CN107306853B - 骨髓增生异常综合征动物模型及转基因斑马鱼在制备该动物模型中的用途 - Google Patents
骨髓增生异常综合征动物模型及转基因斑马鱼在制备该动物模型中的用途 Download PDFInfo
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Abstract
本发明涉及一种转基因斑马鱼在制备骨髓增生异常综合症(MDS)的动物模型中的用途,其中所述转基因斑马鱼为c‑mybhyper突变体。
Description
技术领域
本申请涉及生物技术领域,具体地,涉及斑马鱼用于人类白血病的发病机理研究及其大规模药物筛选的用途。
背景技术
很多调节因子都与正常造血细胞的增殖、分化以及谱系命运决定相关。原癌基因c-MYB是其中一种关键的调节因子(1,2)。c-MYB特异性表达在造血干细胞以及祖细胞中,并且其表达随着细胞分化而降低(2-4)。
骨髓增生异常综合症(MDS)是老年人常见的血液肿瘤疾病,并且可以进展为急性髓系白血病(AML)或者急性淋系白血病(ALL)(38)。然而,MDS进展为AML或者ALL的具体分子机制尚不清楚。其中获得型突变是MDS向急性白血病转化的一个重要因素(39)。
现有的一种c-MYB异常激活小鼠中,其c-Myb是在鼠的淋巴细胞特异性启动子下启动,而不是原有的启动子启动,其表达的时空已经发生改变。结果是,只影响到淋巴细胞的发育,导致淋巴细胞血液疾病,而对髓系细胞发育和髓系血液疾病的影响并无研究(42)。
斑马鱼与人的血液组成以及基因调控机制的相似使得斑马鱼成为研究造血发育的理想动物模型(19)。斑马鱼已经作为疾病模式动物用于阐述一些造血疾病的新的分子机制(20)和筛选新的药物(21)。通过突变或者过表达一些原癌基因建立起与血液肿瘤相关的越来越多的斑马鱼突变体和转基因系(22,23)。利用斑马鱼白血病模型不仅可以进一步研究人白血病发病的分子机制,还可以在体内进行新的小分子化合物的药筛。
斑马鱼是进行药物研发的理想模式动物(21,35)。斑马鱼相比于小鼠更适合做大规模的药物筛选,具有成本低、高效等优势。斑马鱼用于药物筛选可以实现以下益处:高通量:斑马鱼产卵数量多,且受精后的1天开始加药,2-5天后检测,在此期间卵黄可提供胚胎营养而不用喂食,使得可以用96孔或384孔板进行药物筛选。低费用:斑马鱼饲养和消耗的费用远远低于小鼠,平均每天每只消费约为小鼠的1/100。给药方便:可直接将化合物溶于水中,扩散至胚胎,所需化合物的量仅为小鼠的1/100-1/1000。
发明内容
本发明要解决的技术问题是:c-myb转基因斑马鱼在表型、药物敏感性、疾病进展过程等方面与人的白血病具备何种程度的相似性?如何开展新的斑马鱼白血病模型的建立及标准化、高通量新药筛选?
为了解决这些技术问题,本发明人提出了以下发明总体构思:
(1)胚胎时期,利用标记不同阶段髓系细胞的探针进行WISH实验来检测详细的髓系造血改变,用c/ebpɑ标记不成熟的中性粒细胞;用lyz标记成熟的中性粒细胞;用mfap4来标记成熟的巨噬细胞。阐述c-myb在斑马鱼中特异性髓系祖细胞分化以及随后中性粒细胞和巨噬细胞命运选择中的作用。
(2)在成鱼时期,将c-myb异常激活斑马鱼和已经用dsRed标记了髓系祖细胞、中性粒细胞、巨噬细胞的转基因家系鱼进行杂交,用流式细胞仪(FACS)和细胞学方法分析(3M,1Y)杂交子代的肾脏、血液循环***中髓系细胞的数量、亚型、分化阶段的情况,观察白血病的特征是否会在这些鱼中出现。
(3)用Brdu、TUNEL实验确定髓系细胞分化过程中增殖和凋亡的改变。
(4)已知白血病治疗药物对白血病模型的药理学验证:配鱼(将c-myb异常激活斑马鱼Tg(c-myb:GFP)与野生型斑马鱼AB杂交),每条母鱼产卵200-300个。通过绿色荧光挑出c-myb异常激活受精卵。将c-myb异常激活受精卵放入6孔板,每孔放30~50个。选取已知的临床上治疗白血病的药物(主要为临床上已知的且以c-myb为靶点的药物)。将所选的不同的药物加入到含有鱼卵和水的孔中,每孔只加一种药物。孵育一段时间后,通过苏丹黑B(Sudan black B,SB)染色等方法分析,找到可以改变c-myb异常激活鱼卵造血发育表型的药物。
(5)潜在生物活性小分子库的药物筛选:配鱼(将c-myb异常激活斑马鱼Tg(c-myb:GFP)与野生型斑马鱼AB杂交),每条母鱼产卵200-300个。通过绿色荧光挑出c-myb异常激活受精卵。将c-myb异常激活受精卵放入96孔板或384孔板,每孔放3~5个。选取具有潜在生物学活性化合物的小分子库(主要为成药库)。将所选的不同的小分子加入到含有鱼卵和水的孔中,每孔只加一种药物。孵育一段时间后,通过SB染色等方法大规模分析,找到可以改变c-myb异常激活鱼卵造血发育表型的新的化合物。
本发明与现有技术的区别主要在于:本发明是在c-myb时空表达不改变的情况下c-myb被异常激活,继而引起髓系造血异常的表型。发明人首次发现了本发明的转基因斑马鱼在幼年和成年时示出类似人白血病的表型,并证明了可以利用其对治疗白血病的药物进行高通量筛选。
本发明的斑马鱼模型中,c-myb是在原有的核心启动子下启动,时空表达不改变。造成的表型主要表现在血液***上,且有类白血病表型。
本申请提供了一种转基因斑马鱼在制备骨髓增生异常综合症(MDS)的动物模型中的用途,其中所述转基因斑马鱼为c-mybhyper突变体。在一些实施方案中,所述骨髓增生异常综合症(MDS)可以是由c-myb基因的异常激活引起。一些实施方案中,所述骨髓增生异常综合症(MDS)可以在通过阻断细胞周期起作用的药物或c-myb靶向药物治疗后症状缓解。在进一步的实施方案中,所述通过阻断细胞周期起作用的药物为阿糖胞苷,所述c-myb靶向药物为弗拉平度。
本申请还提供了一种转基因斑马鱼在制备用于筛选对骨髓增生异常综合症(MDS)有效的药物的动物模型中的用途,其中所述转基因斑马鱼为c-mybhyper突变体。在一些实施方案中,所述骨髓增生异常综合症(MDS)可以是由c-myb基因的异常激活引起。在一些实施方案中,所述骨髓增生异常综合症(MDS)可以在通过阻断细胞周期起作用的药物或c-myb靶向药物治疗后症状缓解。在一些实施方案中,所述通过阻断细胞周期起作用的药物为阿糖胞苷,所述c-myb靶向药物为弗拉平度。在一些实施方案中,所述筛选可以包括以下步骤:(a)以相同浓度的备选药物处理受精后第一至两天的所述转基因斑马鱼的胚胎和野生型同胞鱼胚胎;(b)在处理后第二至五天观察胚胎尾部造血组织(CHT)区域的血液表型,以确定所述备选药物对所述骨髓增生异常综合症(MDS)的治疗效果。在一些实施方案中,所述血液表型可以包括苏丹黑B(SB)染色阳性粒细胞计数。
本申请还提供了一种利用转基因斑马鱼来筛选对骨髓增生异常综合症(MDS)有效的药物的方法,其中所述转基因斑马鱼为c-mybhyper突变体。在一些实施方案中,所述骨髓增生异常综合症(MDS)可以是由c-myb基因的异常激活引起。在一些实施方案中,所述骨髓增生异常综合症(MDS)可以在通过阻断细胞周期起作用的药物或c-myb靶向药物治疗后症状缓解。在一些实施方案中,所述通过阻断细胞周期起作用的药物为阿糖胞苷,所述c-myb靶向药物为弗拉平度。在一些实施方案中,所述筛选可以包括以下步骤:(a)以相同浓度的备选药物处理受精后第一至两天的所述转基因斑马鱼的胚胎和野生型同胞鱼胚胎;(b)在处理后第二至五天观察胚胎尾部造血组织(CHT)区域的血液表型,以确定所述备选药物对所述骨髓增生异常综合症(MDS)的治疗效果。在一些实施方案中,所述血液表型可以包括苏丹黑B(SB)染色阳性粒细胞计数。
本申请还提供了一种骨髓增生异常综合症(MDS)的动物模型,所述动物模型包括c-mybhyper突变体的转基因斑马鱼。
本文所使用的术语“hpf”指受精后的小时数。本文所使用的术语“dpf”指受精后的天数。举例来说,“3dpf”指受精后三天,“8dpf”指受精后八天。
本文所使用的术语“野生型”或者“WT”都是指野生型斑马鱼。
本文所使用的术语“同胞鱼(sibling)”是指由同一双亲的后代个体。
附图说明
图1.c-myb mRNA在c-mybhyper突变体的胚胎到成鱼时期持续增加。(A)在c-myb-gfp转基因斑马鱼的PAC中发现两段重复序列。第一段(1st,485-bp的小启动子)用实线框标出,第二段重复序列(2nd,pWSMK-T到c-myb内含子10的区域)用实线框标出。每段重复序列的***位点用箭头指示。c-myb的外显子用黑色条指示,pWSMK-T载体包含了GFP、SV40ployA终止信号以及氨苄抗性基因的反向序列。77-bp的未知序列用白色条指示出。(B)c-myb-WT以及c-myb-T1的转录本和蛋白信息。c-myb-WT和c-myb-T1的转录起始位点分别用箭头标出。终止密码子用星号标出。DBD表示DNA结合域,TAD表示转录激活域,NRD表示负性调控域。(C)c-myb在受精后36小时、3天以及5天的原位杂交结果。黑色框为AGM(主动脉性腺中肾区)、CHT(尾部造血组织)以及肾脏区域放大到20倍的结果。图右上方的数字表示表达增加的胚胎比胚胎总数(Fisher’s检验,P≤0.01)。(D)c-myb基因在受精后3天、5天的整体胚胎以及3个月、6个月和1年的肾脏中qPCR结果(*P≤0.05,**P≤0.01,独立样本t检验;整体胚胎每组30个样品,肾脏每组10个样品)。
图2.c-mybhyper胚胎中髓系细胞异常增生。(A-C)c-myb异常激活促进了中性粒细胞的积累以及巨噬细胞的减少。原位杂交显示c/ebpα(A)、mfap4(B)和lyz(C)在受精后3天的c-mybhyper斑马鱼和其同胞鱼中的表达。SB在受精后3天(D)以及7天(E)的幼鱼中的染色。黑色框为CHT以及肾脏区域放大到20倍的结果。D图中的最右边黑色框为单个细胞放大到100倍的结果。图右上方的数字表示表达增加的胚胎比胚胎总数(Fisher’s检验,n≥10,P≤0.01)。
图3.c-mybhyper成鱼表现出类MDS表型:肾脏血异常髓系增生。(A-D)1年的c-mybhyper斑马鱼和其同胞鱼的外周血(PB)细胞(A)和肾脏血(KM)细胞(C)的瑞姬氏染色。箭头、星号、三角形和闪电分别指示红细胞、祖细胞、髓系单核细胞以及淋巴细胞。c-mybhyper斑马鱼(黑色条图)和其同胞鱼(灰色条图)的PB(B)以及KM(D)血细胞计数。黑色星号表示具有统计学差异。(t-检验,1年同胞鱼和c-mybhyper的PB样本量分别为9和5,KM样本量分别为6和5,平均值±SEM;*P≤0.05,**P≤0.01)。(E 和F)流式分析c-mybhyper斑马鱼和其同胞鱼的血液组成。利用FSC表示细胞大小和利用SSC表示细胞内颗粒多少将血液细胞区分计数。(G-K)c-mybhyper斑马鱼出现肾肿大和肝肿大。1年的c-mybhyper斑马鱼肾脏面积为同胞鱼的1.4倍(H),重量为同胞鱼的3.3倍(I)。1年的c-mybhyper斑马鱼肝脏出现SB阳性细胞增多(J)。J图中右边黑色框为左图中的局部放大图。1年的c-mybhyper斑马鱼肝脏重量增加(K)。(t-检验,n≥10;平均值±SEM;*P≤0.05,**P≤0.01)。
图4.c-mybhyper胚胎以及成鱼中髓系细胞的增多是由于细胞增殖增加导致的。(A-H)c-myb异常激活会导致胚胎(A-D)以及成鱼肾脏(E-H)中髓系细胞的增殖增加而凋亡无显著性改变。BrdU/Lcp的免疫荧光双染(A和E)以及TUNEL/Lcp双染(C和G)指示了BrdU+或者TUNEL+在3dpf/3个月的CHT/KM区域与Lcp+细胞共染结果。CHT和KM区域中Lcp+/BrdU+细胞占Lcp+细胞的比例(B和F)以及Lcp+/TUNEL+细胞占Lcp+细胞的比例(D和H)被计算出(t-检验,n>13;平均值±SEM;*P≤0.05,**P≤0.01)。
图5.c-mybhyper MDS样斑马鱼对化疗药物的反应。(A-C)用PBS(A)或者阿糖胞苷(Ara-c,B),柔红霉素(DNR,C)处理受精后1天的c-mybhyper(右边栏)和同胞鱼(左边栏)胚胎5天后进行SB染色。(D和E)受精后1天的胚胎用二甲基亚砜(D)或者弗拉平度(E)处理4天后进行SB染色。(F)药物处理后每条幼鱼的SB阳性细胞计数。(t-检验,n≥10;平均值±SEM;*P≤0.05,**P≤0.01)。(G)利用qPCR检测药物处理后c-myb基因在c-mybhyper(黑色条图)和同胞鱼(灰色条图)中的表达情况(t-检验,n=30;平均值±SEM;*P≤0.05,**P≤0.01)。
图6.c-mybhyper斑马鱼中的重复序列。(A)c-myb小的启动子重复序列包含一个315bp的核心启动子序列以及170bp的外显子1的序列。c-myb小启动子重复序列和其附近序列被标示出来。(B)第二段重复序列中的内含子10的断点位置附近序列被标示出来(黑色框)。c-myb的外显子用黑色条指示,pWSMK-T载体包含了GFP、SV40ployA终止信号以及氨苄抗性基因的反向序列(AmpR)。77-bp的未知序列用白色条指示出。
图7.髓系细胞在3个月的c-mybhyper斑马鱼肾脏血中异常增生。(A-D) 3个月的c-mybhyper斑马鱼和其同胞鱼的外周血(PB)细胞(A)和肾脏血(KM)细胞(C)的瑞姬氏染色。箭头、星号、三角形和闪电分别指示红细胞、祖细胞、髓系单核细胞以及淋巴细胞。c-mybhyper斑马鱼(黑色条图)和其同胞鱼(灰色条图)的PB(B)以及KM(D)血细胞计数。黑色星号表示具有统计学差异。(t-检验,1年同胞鱼和c-mybhyperPB样本量分别为6和9,KM样本量分别为8和8,平均值±SEM;*P≤0.05,**P≤0.01)。
具体实施方式
下面将以实施例的方式对本申请作进一步的详细描述,以使本领域技术人员能够实践本申请。应当理解,可以采用其他实施方式,并且可以做出适当的改变而不偏离本申请的精神或范围。为了避免对于使本领域技术人员能够实践本申请来说不必要的细节,说明书可能省略了对于本领域技术人员来说已知的某些信息。因此,以下详细描述不应以限制性的意义来理解,且本发明的范围仅由所附权利要求界定。
以下的实施例便于更好地理解本申请,但并不用来限制本申请的范围。
下述实施例中所使用的各原料,除特别指出的以外,均可由市售获得。
材料和方法
斑马鱼养殖
斑马鱼的养殖如文献所述(25)。本发明中用到下面的品系:AB、Tg(c-myb-gfp)、Tg(rag2:dsRed)。
5’和3’-RACE
RACE是用RACE 5’/3’Kit(Clontech,CA,USA;634858)进行的,具体的操作步骤参考该试剂盒的说明书。5’-RACE PCR中c-myb基因特异性引物是5’-GATTACGCCAAGCTTTCCATGATCGAACGCCTCAGGTTGGGA-3’,3’-RACE PCR中c-myb基因特异性引物是 5’-GATTACGCCAAGCTTCGAGGCGGCACAGACACAGTGTTTACAG-3’。PCR产物电泳后回收,***到pRACE载体后转染感受态细胞,挑取多于12个的单克隆进行测序。
整体原位杂交
整体原位杂交(WISH)是利用抗地高辛标记的RNA探针进行的,实验步骤如文献所述(28)。图像利用奥林巴斯MVX10显微镜(Shinjuku,Tokyo,Japan)进行拍摄。
实时荧光定量PCR
总RNA利用RNAeasy Kit(Qiagen,Maryland,USA;74004)提取,继而利用反转录试剂盒(Promega,Madison,USA;M1701)合成cDNA。实时荧光定量PCR利用SYBR Green Real-time PCR Master Mix(Applied Biosystems,Austin,USA;4472908)染料在LightCyclerNano SW1.0机器上进行。具体操作步骤参照说明书。内参为衍伸因子1α(ef1a)。所有的引物利用Primer5软件设计(表1)。
表1:qPCR引物
溴脱氧尿嘧啶核苷和末端脱氧核苷酰转移酶介导的dUTP缺口末端标记法
溴脱氧尿嘧啶核苷(BrdU)标记实验操作如文献所述(29)。受精后3 天的Tg(c-myb-egfp)胚胎和同胞鱼对照组用10mM的BrdU(Sigma-Aldrich;B5002)孵育2小时,之后用4%的多聚甲醛固定,再用鼠源性的抗-BrdU(Roche,Germany;10875400)和兔源性的抗-lcp抗体孵育,最后用抗鼠的488(Invitrogen,CA,USA;A21202)以及抗兔的555(Invitrogen,CA,USA;A31572)荧光染色并观察。末端脱氧核苷酰转移酶介导的dUTP缺口末端标记(TUNEL)实验利用In Situ Cell Death Detection Kit TMR red(Roche;12156792910)试剂盒进行。接着使用兔源性的抗-lcp以及抗兔的555染色。图像利用奥林巴斯fluoview1000共聚焦显微镜进行拍摄。
细胞学分析
造血细胞的分离如下:斑马鱼用三卡因麻醉,外周血(PB)通过用10μL肝素化过的微量血细胞比容管从鳃中获取。肾脏血(KM)细胞从肾脏中分离。分离出的PB和KM用含5%的FBS的PBS重悬,接着将重悬的细胞400转3分钟离心至玻片上。最后用姬姆萨(Merck,Germany;1.09204.0500),瑞士姬(Merck;1.01424.0500)染色。根据细胞形态对PB和KM细胞进行计数。
实施例1:C-myb的表达量在c-myb–gfp转基因斑马鱼中从胚胎到成鱼不断增加
North等人首次描述了c-myb–gfp转基因斑马鱼系,它是作为用于标记c-myb阳性的HSCs被建立的(24)(对构建c-myb-gfp所需的重组序列的测序结果如SEQ ID NO:1所示)。然而,当我们将c-myb-gfp和c-mybhkz3/hkz3突变斑马鱼交配后,发现c-mybhkz3/hkz3斑马鱼造血缺陷表型被拯救。
为了寻找c-myb-gfp/c-mybhkz3/hkz3斑马鱼中造血缺陷的恢复的原因,我们将c-myb–gfp转基因斑马鱼中PAC区域进行测序(测序结果如SEQ ID NO:2所示)并发现了两段重复序列,其中一段是在翻译起始位点之前发现的一段485bp的重复序列,它包括了315bp的核心启动子区域以及170bp的5’UTR(图1A和图6A);另一段大的重复区域是从pWSMK-T载体到c-myb内含子10的重复序列,它插在了c-myb内含子10中的断裂位置(图1A和图6B)。3’以及5’RACE实验则发现c-myb–gfp中PAC产生了2种不同的转录本(分别命名为c-myb-WT和c-myb-T1)(图1B)。其中一个转录本是c-myb-WT(其对应的cDNA序列如SEQ ID NO:3所示),它由第二个小启动子启动,并且与野生型斑马鱼的c-myb基因转录本(其对应的cDNA序列如SEQ ID NO:4所示)完全一致(图1B);另一个转录本是4.3kb的c-myb-T1(其对应的cDNA序列如SEQ ID NO:5所示),它由第一个小启动子启动,并且由一个截断的c-myb(外显子1到外显子10)以及一个接近完整的c-myb(外显子2到外显子15)组成(图1B)。c-myb-T1翻译的蛋白是由两段蛋白融合而成的,第一段是缺少完整C端的负性调控区域(NRD)的c-myb蛋白,另一段则是在N端少了8个氨基酸的c-myb蛋白(图1B)。
为了检测c-myb基因在c-myb–gfp转基因斑马鱼中的表达情况,我们对c-myb–gfp转基因斑马鱼的整体胚胎以及成鱼的肾脏进行了WISH和qPCR实验(图1C、D)。结果发现在c-myb–gfp转基因斑马鱼中,以c-myb探针(SEQ ID NO:6)检测,c-myb基因在HSC产生的部位——主动脉性腺中肾区(AGM)、尾部造血组织(CHT)以及肾脏中表达,但是,c-myb基因在c-myb–gfp转基因斑马鱼中的表达水平相比于野生型增强了很多(图1C)。qPCR结果显示从3dpf开始,c-myb基因在c-myb–gfp转基因斑马鱼中的表达量约为野生型中的2倍,并且随着时间推移越来越多(图1D)。c-myb基因在c-myb–gfp转基因斑马鱼中的过表达可能是由于PAC中外源性c-myb基因的表达。发明人据此将c-myb–gfp转基因斑马鱼命名为c-myb基因异常激活斑马鱼(c-myb hyperactivity,c-mybhyper)。
实施例2:早期胚胎时期c-myb基因异常激活导致异常的粒细胞累积
我们在c-mybhyper斑马鱼胚胎中利用谱系特异性标记物通过WISH实验来观察c-myb的异常激活对髓系细胞发育的影响。我们在定向髓系发生的时期用c/ebpα探针(SEQ IDNO:7)检测了c/ebpα基因(一种早期的髓系细胞特异性标记物)的表达情况。结果显示,与对照组相比,c-mybhyper中表达c/ebpα基因的细胞数量明显增多(图2A)。
接下来,我们利用巨噬细胞特异性标记物以及粒细胞特异性标记物通过WISH实验来研究c-mybhyper中累积的髓系细胞的细胞类型。其中表达巨噬细胞特异性标记物mfap4(SEQ ID NO:8)的细胞在3dpf的c-mybhyper中数量显著减少(图2B)。相反的,表达粒细胞特异性标记物lyz(SEQ ID NO:9)的细胞在3dpf的c-mybhyper中数量显著增多(图2C)。这提示在c-mybhyper胚胎中积累的髓系细胞来源为粒细胞。这一结果也被SB染色(SB染色可以特异标记斑马鱼中的粒细胞,在临床上也被用于诊断髓系白血病细胞)进一步证明。在c-mybhyper胚胎以及幼鱼中,被SB染色所标记的粒细胞在CHT以及肾脏(KM)中显著增多(图2D和E)。更重要的是,在c-mybhyper中SB阳性的粒细胞不仅数量增多,而且其大小更大、染色更深(图2D、E)。这提示了c-mybhyper中粒细胞功能的异常。以上的结果显示,在c-mybhyper胚胎中积累的髓系细胞是粒细胞来源而不是巨噬细胞来源,并且这种粒细胞在早期发育时期存在功能和形态上的异常。
实施例3:c-mybhyper成鱼出现MDS样表型
为了观察成鱼的造血改变,我们利用流式细胞仪以及细胞学实验分离c-mybhyper和同胞鱼对照组中的血细胞。对从3个月和1年的c-mybhyper和同胞鱼对照组中分离的外周血(PB)和肾脏血(KM)细胞进行不同细胞类型计数分析。
结果发现:在3个月和1年的c-mybhyper中PB血的组成比例与同胞鱼对照组没有明显区别(图7A、B和图3A、B);而3个月和1年的c-mybhyper中KM血的组成比例区别于同胞鱼对照组。与同胞鱼对照组相比,3个月的c-mybhyper的KM血中髓系细胞数量明显增多,同时伴随祖细胞的减少(图7C、D)。与同胞鱼对照组相比,1年的c-mybhyper的KM血中髓系细胞数量增加了一倍,并伴随其它谱系细胞的减少(图3C、D)。这些表型与髓系异常增生相符合。
流式细胞分析结果与之前的一致,显示在1年的c-mybhyper中髓系细胞数量增多两倍并伴随其它谱系细胞的减少(图3E、F)。并且通过流式细胞分析可以看出髓系细胞不仅数量增多,其大小变大,粒性更强。为了观察c-mybhyper斑马鱼中的内脏是否受到影响,我们将c-mybhyper和同胞鱼对照组的内脏在显微镜下解剖出来。结果发现1年的c-mybhyper斑马鱼肾脏出现肿大,与同胞鱼对照组相比,肾脏的面积增加1.37倍,重量增加了4倍(图3G-I)。肝脏也变重并且与对照组相比有大量的SB阳性细胞浸润(图3J、 K)。这些内脏的缺陷以及浸润同样出现在人粒细胞性血液恶性肿瘤中。
综上所述,c-mybhyper斑马鱼中血液表型和组织浸润表型与人的MDS症状类似。
实施例4:c-mybhyper斑马鱼中粒细胞增多主要由于增殖增多导致
c-mybhyper斑马鱼中粒细胞增多既可能由于粒细胞增殖增多导致也可能由于凋亡减少导致。为了明确c-mybhyper斑马鱼中粒细胞增多的分子机制,我们分别用BrdU和TUNEL实验来监测细胞的增殖和凋亡情况。c-mybhyper胚胎以及成鱼肾脏中髓系细胞的凋亡情况与对照组类似(图4C、D、G和H),提示c-mybhyper斑马鱼中粒细胞增多并不是由于凋亡改变而导致的。然而,BrdU实验结果显示c-mybhyper胚胎以及成鱼肾脏中髓系细胞的增殖显著增多(图4A、B、E和F),显示c-mybhyper斑马鱼中粒细胞增多可能是增殖增多的结果。以上结果提示c-myb异常激活促进了粒细胞的持续增殖。
实施例5:临床化疗药物对c-mybhyper斑马鱼白血病模型有效
我们观察了临床常用抗白血病药物在c-mybhyper斑马鱼中的效果。阿糖胞苷(36)和柔红霉素(37)是治疗白血病的常用化疗药物,它们分别通过阻断细胞周期以及促进累积的白血病细胞凋亡而起作用。将1dpf的c-mybhyper胚胎以及同胞鱼对照组用103mg/L阿糖胞苷或者30mg/L柔红霉素孵育,在6dpf的时候对胚胎CHT区域进行SB阳性粒细胞计数(图5A–C和F)。未经药物处理的c-mybhyper斑马鱼CHT区域SB阳性粒细胞数量很多(图5A和F)。经过5天的阿糖胞苷处理后的c-mybhyper斑马鱼的SB阳性粒细胞数量与未经过药物处理组相比显著减少(图5B和F),而阿糖胞苷处理的同胞鱼对照组与未处理的同胞鱼对照组相比没有明显改变(图5A和F)。然而,柔红霉素对c-mybhyper斑马鱼的粒细胞没有任何作用(图5C和F)。以上结果显示c-myb异常激活诱导的髓系异常增殖表型可以被细胞周期特异性抗增殖药物阿糖胞苷缓解而不能被促凋亡药物柔红霉素缓解。
我们同时观察了c-mybhyper中异常增殖的髓系细胞对c-myb靶向药物CDK9的抑制剂弗拉平度的反应。弗拉平度可以显著减少c-mybhyper中SB 阳性粒细胞的数量,而对同胞鱼对照组没有影响(图5D–F)。同时,与未经药物处理的c-mybhyper相比,经过弗拉平度处理的c-mybhyper中c-myb的RNA表达水平显著降低(图5G)。结果表明临床上的c-myb靶向药可以减少c-mybhyper斑马鱼中的髓系细胞异常扩增。
综上所述,研究结果显示斑马鱼中c-myb异常激活可以引起MDS并随着年龄进展为AML以及ALL,并且c-mybhyper斑马鱼模型的肿瘤表型可以被临床的化疗药物阿糖胞苷、弗拉平度所缓解。提示c-myb可以作为特定类型白血病的一个新的治疗靶点。c-mybhyper斑马鱼模型也为在体内的新药筛选提供有价值的平台,特别是应用于旨在治疗上述特定类型白血病的细胞周期特异性抗增殖药物候选物以及c-myb靶向药候选物的筛选。
综上所述,以上仅为本申请的较佳实施例而已,并非用于限定本申请的保护范围,因此,凡在本申请的精神和原则之内所作的任何修改、等同替换、改进等,均应包含在本申请的保护范围之内。
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Claims (5)
1.一种转基因斑马鱼在制备骨髓增生异常综合症(MDS)的动物模型中的用途,其中所述转基因斑马鱼为c-myb基因异常激活(c-mybhyper)斑马鱼。
2.如权利要求1所述的用途,其中所述骨髓增生异常综合症(MDS)是由c-myb基因的异常激活引起。
3.如权利要求1所述的用途,其中所述骨髓增生异常综合症(MDS)在通过阻断细胞周期起作用的药物或c-myb靶向药物治疗后症状缓解。
4.如权利要求3所述的用途,其中所述通过阻断细胞周期起作用的药物为阿糖胞苷,所述c-myb靶向药物为弗拉平度。
5.一种骨髓增生异常综合症(MDS)的动物模型,所述动物模型包括c-myb基因异常激活(c-mybhyper)的转基因斑马鱼。
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