本文選題：骨肉瘤　切入點(diǎn)：乙�；�　出處：《浙江大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：骨肉瘤是最常見的原發(fā)惡性骨腫瘤,尤以15-19歲的青少年多見,其發(fā)病率為4／百萬,且男性患病率約為女性的1.4倍。目前臨床治療骨肉瘤除手術(shù)切除外,化療仍以阿霉素(ADM)、順鉑(CDDP)、甲氨蝶呤(MTX)為主,隨著新輔助化療的發(fā)展,患者5年生存率已上升到65-75%。盡管如此,對于治療骨肉瘤新藥的研發(fā)卻少之又少,而且,對于肺轉(zhuǎn)移和復(fù)發(fā)的患者,其預(yù)后更差,至今尚無有效可行的方案。因此,研發(fā)高效低毒的新型抗骨肉瘤藥物或化療增敏藥物,可作為目前骨肉瘤治療中的一大方向。 伏立諾他(辛二酰苯胺異羥肟酸,SAHA)是第一個(gè)于2006年經(jīng)美國食品藥品監(jiān)督管理局(Food and Drug Administration, FDA)批準(zhǔn)用于臨床治療皮膚T細(xì)胞淋巴瘤(CTCL)的組蛋白去乙酰化酶抑制劑(HDACi)類藥物,它可通過誘導(dǎo)細(xì)胞分化、阻斷細(xì)胞周期、誘導(dǎo)細(xì)胞調(diào)控而發(fā)揮作用。 順鉑(cisplatin, CDDP),是一種細(xì)胞周期非特異性藥物,其可通過抑制腫瘤細(xì)胞的DNA復(fù)制,損傷腫瘤細(xì)胞膜的結(jié)構(gòu),而發(fā)揮著較強(qiáng)的抗癌作用并被廣泛應(yīng)用于臨床,同時(shí)它也是治療骨肉瘤的一線化療藥物。 鑒于新藥研發(fā)的困難和應(yīng)用增敏藥物的啟示,以及兩類藥物的作用原理,我們設(shè)計(jì)了聯(lián)合用藥的相關(guān)課題,本課題將通過以下三部分來探討：SAHA與CDDP聯(lián)合作用后對骨肉瘤細(xì)胞的增殖抑制情況；體內(nèi)抑瘤效果；以及二者作用的可能分子機(jī)制。這對進(jìn)一步揭示骨肉瘤的發(fā)病機(jī)制具有重要意義,并為骨肉瘤患者的臨床治療帶來新的思路。 本實(shí)驗(yàn)研究主要分：一、通過實(shí)驗(yàn)得出不同細(xì)胞株對SAHA與CDDP作用后在有效聯(lián)合指數(shù)(CI)下的作用濃度,在此濃度前提下觀察藥物聯(lián)合對細(xì)胞的增殖抑制、周期阻滯以及凋亡與自噬；二、在第一部分的基礎(chǔ)上,我們建立了骨肉瘤的異位移植動(dòng)物模型,并將SAHA與CDDP聯(lián)合應(yīng)用于裸鼠,觀察對骨肉瘤的抑瘤效果；三、著重探討二者聯(lián)合作用后可能的分子機(jī)制。本論文的研究結(jié)果表明：SAHA顯著增加了CDDP對骨肉瘤細(xì)胞抑制的敏感性,因此,在減少CDDP應(yīng)用劑量的同時(shí),也降低了其毒副作用。 第一部分SAHA聯(lián)合CDDP對骨肉瘤細(xì)胞增殖抑制的研究 目的： 觀察二者聯(lián)合后對骨肉瘤細(xì)胞的增殖抑制以及細(xì)胞周期變化,凋亡與自噬。 方法： 通過細(xì)胞增殖抑制試驗(yàn)(MTS)來觀察對骨肉瘤細(xì)胞(HOS和U20S)的增殖抑制效果；通過流式細(xì)胞儀(FCM)來分析細(xì)胞周期阻滯與凋亡的變化,通過透射電子顯微鏡(TEM)來觀察細(xì)胞自噬的情況。 結(jié)果： SAHA聯(lián)合CDDP后可顯著抑制HOS與U20S細(xì)胞的增殖,聯(lián)合組與對照組相比有顯著性差異(P0.05),而對正常的人成骨細(xì)胞(hFOB)抑制作用較弱；同時(shí)引起細(xì)胞S期阻滯,細(xì)胞凋亡比例明顯增加；誘導(dǎo)了自噬小體的形成。 結(jié)論： SAHA增敏了CDDP對骨肉瘤細(xì)胞株HOS與U20S的增殖抑制作用,但對hFOB細(xì)胞抑制較弱；誘導(dǎo)了自噬的發(fā)生。 第二部分SAHA聯(lián)合CDDP對荷瘤裸鼠的體內(nèi)抑瘤效果研究 目的： 觀察SAHA聯(lián)合CDDP對異位骨肉瘤裸鼠模型的抑瘤效果 方法： 首先應(yīng)用HOS細(xì)胞株建立骨肉瘤的裸鼠皮下移植瘤模型,并將動(dòng)物分為四組：對照組、SAHA組、CDDP組和SAHA+CDDP聯(lián)合治療組,后經(jīng)腹腔注射給藥,觀察裸鼠體重和腫瘤體積變化。 結(jié)果： SAHA+CDDP聯(lián)合組顯著抑制了腫瘤的生長,與對照組相比,差異有統(tǒng)計(jì)學(xué)意義(P0.01),而體重在CDDP單藥組和聯(lián)合組均出現(xiàn)了先降低后逐漸回升的變化趨勢。 結(jié)論： 在裸鼠體內(nèi)同時(shí)應(yīng)用SAHA與CDDP,可顯著抑制腫瘤生長。 第三部分SAHA聯(lián)合CDDP抑制骨肉瘤細(xì)胞增殖的分子機(jī)制研究 目的： 探討SAHA聯(lián)合CDDP作用于骨肉瘤細(xì)胞引起凋亡與自噬相關(guān)信號通路的變化 方法： 應(yīng)用FCM檢測活性氧(ROS)的變化,real-time PCR檢測相關(guān)周期基因的變化,Western Blot檢測凋亡(Caspases)信號通路、內(nèi)質(zhì)網(wǎng)應(yīng)激(ER stress)信號通路以及自噬相關(guān)蛋白的變化。 結(jié)果： 應(yīng)用抗氧化劑(NAC)后,可部分抵抗二者聯(lián)合對骨肉瘤細(xì)胞的增殖抑制作用；SAHA聯(lián)合CDDP引起了周期相關(guān)基因CyclinA2、CyclinE、CDK2、p21的變化,使得p21和CDK2表達(dá)增加,CyclinA2和CyclinE表達(dá)減少。Western Blot結(jié)果示：Caspases家族蛋白表達(dá)增加；ER stress相關(guān)蛋白PERK與eIF2α變化不明顯；自噬蛋白LC3BⅡ/Ⅰ的比例增加而且加入自噬抑制劑3-MA和CQ后,凋亡蛋白表達(dá)增加。 結(jié)論： 在引起骨肉瘤細(xì)胞凋亡的過程中ROS參與其中,以經(jīng)典的Caspase凋亡信號通路為主；二者聯(lián)合后促進(jìn)了自噬的形成；同時(shí),對ER stress的影響甚微；根據(jù)凋亡與自噬蛋白的表達(dá)變化關(guān)系：抑制自噬而增加凋亡,說明：自噬在其中扮演了部分保護(hù)的角色。
[Abstract]:Osteosarcoma is the most common primary malignant bone tumors, especially in the 15-19 year old adolescents, the incidence rate was 4 per million, and the male prevalence rate is about 1.4 times higher than women. The clinical treatment of osteosarcoma besides surgical resection, chemotherapy with adriamycin (ADM), cisplatin (CDDP), a methotrexate (MTX), with the development of neo adjuvant chemotherapy in patients with 5 year survival rate has risen to 65-75%. though, for the development of new drugs in the treatment of osteosarcoma is rare, and for lung metastasis and recurrence of the patients, the prognosis is worse, there is no effective and feasible scheme. Therefore, drug sensitivity by new anti osteosarcoma drug development with high efficiency and low toxicity or chemotherapy can be used as a major direction in osteosarcoma treatment at present.
Vorinostat (Xin two anilide hydroxamic acid, SAHA) is the first in 2006 by the U.S. Food and Drug Administration (Food and Drug Administration, FDA) approved for clinical treatment of cutaneous T cell lymphoma (CTCL) of the histone deacetylase inhibitor (HDACi) drugs, it can induction of cell differentiation, blocking cell cycle, inducing cell regulation and play a role.
Cisplatin (cisplatin, CDDP), is a cell cycle non-specific drugs, which can inhibit tumor cell DNA replication, damage the membrane of tumor cells, and play a strong anti-cancer effects and has been widely applied in clinical, first-line chemotherapy at the same time it is also the treatment of osteosarcoma.
In view of the difficulties and application of new drug R & D sensitizing drugs and two kinds of enlightenment, drug action principle, we design issues related to the combination of the task through the following three parts: To explore the inhibition of osteosarcoma cell proliferation combined effect of SAHA and CDDP; anti-tumor effect; and the two. The possible molecular mechanism. It has an important significance to further reveal the pathogenesis of osteosarcoma, and bring new ideas for the clinical treatment of patients with osteosarcoma.
This study includes: first, through the experiment of different cell lines of SAHA and CDDP after combination index (CI) in the effective concentration, combination of inhibition at this concentration under the premise of the effect of drugs on cell proliferation, cell cycle arrest and apoptosis and autophagy; two, based on the first part, we establish the ectopic transplantation animal model of osteosarcoma, and SAHA combined with CDDP in nude mice, observe the inhibitory effect of osteosarcoma; three, focuses on the molecular mechanism of joint action of the two post. The research results show that SAHA significantly increased the sensitivity of CDDP in inhibiting osteosarcoma cells therefore reduction in the CDDP dose at the same time, but also to reduce its toxicity.
Study on the inhibition of osteosarcoma cell proliferation by SAHA combined with CDDP in the first part
Objective:
The inhibition of proliferation of osteosarcoma cells, cell cycle changes, apoptosis and autophagy were observed after the combination of the two groups.
Method錛，

本文編號：1616319