【摘要】：目的：研究正常腦組織、不同級(jí)別膠質(zhì)瘤組織以及膠質(zhì)瘤細(xì)胞系U87中H3乙酰化組蛋白的表達(dá)，使用去乙酰化酶抑制劑Apicidin干擾U87膠質(zhì)瘤細(xì)胞系，研究組蛋白H3乙酰化程度改變對(duì)膠質(zhì)瘤細(xì)胞中Nanog的影響，并探索組蛋白H3低乙酰化對(duì)膠質(zhì)瘤細(xì)胞體內(nèi)及體外生物學(xué)活性的影響。為后續(xù)進(jìn)一步研究Nanog的表觀遺傳學(xué)干預(yù)治療奠定基礎(chǔ)。 方法：①使用Western-Blot方法檢測(cè)53例不同級(jí)別腦膠質(zhì)瘤組織及11例取自顱腦損傷行內(nèi)減壓術(shù)患者的正常腦組織中H3乙酰化組蛋白的表達(dá)情況，RealtimePCR檢測(cè)高級(jí)別和低級(jí)別膠質(zhì)瘤組織內(nèi)Nanog表達(dá)情況。②通過(guò)組蛋白去乙�；敢种苿〢picidin干預(yù)U87膠質(zhì)瘤細(xì)胞，不同濃度和時(shí)間干預(yù)過(guò)后，，通過(guò)RT-PCR和Western-blot實(shí)驗(yàn)分別檢測(cè)U87細(xì)胞株中H3乙�；M蛋白和Nanog的mRNA和蛋白表達(dá)水平，ChIP檢測(cè)U87細(xì)胞系中Nanog啟動(dòng)子區(qū)域H3組蛋白乙�；健＂弁ㄟ^(guò)MTT、細(xì)胞劃痕實(shí)驗(yàn)、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡、細(xì)胞周期、透射電鏡檢測(cè)凋亡、裸鼠成瘤實(shí)驗(yàn)、免疫組織化學(xué)染色等方法檢測(cè)組蛋白H3低乙酰化水平抑制Nanog后對(duì)膠質(zhì)瘤細(xì)胞體內(nèi)外生物學(xué)活性的影響。 結(jié)果：①與正常腦組織相比，乙�；M蛋白H3在不同級(jí)別膠質(zhì)瘤中的表達(dá)均明顯上調(diào)（P＜0.05）。WHO Ⅲ～Ⅳ級(jí)膠質(zhì)瘤組織中乙�；M蛋白H3表達(dá)明顯高于WHOⅠ～Ⅱ級(jí)（P＜0.05）。與正常腦組織相比，在膠質(zhì)瘤細(xì)胞系U87中的表達(dá)亦顯著升高（P＜0.05）。②利用組蛋白去乙�；敢种苿〢picidin干預(yù)膠質(zhì)瘤U87細(xì)胞系后，同時(shí)檢測(cè)乙�；M蛋白H3及Nanog表達(dá)水平的變化，發(fā)現(xiàn)Nanog啟動(dòng)子區(qū)域的組蛋白H3乙�；浇档停瑫r(shí)Nanog的表達(dá)亦明顯降低；③與空白對(duì)照組相比，U87-Apicidin膠質(zhì)瘤細(xì)胞的增殖水平和遷移能力均減弱，細(xì)胞凋亡增加； U87-Apicidin皮下膠質(zhì)瘤的體積與質(zhì)量都明顯小于空白對(duì)照組，提示Apicidin處理組U87細(xì)胞裸鼠皮下成瘤能力亦明顯下降（P＜0.05）。 結(jié)論：①組蛋白H3乙�；赡芘c膠質(zhì)瘤的發(fā)生發(fā)展關(guān)系密切。膠質(zhì)瘤級(jí)別越高，乙�；M蛋白H3的表達(dá)量越高；②Nanog啟動(dòng)子區(qū)域的組蛋白H3乙酰化修飾水平可以影響Nanog的表達(dá)；③Apicidin可以特異性的降低Nanog啟動(dòng)子區(qū)域乙酰化組蛋白H3水平，從而影響Nanog的表達(dá)，最終對(duì)膠質(zhì)瘤的生物學(xué)活性產(chǎn)生抑制性作用。
[Abstract]:Objective: to study the expression of H3 acetylated histone in normal brain tissue, glioma tissue of different grades and glioma cell line U87, and to use deacetylase inhibitor Apicidin to interfere with U87 glioma cell line. The effect of acetylation of protein H3 on Nanog in glioma cells and the effect of low acetylation of histone H3 on the biological activity of glioma cells in vivo and in vitro were investigated. To lay a foundation for the further study of epigenetic intervention therapy of Nanog. Methods: 1the expression of H3 acetylated histone was detected by Western-Blot in 53 gliomas with different grades and 11 normal brain tissues from patients with craniocerebral injury undergoing internal decompression. RealtimePCR was used to detect the expression of Nanog in high grade and low grade gliomas. (2) U87 glioma cells were treated with histone deacetylase inhibitor (Apicidin). The mRNA and protein expressions of H3 acetylated histone and Nanog in U87 cell line were detected by RT-PCR and Western-blot assay respectively. The H3 histone acetylation level of Nanog promoter region in U87 cell line was detected by ChIP. 3 the H3 histone acetylation level was detected by MTT, cell scratch assay. Flow cytometry was used to detect cell apoptosis, cell cycle, transmission electron microscopy (TEM), and tumorigenesis in nude mice. The effects of low acetylation of histone H3 on the biological activity of glioma cells in vivo and in vitro were detected by immunohistochemical staining. Results: 1Compared with normal brain tissue, The expression of acetylated histone H3 was significantly up-regulated in different grade gliomas (P < 0. 05). The expression of acetylated histone H3 in gliomas of grade 鈪

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