本文選題：As_4O_6 + EGFR信號(hào)通路　； 參考：《北京中醫(yī)藥大學(xué)》2017年碩士論文

【摘要】：乳腺癌是全球女性患者高發(fā)性的癌癥,這種高轉(zhuǎn)移率的癌癥有著較高的死亡率。雖然乳腺不是維持人類生命活動(dòng)的主要器官,而且原位乳腺癌并不是致命的,但是由于乳腺癌組織的細(xì)胞失去了正常細(xì)胞的特性,這些癌細(xì)胞的粘附能力較低,并且很容易通過(guò)血液循環(huán)或者淋巴循環(huán)進(jìn)行擴(kuò)散轉(zhuǎn)移。到目前為止,盡管在臨床許多治療乳腺癌的方法被應(yīng)用于治療當(dāng)中,但是治療效果并不是令人十分滿意,尤其對(duì)HER-2陰性的乳腺癌患者幾乎沒有作用。曲妥珠單抗是一個(gè)近年來(lái)開發(fā)得比較成功的治療乳腺癌的抗體藥物,臨床上用其治療HER-2陽(yáng)性的乳腺癌患者取得了較好療效,但對(duì)于HER-2陰性的乳腺癌患者無(wú)效。所以研究和開發(fā)一個(gè)針對(duì)HER-2陰性患者的抗腫瘤藥物非常必要。EGFR和HER-2是同一家族膜蛋白受體,并且它們是乳腺癌臨床治療的主要靶點(diǎn)。雖然近年來(lái)有許多靶向EGFR和HER-2的藥物在開發(fā),但是最終在臨床上獲得卓越療效的藥物卻少之又少。另外,隨著不斷的研究發(fā)現(xiàn)(EGF)/EGFR參與了細(xì)胞轉(zhuǎn)移,并在這個(gè)過(guò)程起到促進(jìn)作用,可以誘導(dǎo)上皮-間質(zhì)轉(zhuǎn)化過(guò)程。EGFR的下游信號(hào)通路,EGFR-Akt-mTOR信號(hào)軸,在多種遷移的癌細(xì)胞中被發(fā)現(xiàn)激活,因此以EGFR和HER-2為靶點(diǎn)的有效治療藥物很有必要被開發(fā)。天地散是從砒霜開發(fā)出來(lái)的一種傳統(tǒng)醫(yī)學(xué)藥物,其主要成分As_4O_6是一種砷制劑,其在韓國(guó)被廣泛用于惡性腫瘤的治療。自從上個(gè)世紀(jì)八十年代,As_4O_6就在韓國(guó)當(dāng)做民間偏方來(lái)使用。在近十幾年的研究中,As_4O_6在體外實(shí)驗(yàn)中對(duì)不同的腫瘤細(xì)胞(子宮頸癌、結(jié)腸癌、腦膠質(zhì)瘤和白血病)表現(xiàn)出了顯著的抑制作用,并在抗腫瘤新生血管、提高化療藥物敏感性和聯(lián)合用藥方面有著一定的作用。盡管對(duì)As406抗癌癥的研究報(bào)道頗多,但是其對(duì)乳腺癌方面的研究報(bào)道僅有一篇,該篇文章證明了 As_4O_6可以通過(guò)NF-κB信號(hào)通路對(duì)MCF-7乳腺癌細(xì)胞起到抑制作用,但是As_4O_6對(duì)乳腺癌侵襲轉(zhuǎn)移的影響未見報(bào)道,所以本研究在前期研究的基礎(chǔ)上,查閱相關(guān)的參考文獻(xiàn),選取MCF-7、MDA-MB-231、MDA-MB-453和SKBR3四種不同類型的乳腺癌細(xì)胞為研究對(duì)象,采用MTT法、劃痕實(shí)驗(yàn)、Transwell小室實(shí)驗(yàn)、粘附實(shí)驗(yàn)、RT-PCR和Western blotting分子生物學(xué)實(shí)驗(yàn)方法,探究As406對(duì)乳腺癌細(xì)胞侵襲轉(zhuǎn)移作用的影響并闡明其分子機(jī)制,并建立BABL/C小鼠乳腺癌移植瘤模型,進(jìn)一步探討As406對(duì)動(dòng)物體內(nèi)移植瘤的影響。本文的主要研究結(jié)果如下:(1)As_4O_6可以抑制MCF-7細(xì)胞的增殖。As_4O_6可以降低MCF-7細(xì)胞的遷移和侵襲能力。經(jīng)EGF誘導(dǎo)后,細(xì)胞的EGFR的磷酸化明顯增加,但是經(jīng)As_4O_6干預(yù)后被抑制。現(xiàn)有的數(shù)據(jù)表明,As_4O_6可以有效地通過(guò)抑制EGFR介導(dǎo)的信號(hào)通路,抑制相關(guān)基因(EGFR)的表達(dá),降低MCF-7細(xì)胞的遷移和侵襲能力。(2)As_4O_6可以抑制MDA-MB-231細(xì)胞的增殖呈時(shí)間和劑量依賴性。As_4O_6可以降低MDA-MB-231細(xì)胞的遷移和侵襲能力,并且可以降低MDA-MB-231細(xì)胞的MMP-2和MMP-9的表達(dá),特別是對(duì)EGFR磷酸化的抑制作用。同時(shí),經(jīng)EGF誘導(dǎo)后,EGFR的磷酸化明顯增加,但是經(jīng)As_4O_6干預(yù)后被抑制。現(xiàn)有的數(shù)據(jù)表明,As_4O_6可以有效地通過(guò)抑制EGFR介導(dǎo)的信號(hào)通路,抑制相關(guān)基因(EGFR)的表達(dá),降低MDA-MB-231細(xì)胞的遷移和侵襲能力。(3)As_4O_6可以抑制MDA-MB-453細(xì)胞的增殖,并呈時(shí)間和劑量依賴性。As_4O_6可以降低MDA-MB-453細(xì)胞的遷移和侵襲能力。經(jīng)EGF誘導(dǎo)后,MDA-MB-453細(xì)胞的侵襲和遷移能力明顯增強(qiáng),As_4O_6干預(yù)后明顯被抑制。與Lapatinib對(duì)比,發(fā)現(xiàn)As_4O_6抑制MDA-MB-453細(xì)胞遷移和侵襲能力的效果要優(yōu)于Lapatinib,兩藥進(jìn)行等劑量的聯(lián)合使用后,對(duì)MDA-MB-453細(xì)胞遷移和侵襲能力的抑制作用要優(yōu)于兩藥單獨(dú)使用。(4)As_4O_6可以抑制SKBR3細(xì)胞的增殖,并呈時(shí)間和劑量依賴性。As_4O_6能有效抑制HER-2陽(yáng)性乳腺癌SKBR3細(xì)胞的侵襲和遷移,并且SKBR3細(xì)胞的粘附能力在As_4O_6的干預(yù)下也有所減弱。實(shí)驗(yàn)結(jié)果表明,As_4O_6抗腫瘤效果與HER-2/EGFR信號(hào)通路有關(guān),通過(guò)調(diào)節(jié)在HER-2/EGFR信號(hào)通路中的細(xì)胞因子(EGFR,HER-2,Akt,MMP-9)和其他關(guān)鍵分子,實(shí)現(xiàn)對(duì)HER-2陽(yáng)性乳腺癌細(xì)胞遷移和侵襲的抑制作用。總之,As_4O_6抑制HER-2陽(yáng)性乳腺癌SKBR3細(xì)胞的侵襲和遷移能力是通過(guò)HER-2/EGFR信號(hào)通路的負(fù)調(diào)節(jié)實(shí)現(xiàn)的。綜上,As406對(duì)人三陰性乳腺細(xì)胞MDA-MB-231作用較明顯,人三陰性乳腺細(xì)胞MDA-MB-231 對(duì) As_4O_6 較敏感。(5)天地散可以抑制BABL/C裸鼠乳腺癌腫瘤生長(zhǎng),在本實(shí)驗(yàn)劑量下其抑瘤率可達(dá)40%以上,并且裸鼠給藥前后的體重沒有顯著性差異(P0.05),說(shuō)明天地散比較安全,未見明顯的毒性作用。
[Abstract]:Breast cancer is the high incidence of cancer in women worldwide, the high metastasis rate of cancer has a high mortality. Although the main organs of breast not sustain human life activities, and in situ breast cancer is not fatal, but because breast cancer cells lose the characteristics of normal cells, the adhesion ability of these cancer cells low and easily through blood circulation or lymph circulation by diffusion transfer. So far, although many methods in clinical treatment of breast cancer was used in the treatment, but the treatment effect is not very satisfactory, especially for patients with HER-2 negative breast cancer almost no role. Trastuzumab is an antibody drug in recent years the development of more successful treatment of breast cancer, the clinical use in the treatment of HER-2 positive breast cancer patients has good curative effect, but for HER-2 negative breast Invalid patients with adenocarcinoma. So the research and development of an HER-2 for patients with negative anti tumor drugs necessary to.EGFR and HER-2 are the same membrane protein receptor family, and they are the main target for the clinical treatment of breast cancer. In recent years there are many drugs targeting EGFR and HER-2 in the development of drugs, but ultimately excellent clinical efficacy is less and less. In addition, with the continuous study found that (EGF) /EGFR is involved in cell migration, and to promote the role in this process, can induce epithelial mesenchymal transition process downstream of.EGFR signaling pathway, EGFR-Akt-mTOR signaling axis, in a variety of cancer cell migration was found with EGFR and HER-2 activation, so it is necessary for the effective treatment of drug targets is developed. The world is scattered from a traditional medicine developed arsenic, the main component of As_4O_6 is a kind of arsenic, which was in South Korea Widely used in the treatment of malignant tumors. Since the last century in 80s, As_4O_6 in South Korea as folk remedies to use. In the recent decade, As_4O_6 in vitro experiments on different tumor cells (Zi Gongjing cancer, colon cancer, glioma and leukemia) showed significant inhibitory effect, and in tumor angiogenesis, has a certain role to improve the sensitivity of chemotherapy drugs and combination therapy. Although the research reports on As406 cancer a lot, but the breast cancer study reported only one article, the article proves that As_4O_6 can through the NF- B pathway of MCF-7 breast cancer cells to inhibit effect of As_4O_6 on invasion and metastasis, but no reports of breast cancer, so the study on the basis of previous research, access to relevant references, including MCF-7, MDA-MB-231, MDA-MB-453 and SKBR3 four Breast cancer cells of different types as the research object, using the method of MTT, scratch test, Transwell assay, adhesion assay, RT-PCR blotting and Western experimental method of molecular biology, to explore the impact of As406 on invasion and metastasis of breast cancer cells and to elucidate its molecular mechanism, and the establishment of BABL/C mice xenograft model of breast cancer, to further explore the effect of As406 the animal transplanted tumor. The main results are as follows: (1) As_4O_6 can inhibit the proliferation of MCF-7 cells.As_4O_6 can reduce the migration and invasion of MCF-7 cells. After induced by EGF cells, the phosphorylation of EGFR increased significantly, but the intervention of As_4O_6 was inhibited. The existing data show that As_4O_6 can effectively through the signal pathway of EGFR mediated inhibition, inhibition of gene expression (EGFR), reduced the migration and invasion of MCF-7 cells. (2) As_4O_6 can inhibit MDA-MB- 231 cell proliferation in a time and dose dependent.As_4O_6 can reduce the migration and invasion of MDA-MB-231 cells, and MDA-MB-231 cells can reduce the expression of MMP-2 and MMP-9, especially the inhibition of the phosphorylation of EGFR. At the same time, after induced by EGF, the phosphorylation of EGFR increased significantly, but the intervention of As_4O_6 was inhibited. The existing data show that As_4O_6 can effectively inhibit EGFR mediated by signaling pathways, inhibition of gene expression (EGFR), reduced the migration and invasion of MDA-MB-231 cells. (3) As_4O_6 can inhibit the proliferation of MDA-MB-453 cells in dose and time dependent.As_4O_6 can reduce the migration and invasion of MDA-MB-453 cells. After EGF induction, the migration and invasion of MDA-MB-453 cells was significantly enhanced, the intervention of As_4O_6 was significantly inhibited. Compared with Lapatinib, As_4O_6 was found to inhibit MDA-MB-453 cell Cell migration and invasion ability is better than the Lapatinib, the two drugs used in combination group, inhibited the invasion and migration of MDA-MB-453 cells is superior to the two drugs used alone. (4) As_4O_6 can inhibit the proliferation of SKBR3 cells in dose and time dependent.As_4O_6 can effectively inhibit the cell invasion and migration HER-2 positive breast cancer SKBR3, and the adhesion ability of SKBR3 cells was also reduced in the intervention of As_4O_6. Experimental results show that the antitumor effect of As_4O_6 HER-2/EGFR pathway, through the regulation of cytokines in HER-2/EGFR signal pathway in (EGFR, HER-2, Akt, MMP-9) and other key molecules that inhibit the migration and invasion of HER-2 positive breast cancer cells. In conclusion, As_4O_6 inhibited the invasion and migration of HER-2 positive breast cancer cell SKBR3 is a negative regulator of HER-2/EGFR signal through the path The summary, As406 on three breast MDA-MB-231 cells obviously, MDA-MB-231 three negative breast cells are sensitive to As_4O_6. (5) and San BABL/C can inhibit the growth of breast carcinoma in nude mice, the dose of the experiment the inhibition rate can reach more than 40%, and nude mice before and after administration of no significant weight the difference (P0.05), and that scattered relatively safe, no toxicity.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285

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