CN113355326A - Zebra fish model for giant platelet syndrome - Google Patents
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Abstract
The invention discloses a zebra fish model for giant platelet syndrome. The zebra fish model is gp9SMU15Mutant zebrafish. In the invention, the fact that the gp9 mutation on the zebra fish is related to the proliferation of the platelet is found for the first time, and the zebra fish gp9SMU15The mutant had similar symptoms to human giant platelet syndrome and demonstrated that gp9 was presentSMU15Mutant zebrafish blood coagulation dysfunction. In addition, the Bernard-Soulier Syndrome disease model for thrombocytopenia and blood coagulation dysfunction is established for the first time according to the zebra fish model, and the zebra fish model can become a new carrier for researching the Bernard-Soulier Syndrome disease and drug screening.
Description
Technical Field
The invention belongs to the technical field of biology, particularly relates to the field of research on development of platelets, and particularly relates to a zebra fish model for giant platelet syndrome.
Background
Platelets are a major component of the blood system. When the blood vessel is damaged, the blood platelet plays the role of hemostasis and blood coagulation. When the number of platelets is reduced or the function of the platelets is reduced, the hemostatic dysfunction and thrombosis of the body are caused, and related platelet diseases are caused. Giant platelet Syndrome, also known as Bernard-Soulier Syndrome, is a rare hereditary hemorrhagic disease, the incidence rate of which is less than 1/100 ten thousand, and is mainly caused by the synthesis and expression defect of glycoprotein complex GPIb-IX-V on platelet membrane or the gene defect of any subunit GPIb, GPIX and GPV. The defect of GPIb-IX-V causes that the blood platelet can not normally adhere to the vascular endothelium to cause skin, mucosal hemorrhage or visceral hemorrhage, and the bleeding is not uniform. The patient has a large number of platelets in the peripheral blood and a mild or moderate reduction in platelet number. (Anna Savoia, Annalisa store, Daniela De Rocco, et al (2011) Clinical and genetic aspects of Bernard-Soulier syndrome: searching for genetic/phenotypic conjugates. Haematologica 96(3) 417. 423) in the mouse model, abnormality also occurs in the morphology of platelets in peripheral blood, the number of platelets capable of forming pseudopodoids is reduced, indicating that GPIb-IX-V plays an important role in the maintenance and maturation of the morphology of platelets. (C.Strassel, C.Nonne, A.Eckly, T.David, C.Leon, et al (2007) classified Thrombotic testing in Mouse Models of the Bernard-Soulier syndrome, ArteriosclerThromb Vasc biol.27:241- "247.) in 13 Bernard-Soulier cases, patients with missense mutations in the GPIX gene were found.
In order to better study the role of GPIX in platelets and the pathogenesis of Bernard-Soulier Syndrome giant platelets, the development of embryos and large-scale drug screening need to be observed. Therefore, there is a need to establish a relevant animal model.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a zebrafish model with giant platelet syndrome. The zebra fish model of giant platelet syndrome provided by the invention is a zebra fish model with thrombocytopenia caused by GPIX gene mutation.
In order to better research the role of gp9 in early development of platelets and related regulation and control mechanisms, the invention constructs a zebra fish mutant gp9 and applies the zebra fish mutant gp9 to preparation of a corresponding disease model (a zebra fish model with giant platelet syndrome). The zebra fish has the characteristics of high spawning, in-vitro fertilization and transparent early embryo development, and is favorable for observing the behavior change of the platelets. Meanwhile, the method is also beneficial to large-scale drug screening for treating the giant platelet syndrome.
The invention aims to provide a zebra fish model for giant platelet syndrome. The zebra fish model for reducing thrombopenia caused by GPIX gene mutation can be applied to the research of pathogenesis of Bernard-Soulier giant platelet syndrome.
The purpose of the invention is realized by at least one of the following technical solutions.
The zebra fish model of the giant platelet syndrome provided by the invention is gp9SMU15Mutant zebrafish.
Further, gp9SMU15The phenotype of the mutant zebrafish is caused by abnormal expression of GP9 gene, namely GP9SMU15The mutation causes.
Further, said gp9SMU15The mutant zebra fish is a mutant zebra fish with 17 basic groups deleted from the zebra fish gp9 gene, so that the gp9 gene stops expression early and gp9 protein expression is abnormal. The gp9SMU15The mutant zebra fish gp9 gene translation is terminated early, and is a mutant with deletion of important functional domains.
Further, said gp9SMU15The mutant zebra fish is obtained by target gene knockout of 17 bases on gp9 gene through CRISPR/Cas 9.
Further, said gp9SMU15The mutant zebra fish is subjected to CRISPR/Cas9 targeted gene knockout on 17 bases behind the ATG start codon of No. 2 exon sequence of zebra fish GP9 geneAnd (4) obtaining the product.
Further, said gp9SMU15The mutant zebra fish gp9 protein lacks the functional domain of LRRNT relative to the wild zebra fish gp9 protein.
Further, said gp9SMU15The mutant zebra fish gp9 protein lacks the functional domain of LRRCT relative to the wild zebra fish gp9 protein.
The invention provides application of a zebra fish model with thrombocytopenia caused by GPIX gene mutation in preparation of an animal model of Bernard-Soulier Syndrome giant platelet Syndrome.
Further, the giant platelet Syndrome is Bernard-Soulier Syndrome; the symptoms of the giant platelet syndrome are thrombocytopenia and reduced platelet proliferation.
Further, the symptoms of the giant platelet syndrome are a decrease in the number of platelets in the embryonic stage, a decrease in the proliferation of platelet precursor cells;
further, the symptom of giant platelet syndrome is coagulation dysfunction in adult stage, and the number of peripheral platelets is reduced. Some zebrafish models have increased peripheral platelet size and bleeding from the skin mucosa.
The general concept of the invention is as follows:
(1) the mutant zebra fish with gp9 gene sequence deletion is obtained by a CRISPR/Cas9 targeted gene knockout technology.
(2) In the embryonic period, in-situ hybridization tests are carried out by using probes for marking the platelets at different stages to detect the change of platelet molecular markers and the expression change of platelet related genes by a Q-PCR method.
(3) Detecting changes of proliferation and apoptosis in the platelet differentiation process by using a BrdU proliferation experiment and a TUNEL apoptosis experiment;
(4) in zebrafish gp9SMU15Adult-age mutants, flow cytometry for gp9SMU15The number of mutant platelets varied.
(5) In adult fish stage, gp9 is damagedSMU15The tail of the mutant is detected by measuring the hemostasis timeTesting whether the coagulation function of the mutant is normal; the mutants were observed for the clinical symptoms of Bernard-Soulier Syndrome such as mucosal bleeding and visceral bleeding.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the mutant zebra fish provided by the invention can be applied to screening of medicines effective to the giant platelet syndrome; the zebra fish gp9SMU15The number of platelets in the embryonic stage of the mutant is reduced, and the coagulation function in the adult stage is abnormal, and some gp9SMU15The skin mucosa of the mutant has bleeding symptoms, and has similar symptoms with human BSS diseases; due to the characteristics of large egg laying amount of the zebra fish, convenient culture and lower breeding cost than that of the mice, the method is beneficial to screening the medicines for treating the giant platelet syndrome with high flux and has low cost; in addition, gp9SMU15The number of the mutant embryonic-stage platelets is reduced, the zebra fish can be subjected to drug treatment at the embryonic stage to screen effective drugs, and the experimental period is short.
(2) The zebrafish model of giant platelet syndrome in the invention can be used for tracking the pathogenesis of diseases for a long time due to the stable inheritance, and the disease progresses from an embryonic stage to an adult stage. And gp9SMU15The mutant has a stably inherited thrombocytopenic phenotype, gp9SMU15Mutants can also be used to expand the mechanisms of studying platelet coagulation dysfunction and platelet-related diseases.
(3) In a mouse, the existing Bernard-Soulier Syndrome disease model is mainly caused by the defect of one subunit GPIb gene of a GPIb-IX-V complex on a platelet membrane; however, a Bernard-Soulier Syndrome mouse model caused by GPIX (general purpose interface X), namely gp9 gene defect does not exist so far; zebra fish gp9SMU15The mutant is caused by the genetic defect of one subunit GPIX of the GPIb-IX-V complex, and some patients with BSS are caused by the genetic defect of GPIX, zebrafish gp9SMU15The mutant can provide a disease model of BSS caused by gene deficiency aiming at GPIX; meanwhile, the function of the platelet membrane protein gp9 in the blood system can be researched.
(4) Gp9 of the inventionSMU15The mutants had the following phenotypes: (. g) gp9SMU15The number of platelets in the mutant embryonic stage is reduced, gp9SMU15The proliferation of the mutant blood platelet is reduced, and the apoptosis is unchanged; ② adult period, gp9SMU15Mutant blood coagulation disorder, after injury, the bleeding time is obviously prolonged; ③ adulthood, about 1/5gp9SMU15The mutant fish has the symptoms of bleeding of skin mucous membrane, which is similar to the symptoms of Bernard-Soulier Syndrome patients; (iv) adult period, gp9SMU15The proportion of platelets in peripheral blood of the mutant decreases.
Drawings
FIG. 1 shows that a zebra fish mutant gp9 is obtained by CRISPR/Cas9 targeted gene knockout technologySMU15A schematic diagram; the Ai part in the figure 1 is a CRISPR/Cas9 targeted gene knockout design scheme, and a No. 2 exon sequence of zebra fish gp9 gene is selected as a target point for gene knockout. Wherein the Aii part in FIG. 1 is a mutant gp9 sequencedSMU1517 bases are deleted at the target position, after deletion, the Gp9 protein is terminated early, and an important functional domain is lacked. The Aii portion of FIG. 1 is gp9SMU15The mutant protein is lack of LRRNT (leucoine-rich repeat N-terminal domain) relative to the wild type and is an N-terminal leucine-rich repeat domain; LRRCT (Leucine-rich repeat C-terminal domain) is a C-terminal leucine-rich repeat domain.
FIG. 2 is a graph of fish blood phenotype results for 3 dpf; FIG. 2, part A, is a graph showing the results of flow assays, and part B, is a flow chart, showing 3dpf wild type synechocystis and mutant gp9SMU15The number of cd41GFPhigh cells was significantly reduced, with the portion C of FIG. 2 being a WISH global in situ hybridization pattern and the portion D of FIG. 2 being a statistical pattern showing the mutant gp9SMU15The expression of the cd41 gene is obviously reduced compared with that of the wild type sibling fish; FIG. 2E shows the Qpcr assay for gene expression level and shows the mutant gp9SMU15Reduced expression of platelet-associated genes (t-test, P)<0.05; mean ± sem, n represents the amount of zebrafish sample in the respective group).
FIG. 3 shows the signal at 56hpf, WT (gp 9)+/+) And gp9SMU15Mutant zebra fish blood is littlePlate proliferation and apoptosis test result graph; wherein, part A of FIG. 3 is WT (gp 9)+/+) And gp9SMU15Mutant platelet precursor cells CD41, GFP and BrdU double staining experimental result picture (upper side: CD41 staining, middle: Brdu, lower side: synthesis picture), arrow shows CD41, GFP and Brdu co-staining positive cells; parts B of FIG. 3 are WT and gp9SMU15Mutant CD41GFP+Statistical map of platelet-proliferating cell proportion (t-test, P)<0.01; mean ± standard error); section C of FIG. 3 is at 3dpf, WT and gp9SMU15Mutant platelet CD41 double staining experiments with GFP and TUNEL (upper panel: CD41 staining, middle panel: TUNEL staining, lower panel: FIG. A).
FIG. 4 shows WT and gp9 in adulthoodSMU15A ratio result chart of platelets in peripheral blood of the mutant zebra fish; wherein the side of part A of FIG. 4 is a flow analysis chart, and the side of part B of FIG. 4 is CD41: GFP+Platelet ratio statistics.
FIG. 5 is a graph of injury WT and gp9 during adulthoodSMU15Statistical analysis of clotting time of mutant zebrafish tails (t-test, P)<0.001; mean ± standard error).
Detailed Description
The following examples are presented to further illustrate the practice of the invention, but the practice and protection of the invention is not limited thereto. It is noted that the processes described below, if not specifically described in detail, are all realizable or understandable by those skilled in the art with reference to the prior art. The reagents or apparatus used are not indicated to the manufacturer, and are considered to be conventional products available by commercial purchase.
The terms "wild type" or "WT" as used herein both refer to wild type zebrafish.
The term "dpf" as used herein refers to the number of days after fertilization.
Example 1
1. Materials and methods:
(1) zebra fish culture
Zebra fish are cultivated as described in The literature (Westerfield M: The zebrafish: guide for The laboratory use of zebrafish (Brachdanio relay).
(2) The following lines were used in the present invention: AB wild type zebra fish, gp9SMU15Mutant zebrafish, Tg (CD41: GFP) transgenic zebrafish. Wherein each transgenic zebrafish line is contacted with gp9SMU15Gp9 with CD41 transgenic background is obtained by selfing after hybridization of mutant zebra fishSMU15Mutant zebrafish, such as Tg (CD41: GFP); gp9-/-。
(3) CRISPR/Cas 9-Targeted Gene knockout gp9 Gene (see FIG. 1)
Selecting a sequence behind an ATG start codon of an exon sequence No. 2 of a gp9 gene as a targeted knockout sequence, predicting a target sequence GGGCAAAGTCACGCACCTGC with high score, high target efficiency and low off-target efficiency according to http:// www.crisprscan.org/website, wherein AGG behind the sequence forms a PAM region, and the sequence of a finally synthesized primer is a T7(17bp) + target + gRNA FP (20bp) sequence: 5 '-3' TAATACGACTCACTATAGGGCAAAGTCACGCACCTGCGTTTTAGAGCT AGAAATAG, gRNA RP sequence 5 '-3' AGCACCGACTCGGTGCCACT, using Z-cas9 plasmid as template, synthesized in vitro with T7 RNA polymerase (Thermo, EP 0111).
The obtained mutant is a mutant with 17 bases deleted from No. 2 exon of gp9 gene, which causes frame shift mutation and premature termination to generate truncated gp9 protein; relative to the wild gp9 protein, important functional domains of LRRNT and LRRCT are deleted.
Example 2
Detection of gp9 by Whole-mount in situ hybridizationSMU15Expression of mutant platelet-associated genes
This was done according to the following standard experimental protocol (ThisseC, Thisse B. (2008). High-resolution in situ hybridization to white-mount zebraffinh emulsions. NATURE PROTOCOLS. VOL.3NO.1).
The results are shown in FIG. 2, part C, and FIG. 2, part D, at 3dpf, gp9SMU15The signal of the platelet marker molecule cd41 in the mutant was less compared to the wild type, indicating gp9SMU15The number of mutant platelets is likely to decrease.
EXAMPLE 3Q-PCR detection of gp9SMU15Mutant platelet-related proteinsExpression of genes
Collection of 3dpf WT and 3dpf gp9SMU15Mutant fish were treated with Tripure RNA Isolation Reagent (Thermo,15596018) to extract total RNA from embryos, according to the protocol described in the specification. The cDNA was reverse-transcribed using M-MLV reverse transcriptase (Promega, M1701). Q-PCR detection was performed using zebrafish elf1a as an internal reference gene, wherein the sequence information of the primers is shown in Table 1 below.
TABLE 1
The results are shown in section E of FIG. 2 at 3dpf, gp9SMU15The mutant has reduced expression of CD41, nfe2 and fog1 relative to wild type, and combines the result of CD41 whole in situ hybridization to further illustrate that gp9 is in embryonic stageSMU15The number of platelets in the mutant decreased.
Example 4 proliferation and apoptosis assay of embryonic platelets
The bromodeoxyuridine (Brdu) experiments were performed with reference to the specification. 56hpf transgenic line Tg (CD41: GFP) gp9 was soaked with 10mM Brdu (Sigma-Aldrich; B5002) concentration+/+And gp9SMU15The mutants were incubated for 1 hour with mouse-derived Brdu antibody (Roche; 10875400) and goat-derived GFP antibody (Abcam; ab66580) for 12-14 hours, and with anti-mouse 555 (Thermo; a31570 and anti-goat 488 (Thermo; A11055) were stained with fluorescence, and the labeling results are shown in part A of FIG. 3, respectively. Assays for detecting apoptosis using terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) were also performed. Detection was performed with In Situ Cell Death Detection kit (Roche; 12156792910), and staining was performed with goat-derived GFP antibody (Abcam; ab66580) and 488 fluorescence against goat. As shown in part B of fig. 3: gp9 relative to wild typeSMU15Co-localization of Medium GFP Signal number and BrduThe percentage of cells in total GFP signal decreased, as indicated in gp9SMU15The proliferation of middle platelets is reduced. The TUNEL results are shown in section C of FIG. 3, with no co-localized apoptotic cells, indicating gp9SMU15Apoptosis of the middle platelet was unchanged.
Example 5 flow cytometry detection of the number of platelets in larvae and adult peripheral blood
And (3) juvenile fish treatment: tg (CD41: GFP) wild type and Tg (CD41: GFP) gp9 were chosenSMU153dpf larvae, tissue ground and resuspended in 5% FBS suspension. Adult fish treatment: tg (CD41: GFP) wild type and Tg (CD41: GFP) gp9 were chosenSMU15Adult fish, anesthetized with ice to adult fish, peripheral blood was taken by gill puncture, peripheral blood was suspended in 5% FBS, and wild type and gp9 were detected by flow analysisSMU15The GFP, i.e. the percentage size of platelets in peripheral blood, in the mutant was analyzed statistically. Detection of wild type and gp9 by flow analysisSMU15Mutant GFP, the size of platelets as a percentage of cells in peripheral blood, was analyzed statistically. For larvae at 3dpf, gp9, relative to wild type, as shown in part A of FIG. 2, part B of FIG. 2SMU15Middle GFPhighThe number of platelets represented decreased. For adult fish, as shown in fig. 4, part a, and fig. 4, part B: gp9 relative to wild typeSMU15The number of platelets in the peripheral blood decreases.
Example 6 clotting experiments and conditions of spontaneous bleeding
Blade pair adult fish wild type and gp9SMU15The tail of the fish is cut, timing is started after cutting, and the time required for bleeding of the adult fish to coagulation is measured. As shown in FIG. 5, gp9 is present relative to wild typeSMU15The longer time required for adult fish of the mutant indicates gp9SMU15The mutant develops a disorder in blood coagulation function. And tail mucosal bleeding was observed in the mutants, 19 gp9SMU15In 4 of the mutants, bleeding was observed in the mucous membrane of the tail.
The above examples are only preferred embodiments of the present invention, which are intended to be illustrative and not limiting, and those skilled in the art should understand that they can make various changes, substitutions and alterations without departing from the spirit and scope of the invention.
Claims (10)
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