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A治疗增加了细胞弹性,正如原子力显微镜(AFM)试验所揭示的那样,而不会对小鼠卵巢癌细胞系MOSE-E和MOSE-L中的肌动蛋白微丝或微管网络发生剧烈变化[4]。",{"key":224,"value":225},"体内研究","每日治疗0.5mg\u002Fkg的Tubastatin A抑制HDAC6在炎症和自身免疫小鼠模型中促进Tregs抑制活性,包括多种形式的实验性结肠炎和完全主要组织相容性复合物(MHC)不相容的心脏同种异体移植物排斥[2]。",{"key":227,"value":228},"激酶实验","使用Reaction Biology HDAC Spectrum平台进行酶抑制测定。 HDAC1,2,4,5,6,7,8,9,10和11测定使用分离的重组人蛋白质; HDAC3 \u002F NcoR2复合物用于HDAC3测定。用于HDAC1,2,3,6,10和11测定的底物是来自p53残基379-382（RHKKAc）的荧光肽; HDAC8的底物是基于p53的残基379-382（RHKAcKAc）的荧光二酰基肽。乙酰基-Lys（三氟乙酰基）-AMC底物用于HDAC4,5,7和9测定。将Tubastatin A溶解于DMSO中，并以10剂量IC50模式进行测试，以30μM开始进行3倍连续稀释。对照化合物曲古抑菌素A（TSA）在10个剂量的IC50中进行测试，其中3倍连续稀释从5μM开始。通过曲线拟合剂量\u002F响应斜率来提取IC 50值。",{"key":230,"value":231},"细胞实验","如前所述，从胎儿Sprague-Dawley大鼠（胚胎第17天）的大脑皮层获得原代皮质神经元培养物。所有实验均在接种后24小时开始。在这些条件下，细胞不易受谷氨酸介导的兴奋毒性的影响。对于细胞毒性研究，用温PBS冲洗细胞，然后置于含有5.5g \u002F L葡萄糖，10％胎牛血清，2mM L-谷氨酰胺和100μM胱氨酸的最低必需培养基中。通过向培养基中加入谷氨酸类似物高半胱氨酸（HCA; 5mM）诱导氧化应激。将HCA从100倍浓缩的溶液中稀释，将其调节至pH 7.5。与HCA组合，用指定浓度的Tubastatin A处理神经元。通过MTT测定（溴化3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑）方法在24小时后评估生存力。",{"key":233,"value":234},"动物实验","评估HDAC6靶向葡聚糖硫酸钠（DSS）和结肠炎的过继转移模型的效果，每组使用10只小鼠。每天将新鲜制备的4％（wt \u002F vol）DSS（MP Biomedicals）加入WT B6小鼠的pH平衡自来水中5天。用tubacin或niltubacin（0.5mg \u002F kg体重\u002F天，ip）每天治疗小鼠7天，并通过每日监测体重，粪便稠度和粪便血液来评估结肠炎。将粪便稠度评分为0（硬），2（软）或4（腹泻），并将粪便血液（Hemoccult）评分为0（不存在），2（隐匿）或4（粗）。为了评估T细胞依赖性模型中结肠炎的预防，将使用磁珠（> 95％细胞纯度，流式细胞术）从WT小鼠分离的CD4 + CD45RBhi T细胞（1×106）腹膜内注射到B6 \u002F Rag1  -  \u002F  - 小鼠中加上使用来自HDAC6  -  \u002F  - 或WT小鼠的磁珠（> 90％Treg纯度，流式细胞术）分离的CD4 + CD25 + Tregs（1.25×105），并且每两周监测小鼠的结肠炎的临床证据。为了评估已建立的T细胞依赖性结肠炎的治疗，用CD4 + CD45RBhi细胞（1×106）腹膜内注射B6 \u002F Rag1  -  \u002F  - 小鼠。一旦结肠炎发展，小鼠还接受如上所述从HDAC6  -  \u002F  - 或WT小鼠分离的CD4 + CD25 + Tregs（5×10 5个细胞）或用HDAC6i（tubastatin A）或HSP90i（17-AAG）处理。监测小鼠的持续体重减轻和粪便稠度。在研究停止时，用Alcian Blue或苏木精和曙红染色的结肠的石蜡切片在组织学上分级或通过免疫过氧化物酶染色评估Foxp3 + Treg浸润。",{"key":236,"value":237},"参考文献","[1].\nKyle V. Butler et al. Rational Design and Simple Chemistry Yield a Superior, Neuroprotective HDAC6 Inhibitor, Tubastatin A J. Am. Chem. Soc., 2010, 132 (31), pp 10842-10846\n[2].\nde Zoeten EF, et al. Histone deacetylase 6 and heat shock protein 90 control the functions of Foxp3(+) T-regulatory cells. Mol Cell Biol. 2011 May;31(10):2066-78.\n[3].\nDi Fulvio S, et al. Dysferlin interacts with histone deacetylase 6 and increases alpha-tubulin acetylation. PLoS One. 2011;6(12):e28563\n[4].\nKetene AN, et al. Actin filaments play a primary role for structural integrity and viscoelastic response in cells. Integr Biol (Camb). 2012 May;4(5):540-9.\n[5].\nKozyreva VK, et al. NEDD9 regulates actin dynamics through cortactin deacetylation in an AURKA\u002FHDAC6-dependent manner. Mol Cancer Res. 2014 May;12(5):681-93.\n[6].\nBrijmohan, Angela, et al. Role of HDAC6 in Transcription Factor EB Mediated Clearance of Misfolded Proteins in Chronic Kidney Disease. 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