application. To find a new drug to replace BRAFV600E inhibitor and study its biological activity and anti-tumor mechanism, so as to lay a foundation for its clinical application in the future.Methods: In order to find a small molecule compound which can inhibit the activity of thyroid cancer cells, we used high-throughput drug screening method to select two kinds of drugs containing BRAFV600E. Human thyroid cancer cell lines 8505C and KTC-1 were selected as the screening targets, while another non-thyroid cancer cell line Malme-3M with BRAFV600E gene mutation was used as the reference. Cell viability was detected by Alamar Blue staining, cell proliferation was detected by cell counting, and the biological characteristics of the drug were determined by flow cytometry; apoptosis was detected by Western blot, Annexin V-FITC and PI double staining combined with flow cytometry; and apoptosis was detected by Western blot. Methods The mechanism of apoptosis induced by colchicine-resistant cells was investigated by establishing stable clone of colchicine-resistant cells. The inoculation model of mouse thyroid cancer cells (8505C and WRO) was established to determine the validity of colchicine in vivo animal test and its toxicity in mice. AV-412, one of the candidate drugs, was validated with PLX4032 for Malme-3M. The results were in good agreement with high throughput screening experiments, which confirmed the reliability and reliability of the system. Colchicine as one of the candidate drugs was the main research object of this project. Colchicine was cocoa through Alamar Blue staining experiment and cell counting experiment. The viability and proliferation of BRAFV600E (8505C, KTC-1) and BRAFWT (WRO, TPC-1) thyroid cancer cells were significantly inhibited by colchicine. Flow cytometry showed that colchicine could induce 8505C and WRO cells to block at G2/M phase, but the number of cells entering G1 phase was significantly decreased. The apoptosis of 8505C and WRO cells induced by colchicine was detected by Annexin V-FITC and PI staining. In 8505C cells, colchicine induced PARP cleavage to form apoptotic fragments in a concentration-and time-dependent manner, and activated AKT, MEK/ERK, P38 and JNK/c-Jun pathways simultaneously. However, selective inhibitors U0126 and SP600125 were used to inhibit MEK1/2 and JNK pathways respectively, interfering with them. In contrast, SB203580 and LY294002 inhibited p38 MAPK and AKT pathways respectively, but had no effect on colchicine-induced apoptosis in WRO cells. The phosphorylation levels of MEK, P38 and JNK/c-Jun were significantly up-regulated. AKT phosphorylation was down-regulated after colchicine treatment for 24 hours, but did not change significantly at 48 and 72 hours, and ERK did not change significantly. The selective inhibitors U0126 and SP600125 were used to treat MEK, respectively. 1/2 and JNK inhibited the ability of colchicine to induce apoptosis, resulting in increased cell viability, decreased the ability of caspase 3 and PARP to form cleavage fragments, and decreased the number of apoptotic cells. SB203580 inhibited the p38 MAPK pathway, only slightly interfered with the ability of colchicine to induce apoptosis. The phosphorylation levels of MEK1/2, ERK1/2 and JNK in the parental cells were significantly higher than those in the drug-resistant cells R2 and R4. The phosphorylation of p38 was different in the drug-resistant cells. The tumor volume and weight in the treatment group were significantly lower than those in the control group, and the tumor volume and weight were in a concentration-and time-dependent manner. The number of mitotic cells in the treatment group was significantly lower than that in the control group, which was concentration-dependent. TUNEL staining showed that the number of apoptotic cells in the treatment group was significantly higher than that in the control group. Conclusion: 1. High-throughput drug screening provides a new platform for characterizing the biological activity of drugs as an effective method. Colchicine, a hot drug, was successfully screened out by high-throughput screening method, and it is also a drug for thyroid cancer. Colchicine can inhibit not only BRAFV600E gene mutation but also other BRAFV600E wild-type thyroid cancers. 3. Colchicine inhibits a variety of thyroid cancers by blocking G2/M cell cycle and inducing apoptosis. Colchicine induces apoptosis by activating MEK/ERK and JNK/c-Jun phosphorylation pathways. The phosphorylation of p38 only partially induces apoptosis in WRO cells, but not in 8505C cells. AKT phosphorylation does not participate in the process of apoptosis. Colchicine has been shown to be effective in inhibiting the growth of transplanted thyroid cancer in 8505C and WRO mice, and has no significant toxic and side effects on animal models.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R736.1

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3 副主任醫(yī)師 韓詠霞;不耐受秋水仙堿怎么辦[N];醫(yī)藥經(jīng)濟(jì)報(bào);2006年

4 程書權(quán);百年老藥 活力不減[N];醫(yī)藥經(jīng)濟(jì)報(bào);2001年

5 教授 呂群;“是痛風(fēng)就上秋水仙堿”欠妥[N];家庭醫(yī)生報(bào);2003年

6 阿勝;最佳劑量仍難琢磨[N];醫(yī)藥經(jīng)濟(jì)報(bào);2004年

7 董江萍;FDA停止生產(chǎn)銷售秋水仙堿類注射劑[N];中國(guó)醫(yī)藥報(bào);2008年

8 吳遼;CORP研究證實(shí) 秋水仙堿可防止心包炎復(fù)發(fā)[N];中國(guó)醫(yī)藥報(bào);2011年

9 ;抗痛靈治療急性痛風(fēng)性關(guān)節(jié)炎[N];中國(guó)中醫(yī)藥報(bào);2005年

10 保健時(shí)報(bào)特約專家 車會(huì)蓮;通緝蔬菜中的毒素[N];保健時(shí)報(bào);2006年

相關(guān)博士學(xué)位論文 前3條

1 張樂(lè);高通量藥物篩選識(shí)別秋水仙堿作為甲狀腺癌新型抑制劑的研究[D];吉林大學(xué);2016年

2 黃文彥;腎間質(zhì)纖維化的分子機(jī)制探討以及秋水仙堿防治作用的研究[D];南京醫(yī)科大學(xué);2003年

3 王文蘋;秋水仙堿口服緩控釋劑型研究[D];成都中醫(yī)藥大學(xué);2008年

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1 肖惠;秋水仙堿配合SrCl_2對(duì)小鼠卵母細(xì)胞去核效果及其機(jī)制研究[D];華中農(nóng)業(yè)大學(xué);2015年

2 劉俊;秋水仙堿治療斯氏貍殖吸蟲感染大鼠肝臟損傷的分子機(jī)制研究[D];昆明醫(yī)科大學(xué);2015年

3 曹福悅;秋水仙堿的電化學(xué)行為及其應(yīng)用研究[D];中南大學(xué);2012年

4 何純蓮;百合中秋水仙堿的分離應(yīng)用研究[D];湖南大學(xué);2003年

5 賀世洪;百合中秋水仙堿及其有效成分分析研究[D];中南大學(xué);2002年

6 李谷才;超臨界流體萃取百合中的秋水仙堿[D];中南大學(xué);2004年

7 杜航航;秋水仙堿對(duì)人瘢痕疙瘩成纖維細(xì)胞的影響[D];重慶醫(yī)科大學(xué);2011年

8 王葉新;局部注射秋水仙堿治療急性痛風(fēng)性關(guān)節(jié)炎的實(shí)驗(yàn)研究[D];重慶醫(yī)科大學(xué);2007年

9 雷三喜;秋水仙堿對(duì)小鼠膠質(zhì)瘤母細(xì)胞抑制作用的體外實(shí)驗(yàn)研究和對(duì)運(yùn)動(dòng)功能的影響[D];昆明醫(yī)學(xué)院;2008年

10 陳雪梅;維吾爾藥材秋水仙體外肝毒性評(píng)價(jià)及其機(jī)制探討[D];新疆大學(xué);2011年

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