【摘要】：研究背景及目的:表觀遺傳學(xué)是近年來遺傳學(xué)的研究熱點(diǎn)。組蛋白氨基酸殘端的甲基化、乙�；�、磷酸化、泛素化修飾等可以通過改變組蛋白和DNA的相互親和力從而導(dǎo)致基因轉(zhuǎn)錄發(fā)生改變。多項(xiàng)研究表明,腫瘤的發(fā)生發(fā)展與組蛋白乙酰化狀態(tài)(由組蛋白乙酰轉(zhuǎn)移酶HAT和組蛋白去乙�；窰DAC共同調(diào)控)關(guān)系密切。組蛋白去乙�；敢种苿�(HDACi)作為一種特異性靶向抗腫瘤藥物越來越引起人們的關(guān)注,目前FDA已批準(zhǔn)HDAC抑制劑伏立諾他(Vorinostat,SAHA)和羅咪酯肽(Romidepsin)用于治療皮膚及外周T細(xì)胞淋巴瘤,批準(zhǔn)帕比司他(Panobinostat)治療多發(fā)性骨髓瘤。多發(fā)性骨髓瘤(multiple myeloma,MM)是一種漿細(xì)胞惡性腫瘤,臨床上經(jīng)過DNA損傷性化療、蛋白酶體抑制劑、免疫調(diào)節(jié)劑(沙利度胺或來那度胺)及大劑量化療聯(lián)合自體干細(xì)胞移植等治療后,大多數(shù)患者最終仍出現(xiàn)疾病復(fù)發(fā)或者進(jìn)展,因此,尋找更有效的治療手段一直是MM治療領(lǐng)域的研究熱點(diǎn)。西達(dá)本胺是我國第一個(gè)自主研發(fā)的組蛋白去乙�；敢种苿�(HDACi),可以抑制HDAC1、2、3和10亞型的活性。在晚期腫瘤患者的Ⅰ期臨床研究中,西達(dá)本胺體現(xiàn)了良好的耐受性,目前已有研究證明,西達(dá)本胺對肺癌、結(jié)腸癌、乳腺癌、肝癌、前列腺癌及淋巴瘤有抑制或殺傷活性,我國正在開展針對皮膚T細(xì)胞淋巴瘤(PTCL)及外周T細(xì)胞淋巴瘤(CTCL)的Ⅱ/Ⅲ期臨床試驗(yàn),初步統(tǒng)計(jì)結(jié)果提示療效十分顯著。綜上述,HDACi(如伏立諾他、羅咪酯肽、帕比司他)均有抗MM活性,但單藥治療顯示出了劑量依賴性的副作用,使得HDACi治療骨髓瘤收到了限制;同時(shí)西達(dá)本胺治療難治復(fù)發(fā)型PTCL顯示出了極好的療效。因此,我們設(shè)想MM的乙�；癄顟B(tài)與MM細(xì)胞活性密切相關(guān),目前有關(guān)西達(dá)本胺抗MM活性尚未見相關(guān)報(bào)道,故本課題選擇人多發(fā)性骨髓瘤細(xì)胞系RPMI8226、H929為實(shí)驗(yàn)對象,觀察西達(dá)本胺對人多發(fā)性骨髓瘤細(xì)胞的抑制作用并探索其可能的機(jī)制。方法:①M(fèi)TT法研究組蛋白去乙�；敢种苿┪鬟_(dá)本胺對RPMI8226及H929細(xì)胞增殖的影響;②流式細(xì)胞術(shù)檢測西達(dá)本胺作用后細(xì)胞凋亡率的變化及細(xì)胞周期分布情況;③RT-PCR檢測西達(dá)本胺對MM細(xì)胞SIRT1基因、自噬相關(guān)基因m RNA表達(dá)的影響;④Western blot法檢測西達(dá)本胺處理MM細(xì)胞48h后周期相關(guān)蛋白p21、自噬相關(guān)蛋白、DNA損傷相關(guān)蛋白及凋亡相關(guān)蛋白表達(dá)情況;⑤采用ATM激酶特異性抑制劑KU-55933抑制ATM激酶活性,研究ATM在西達(dá)本胺調(diào)控MM細(xì)胞增殖中的作用。結(jié)果:①西達(dá)本胺顯著抑制MM細(xì)胞的增殖;②西達(dá)本胺上調(diào)p21蛋白,使細(xì)胞周期阻滯于G0/G1期;③西達(dá)本胺處理后的MM細(xì)胞DNA損傷相關(guān)蛋白γH2AX、p ATM及凋亡相關(guān)蛋白Cleaved Caspase-3表達(dá)水平升高;④KU-55933抑制ATM激酶活性后,部分逆轉(zhuǎn)由西達(dá)本胺誘導(dǎo)的MM細(xì)胞DNA損傷和凋亡反應(yīng);⑤西達(dá)本胺抑制SIRT1基因的轉(zhuǎn)錄及表達(dá),上調(diào)組蛋白H4K16的乙�；�;⑥西達(dá)本胺下調(diào)自噬相關(guān)基因ATG7和LC3的轉(zhuǎn)錄和表達(dá)。結(jié)論:①達(dá)本胺誘導(dǎo)MM細(xì)胞凋亡部分涉及DNA損傷反應(yīng);②西達(dá)本胺通過上調(diào)組蛋白H4K16乙�；种芃M細(xì)胞的自噬反應(yīng)。
[Abstract]:The background and purpose of the study are as follows: Epigenetics is a hot topic of genetics in recent years. Methylation, ethylation, phosphorylation, ubiquitination modification, etc. of histone amino acid residues can lead to a change in gene transcription by changing the mutual affinity of histone and DNA. A number of studies have shown that the development of the tumor is closely related to histone ethylation status (which is co-regulated by histone B-transferase HAT and histone de-ethylation enzyme HDAC). Histone deethanolase inhibitors (HDACi) are increasingly concerned as a specific targeted anti-tumor drug, and currently the FDA has approved the HDAC inhibitor of VORinostat, SAHA and Romidepsin for the treatment of skin and peripheral T-cell lymphomas, Panobinstat was approved for multiple myeloma. Multiple myeloma (MM) is a plasma cell malignancy, and is clinically treated with DNA damage chemotherapy, proteasome inhibitor, immunomodulator (thalidomide or lenalidomide) and large-dose chemotherapy combined with autologous stem cell transplantation. In most patients, there is still a recurrence or progression of the disease, and therefore finding more effective means of treatment has been a hot spot in the field of MM therapy. Sida Benamine is the first histone de-ethylation enzyme inhibitor (HDACi) developed by China's first self-developed histone, which can inhibit the activity of HDAC1,2,3 and 10 subtypes. In the first clinical study of late-stage tumor patients, Sida Benamine is well tolerated and has been shown to have been shown to have inhibitory or anti-killing activity on lung cancer, colon cancer, breast cancer, liver cancer, prostate cancer and lymphoma, Our country is carrying out the 鈪，

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