本文選題：Notch調(diào)節(jié)錨蛋白 + 甲狀腺未分化癌�。� 參考：《第二軍醫(yī)大學(xué)》2016年博士論文

【摘要】：背景與目的甲狀腺未分化癌(anaplastic thyroid cancer,ATC)約占甲狀腺癌7%~8%,惡性程度極高,發(fā)展迅速,浸潤性生長,早期轉(zhuǎn)移,多數(shù)患者在確診時(shí)已經(jīng)出現(xiàn)了遠(yuǎn)處轉(zhuǎn)移,與分化較好的甲狀腺乳頭狀癌和濾泡狀癌相比預(yù)后較差,一經(jīng)診斷生存期通常一年左右。目前未分化癌尚無有效的治療方法,治療方式亦無統(tǒng)一標(biāo)準(zhǔn)。不管是手術(shù)治療,還是放療、化療,單獨(dú)或聯(lián)合應(yīng)用治療甲狀腺未分化癌都難以獲得較好療效。甲狀腺未分化癌的治療仍然是世界各醫(yī)療中心所面臨的挑戰(zhàn),盡管進(jìn)行了手術(shù)、放療、化療等各項(xiàng)臨床治療相關(guān)的研究,但目前為止未找到統(tǒng)一、有效的治療方法。由于疾病發(fā)生率低,幾乎所有有關(guān)甲狀腺未分化癌的治療結(jié)果都是回顧性的小樣本資料,缺乏大樣本前瞻性治療方法的總結(jié)。當(dāng)今隨著分子生物學(xué)技術(shù)及免疫學(xué)技術(shù)的迅猛發(fā)展和人類對惡性腫瘤的發(fā)展機(jī)制認(rèn)識不斷深入,生物醫(yī)學(xué)療法已經(jīng)成為第四種腫瘤的治療模式,其中通過制定個(gè)體化的腫瘤靶向治療,及其相應(yīng)靶點(diǎn)檢測,提高腫瘤的治療效果,具有關(guān)鍵的臨床意義�；诩谞钕傥捶只﹩渭兪中g(shù)、放療、化療療效差的特點(diǎn),各個(gè)腫瘤中心探索治療未分化癌的新方法,其中甲狀腺未分化腫瘤的靶向治療為提高患者預(yù)后提供了新的思路。Notch信號在腫瘤的發(fā)生和發(fā)展過程中起著非常關(guān)鍵的作用,Notch信號紊亂不僅能夠直接導(dǎo)致惡性腫瘤的發(fā)生,而且通過與其他信號通路的相互作用,可以間接地誘導(dǎo)腫瘤的最終形成。在很多惡性腫瘤的發(fā)生發(fā)展過程中均報(bào)道Notch信號通路的異常信號表達(dá),如前列腺癌、子宮頸癌、乳腺癌等。Notch信號通路成員存在于甲狀腺組織中,且Notch受體表達(dá)受促甲狀腺激素的調(diào)節(jié)。甲狀腺癌中Notch信號通路存在異常表達(dá),如在乳頭狀癌和濾泡狀癌中,Notch基因表達(dá)較正常甲狀腺組織及癌旁組織高。Notch調(diào)節(jié)錨蛋白(NOTCH-regulated ankyrin repeat protein,NRARP)是Notch信號通路的重要因子�？赡軈⑴c甲狀腺未分化癌的發(fā)生發(fā)展。NRARP基因編碼114氨基酸的殘基蛋白,有兩個(gè)錨蛋白重復(fù)序列,是Notch信號通路上的一個(gè)成員。NRARP可以阻礙Notch誘導(dǎo)的CBF-1活化,能促進(jìn)胞內(nèi)段降解,抑制Notch信號通路激活。NRARP表達(dá)受Notch基因調(diào)控,Notch基因表達(dá)升高可以誘導(dǎo)NRARP表達(dá)增高,說明NRARP作為機(jī)體的一種反饋機(jī)制,可限制Notch信號過度增強(qiáng)和持續(xù)表達(dá)。至今尚無關(guān)于notch基因和nrarp基因在甲狀腺未分化癌的細(xì)胞和組織表達(dá)的臨床及實(shí)驗(yàn)報(bào)道。本課題探討nrarp基因在甲狀腺未分化癌組織和細(xì)胞中的表達(dá)情況,并評估其對甲狀腺未分化癌細(xì)胞增殖、凋亡、周期、遷移和侵襲的作用,為確定治療未分化癌的關(guān)鍵靶點(diǎn)提供實(shí)驗(yàn)依據(jù)。方法收集甲狀腺未分化癌標(biāo)本34例,采用免疫組化方法檢測并比較nrarp蛋白在癌組織和癌旁組織中的表達(dá)水平,并根據(jù)表達(dá)水平分成高表達(dá)和低表達(dá)組,進(jìn)行預(yù)后生存分析;采用攜帶nrarp-shrna的慢病毒(lenti-nrarp-shrna)抑制nrarp基因mrna和蛋白的表達(dá)水平并進(jìn)行驗(yàn)證;采用wst-1方法檢測lenti-nrarp-shrna對甲狀腺癌細(xì)胞株bht-101和8305c體外增殖的影響,并通過裸鼠荷瘤模型檢測lenti-nrarp-shrna對細(xì)胞株體內(nèi)增殖的影響;采用細(xì)胞流式儀技術(shù)檢測lenti-nrarp-shrna對細(xì)胞株周期和凋亡的影響;采用transwell法檢測lenti-nrarp-shrna對細(xì)胞遷移和侵襲的影響;采用westernblot技術(shù)檢測lenti-nrarp-shrna處理后一系列與周期蛋白(p21、cyclind1)、凋亡蛋白(bax、bcl-2、caspase-3)、遷移調(diào)控有關(guān)蛋白的表達(dá)水平。結(jié)果nrarp蛋白在甲狀腺癌組織中表達(dá)水平顯著高于正常組織,lenti-nrarp-shrna顯著抑制bht-101和8305c細(xì)胞體內(nèi)和體外增殖;lenti-nrarp-shrna誘導(dǎo)p21蛋白表達(dá)、抑制cyclind1蛋白表達(dá)進(jìn)而誘導(dǎo)細(xì)胞發(fā)生g1期阻滯;lenti-nrarp-shrna誘導(dǎo)bax蛋白表達(dá)、抑制bcl-2蛋白表達(dá),并激活caspase-3蛋白,導(dǎo)致細(xì)胞凋亡;lenti-nrarp-shrna抑制mmp-9蛋白表達(dá),抑制細(xì)胞遷移和侵襲。結(jié)果一、notch蛋白和nrarp蛋白在甲狀腺癌組織及轉(zhuǎn)移灶中較癌旁組織高表達(dá)。nrarp高表達(dá)與患者預(yù)后呈顯著負(fù)性相關(guān),提示nrarp可作為甲狀腺癌患者靶向治療的潛在靶點(diǎn)。二、干擾nrarp表達(dá)可顯著抑制細(xì)胞株體內(nèi)和體外增殖;可提高腫瘤周期相關(guān)抑癌基因p21表達(dá),抑制促癌基因cyclind1,從而誘導(dǎo)腫瘤細(xì)胞g1期阻滯;使凋亡相關(guān)bax蛋白表達(dá)增高,bcl-2表達(dá)降低,caspase-3被激活,從而提高凋亡細(xì)胞比例;并抑制mmp-9表達(dá)進(jìn)而減弱未分化癌細(xì)胞的侵襲能力。結(jié)論NRARP與甲狀腺未分化癌發(fā)生發(fā)展密切相關(guān),降低其表達(dá)水平可以誘導(dǎo)周期阻滯、促進(jìn)細(xì)胞凋亡進(jìn)而抑制甲狀腺癌細(xì)胞遷移、增殖和侵襲,可成為甲狀腺未分化癌靶向治療的潛在靶點(diǎn)。
[Abstract]:Background and objective anaplastic thyroid cancer (ATC) accounts for about 7%~8% of thyroid cancer. It has a high degree of malignancy, rapid development, invasive growth and early metastasis. Most patients have had distant metastases at the time of diagnosis. Compared with the poorly differentiated thyroid papillary and follicular carcinomas, the prognosis is poor. It is usually about a year or so. There is no effective treatment for undifferentiated carcinoma and there is no unified standard for treatment. It is difficult to achieve better curative effect, whether it is surgical treatment, radiotherapy, chemotherapy, alone or combined with treatment of undifferentiated thyroid cancer. The treatment of undifferentiated thyroid cancer is still a challenge to various medical centers in the world, though Studies related to surgery, radiotherapy, chemotherapy and other clinical treatments were carried out, but the unified and effective treatment has not been found so far. Due to the low incidence of disease, almost all the results of the treatment of undifferentiated thyroid cancer are retrospective and small sample data, lack of a summary of large sample prospective treatment methods. The rapid development of physical and immunological technology and the deepening of human understanding of the development mechanism of malignant tumor. Biomedical therapy has become the treatment mode of fourth kinds of tumor. It is of key clinical significance to improve the therapeutic effect of tumor by formulating individualized tumor targeting therapy and its corresponding target detection. A new approach to the treatment of undifferentiated cancer in the center of the thyroid undifferentiated carcinoma with simple surgery, radiotherapy and chemotherapy, and the targeted treatment of undifferentiated thyroid tumors provides a new way of thinking to improve the prognosis of the patients. The.Notch signal plays a very important role in the occurrence and development of the tumor, Notch signal. The disorder can not only directly lead to the occurrence of malignant tumors, but also indirectly induce the final formation of the tumor by interaction with other signaling pathways. In the course of the development of many malignant tumors, the abnormal signal expression of Notch signaling pathway, such as prostate cancer, cervical cancer, breast cancer and other.Notch signaling pathways, is reported. In thyroid tissue, the expression of Notch receptor is regulated by thyroid stimulating hormone. There is an abnormal expression of Notch signaling pathway in thyroid carcinoma, such as in papillary and follicular carcinomas, Notch gene expression is higher than normal thyroid tissue and paracancerous tissue.Notch regulated anchorage white (NOTCH-regulated ankyrin repeat protein, NRARP). The important factor of Notch signaling pathway may be involved in the development and development of the.NRARP gene encoding 114 amino acid residues in the undifferentiated thyroid carcinoma, and there are two anchorage repeat sequences. A member of the Notch signaling pathway,.NRARP, can impede the activation of CBF-1 induced by Notch, promote the degradation of the intracellular segments and inhibit the activation of.NRAR by the Notch signaling pathway. The expression of P is regulated by Notch gene, and the increase of Notch gene expression can induce the increase of NRARP expression. As a feedback mechanism of the body, NRARP can restrict the excessive enhancement and continuous expression of Notch signal. There is no clinical and experimental report on the expression of Notch gene and nrarp gene in undifferentiated thyroid carcinoma. To investigate the expression of nrarp gene in the tissues and cells of undifferentiated thyroid carcinoma and evaluate its effect on the proliferation, apoptosis, cycle, migration and invasion of undifferentiated thyroid cancer cells, and provide experimental basis for determining the key targets for undifferentiated carcinoma. Methods 34 specimens of undifferentiated thyroid carcinoma were collected and detected by immunohistochemical method. The expression level of nrarp protein in cancer tissue and para cancer tissue was compared, and the expression level was divided into high expression and low expression group according to the expression level, and the prognosis survival analysis was carried out. The expression level of nrarp gene mRNA and protein was inhibited by the lentivirus (lenti-nrarp-shrna) carrying nrarp-shrna and the WST-1 method was used to detect lenti-nrarp-shrna. The effects of bht-101 and 8305c on the proliferation of thyroid cancer cell lines in vitro, and the effect of lenti-nrarp-shrna on the proliferation of cell lines in nude mice were detected by the nude mouse model. The effect of lenti-nrarp-shrna on cell cycle and apoptosis was detected by cell flow cytometry, and lenti-nrarp-shrna was used to detect the migration and invasion of cells by the Transwell method. Westernblot technique was used to detect the expression level of protein in lenti-nrarp-shrna, p21, CyclinD1, apoptosis protein (Bax, Bcl-2, caspase-3), and the expression level of nrarp protein in thyroid carcinoma tissue was significantly higher than that of normal tissue, lenti-nrarp-shrna significantly inhibited bht-101 and 8305. The proliferation of C cells in vivo and in vitro, lenti-nrarp-shrna induced p21 protein expression, inhibition of cyclinD1 protein expression and induced G1 phase block, lenti-nrarp-shrna induced Bax protein expression, inhibition of Bcl-2 protein expression, and activation of caspase-3 protein, leading to cell apoptosis; lenti-nrarp-shrna inhibits the expression of MMP-9 protein, inhibits cell migration and inhibits cell migration. The high expression of Notch protein and nrarp protein in thyroid carcinoma tissues and metastatic foci was significantly negatively correlated with the prognosis of the patients, suggesting that nrarp could be a potential target for the target treatment of thyroid cancer patients. Two, interference nrarp expression could inhibit the proliferation of cell lines in vivo and in vitro, and improve the swelling of the cells. The expression of tumor suppressor gene p21, inhibiting the oncogene CyclinD1, inducing the G1 block of tumor cells, increasing the expression of Bax protein, decreasing the expression of Bcl-2, activating the Caspase-3, and increasing the percentage of apoptotic cells, and inhibiting the MMP-9 expression to weaken the invasion ability of undifferentiated cancer cells. Conclusion NRARP and thyroid are not. It is closely related to the development and development of differentiated cancer. Reducing its expression level can induce cell cycle arrest, promote cell apoptosis and inhibit the migration, proliferation and invasion of thyroid cancer cells, which may be a potential target for targeted therapy for undifferentiated thyroid cancer.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R736.1

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