【摘要】：目的:甲狀腺未分化癌(anaplastic thyroid carcinoma,ATC)是甲狀腺癌中發(fā)病率較低但惡性度很高預(yù)后極差的惡性腫瘤,目前治療的方法主要有傳統(tǒng)手術(shù)和放化療結(jié)合手術(shù)的方式,但效果都不理想。隨著ATC遺傳和分子發(fā)病機(jī)制研究的不斷深入,基因靶向治療也逐漸興起,并有望成ATC治療的發(fā)展方向之一。Notch信號(hào)通路在進(jìn)化上高度保守,通過相鄰細(xì)胞之間的相互作用調(diào)節(jié)細(xì)胞、組織、器官的分化和發(fā)育。軟木肟酸(Suberoyl bis-hydroxamic acid,SBHA)屬于第二代異羥肟酸類組蛋白乙酰化酶抑制劑,它可以誘導(dǎo)激活Notch1信號(hào)通路,在神經(jīng)內(nèi)分泌腫瘤中起到抑癌作用。本研究探討在甲狀腺未分化癌細(xì)胞中應(yīng)用軟木肟酸激活Notch1信號(hào)通路,是否可以抑制腫瘤細(xì)胞增殖誘導(dǎo)細(xì)胞凋亡,在此基礎(chǔ)上探討Notch1信號(hào)通路是否通過P53通路發(fā)揮作用。方法:1.收集天津醫(yī)科大學(xué)腫瘤醫(yī)院甲狀腺未分化癌組織標(biāo)本16例,應(yīng)用免疫組織化學(xué)染色檢測(cè)Notch1和P53蛋白表達(dá)情況。應(yīng)用Western blot技術(shù)檢測(cè)正常甲狀腺細(xì)胞系、甲狀腺乳頭狀癌系和甲狀腺未分化癌細(xì)胞系中Notch1蛋白的表達(dá)情況。2.應(yīng)用不同濃度SBHA作用于甲狀腺未分化癌細(xì)胞系,應(yīng)用MTT實(shí)驗(yàn)判斷SBHA對(duì)甲狀腺未分化癌細(xì)胞增殖情況的影響,應(yīng)用流式細(xì)胞術(shù)判斷SBHA對(duì)甲狀腺未分化癌細(xì)胞促凋亡效應(yīng),應(yīng)用Western blot技術(shù)檢測(cè)Notch1、cleaved caspase3、cleaved PRAP和Bax蛋白的表達(dá)情況,來驗(yàn)證細(xì)胞凋亡情況。應(yīng)用Real-time PCR技術(shù)檢測(cè)Notch1和Hes1的m RNA水平,來判斷Notch1信號(hào)通路的激活情況。應(yīng)用siRNA干擾細(xì)胞系Notch1的表達(dá),再應(yīng)用SBHA激活該信號(hào)通路,判斷SBHA是否作用于Notch1通路。3.應(yīng)用不同濃度SBHA作用于甲狀腺未分化癌細(xì)胞系,應(yīng)用Western blot技術(shù)檢測(cè)P53和P21蛋白的表達(dá)情況,來判斷細(xì)胞凋亡是否通過該通路完成,并分析和Notch1通路是否有關(guān)。應(yīng)用Real-time PCR技術(shù)檢測(cè)P53和P21的mRNA情況驗(yàn)證P53和P21激活情況。應(yīng)用免疫共沉淀實(shí)驗(yàn)檢測(cè)Notch1蛋白和P53蛋白是否存在聯(lián)系。應(yīng)用shRNA干擾細(xì)胞系P53的表達(dá),再應(yīng)用SBHA激活Notch1通路,驗(yàn)證Notch1和P53兩者的關(guān)系。結(jié)果:1.免疫組織化學(xué)結(jié)果顯示,16例組織切片中僅有1例切片呈現(xiàn)Notch1和P53陽性結(jié)果,且為同一患者組織。Western blot檢測(cè)結(jié)果顯示正常甲狀腺細(xì)胞系中有Notch1蛋白表達(dá),甲狀腺乳頭狀癌系中Notch1蛋白低表達(dá)而甲狀腺未分化癌細(xì)胞系中Notch1蛋白不表達(dá)。2.應(yīng)用不同濃度SBHA作用于甲狀腺未分化癌細(xì)胞系,可以誘導(dǎo)Notch1mRNA和蛋白的表達(dá),活化Notch1信號(hào)通路,有效的抑制細(xì)胞的增殖,誘導(dǎo)促凋亡蛋白的表達(dá),促進(jìn)細(xì)胞凋亡,并且呈時(shí)間和劑量雙重依賴性。3.在甲狀腺未分化癌細(xì)胞系中應(yīng)用不同濃度SBHA可以誘導(dǎo)P53和P21蛋白和mRNA時(shí)間和劑量依賴性的表達(dá),這與Notch1一致。免疫共沉淀實(shí)驗(yàn)提示兩者存在關(guān)聯(lián),shRNA干擾實(shí)驗(yàn)提示SBHA激活Notch1信號(hào)通路是通過P53通路誘導(dǎo)細(xì)胞凋亡的。結(jié)論:1.Notch1和P53蛋白在甲狀腺未分化癌組織中處于低表達(dá)狀態(tài),Notch1蛋白在甲狀腺未分化癌細(xì)胞系中低表達(dá)或不表達(dá)。2.SBHA在甲狀腺未分化癌細(xì)胞系中可以激活Notch1信號(hào)通路,抑制細(xì)胞的增殖,并且呈時(shí)間和劑量雙重依賴誘導(dǎo)細(xì)胞凋亡。3.在甲狀腺未分化癌細(xì)胞系中,Notch1和P53兩者存在聯(lián)系,SBHA激活Notch1信號(hào)通路是通過P53通路誘導(dǎo)細(xì)胞凋亡的。
[Abstract]:Objective: Undifferentiated thyroid carcinoma (ATC) is a malignant tumor with a low incidence but a high degree of malignancy and a poor prognosis. Currently, the main treatment methods are traditional surgery combined with radiotherapy and chemotherapy, but the effect is not satisfactory. Notch signaling pathway is highly conserved in evolution and regulates the differentiation and development of cells, tissues and organs through the interaction between adjacent cells. Suberoyl bis-hydroxamic acid (SBHA) belongs to the second generation of isohydroxamic acid histone acetylation. Enzyme inhibitors, which can induce and activate Notch1 signaling pathway, play an inhibitory role in neuroendocrine tumors. This study was to investigate whether pyramidal acid activates Notch1 signaling pathway in undifferentiated thyroid cancer cells and inhibits proliferation and induces apoptosis of tumor cells. On this basis, we investigated whether Notch1 signaling pathway is mediated by P53. Methods: 1. The expression of Notch1 and P53 proteins in 16 undifferentiated thyroid carcinoma tissues collected from the Tumor Hospital of Tianjin Medical University was detected by immunohistochemical staining. The expression of Notch1 protein in normal thyroid cell line, thyroid papillary carcinoma line and thyroid undifferentiated carcinoma cell line was detected by Western blot. Expression of SBHA in undifferentiated thyroid carcinoma cell lines was determined by MTT assay. The effect of SBHA on the proliferation of undifferentiated thyroid carcinoma cells was evaluated by flow cytometry. Notch 1, cleaved caspase 3, cleaved PRA were detected by Western blot. Real-time PCR was used to detect the level of Notch1 and Hes1 m RNA and to determine the activation of Notch1 signaling pathway. Western blot was used to detect the expression of P53 and P21 proteins in undifferentiated thyroid carcinoma cell lines to determine whether apoptosis was completed through this pathway and whether there was any relationship between the pathway and Notch1. Real-time PCR was used to detect the mRNA of P53 and P21 to verify the activation of P53 and P21. Immunocoprecipitation assay was used to detect the expression of P53 and P21 proteins. The relationship between Notch1 and P53 was examined by interfering with the expression of P53 by shRNA and activating Notch1 pathway by SBHA. Results: 1. Immunohistochemistry showed that only one of the 16 tissue sections showed Notch1 and P53 positive results, and were the same patient tissue. The results showed that Notch1 protein was expressed in normal thyroid cell lines, but not in undifferentiated thyroid carcinoma cell lines. 2. The expression of Notch1 mRNA and protein was induced and Notch1 signaling pathway was activated by different concentrations of SBHA in undifferentiated thyroid carcinoma cell lines. 3. Different concentrations of SBHA could induce time and dose dependent expression of P53 and P21 proteins and mRNA in undifferentiated thyroid carcinoma cell lines, which was consistent with Notch1. Conclusion: 1. Notch1 and P53 proteins are low expressed in undifferentiated thyroid carcinoma tissues, and Notch1 protein is low or not expressed in undifferentiated thyroid carcinoma cell lines. 2. SBHA is not expressed in undifferentiated thyroid carcinoma cell lines. In undifferentiated thyroid carcinoma cell lines, Notch1 and P53 are related, and SBHA activates Notch1 signaling pathway to induce apoptosis through P53 pathway.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R736.1

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