本文選題：SAHA + CTSB��； 參考：《沈陽(yáng)醫(yī)學(xué)院》2017年碩士論文

【摘要】：目的乳腺癌(Breast Cancer,BC)是女性中最常見的惡性腫瘤之一,其發(fā)病人群主要集中在40歲～60歲之間的圍絕經(jīng)期女性。在歐美等發(fā)達(dá)國(guó)家中,乳腺癌發(fā)病率位居女性惡性腫瘤的首位。近年來(lái),隨著我國(guó)現(xiàn)代化城市建設(shè)的不斷加快以及生活壓力的不斷增加,乳腺癌在我國(guó)大中城市的發(fā)病率逐年增加,且呈現(xiàn)年輕化趨勢(shì),嚴(yán)重威脅著女性的身心健康。在真核生物中,組蛋白的乙�；c去乙�；揎椩诨虮磉_(dá)中起到重要的調(diào)控作用。染色質(zhì)的高乙�；癄顟B(tài)能導(dǎo)致腫瘤抑制基因或促凋亡基因高表達(dá)。組蛋白去乙�；敢种苿�(Histone Deacetylase Inhibitor,HDACi)辛二酰苯胺異羥肟酸(SuberoylanilideHydroxamicAcid,SAHA)可通過(guò)提高染色質(zhì)特定區(qū)域組蛋白乙�；接绊懟虻谋磉_(dá),已成為一類新的抗腫瘤藥物。大量的研究發(fā)現(xiàn),SAHA能夠引發(fā)多種腫瘤細(xì)胞生長(zhǎng)停滯和死亡,其中包括多種乳腺癌細(xì)胞系MCF-7、MDA-MB-231、MDA-MB-435、SKBr-3 等。組織蛋白酶B(CathepsinB,CTSB)是一個(gè)溶酶體內(nèi)的半胱氨酸蛋白酶。近年來(lái)的研究表明,CTSB表達(dá)水平的改變與腫瘤細(xì)胞(乳腺癌、卵巢癌、膀胱癌、肺癌、腸癌、黑色素瘤、胃癌等)的增殖、侵襲、轉(zhuǎn)移和血管生成密切相關(guān)。因此CTSB在腫瘤細(xì)胞中的過(guò)表達(dá)被認(rèn)為與臨床腫瘤的侵襲性強(qiáng)和不良預(yù)后有關(guān)。但也有研究表明,CTSB的表達(dá)對(duì)某些腫瘤細(xì)胞的凋亡和自噬也有誘導(dǎo)作用。自噬(Autophagy)是一種能夠清除機(jī)體內(nèi)異常蛋白質(zhì)和損傷細(xì)胞器而有利于細(xì)胞內(nèi)各物質(zhì)保持穩(wěn)態(tài)的機(jī)制。在缺氧、能量缺乏、感染炎癥等應(yīng)激狀態(tài)下,自噬可抑制有毒甚至致癌的受損蛋白質(zhì)和細(xì)胞器的聚集,保護(hù)細(xì)胞進(jìn)而抑制細(xì)胞癌變;然而在腫瘤后期時(shí),自噬則給腫瘤細(xì)胞提供無(wú)限生長(zhǎng)的養(yǎng)分,促進(jìn)癌細(xì)胞增殖。近年來(lái)隨著對(duì)自噬研究的不斷深入,學(xué)者們發(fā)現(xiàn)自噬及其相關(guān)分子在乳腺癌的增殖和治療中扮演重要的角色,因此越來(lái)越受到人們的重視。本研究旨在初步探討SAHA對(duì)兩種乳腺癌細(xì)胞系MCF-7和MDA-MB-231細(xì)胞增殖能力的影響;以及對(duì)兩種乳腺癌細(xì)胞CTSB表達(dá)變化的影響。由于CTSB是溶酶體中的半胱氨酸蛋白酶,然而參與自噬發(fā)生的關(guān)鍵酶主要集中在溶酶體中,因此我們推測(cè)SAHA-CTSB與某種信號(hào)通路間存在分子串說(shuō),進(jìn)而促進(jìn)或抑制自噬來(lái)調(diào)控乳腺癌的發(fā)生和發(fā)展。本研究為明確SAHA-CTSB對(duì)乳腺癌細(xì)胞增殖及自噬產(chǎn)生的作用機(jī)制提供了良好的實(shí)驗(yàn)基礎(chǔ),也為SAHA-CTSB能夠成為乳腺腫瘤基因治療研究的一個(gè)新靶標(biāo)提供理論依據(jù)。方法1.培養(yǎng)乳腺癌細(xì)胞MCF-7和MDA-MB-231。2.應(yīng)用RTCA檢測(cè)SAHA對(duì)MCF-7和MDA-MB-231細(xì)胞增殖能力的影響。3.應(yīng)用BioStationIM活細(xì)胞工作站,形態(tài)學(xué)觀察SAHA作用MCF-7和MDA-MB-231細(xì)胞后細(xì)胞增殖的變化情況。4.利用CTSB抑制劑Cystatin C阻斷CTSB的表達(dá)。5.Western Blot、ELISA 和 In Cell Western 檢測(cè) SAHA 和(或)Cystatin C 作用于MCF-7和MDA-MB-231乳腺癌細(xì)胞后,CTSB蛋白表達(dá)變化的情況。6.MuseTM流式細(xì)胞技術(shù)檢測(cè)SAHA和(或)Cystatin C作用于MCF-7和MDA-MB-231乳腺癌細(xì)胞后細(xì)胞活力變化的情況。7.應(yīng)用細(xì)胞免疫熒光技術(shù),熒光顯微鏡下觀察SAHA和(或)Cystatin C作用MCF-7和MDA-MB-231細(xì)胞后細(xì)胞內(nèi)LC3 Ⅱ的表達(dá)情況。8.應(yīng)用 RT-QPCR Array、Western Blot 檢測(cè) SAHA 和(或)Cystatin C 作用乳腺癌細(xì)胞后自噬相關(guān)因子表達(dá)的變化。9.應(yīng)用 Human MAPK antibody Array 檢測(cè) SAHA 和(或)Cystatin C 作用MCF-7和MDA-MB-231細(xì)胞后MAPK信號(hào)通路相關(guān)因子的表達(dá)情況。結(jié)果1.RTCA、BioStationIM結(jié)果顯示,SAHA能夠抑制乳腺癌MCF-7和MDA-MB-231細(xì)胞的增殖。2.Western Blot、ELISA、In Cell Western及MuseTM流式細(xì)胞活力檢測(cè)結(jié)果顯示,SAHA在抑制乳腺癌細(xì)胞增殖時(shí)CTSB高表達(dá),相反的是CTSB功能失活時(shí)則抑制SAHA的抗腫瘤作用。3.細(xì)胞免疫熒光、RT-QPCR Array、Western Blot 及 Human MAPK antibody Array檢測(cè)結(jié)果顯示,SAHA通過(guò)CTSB的介導(dǎo)誘導(dǎo)乳腺癌細(xì)胞自噬的發(fā)生,在此過(guò)程中MAPK信號(hào)通路的激活起著重要的作用。結(jié)論1.SAHA能夠抑制乳腺癌MCF-7和MDA-MB-231細(xì)胞增殖,并且SAHA在抑制乳腺癌細(xì)胞增殖時(shí)伴隨著CTSB的高表達(dá)。2.CTSB功能失活抑制SAHA的抗腫瘤作用。3.SAHA通過(guò)激活CTSB介導(dǎo)乳腺癌細(xì)胞自噬,而這一過(guò)程可能主要通過(guò)MAPK信號(hào)通路調(diào)節(jié)。
[Abstract]:Breast Cancer (BC) is one of the most common malignant tumors in women. Its incidence is mainly concentrated in perimenopausal women between 40 and 60 years old. In Europe and America, the incidence of breast cancer is the first of the female malignant tumors. In recent years, with the rapid development of modern urban construction in China and the life of the country, The increase of pressure is increasing, the incidence of breast cancer in China's large and middle cities is increasing year by year, and it is becoming younger and threatening the physical and mental health of women. In eukaryotes, the acetylation and deacetylation modification of histone plays an important role in gene expression. The high acetylation state of chromatin can lead to tumor inhibition. Gene or apoptotic genes are highly expressed. Histone deacetylase inhibitor (Histone Deacetylase Inhibitor, HDACi) symplectic two aniline oxime (SuberoylanilideHydroxamicAcid, SAHA) can improve the expression of gene by increasing the level of histone acetylation in a specific chromatin region. It has become a new type of antitumor drug. It is found that SAHA can cause a variety of tumor cell growth stagnation and death, including a variety of breast cancer cell lines MCF-7, MDA-MB-231, MDA-MB-435, SKBr-3 and so on. Cathepsin B (CathepsinB, CTSB) is a cysteine protease in a soluble enzyme. Recent studies have shown that the level of CTSB expression changes with the tumor cells (breast cancer, eggs). The proliferation, invasion, metastasis and angiogenesis are closely related to the proliferation, invasion, metastasis and angiogenesis of nests, bladder cancer, lung cancer, colon cancer, melanoma, gastric cancer and so on. So the overexpression of CTSB in tumor cells is considered to be related to the aggressive and bad prognosis of clinical tumors. However, some studies have also shown that the expression of CTSB also induces the apoptosis and autophagy of some tumor cells. Autophagy (Autophagy) is a mechanism that can remove abnormal proteins and damage organelles in the body, which is beneficial to the homeostasis of all substances in the cells. In the stress state of hypoxia, energy shortage, infection and inflammation, autophagy can inhibit the accumulation of toxic and even carcinogenic proteins and cell organelles, and protect the cells to inhibit cell carcinogenesis. In the late stage of the tumor, autophagy provides the tumor cells with unlimited growth nutrients and promote the proliferation of cancer cells. In recent years, scholars have found that autophagy and its related molecules play an important role in the proliferation and treatment of breast cancer. The effects of SAHA on the proliferation of two breast cancer cell lines, MCF-7 and MDA-MB-231 cells, and the effect on the CTSB expression in two breast cancer cells. Because CTSB is a cysteine protease in the lysosome, however, the key enzymes involved in autophagy are mainly concentrated in the lysosomes. Therefore, we speculate that SAHA-CTSB and some signal pathway are in the lysosome. This study provides a good experimental basis for SAHA-CTSB on the mechanism of breast cancer cell proliferation and autophagy, and provides a theoretical basis for SAHA-CTSB to be a new target for the study of breast cancer gene therapy. Method 1 The effect of MCF-7 and MDA-MB-231.2. on the proliferation of MCF-7 and MDA-MB-231 cells by RTCA and the effect of SAHA on the proliferation of MCF-7 and MDA-MB-231 cells. The morphological changes of the cell proliferation of MCF-7 and MDA-MB-231 cells were observed by morphology. ISA and In Cell Western detection of the changes in the expression of CTSB protein after SAHA and / or Cystatin C acts on MCF-7 and MDA-MB-231 breast cancer cells The expression of LC3 II in cells after the action of SAHA and / or Cystatin C on MCF-7 and MDA-MB-231 cells.8. application RT-QPCR Array, Western Blot detection of SAHA and / or the changes in the expression of autophagy related factors after breast cancer cells The expression of MAPK signaling pathway related factors after MCF-7 and MDA-MB-231 cells. Results 1.RTCA, BioStationIM results showed that SAHA could inhibit the proliferation of MCF-7 and MDA-MB-231 cells of breast cancer,.2.Western Blot, ELISA. The expression, on the contrary, inhibits the anti-tumor effect of SAHA on the.3. cell immunofluorescence, and the Array detection results of the RT-QPCR Array, Western Blot and Human MAPK antibody show that the activation of the mammary cancer cells is induced by the mediation of SAHA, and the activation of the signal pathway plays an important role in this process. A inhibits the proliferation of MCF-7 and MDA-MB-231 cells in breast cancer, and SAHA inhibits the proliferation of breast cancer cells with the high expression of CTSB, which inhibits the anti tumor action of SAHA, and.3.SAHA activates the autophagy of breast cancer cells by activating CTSB, which may be regulated by the MAPK signaling pathway.
【學(xué)位授予單位】：沈陽(yáng)醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.9

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