CN101653449A - ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用 - Google Patents
ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用 Download PDFInfo
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Abstract
本发明属于生物医药技术领域,公开了ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用,为β-地中海贫血、镰刀型细胞贫血病、造血系统肿瘤以及造血功能障碍引起的贫血的治疗提供了候选药物。
Description
技术领域
本发明属于生物医药技术领域,涉及ADDA(adenosine-2’,3’-dialdehyde,2’,3’-二醛腺苷)和/或DMTA(5’-deoxy-5’(methyl-thio)adenosine,5’-脱氧-5’-甲硫羟基腺苷)的新用途,特别是涉及ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用,以及在制备治疗β-地中海贫血和镰刀型细胞贫血、造血系统障碍或肿瘤引起的贫血的药物中的应用。
背景技术
成人体内血红蛋白由两个α-珠蛋白亚基和两个β-珠蛋白亚基(α2β2)组成。人β-样珠蛋白基因簇位于11号染色体短臂,含有五个顺次排列的珠蛋白基因(ε,Gγ,Aγ,δ,β),编码β-样珠蛋白以合成血红蛋白。在人的个体发育过程中,各个珠蛋白基因严格按照特定的发育时期和组织特异性开启表达和关闭。从受孕到妊娠五周内,胚胎型ε-珠蛋白在卵黄囊中表达并逐步降低。此后,第一次珠蛋白亚型的切换发生,胎肝造血细胞中胎儿型珠蛋白(Gγ,Aγ)开始表达并逐步升高,胚胎型ε-珠蛋白表达逐渐下降。胎儿型珠蛋白表达一直延续到妊娠晚期胎儿即将出生,第二次珠蛋白亚型的切换开始。成体型β-珠蛋白表达开始升高并延续一生,伴随着胎儿型珠蛋白(γ-珠蛋白)表达降低(在新生儿出生9个月内下降至~2%),骨髓成为主要的造血器官。在红系细胞发育过程中,α-、β-两类珠蛋白基因簇协同表达,始终保持产物的平衡,分别产生胚胎型、胎儿型和成年型血红蛋白四聚体。在不同的发育时期,如胚胎、胎儿和成体中,高水平的基因表达受到ε-珠蛋白基因前侧的具有多个DNA酶I超敏感点位点调控区域(LCR)的调节。但是,基因突变研究表明控制珠蛋白样基因阶段特异表达的序列很可能位于每一单个基因的近端(Bank A,Blood,107:435-43,2006)。
β-地中海贫血和镰刀型细胞贫血是由于β-珠蛋白基因突变或表达缺损所引起,然而重新激活成人体内已关闭了的γ-珠蛋白的表达就能显著减轻或治疗上述贫血病人的临床症状。药物诱导胎儿型血红蛋白基因的表达是缓解和治疗这类疾病的重要举措。因为α-珠蛋白亚基的过量与β-珠蛋白表达的不足所引起的两类终产物的不平衡是β-地中海贫血的主要发病原因,γ-珠蛋白的增强表达形成α2γ2血红蛋白四聚体弥补了这种不平衡,进而缓解了病情。而脱氧产生的镰刀型血红蛋白多聚体所引起的血管阻塞是镰刀型细胞贫血发病的主要因素,γ-珠蛋白的增强表达可以缓解镰刀型血红蛋白分子的聚化。此外,造血系统肿瘤,如急、慢性白血病、淋巴瘤、骨髓瘤等以及造血功能障碍,如再生障碍性贫血、骨髓增生异常综合征等引起的贫血亦可以由升高的γ-珠蛋白达到缓解。
因此,研究γ-珠蛋白的表达调控机制显得尤其重要。多年来一直有多个假说试图阐明γ、β-珠蛋白基因表达切换的调节机理,其中包括:1.β-样珠蛋白基因簇位点内的基因次序;2.各个珠蛋白基因启动子对LCR竞争结合;3.不同发育期的转录因子氛围;4.活性基因的核区域化;5.组织和时期特异的染色质再塑(chromatin remodeling)调节。虽然目前仍难决断哪一因素起着决定性作用,随着表观遗传学研究的兴起,染色质再塑对β-样珠蛋白基因表达的调控备受重视(Zhao Q.et al,Blood,107:2138-45,2006)。
近来,越来越多的证据表明组蛋白的共价修饰对染色体的结构变化和功能调节具有重要作用。其中,组蛋白的乙酰化研究最为清楚。它能松散染色质的结构,使转录活化易于发生,是一个可逆的动态过程。此外,组蛋白还可以被磷酸化、泛素化、ADP核糖基化以及甲基化。有研究表明,组蛋白H4的乙酰化与基因位点的核酸酶敏感性普遍变化有关,而H3乙酰化和H3K4的甲基化则与基因的高水平转录包括激活启动子和LCR相联系。近来,组蛋白H4R3的甲基化也显示与活性染色质直接有关,并且能通过控制DNA甲基化参与调控珠γ-蛋白的基因表达。一般认为,DNA甲基化以及组蛋白H3K9、H3K27和H4K20的甲基化与基因的负调控相关(Jenuwein T.et al,Science,293:1074-80,2001;Barski A.et al,Cell,129:823-37,2007)
目前,一些DNA甲基化或组蛋白乙酰化靶向试剂如:5-azacytidine、decitabine、butyrate以及HDAC抑制剂等已初步应用于贫血病人的临床试验治疗,用来重新激活成体中已关闭的γ-珠蛋白基因表达来部分弥补β-珠蛋白基因表达缺损。也有中药提取物如黄芪多糖对K562细胞γ-珠蛋白基因表达具有诱导作用的报导。但是由于这些化合物具有毒副作用、剂量大和有色有味等缺点,它们的广泛使用受到限制(Borgna-Pignatti C.BrJ haematol,138:291-304,2007)。因此,寻找新的γ-珠蛋白诱导剂将是一条出路。
DNA或组蛋白甲基化需甲基供体S-腺苷甲硫氨酸(S-Adenosylmethionine,SAM)。SAM在甲基转移酶的作用下催化甲基化后,转变为S-腺苷高丝氨酸(S-Adenosylhomocysteine),随后被S-腺苷高丝氨酸水解酶水解。ADDA能抑制S-腺苷高丝氨酸水解酶的活性,增加S-腺苷高丝氨酸的浓度从而反馈性抑制甲基化反应。DMTA为SAM的类似物从而竞争抑制甲基化反应。
目前尚没有将ADDA或DMTA用于制备γ-珠蛋白诱导剂的报导。
发明内容
针对上述技术问题,本发明的目的是提供了一种效果较好的新的γ-珠蛋白诱导剂,提出了ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用,以及在制备治疗β-地中海贫血和镰刀型细胞贫血等疾病的药物中的应用。
本发明的目的是通过下列技术方案实现的:
ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用。
ADDA和/或DMTA在制备治疗β-地中海贫血或镰刀型细胞贫血,造血系统肿瘤,如急、慢性白血病、淋巴瘤、骨髓瘤等以及造血功能障碍,如再生障碍性贫血、骨髓增生异常综合征等引起的贫血的药物中的应用。这些类型的贫血可以由升高的γ-珠蛋白得到缓解。
本发明有益效果:
ADDA或DMTA具有同时抑制DNA甲基化和组蛋白甲基化的特性,发明人以人红系细胞系K562细胞和人骨髓细胞为研究对象,运用现代分子生物学方法和实验手段,发现它们能特异地提高K562细胞和人骨髓细胞胎儿型珠蛋白(γ-珠蛋白)的表达水平。本发明首次阐明ADDA或DMTA这两种化合物在人血红蛋白合成中作用,为β-地中海贫血和镰刀型细胞贫血、造血系统障碍或肿瘤引起的贫血的治疗提供了候选药物。ADDA和DMTA(尤其是ADDA)无色无味、用量少(微摩尔级)、易溶解、作用浓度低,具有广阔的应用前景。
附图说明
图1.ADDA或DMTA提高人K562细胞中γ-珠蛋白的表达。
A,B:与对照(PBS或DMSO)相比,实时PCR分析发现经ADDA或DMTA处理的K562细胞中胎儿型珠蛋白的转录表达水平增加8-10倍(*P<0.01)。C:联苯胺染色显示ADDA处理的K562细胞中胎儿型珠蛋白显著增加(棕色细胞,黑白图片中显示为深黑色细胞增多)。
图2.ADDA或DMTA提高人骨髓细胞CD34+中γ-珠蛋白的表达。
A,B:与对照(PBS或DMSO)相比,实时PCR分析发现经ADDA或DMTA处理的人骨髓细胞CD34+中胎儿型珠蛋白的转录表达水平增加2-6倍(*P<0.01)。C:HPLC分析发现ADDA处理骨髓细胞中胎儿型血红蛋白水平(HbF)升高2.7倍。
具体实施方式
以下通过实施例对本发明作进一步的阐述。
实施例1
一、材料与方法
1)化合物
ADDA(adenosine-2’,3’-dialdehyde),DMTA(5’-deoxy-5’(methyl-thio)-adenosine)均购自美国Sigma公司。ADDA溶解于去离子水中配成10mM储备液;DMTA溶解于DMSO(二甲亚砜)配成0.5M储备液。
2)细胞培养
人红系K562细胞在RPMI1640培养基中37℃培养,10%小牛血清,105U/L青霉素,100mg/L链霉素,5%CO2。K562细胞用终浓度50μM的ADDA或0.1mM DMTA处理72小时,收集细胞。
人骨髓CD34+细胞用CD34免疫磁珠(美国Miltenyi Biotec公司)分离,加入50μMADDA或100μM DMTA,对照组实验加入生理盐水或DMSO(体积比<0.01%),然后在红系细胞增生分化条件下培养3周,即在IMDM(Isove’s modified Dulbecco’s medium,依素唯修饰培养基)中培养,15%小牛血清及上述抗菌素,37℃,5%CO2。具体地,先在培养基中加入细胞因子SCF(100ng/ml),IL-3(10ng/ml)和Flt-3(10ng/ml)培养1周后,换成只加细胞因子EPO(5U/ml)再培养2周后,收集细胞。
3)RNA提取、实时PCR分析
适量细胞加入1ml Trizol试剂(美国Invitrogen公司),充分破碎裂解细胞后,加入200μl氯仿,13000rpm离心10分钟。取上清加入等体积的异丙醇,室温静置15分钟,13000rpm离心10分钟,沉淀用75%酒精洗涤,干燥,20μl无RNA酶的水溶解。1μgRNA进行cDNA逆转录反应,按说明书操作(SuperScript III First-Strand Synthesis Systemfor RT-PCR,美国Invitrogen公司)。取1μl cDNA分别进行胎儿型珠蛋白和GAPDH水平测定。实时PCR反应条件:95℃5分钟,95℃30秒,60℃30秒,72℃ 1分钟,40个循环。胎儿型珠蛋白:上游引物AATGTGGAAGATGCTGGAGGAGAA,下游引物CTTCTTGCCATGTGCCTTGACTT;GAPDH:上游引物GAAGGTGAAGGTCGGAGTC,下游引物GAAGATGGTGATGGGATTTC。
4)联苯胺染色
收集K562细胞(1000rpm,5分钟),PBS洗涤一次。新鲜配制联苯胺液体(5mg 3,3’-二氨基联苯胺溶解于10ml PBS,加入10μl 30%H2O2),吸取1ml悬浮细胞沉淀,染色15分钟。离心,PBS洗涤,悬浮细胞1-5×105/ml,用细胞涂片机将细胞置于载玻片上,空气干燥细胞。用1.25%戊二醛1%甲酰胺(PBS配制)固定。PBS漂洗,苏木精染色2分钟,自来水漂洗,干燥封片。
5)HPLC分析
人骨髓细胞(5×106)收集后,PBS充分洗涤,加入200μl去离子水,反复冻融2-3次,离心取上清,按美国Bio-Rad公司Variant Hemoglobin Testing System程序(Variant-thalassemia Short Program)进行HPLC分析胎儿型珠蛋白测定。
结果数据
1.ADDA或DMTA提高人K562细胞中胎儿型珠蛋白的表达:与对照相比,分析发现经ADDA或DMTA处理的K562细胞中胎儿型珠蛋白的转录表达水平增加8-10倍(图1A,1B)。联苯胺染色显示ADDA处理的K562细胞胎儿型珠蛋白显著增加(图1C)。
2.ADDA或DMTA提高人骨髓细胞胎儿型珠蛋白的表达:与对照相比,分析发现经ADDA或DMTA处理的骨髓细胞中胎儿型珠蛋白的转录表达增加2-6倍(图2A,2B);ADDA处理骨髓细胞胎儿型血红蛋白水平(HbF)升高2.7倍(图2C)。
结论:以上结果表明ADDA或DMTA能显著升高胎儿型珠蛋白的表达。
序列表
<110>南京大学
<120>ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用
<160>4
<210>1
<211>24
<212>DNA
<213>人工序列
<220>
<223>胎儿型珠蛋白上游引物
<400>1
aatgtggaag atgctggagg agaa 24
<210>2
<211>23
<212>DNA
<213>人工序列
<220>
<223>胎儿型珠蛋白下游引物
<400>2
cttcttgcca tgtgccttga ctt 23
<210>3
<211>19
<212>DNA
<213>人工序列
<220>
<223>GAPDH上游引物
<400>3
gaaggtgaag gtcggagtc 19
<210>4
<211>20
<212>DNA
<213>人工序列
<220>
<223>GAPDH下游引物
<400>4
gaagatggtg atgggatttc 20
Claims (3)
1、ADDA和/或DMTA在制备提高γ-珠蛋白表达量的药物中的应用。
2、ADDA和/或DMTA在制备治疗β-地中海贫血或镰刀型细胞贫血、造血系统肿瘤以及造血功能障碍引起的贫血的药物中的应用。
3、根据权利要求2所述的应用,其特征在于造血系统肿瘤是指急性白血病、慢性白血病、淋巴瘤、骨髓瘤;造血功能障碍是指再生障碍性贫血、骨髓增生异常综合征。
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CN111481532A (zh) * | 2020-06-28 | 2020-08-04 | 南京中澳转化医学研究院有限公司 | 化合物28d在制备提高γ-珠蛋白表达量的药物中的应用 |
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CN110623960B (zh) * | 2018-06-22 | 2022-08-19 | 成都山权江生物科技有限公司 | 一种小分子化合物在制备治疗阿尔茨海默病的药物中的应用 |
CN111481532A (zh) * | 2020-06-28 | 2020-08-04 | 南京中澳转化医学研究院有限公司 | 化合物28d在制备提高γ-珠蛋白表达量的药物中的应用 |
CN111481532B (zh) * | 2020-06-28 | 2020-09-22 | 南京中澳转化医学研究院有限公司 | 化合物28d在制备提高γ-珠蛋白表达量的药物中的应用 |
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