CN107306853B - Myelodysplastic syndrome animal model and application of transgenic zebra fish in preparation of animal model - Google Patents
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Abstract
The invention relates to application of transgenic zebra fish in preparation of an animal model of myelodysplastic syndrome (MDS), wherein the transgenic zebra fish is c-mybhyperAnd (3) mutants.
Description
Technical Field
The application relates to the field of biotechnology, in particular to application of zebra fish in pathogenesis research of human leukemia and large-scale drug screening.
Background
Many regulatory factors are involved in the proliferation, differentiation and lineage fate determination of normal hematopoietic cells. The protooncogene c-MYB is one of the key regulators (1, 2). c-MYB is specifically expressed in hematopoietic stem and progenitor cells, and its expression decreases as the cells differentiate (2-4).
Myelodysplastic syndrome (MDS) is a common hematological neoplastic disease in the elderly and can progress to Acute Myeloid Leukemia (AML) or Acute Lymphoid Leukemia (ALL) (38). However, the specific molecular mechanism by which MDS progresses to AML or ALL is not clear. Wherein acquired mutations are an important factor in the transformation of MDS into acute leukemia (39).
In the existing c-MYB abnormally activated mouse, c-Myb is started under a mouse lymphocyte specific promoter instead of the original promoter, and the expression space time is changed. As a result, only the development of lymphocytes was affected, resulting in a lymphocytic hematological disease, and the effects on the development of myeloid cells and myeloid hematological diseases were not studied (42).
The similarity of blood composition and gene regulation mechanism between zebrafish and human makes zebrafish an ideal animal model for studying hematopoietic development (19). Zebrafish have been used as disease model animals to elucidate some novel molecular mechanisms of hematopoietic diseases (20) and to screen for new drugs (21). More and more zebrafish mutants and transgenic lines associated with hematologic tumors are established by mutating or overexpressing some protooncogenes (22, 23). The zebra fish leukemia model can be used for further researching the molecular mechanism of human leukemia pathogenesis and can also be used for carrying out drug screening on a new small molecular compound in vivo.
Zebrafish are ideal model animals for drug development (21, 35). Compared with a mouse, the zebra fish is more suitable for large-scale drug screening, and has the advantages of low cost, high efficiency and the like. The use of zebrafish for drug screening may achieve the following benefits: high flux: the number of eggs laid by zebrafish is large, and the drug is added 1 day after fertilization, and the detection is carried out 2-5 days later, during which the yolk can provide embryo nutrition without feeding, so that drug screening can be carried out with 96-well or 384-well plates. Low cost: zebrafish are kept and consumed at a much lower cost than mice, on average about 1/100 per mouse per day. The administration is convenient: the compounds can be dissolved directly in water and diffused to the embryo, the amount of compound required being only 1/100-1/1000 for mice.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: what degree do c-myb transgenic zebrafish share similarities with human leukemia in phenotype, drug susceptibility, disease progression, etc.? How to develop the establishment and standardization of a new zebra fish leukemia model and high-throughput new drug screening?
In order to solve these technical problems, the present inventors have proposed the following general inventive concept:
(1) in the embryonic period, WISH experiments are carried out by using probes for marking the myeloid cells in different stages to detect detailed hematopoietic changes of the myeloid lines, and immature neutrophils are marked by using c/ebp alpha; labeling mature neutrophils with lyz; mature macrophages were labeled with mfap 4. The role of c-myb in the differentiation of specific myeloid progenitor cells in zebrafish and the subsequent selection of neutrophil and macrophage fate is elucidated.
(2) In the adult fish stage, c-myb abnormally activated zebrafish and transgenic domestic fish which have been marked with dsRed for myeloid progenitor cells, neutrophils and macrophages are hybridized, and the filial generation of the hybrids is analyzed by flow cytometry (FACS) and cytology (3M, 1Y) for the number, subtype and differentiation stage of myeloid cells in kidney and blood circulation system, and whether the characteristics of leukemia will appear in the fish is observed.
(3) Changes in proliferation and apoptosis during myeloid cell differentiation were determined using Brdu, TUNEL experiments.
(4) Pharmacological validation of known leukemia therapeutic drugs on leukemia models: for gametophyte (the c-myb abnormally activated zebrafish Tg (c-myb: GFP) is hybridized with the wild type zebrafish AB), 200 eggs are laid by each mother fish. C-myb is picked out by green fluorescence to activate the fertilized egg abnormally. And (3) putting the c-myb abnormally activated fertilized eggs into a 6-hole plate, wherein 30-50 fertilized eggs are put in each hole. Selecting the known drugs for treating leukemia clinically (mainly the drugs which are known clinically and take c-myb as a target). The different drugs selected were added to wells containing roe and water, one drug per well. After a period of incubation, the drugs capable of changing the phenotype of abnormal activation of c-myb in fish egg hematopoiesis are found by means of analysis such as Sudan black B (Sudan black B, SB) staining and the like.
(5) Drug screening of a potential bioactive small molecule library: for gametophyte (the c-myb abnormally activated zebrafish Tg (c-myb: GFP) is hybridized with the wild type zebrafish AB), 200 eggs are laid by each mother fish. C-myb is picked out by green fluorescence to activate the fertilized egg abnormally. And (3) putting the c-myb abnormally activated fertilized eggs into a 96-well plate or a 384-well plate, wherein 3-5 fertilized eggs are put in each well. Selecting a small molecule library (mainly a drug library) with potential biological active compounds. The different small molecules of choice were added to wells containing roe and water, with only one drug per well. After a period of incubation, large-scale analysis is carried out by methods such as SB staining and the like, and a novel compound which can change the phenotype of c-myb abnormally activated roe hematopoiesis and development is found.
The invention is mainly different from the prior art in that: the present invention is a phenotype in which c-myb is abnormally activated without altering its spatiotemporal expression, thereby causing abnormal myeloid hematopoiesis. The inventor firstly discovers that the transgenic zebrafish of the invention shows a phenotype similar to human leukemia in young and adult years, and proves that the transgenic zebrafish can be used for high-throughput screening of drugs for treating leukemia.
In the zebra fish model, c-myb is started under the original core promoter, and the spatiotemporal expression is not changed. The resulting phenotype is mainly manifested in the blood system and has a leukemia-like phenotype.
The application provides application of transgenic zebra fish in preparation of an animal model of myelodysplastic syndrome (MDS), wherein the transgenic zebra fish is c-mybhyperAnd (3) mutants. In some embodiments, the myelodysplastic syndrome (MDS) may be caused by abnormal activation of the c-myb gene. In some embodiments, the myelodysplastic syndrome (MDS) may be alleviated after treatment by a drug that acts to block the cell cycle or a c-myb-targeted drug. In a further embodiment, the drug that acts by blocking the cell cycle is cytarabine and the c-myb targeting drug is frataxin.
The application also provides an application of the transgenic zebrafish in preparing an animal model for screening drugs effective on myelodysplastic syndrome (MDS), wherein the transgenic zebrafish is c-mybhyperAnd (3) mutants. In some embodiments, the myelodysplastic syndrome (MDS) may be caused by abnormal activation of the c-myb gene. In some embodiments, the myelodysplastic syndrome (MDS) may be alleviated after treatment by a drug that acts by blocking the cell cycle or a c-myb-targeted drug. In some embodiments, the drug that acts by blocking the cell cycle is cytarabine and the c-myb targeting drug is frataxin. In some embodiments, the screening may comprise the steps of: (a) treating the embryos of the transgenic zebrafish and the wild type sibling fish embryos in the first to two days after fertilization with the same concentration of the candidate drug; (b) observing the blood phenotype of the embryonic tail hematopoietic tissue (CHT) region the second to five days after treatment to determine saidTherapeutic effect of a drug candidate on said myelodysplastic syndrome (MDS). In some embodiments, the blood phenotype can include a sudan black b (sb) staining positive granulocyte count.
The present application also provides a method for screening for agents effective on myelodysplastic syndrome (MDS) using transgenic zebrafish, which is c-mybhyperAnd (3) mutants. In some embodiments, the myelodysplastic syndrome (MDS) may be caused by abnormal activation of the c-myb gene. In some embodiments, the myelodysplastic syndrome (MDS) may be alleviated after treatment by a drug that acts by blocking the cell cycle or a c-myb-targeted drug. In some embodiments, the drug that acts by blocking the cell cycle is cytarabine and the c-myb targeting drug is frataxin. In some embodiments, the screening may comprise the steps of: (a) treating the embryos of the transgenic zebrafish and the wild type sibling fish embryos in the first to two days after fertilization with the same concentration of the candidate drug; (b) observing a blood phenotype of a hematopoietic tissue of embryonic tail (CHT) region on days two to five after treatment to determine the therapeutic effect of the candidate agent on the myelodysplastic syndrome (MDS). In some embodiments, the blood phenotype can include a sudan black b (sb) staining positive granulocyte count.
The present application also provides an animal model of myelodysplastic syndrome (MDS) comprising c-mybhyperMutant transgenic zebrafish.
The term "hpf" as used herein refers to the number of hours after fertilization. The term "dpf" as used herein refers to the number of days after fertilization. For example, "3 dpf" means three days after fertilization and "8 dpf" means eight days after fertilization.
The terms "wild type" or "WT" as used herein both refer to wild type zebrafish.
The term "sib fish (sibling)" as used herein refers to an individual who is the offspring of the same parent.
Drawings
FIG. 1C-myb mRNA at c-mybhyperThe embryo to adult fish stage of the mutant continues to increase. (A) Two repeats were found in PAC of c-myb-gfp transgenic zebrafish. First stage (1)st485-bp minipromoter) is indicated by the solid line box, the second repeat sequence (2)ndThe region of the intron 10 of pWSMK-T through c-myb) is marked with a solid box. The insertion site of each repeat is indicated by an arrow. The c-myb exon is indicated by a black bar and the pWSMK-T vector contains the GFP, SV40ployA termination signal and the reverse sequence of the ampicillin resistance gene. The unknown sequence of 77-bp is indicated by a white bar. (B) Transcript and protein messages for c-myb-WT and c-myb-T1. The transcription start sites for c-myb-WT and c-myb-T1 are indicated by arrows, respectively. Stop codons are marked with an asterisk. DBD represents a DNA binding domain, TAD represents a transcriptional activation domain, and NRD represents a negative regulatory domain. (C) Results of in situ hybridization of c-myb at 36 hours, 3 days and 5 days post fertilization. The black frame is a result of 20-fold enlargement of AGM (renal area in aortic gonads), CHT (tail hematopoietic tissue) and the renal area. The numbers at the top right of the graph indicate the total number of embryos to embryos expressing increased expression (Fisher's test, P.ltoreq.0.01). (D) The qPCR results for the c-myb gene in 3-day, 5-day whole embryos and 3-month, 6-month and 1-year kidney after fertilization (P.ltoreq.0.05, P.ltoreq.0.01, independent samples t test; 30 samples per group of whole embryos, 10 samples per group of kidney).
FIG. 2.c-mybhyperAbnormal proliferation of myeloid lineage cells in embryos (A-C) abnormal activation of C-myb promotes neutrophil accumulation and macrophage depletion in situ hybridization shows C/ebp α (A), mfap4(B) and lyz (C) C-myb 3 days after fertilizationhyperExpression in zebrafish and its siblings. Staining of SB in young fish 3 days (D) and 7 days (E) after fertilization. The black box is the result of CHT and a 20-fold enlargement of the kidney area. The rightmost black box in panel D is the result of a single cell magnification of 100. The numbers at the top right of the graph indicate the total number of embryos expressing increased embryo ratio (Fisher's test, n.gtoreq.10, P.ltoreq.0.01).
FIG. 3.c-mybhyperAdult fish exhibit MDS-like phenotype: abnormal medullary hyperplasia of kidney blood. (A-D)1 year c-mybhyperPeripheral Blood (PB) cells (A) and kidneys of zebrafish and its siblingsRamie staining of blood (KM) cells (C). Arrows, asterisks, triangles and lightning indicate erythrocytes, progenitor cells, myeloid monocytes and lymphocytes, respectively. c-mybhyperPB (B) and KM (D) blood cell counts of zebrafish (black bar) and their siblings (grey bar). Black asterisks indicate statistical differences. (t-test, 1 year sibling and c-mybhyperThe PB sample size of (a) was 9 and 5, respectively, the KM sample size was 6 and 5, respectively, mean ± SEM; p is less than or equal to 0.05, P is less than or equal to 0.01). (E and F) flow analysis of c-mybhyperBlood composition of zebra fish and its siblings. The cells are differentiated and counted by FSC and SSC for how much intracellular particles are present. (G-K) c-mybhyperZebrafish develop kidney and liver enlargement. 1 year c-mybhyperThe kidney area of the zebra fish is 1.4 times (H) of that of the siblings, and the weight of the zebra fish is 3.3 times (I) of that of the siblings. 1 year c-mybhyperZebrafish liver developed SB positive cytosis (J). The black frame on the right side of the J is a partial enlarged view in the left image. 1 year c-mybhyperZebrafish liver weight gain (K). (t-test, n is more than or equal to 10; mean value + -SEM; P is less than or equal to 0.05; P is less than or equal to 0.01).
FIG. 4.c-mybhyperThe increase in myeloid lineage cells in embryos and adult fish is due to increased cell proliferation. Abnormal activation of (A-H) c-myb results in increased proliferation of myeloid cells without significant changes in apoptosis in the embryo (A-D) and adult fish kidney (E-H). Immunofluorescence double staining for BrdU/Lcp (A and E) and TUNEL/Lcp double staining (C and G) indicate BrdU+Or TUNEL+CHT/KM area and Lcp at 3dpf/3 months+And (5) cell co-staining results. Lcp in CHT and KM regions+/BrdU+Cell occupancy of Lcp+Proportion of cells (B and F) and Lcp+/TUNEL+Cell occupancy of Lcp+The proportion of cells (D and H) was calculated (t-test, n)>13; mean ± SEM; p is less than or equal to 0.05, P is less than or equal to 0.01).
FIG. 5.c-mybhyperMDS-like zebrafish response to chemotherapeutic drugs. (A-C) treatment of C-myb 1 day after fertilization with PBS (A) or Cytarabine (Ara-C, B), daunorubicin (DNR, C)hyper(right bar) and sibling (left bar) embryos were SB stained 5 days later. (D andE) embryos 1 day after fertilization were treated with either dimethyl sulfoxide (D) or Flavering (E) for 4 days before SB staining. (F) SB positive cells were counted per young fish after drug treatment. (t-test, n is more than or equal to 10; mean value + -SEM; P is less than or equal to 0.05; P is less than or equal to 0.01). (G) qPCR (quantitative polymerase chain reaction) is utilized to detect the presence of the c-myb gene in the c-myb after drug treatmenthyper(black bars) and in siblings (grey bars) (t-test, n 30; mean. + -. SEM;. P.ltoreq.0.05. ltoreq. P.ltoreq.0.01).
FIG. 6.c-mybhyperRepetitive sequences in zebrafish. (A) The c-myb small promoter repeat contains a315 bp core promoter sequence and 170bp exon 1 sequence. The c-myb mini-promoter repeat and its adjacent sequences are indicated. (B) The sequence near the breakpoint position of intron 10 in the second repeated sequence is indicated (black box). The exon of c-myb is indicated by a black bar, and the pWSMK-T vector contains GFP, the SV40ployA termination signal, and the reverse sequence of the ampicillin resistance gene (AmpR). The unknown sequence of 77-bp is indicated by a white bar.
FIG. 7C-myb of myeloid lineage cells at 3 monthshyperAbnormal proliferation in the blood of the kidney of zebrafish. (A-D) 3 months of c-mybhyperThe RAIL-JI staining of Peripheral Blood (PB) cells (A) and renal blood (KM) cells (C) of zebrafish and its siblings. Arrows, asterisks, triangles and lightning indicate erythrocytes, progenitor cells, myeloid monocytes and lymphocytes, respectively. c-mybhyperPB (B) and KM (D) blood cell counts of zebrafish (black bar) and their siblings (grey bar). Black asterisks indicate statistical differences. (t-test, 1 year sibling and c-mybhyperPB sample sizes were 6 and 9, respectively, KM sample sizes were 8 and 8, respectively, mean ± SEM; p is less than or equal to 0.05, P is less than or equal to 0.01).
Detailed Description
The present application is described in further detail below by way of examples to enable those skilled in the art to practice the present application. It is to be understood that other embodiments may be utilized and that changes may be made without departing from the spirit or scope of the present application. To avoid detail not necessary to enable those skilled in the art to practice the application, the description may omit certain information known to those skilled in the art. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims.
The following examples are presented to facilitate a better understanding of the present application and are not intended to limit the scope of the present application.
The respective raw materials used in the following examples are commercially available except for those specifically mentioned.
Materials and methods
Zebra fish culture
The culture of zebra fish is described in literature (25). The following lines were used in the present invention: AB. Tg (c-myb-gfp), Tg (rag2: dsRed).
5 'and 3' -RACE
RACE is derived from
RACE 5 '/3' Kit (Clontech, CA, USA; 634858) and the specific procedures are described in the Kit. The c-myb gene specific primer in 5 '-RACE PCR was 5'-GATTACGCCAAGCTTTCCATGATCGAACGCCTCAGGTTGGGA-3', and the c-myb gene specific primer in 3' -RACE PCR was 5'-GATTACGCCAAGCTTCGAGGCGGCACAGACACAGTGTTTACAG-3'. And recovering the PCR product after electrophoresis, transfecting competent cells after inserting the PCR product into a pRACE vector, and picking more than 12 monoclonals for sequencing.
Whole in situ hybridization
Whole In Situ Hybridization (WISH) was performed using anti-digoxigenin-labeled RNA probes, and the experimental procedure was as described in the literature (28). Images were taken using an olympus MVX10 microscope (Shinjuku, Tokyo, Japan).
Real-time fluorescent quantitative PCR
Total RNA was extracted using the RNAeasy Kit (Qiagen, Maryland, USA; 74004), followed by cDNA synthesis using the reverse transcription Kit (Promega, Madison, USA; M1701). Real-time fluorescent quantitative PCR was performed on a LightCyclerNano SW1.0 machine using SYBR Green Real-time PCR Master Mix (Applied Biosystems, Austin, USA; 4472908) dye. The specific operation steps refer to the specification. The internal reference is the extension factor 1 α (ef1 a). All primers were designed using Primer5 software (table 1).
Table 1: qPCR primer
Bromodeoxyuridine and terminal deoxynucleotidyl transferase mediated dUTP nick end labeling
The procedures for the bromodeoxyuridine (BrdU) labeling experiments were as described in the literature (29). Tg (c-myb-egfp) embryos 3 days after fertilization and a sibling fish control group were incubated with 10mM BrdU (Sigma-Aldrich; B5002) for 2 hours, then fixed with 4% paraformaldehyde, incubated with mouse-derived anti-BrdU (Roche, Germany; 10875400) and rabbit-derived anti-lcp antibodies, and finally fluorescently stained with anti-mouse 488(Invitrogen, CA, USA; A21202) and anti-rabbit 555(Invitrogen, CA, USA; A31572) and observed. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) experiments were performed using the In Situ Cell Death Detection Kit TMR red (Roche; 12156792910) Kit. Followed by 555 staining with rabbit derived anti-lcp and anti-rabbit. Images were taken using an olympus fluoview1000 confocal microscope.
Cytological analysis
Hematopoietic cells were isolated as follows: zebrafish were anesthetized with tricaine and Peripheral Blood (PB) was obtained from gills by using 10 μ L heparinized microphotohematocrit tubes. Renal blood (KM) cells were isolated from the kidney. The separated PB and KM were resuspended in PBS containing 5% FBS, and then the resuspended cells were centrifuged onto a slide glass at 400 rpm for 3 minutes. Finally, staining was performed with Jiemsa (Merck, Germany; 1.09204.0500), Swiss Ji (Merck; 1.01424.0500). PB and KM cells were counted according to cell morphology.
Example 1: the expression level of C-myb is increased from embryo to adult in the C-myb-gfp transgenic zebra fish
North et al describe for the first time a c-myb-gfp transgenic zebrafish line, established as HSCs for labeling c-myb positivity (24) (recombination required for construction of c-myb-gfp)The sequencing result of the sequence is shown as SEQ ID NO: 1) are shown. However, when we refer to c-myb-gfp and c-mybhkz3/hkz3After mating of mutant zebrafish, c-myb was foundhkz3/hkz3The hematopoietic deficient phenotype of zebrafish was rescued.
To look for c-myb-gfp/c-mybhkz3/hkz3The reason for the recovery of hematopoietic deficiency in zebrafish, we sequenced the PAC region in c-myb-gfp transgenic zebrafish (sequencing result shown in SEQ ID NO: 2) and found two repeats, one of which was a 485bp repeat found before the translation start site, which included a315 bp core promoter region and a 170bp 5' UTR (FIGS. 1A and 6A); another large repeat region is the repeat sequence from the pWSMK-T vector to the c-myb intron 10, which is inserted at the break in the c-myb intron 10 (FIGS. 1A and 6B). 3 'and 5' RACE experiments revealed that PAC produced 2 different transcripts in c-myb-gfp (designated c-myb-WT and c-myb-T1, respectively) (FIG. 1B). One of the transcripts was c-myb-WT (whose corresponding cDNA sequence is shown in SEQ ID NO: 3), which was initiated by a second, small promoter and was completely identical to the wild-type zebrafish c-myb gene transcript (whose corresponding cDNA sequence is shown in SEQ ID NO: 4) (FIG. 1B); another transcript was 4.3kb of c-myb-T1 (the cDNA sequence for which is shown in SEQ ID NO: 5), which was initiated by the first small promoter and consisted of a truncated c-myb (exon 1 through exon 10) and a nearly complete c-myb (exon 2 through exon 15) (FIG. 1B). The protein translated by C-myb-T1 was fused from two fragments, the first being a C-myb protein lacking the entire C-terminal negative regulatory region (NRD) and the other being a C-myb protein with 8 amino acids less at the N-terminus (FIG. 1B).
To examine the expression of the c-myb gene in c-myb-gfp transgenic zebrafish, we performed WISH and qPCR experiments on the whole embryo of c-myb-gfp transgenic zebrafish as well as the adult fish kidneys (fig. 1C, D). As a result, it was found that in the c-myb-gfp transgenic zebrafish, the c-myb gene was expressed in the renal region (AGM) in the aortic gonad, the Caudal Hematopoietic Tissue (CHT) and the kidney, which are sites where HSC were produced, as detected by the c-myb probe (SEQ ID NO: 6), but the c-myb gene was expressed in the c-myb-gfp transgenic zebrafishThe expression level in zebrafish was much enhanced compared to wild type (fig. 1C). The qPCR results showed that the expression of the c-myb gene in c-myb-gfp transgenic zebrafish was approximately 2-fold higher starting at 3dpf and increased over time (FIG. 1D). Overexpression of the c-myb gene in c-myb-gfp transgenic zebrafish may be due to expression of an exogenous c-myb gene in PAC. The inventor hereby named the c-myb-gfp transgenic zebrafish as a c-myb gene abnormally activated zebrafish (c-myb hyperactity, c-myb)hyper)。
Example 2: abnormal activation of the c-myb gene during early embryonic stages leads to abnormal granulocyte accumulation
We are at c-mybhyperWe examined the expression of the c/ebp α gene (an early myeloid cell-specific marker) using the c/ebp α probe (SEQ ID NO: 7) at the time of committed myeloid developmenthyperThe number of cells expressing the c/ebp α gene is obviously increased (FIG. 2A).
Next, we investigated c-myb by WISH experiments using macrophage specific markers and granulocyte specific markershyperThe cell type of myeloid lineage cells accumulated. Wherein cells expressing the macrophage specific marker mfap4(SEQ ID NO: 8) were c-myb at 3dpfhyperThe number of (d) was significantly reduced (fig. 2B). In contrast, cells expressing the granulocyte specific marker lyz (SEQ ID NO: 9) were c-myb at 3dpfhyperThe number of the median increases significantly (fig. 2C). This suggests that in c-mybhyperThe source of myeloid lineage cells accumulated in the embryo are granulocytes. This result was further demonstrated by SB staining (which specifically labels granulocytes in zebrafish and is also used clinically for the diagnosis of myeloid leukemia cells). In c-mybhyperGranulocytes, marked by SB staining, were significantly increased in CHT and Kidney (KM) in embryos and young fish (fig. 2D and E). More importantly, in c-mybhyperThe SB positive granulocytes were not only increased in number, but also larger in size and stained more deeply (fig. 2D, E). This suggestsC-mybhyperAbnormalities in neutrophil function. The above results show that in c-mybhyperThe myeloid lineage cells that accumulate in the embryo are of granulocyte origin and not of macrophage origin, and there are functional and morphological abnormalities of such granulocytes during early developmental stages.
Example 3: c-mybhyperAdult fish exhibit MDS-like phenotype
To observe hematopoietic changes in adult fish, we isolated c-myb using flow cytometry and cytological experimentshyperAnd blood cells in a control group of siblings. For c-myb from 3 months and 1 yearhyperAnd Peripheral Blood (PB) and renal blood (KM) cells isolated from the control group of synechoic fish.
As a result, it was found that: c-myb at 3 months and 1 yearhyperThe composition ratio of the medium PB blood was not significantly different from the sibling fish control group (fig. 7A, B and fig. 3A, B); and 3 months and 1 year c-mybhyperThe composition ratio of the medium KM blood is different from that of the control group of the sibling fish. C-myb at 3 months compared to a control cohort of siblingshyperThe KM blood of (1) showed a significant increase in the number of myeloid cells accompanied by a decrease in progenitor cells (FIG. 7C, D). 1 year c-myb compared to a control cohort of siblingshyperThe number of myeloid cells in KM blood was doubled with a concomitant decrease in other lineage cells (fig. 3C, D). These phenotypes are consistent with abnormal proliferation of the myeloid lineage.
Flow cytometry results were consistent with those before, showing c-myb at 1 yearhyperThe number of cells of the mesomedullary line increased two-fold with a concomitant decrease in cells of other lineages (FIG. 3E, F). Furthermore, flow cytometry analysis shows that the number of the myeloid cells is increased, the size of the myeloid cells is increased, and the myeloid cells have stronger granularities. To observe c-mybhyperWhether the viscera in zebrafish are affected, we will use c-mybhyperAnd the viscera of the control group of the siblings were dissected under a microscope. As a result, 1 year c-myb was foundhyperThe zebrafish kidney was enlarged, and the area of the kidney was increased 1.37 times and the weight was increased 4 times as compared with the control group of the siblings (fig. 3G-I). The liver also became heavy and had a large number of SB positive cells infiltrating compared to the control group (fig. 3J, K). Of these internal organsDefects and infiltrations also occur in human granulocytic hematological malignancies.
In summary, c-mybhyperThe blood phenotype and tissue infiltration phenotype in zebrafish is similar to MDS symptoms in humans.
Example 4: c-mybhyperGranulocytosis in zebrafish mainly due to increased proliferation
c-mybhyperGranulocytosis in zebrafish may result both from increased granulocytosis and from decreased apoptosis. To clarify c-mybhyperThe molecular mechanism of granulocytosis in zebrafish, we monitored proliferation and apoptosis in cells using BrdU and TUNEL experiments, respectively. c-mybhyperApoptosis of myeloid cells in embryonic and adult fish kidneys was similar to that of the control group (FIGS. 4C, D, G and H), suggesting c-mybhyperGranulocytosis in zebrafish is not due to apoptotic changes. However, the results of the BrdU experiments showed c-mybhyperProliferation of myeloid cells in the kidney of embryos and adult fish was significantly increased (FIGS. 4A, B, E and F), indicating c-mybhyperGranulocytosis in zebrafish may be the result of increased proliferation. These results suggest that abnormal activation of c-myb promotes continued proliferation of granulocytes.
Example 5: clinical chemotherapeutic drug pair c-mybhyperEffectiveness of Zebra Fish leukemia model
We have observed that the commonly used anti-leukemic drugs in c-mybhyperEffects in zebrafish. Cytarabine (36) and daunorubicin (37) are common chemotherapeutic drugs for treating leukemia, which act by blocking the cell cycle and promoting the accumulation of leukemia apoptosis, respectively. 1dpf of c-mybhyperEmbryo and synechocystis control group 103mg/L cytarabine or 30mg/L daunorubicin and SB positive granulocytic counts were performed on the embryonic CHT regions at 6dpf (FIGS. 5A-C and F). Untreated c-mybhyperThe zebrafish CHT region had a high number of SB-positive granulocytes (fig. 5A and F). C-myb after 5 days of cytarabine treatmenthyperThe number of SB-positive granulocytes in zebrafish was significantly reduced compared to the untreated group (FIGS. 5B and F), whereasThe glycocytidine-treated control cohorts of synaptophia did not significantly change compared to the untreated control cohorts of synaptophia (fig. 5A and F). However, daunorubicin is para-c-mybhyperThe granulocytes of zebrafish did not have any effect (fig. 5C and F). The above results show that the abnormal proliferation phenotype of myeloid lines induced by abnormal activation of c-myb can be alleviated by cytarabine, a cell cycle specific antiproliferative drug, but not by daunorubicin, a pro-apoptotic drug.
We have also observed c-mybhyperIn the abnormal proliferating myeloid cells against the inhibitor fra-levelness of the c-myb targeting drug CDK 9. The degree of frataxin can obviously reduce c-mybhyperThe number of SB-positive granulocytes in (C), while there was no effect on the sibling fish control group (FIGS. 5D-F). At the same time, with c-myb which has not been treated with a drughyperIn contrast, furameter-treated c-mybhyperThe RNA expression level of c-myb was significantly reduced (FIG. 5G). The results indicate that clinical c-myb-targeting drugs can reduce c-mybhyperAbnormal expansion of myeloid cells in zebrafish.
Taken together, the results of the study show that abnormal activation of c-myb in zebrafish can cause MDS and progress to AML as well as ALL with age, and that c-mybhyperThe tumor phenotype of the zebrafish model can be relieved by clinical chemotherapy drugs such as cytarabine and frataxin. Suggesting that c-myb may be a new therapeutic target for a particular type of leukemia. c-mybhyperThe zebrafish model also provides a valuable platform for in vivo new drug screening, particularly for the screening of cell cycle specific antiproliferative drug candidates as well as c-myb targeted drug candidates aimed at treating the specific types of leukemia mentioned above.
Therefore, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Reference to the literature
1.Sheiness D,Gardinier M:Expression of a proto-oncogene(proto-myb)inhemopoietic tissues of mice,Molecular and cellular biology 1984,4:1206-1212
2.Westin EH,Gallo RC,Arya SK,Eva A,Souza LM,Baluda MA,Aaronson SA,Wong-Staal F:Differential expression of the amv gene in human hematopoieticcells,Proceedings of the National Academy of Sciences of the United States ofAmerica 1982,79:2194-2198
3.Bender TP,Thompson CB,Kuehl WM:Differential expression of c-mybmRNA in murine B lymphomas by a block to transcription elongation,Science1987,237:1473-1476
4.Mukai HY,Motohashi H,Ohneda O,Suzuki N,Nagano M,Yamamoto M:Transgene insertion in proximity to the c-myb gene disrupts erythroid-megakaryocytic lineage bifurcation,Molecular and cellular biology 2006,26:7953-7965
5.Lieu YK,Reddy EP:Conditional c-myb knockout in adult hematopoieticstem cells leads to loss of self-renewal due to impaired proliferation andaccelerated differentiation,Proceedings of the National Academy of Sciencesof the United States of America 2009,106:21689-21694
6.Vegiopoulos A,Garcia P,Emambokus N,Frampton J:Coordination oferythropoiesis by the transcription factor c-Myb,Blood 2006,107:4703-4710
7.Sumner R,Crawford A,Mucenski M,Frampton J:Initiation of adultmyelopoiesis can occur in the absence of c-Myb whereas subsequent developmentis strictly dependent on the transcription factor,Oncogene 2000,19:3335-3342
8.Bender TP,Kremer CS,Kraus M,Buch T,Rajewsky K:Critical functionsforc-Myb at three checkpoints during thymocyte development,Nature immunology2004,5:721-729
9.Thomas MD,Kremer CS,Ravichandran KS,Rajewsky K,Bender TP:c-Myb iscritical for B cell development and maintenance of follicular B cells,Immunity 2005,23:275-286
10.Shen-Ong GL:The myb oncogene,Biochimica et biophysica acta 1990,1032:39-52
11.Nazarov V,Wolff L:Novel integration sites at the distal 3'end ofthe c-myb locus in retrovirus-induced promonocytic leukemias,Journal ofvirology 1995,69:3885-3888
12.Grasser FA,Graf T,Lipsick JS:Protein truncation is required forthe activation of the c-myb proto-oncogene,Molecular and cellular biology1991,11:3987-3996
13.Siegert W,Beutler C,Langmach K,Keitel C,Schmidt CA:Differentialexpression of the oncoproteins c-myc and c-myb in human lymphoproliferativedisorders,Eur J Cancer1990,26:733-737
14.Machova Polakova K,Lopotova T,Klamova H,Burda P,Trneny M,Stopka T,Moravcova J:Expression patterns of microRNAs associated with CML phases andtheir disease related targets,Molecular cancer 2011,10:41
15.Clappier E,Cuccuini W,Kalota A,Crinquette A,Cayuela JM,Dik WA,Langerak AW,Montpellier B,Nadel B,Walrafen P,Delattre O,Aurias A,Leblanc T,Dombret H,Gewirtz AM,Baruchel A,Sigaux F,Soulier J:The C-MYB locus isinvolved in chromosomal translocation and genomic duplications in human T-cell acute leukemia(T-ALL),the translocation defining a new T-ALL subtype invery young children,Blood 2007,110:1251-1261
16.Lahortiga I,De Keersmaecker K,Van Vlierberghe P,Graux C,CauwelierB,Lambert F,Mentens N,Beverloo HB,Pieters R,SpelemanF,Odero MD,Bauters M,Froyen G,Marynen P,Vandenberghe P,Wlodarska I,Meijerink JP,Cools J:Duplication of the MYB oncogene in T cell acute lymphoblastic leukemia,Naturegenetics 2007,39:593-595
17.Tomita A,Watanabe T,Kosugi H,Ohashi H,Uchida T,Kinoshita T,Mizutani S,Hotta T,Murate T,Seto M,Saito H:Truncated c-Myb expression in thehuman leukemia cell line TK-6,Leukemia 1998,12:1422-1429
18.Zuber J,Rappaport AR,Luo W,Wang E,Chen C,Vaseva AV,Shi J,Weissmueller S,Fellmann C,Taylor MJ,Weissenboeck M,Graeber TG,Kogan SC,VakocCR,Lowe SW:An integrated approach to dissecting oncogene addiction implicatesa Myb-coordinated self-renewal program as essential for leukemia maintenance,Genes&development 2011,25:1628-1640
19.Orkin SH,Zon LI:Hematopoiesis:an evolving paradigm for stem cellbiology,Cell2008,132:631-644
20.Stoletov K,Klemke R:Catch of the day:zebrafish as a human cancermodel,Oncogene 2008,27:4509-4520
21.Ridges S,Heaton WL,Joshi D,Choi H,Eiring A,Batchelor L,Choudhry P,Manos EJ,Sofla H,Sanati A,Welborn S,Agarwal A,Spangrude GJ,Miles RR,Cox JE,Frazer JK,Deininger M,Balan K,Sigman M,Muschen M,Perova T,Johnson R,Montpellier B,Guidos CJ,Jones DA,Trede NS:Zebrafish screen identifies novelcompound with selective toxicity against leukemia,Blood 2012,119:5621-5631
22.Langenau DM,Traver D,Ferrando AA,Kutok JL,Aster JC,Kanki JP,Lin S,Prochownik E,Trede NS,Zon LI,Look AT:Myc-induced T cell leukemia intransgenic zebrafish,Science 2003,299:887-890
23.Sun J,Liu W,Li L,Chen J,Wu M,Zhang Y,Leung AY,Zhang W,Wen Z,LiaoW:Suppression of Pu.1 function results in expanded myelopoiesis in zebrafish,Leukemia2013,27:1913-1917
24.North TE,Goessling W,Walkley CR,Lengerke C,Kopani KR,Lord AM,WeberGJ,Bowman TV,Jang IH,Grosser T,Fitzgerald GA,Daley GQ,Orkin SH,Zon LI:Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis,Nature 2007,447:1007-1011
25.Westerfield M:The zebrafish book:a guide for the laboratory use ofzebrafish(Brachydanio rerio).Edited by Eugene,OR,M.Westerfield,1993,
26.Hall C,Flores MV,Storm T,Crosier K,Crosier P:The zebrafishlysozyme C promoter drives myeloid-specific expression in transgenic fish,BMCdevelopmental biology 2007,7:42
27.Willett CE,Cherry JJ,Steiner LA:Characterization and expression ofthe recombination activating genes(rag1 and rag2)of zebrafish,Immunogenetics1997,45:394-404
28.Wang K,Huang Z,Zhao L,Liu W,Chen X,Meng P,Lin Q,Chi Y,Xu M,Ma N,Zhang Y,Zhang W:Large-scale forward genetic screening analysis of developmentof hematopoiesis in zebrafish,Journal of genetics and genomics＝Yi chuan xuebao 2012,39:473-480
29.Jin H,Li L,Xu J,Zhen F,Zhu L,Liu PP,Zhang M,Zhang W,Wen Z:Runx1regulates embryonic myeloid fate choice in zebrafish through a negativefeedback loop inhibiting Pu.1 expression,Blood 2012,119:5239-5249
30.Zhang Y,Jin H,Li L,Qin FX,Wen Z:cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish hematopoiesis,Blood 2011,118:4093-4101
31.Sakamoto H,Dai G,Tsujino K,Hashimoto K,Huang X,Fujimoto T,MucenskiM,Frampton J,Ogawa M:Proper levels of c-Myb are discretely defined atdistinct steps of hematopoietic cell development,Blood 2006,108:896-903
32.Rosson D,Tereba A:Transcription of hematopoietic-associatedoncogenes in childhood leukemia,Cancer research 1983,43:3912-3918
33.Murati A,Gervais C,Carbuccia N,Finetti P,Cervera N,Adelaide J,Struski S,Lippert E,Mugneret F,Tigaud I,Penther D,Bastard C,Poppe B,SpelemanF,Baranger L,Luquet I,Cornillet-Lefebvre P,Nadal N,Nguyen-Khac F,Perot C,Olschwang S,Bertucci F,Chaffanet M,Lessard M,Mozziconacci MJ,Birnbaum D:Genome profiling of acute myelomonocytic leukemia:alteration of the MYB locusin MYST3-linked cases,Leukemia 2009,23:85-94
34.Le Guyader D,Redd MJ,Colucci-Guyon E,Murayama E,Kissa K,Briolat V,Mordelet E,Zapata A,Shinomiya H,Herbomel P:Origins and unconventionalbehavior of neutrophils in developing zebrafish,Blood 2008,111:132-141
35.Gutierrez A,Pan L,Groen RW,Baleydier F,Kentsis A,Marineau J,Grebliunaite R,Kozakewich E,Reed C,Pflumio F,Poglio S,Uzan B,Clemons P,VerPlank L,An F,Burbank J,Norton S,Tolliday N,Steen H,Weng AP,Yuan H,BradnerJE,Mitsiades C,Look AT,Aster JC:Phenothiazines induce PP2A-mediated apoptosisin T cell acute lymphoblastic leukemia,The Journal of clinical investigation2014,124:644-655
36.Pigneux A,Perreau V,Jourdan E,Vey N,Dastugue N,Huguet F,Sotto JJ,Salmi LR,Ifrah N,Reiffers J:Adding lomustine to idarubicin and cytarabine forinduction chemotherapy in older patients with acute myeloid leukemia:the BGMT95 trial results,Haematologica 2007,92:1327-1334
37.Rabbani A,Finn RM,Ausio J:The anthracycline antibiotics:antitumordrugs that alter chromatin structure,BioEssays:news and reviews in molecular,cellular and developmental biology 2005,27:50-56
38.Disperati P,Ichim CV,Tkachuk D,Chun K,Schuh AC,Wells RA:Progression of myelodysplasia to acute lymphoblastic leukaemia:implicationsfor disease biology,Leukemia research 2006,30:233-239
39.Walter MJ,Shen D,Ding L,Shao J,Koboldt DC,Chen K,Larson DE,McLellan MD,Dooling D,Abbott R,Fulton R,Magrini V,Schmidt H,Kalicki-Veizer J,O'Laughlin M,Fan X,Grillot M,Witowski S,Heath S,Frater JL,Eades W,Tomasson M,Westervelt P,DiPersio JF,Link DC,Mardis ER,Ley TJ,Wilson RK,Graubert TA:Clonal architecture of secondary acute myeloid leukemia,The New Englandjournal of medicine 2012,366:1090-1098
40.Mitra P,Pereira LA,Drabsch Y,Ramsay RG,Gonda TJ:Estrogen receptor-alpha recruits P-TEFb to overcome transcriptional pausing in intron 1 of theMYB gene,Nucleic acids research 2012,40:5988-6000
41.Karp JE,Blackford A,Smith BD,Alino K,Seung AH,Bolanos-Meade J,Greer JM,Carraway HE,Gore SD,Jones RJ,Levis MJ,McDevitt MA,Doyle LA,WrightJJ:Clinical activity of sequential flavopiridol,cytosine arabinoside,andmitoxantrone for adults with newly diagnosed,poor-risk acute myelogenousleukemia,Leukemia research 2010,34:877-882
42.Badiani PA,Kioussis D,Swirsky DM,Lampert IA,Weston K:T-celllymphomas in v-Myb transgenic mice,Oncogene 1996,13:2205-2212。
Claims (5)
1. Use of transgenic zebrafish in preparation of animal model of myelodysplastic syndrome (MDS), wherein the transgenic zebrafish is abnormally activated (c-myb) by c-myb genehyper) Zebra fish.
2. The use of claim 1, wherein the myelodysplastic syndrome (MDS) is caused by abnormal activation of the c-myb gene.
3. The use of claim 1, wherein the myelodysplastic syndrome (MDS) is alleviated after treatment by a drug that acts by blocking the cell cycle or a c-myb-targeted drug.
4. The use of claim 3, wherein the drug that acts by blocking the cell cycle is cytarabine and the c-myb targeting drug is frataxin.
5. An animal model of myelodysplastic syndrome (MDS) comprising aberrant activation of the c-myb gene (c-myb)hyper) The transgenic zebrafish of (3).
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| CN105238742A (en) * | 2015-11-19 | 2016-01-13 | 湖南师范大学 | Inducing method for danio-rerio inductivity multi-potential stem cells and inductive culture medium and iPS culture medium used for the same |
| CN108601356A (en) * | 2015-12-03 | 2018-09-28 | 布里格姆妇女医院 | The method of generating functionality candidate stem cell |
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| CN105238742A (en) * | 2015-11-19 | 2016-01-13 | 湖南师范大学 | Inducing method for danio-rerio inductivity multi-potential stem cells and inductive culture medium and iPS culture medium used for the same |
| CN108601356A (en) * | 2015-12-03 | 2018-09-28 | 布里格姆妇女医院 | The method of generating functionality candidate stem cell |
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