CN105854017A - Reagent for treating beta-thalassemia and application thereof - Google Patents

Abstract

The invention discloses a reagent for treating β-thalassemia and application thereof. The reagent treats β-thalassemia by reducing the expression level of miR-144/451 gene or the amount of miR-144/451. The present invention constructs the miR-144/451 gene knockout β-thalassemia mouse model, and the β-globin gene knockout β-thalassemia mice and miR-144/451 gene knockout mice are genetically cultivated, and the genotype The identified miR-144/451 gene knockout β-thalassemia mouse model, the anemia of this type of β-thalassemia mice changed from severe to mild, and the complete blood cell analysis showed that the red blood cell count and hemoglobin level increased significantly, and the spleen volume β-thalassemia mice with miR-144/451 gene knockout provide a reliable in vivo model for studying the molecular mechanism of anemia improvement. Treatment of β-thalassemia by reducing miR-144/451 in β-thalassemia blood cells has potential huge economic and social benefits.

Description

A reagent for treating beta-thalassemia and its application

technical field

The invention belongs to the field of biotechnology, and more specifically relates to a reagent for treating human beta-thalassemia and its application.

Background technique

Human β-thalassemia is a genetic disease caused by mutations in the β-globin gene. It is the most common single-gene genetic disease in humans. It is mainly distributed in the Mediterranean region, Central Africa, Asia, the South Pacific region and southern China. In my country, Guangdong, Guangxi, Hainan, Macau and Hong Kong are high-incidence areas. More than 200 mutations of β-thalassemia have been found so far. Epidemiological surveys show that there are currently more than 75 million carriers of the β-globin mutation gene in the world. In 2009, the maternal and child health monitoring in Guangxi Zhuang Autonomous Region found that the incidence of severe β-thalassemia was 2.58%. The pathogenesis of β-thalassemia is that the synthesis of β-globin peptide chains is reduced or completely absent due to mutations in the β-globin gene, resulting in a relative excess of matching α-chains, and the excess α-chains precipitate on the red blood cell membrane to form inclusion bodies. Lead to increased apoptosis of erythrocyte precursor cells and cause severe chronic hemolytic anemia.

Clinically, β-thalassemia is divided into three types: mild, moderate and severe. Mild, medium-sized asymptomatic or mild symptoms. Thalassemia major, also known as Cooley anemia, is characterized by growth retardation, head deformation showing a typical "thalassemia appearance", progressive enlargement of the liver and spleen due to compensatory hematopoiesis, abnormal blood routine and red blood cell morphology, and genetic testing can identify β-beads Point mutations or deletions in proteins. Currently, regular blood transfusions and oral iron chelation agents are routinely used in the treatment of β-thalassemia, and are widely used clinically. However, both long-term blood transfusion and β-thalassemia disease itself lead to increased intestinal iron absorption and iron overload, and iron deposition in substantial organs such as the myocardium, liver, pancreas, and brain, as well as secondary infections can cause dysfunction of related organs and failure, ultimately threatening the life of the patient. Bone marrow transplantation and allogeneic hematopoietic stem cell transplantation are effective methods for the treatment of β-thalassemia, which can achieve a complete cure. However, due to high requirements for gametes, low success rate, long-term anti-immune therapy after surgery, high toxicity and side effects, and high cost, the transplantation treatment for most patients, especially those in developing countries and poor areas, is limited.

In recent years, the use of lentiviral vectors to introduce normal β-globin genes or correct β-globin mutant genes in patients has made great progress. Studies have shown that the regulation of the β-globin genome control region (β-LCR) is abnormal and the gene Mutations can cause β-globin synthesis disorders and specifically affect the production of hemoglobin. The expression of β-globin can be promoted by introducing normal β-globin gene, promoter or promoting the function of β-LCR, which can improve β-thalassemia. Cavazzana et al. used viral vectors to introduce the normal β-globin gene into severe βE/β0 adult thalassemia, activate the transcription of high-mobility group A2 (HMGA2) in erythroid cells, increase the expression of γ-globin, and maintain the hemoglobin level at 9-10 grams / deciliter, about 1/3 of the hemoglobin is produced by the β-globin gene encoded by the viral vector. After transferring the β-globin gene into hematopoietic progenitor cells through different ways, there are many reports on the increase of β-globin expression in animal models or patients, which can improve anemia. Among them, gene mutations in patients may induce tumors, and there are difficulties in how to select and introduce alternative hematopoietic stem cells, the stability of related viral vectors is not good, whether the stem cells with β gene can survive in patients for a long time, etc. The further clinical application of this kind of technical method is proposed.

The human β-like globin locus contains the ε, γ, δ, and β globin genes. The ε and γ globin genes were highly expressed in the yolk sac and hematopoietic liver tissues of early embryonic hematopoietic tissues respectively. As the fetus matures, the expression of ε and γ globin genes is silenced, while the expression of β globin increases, and fetal hemoglobin (HbF) is gradually replaced by hemoglobin A (HbA). Current studies have confirmed that infants with β-thalassemia have no obvious anemia within 3 to 6 months of birth, and the anemia is obvious when γ-globin synthesis decreases after 3 to 6 months, and it has been confirmed that increasing the synthesis of HbF can improve β-thalassemia. Studies have also suggested that the level of HbF in erythrocytes in patients with β-thalassemia is negatively correlated with the degree of anemia in patients, and in patients with β-thalassemia intermedia who have not undergone blood transfusion and HbF induction therapy, the level of HbF is positively correlated with the level of total hemoglobin in patients. More and more evidences show that promoting the expression of HbF can effectively treat β-thalassemia. At present, HbF induction therapy mainly includes γ-globin drug induction therapy and γ-globin gene reactivation. Drug induction therapy has more or less toxic side effects, and its safety has been controversial. For example, Hydroxyurea has an effect on fertility and has the possibility of causing malignant tumor formation. γ-globin gene reactivation also induces gene mutations and induces tumors. Therefore, due to the uncertainty of the therapeutic effect and high treatment cost of this kind of HbF induction therapy, the effective data of its clinical application are limited, which limits the application of this kind of drug.

For more than ten years, microRNA (miRNA) has been a research hotspot in the field of molecular biology. It has been found that miRNAs are a kind of stem-loop structure precursor formed by non-protein-coding gene transcripts, which are formed after enzymatic cleavage and maturation. Small RNA molecules of 18-25 nucleotides. miRNAs can participate in the regulation of gene expression in eukaryotes at the post-transcriptional level. They act on specific target genes through incomplete base complementation to form a powerful regulatory network, resulting in decreased stability or expression of target genes. It plays a pivotal role in the process of cell development, proliferation, differentiation and cell death. There is ample evidence in erythropoiesis that miRNAs are critical for erythroid development. A large number of in vitro and in vivo experiments have found that miR-144/451 plays an important role in erythrocyte development and globin synthesis. miR-144 and miR-451 are a bicistronic miRNA gene locus with a distance of about 100 bases. This gene cluster shares the same transcriptional regulatory promoter and is most expressed in mature erythrocytes. RNA-sequencing studies have shown that the expression of miR-451 accounts for about 50% of the total miRNA pool in late erythrocytes. Our in vivo model confirmed that miR-144/451 plays an important role in erythroid maturation. The disordered expression of miR-144/451 can cause corresponding red blood cell diseases.

The quality of life of β-thalassemia patients is poor, and the average life expectancy is short, causing serious burdens to both the family and the society. Early diagnosis and effective treatment methods are of great value to the entire human race. Therefore, the research and development of relatively safe targeted therapy drugs has become a direction for the treatment of β-thalassemia.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for treating β-thalassemia, by reducing the expression level of miR-144/451 gene or reducing the amount of functional mature miR-144/451, improving β-thalassemia Anemia symptoms of anemia.

In order to solve the above technical problems, the embodiment of the present invention discloses a method for treating β-thalassemia, including: by finally reducing the expression level or/and function of the miR-144/451 gene in red blood cells, it can treat β-thalassemia anemia.

Further, the method for reducing the expression level of miR-144/451 gene includes: reducing the expression or/and function of miR-144 and miR-451 mature sequences, regulating precursor or original miR-144 and miR-451 RNA expression methods, methods of regulating miR-144/451 gene regulation to reduce the expression of mature miR-144/451, and methods of changing the molecular biological pathways regulated by miR-144 and miR-451 to change the expression and function of miR-144/451.

Further, the method for reducing the expression level of miR-144/451 gene includes: small nucleic acid drugs, chemical drugs, biogenetic methods, gene therapy (including targeting hematopoietic stem cells, embryonic stem cells and pluripotent induced stem cells) and gene knockout In addition to technology and other methods.

Further, the method for regulating the expression of miR-144 and miR-451 precursors or original RNA includes: changing the sequence and function of the promoter, and changing the activity of transcription factors such as Gata-1 and KLF1.

Further, the method for reducing miR-144 and miR-451 mature sequence expression or/and function is shRNA, siRNA, RNAi, antisense sequence, LNA technology (Locked nucleicAcids) and Crispr-Cas9 combined miR-144/451 sequence Inhibits mature sequence expression and function of miR-144/451.

Furthermore, an experimental method for treating β-thalassemia according to the present method comprises the following steps:

S1: Prepare relevant experimental reagents;

S2: Two β-globin-/+ and miR-144/451-/- gene knockout male mice, several C57/BL6 female mice, a large number of gene knockout β-thalassemia mice were obtained by breeding , miR-144/451 knockout mice and miR-144/451 knockout β-thalassemia mouse models;

S3: Raise the mice according to the SPF (Specific pathogen Free) level feeding standard, control the humidity at 40%-70%, and control the temperature at 20-25°C, and replace them with sterilized feed, bedding and drinking water once a week two to three times;

S4: The method of housing is 1 gene knockout male mouse and 1-5 gene knockout female mice for breeding, and if the female mouse is found to be pregnant, it is immediately divided into cages for birth, and the source of the parental line of the mice is recorded in time;

S5: Mouse DNA extraction: Take the tail end tissue of a 3-week-old mouse with a size of about 0.5cm, put it into a 1.5ml EP tube, add 100μl/95°C lysate to each tube, let it stand for 20 minutes, add 100μl after cooling The neutralizing solution was mixed, and the above supernatant containing mouse DNA was used for PCR (polymerase chain reaction) to identify the mouse genotype;

S6: PCR of mouse DNA. The following reactants constitute a 20 μl PCR reaction system: 2 μl mouse tail DNA, 1 μl primer, 10 μl DNA polymerase mixture, and 7 μl double distilled water. After mixing and centrifuging, set the conditions in a PCR instrument for cycle amplification: pre-denaturation, 95°C, 5 minutes; denaturation, 95°C, 40 seconds; annealing, 60.5°C, 40 seconds; extension, 72°C, 1 minute, cycle; 72°C, 5 minutes;

S7: Perform agarose gel electrophoresis: Take 0.75 g of agarose powder and add it to 50 ml of TAE electrophoresis buffer, heat it to boiling in a microwave oven, cool the dissolved and clarified agar to 60°C at room temperature, add 5 μl of Goldview or 3 μl of EB and mix well. Slowly pour it into the gel plate where the comb has been placed, and leave it at room temperature for 30 minutes until the gel is completely solidified, then gently pull out the comb; put the prepared 1.5% agarose gel into the electrophoresis tank, add 1xTAE solution until it is higher than the gel Surface: Take 10 μl of the final product of the PCR reaction and add it to the gel hole, electrophoresis with a voltage of 100V, and the time is 30 minutes;

S8: Gel imaging: After the electrophoresis is over, take out the gel and place it under the gel imaging analyzer to take pictures. The electrophoresis genotype fragment of the mouse is: β-thalassemia mouse identification, a size of 567bp (base pair, base pair) ) bands are wild-type normal mice, one band with a size of 1000bp is homozygous β-thalassemia mice; the above two bands are β-thalassemia heterozygotes; miR-144/451 wild type small Mouse is 290bp; homozygous 190bp; heterozygous (-/+) 250bp and 190bp.

Described lysate is the mixture that 0.5M EDTA40 μ l, 1ON NaOH5 μ l and 100ml distilled water mix when pH is 8; Described neutralization liquid is the mixed solution that 1M Tris-HCl4ml and distilled water are settled to 100ml; Described polymerase mixture Includes: Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.

Application of the application model in the treatment of β-thalassemia by reducing miR-144/451 gene expression according to this method in anemia research.

According to the application of this method in the screening of drugs for treating anemia in the gene knockout β-thalassemia mouse model for reducing the expression level of miR-144/451.

According to this method, the miR-144/451 gene knockout β-thalassemia mouse model was constructed.

Compared with the prior art, the beneficial effects of the present invention are:

After knocking out miR-144/451 in the β-thalassemia model mice through genetic cultivation, various indicators in the peripheral blood of the mice were detected, and it was found that the β-thalassemia model mice changed from severe anemia to mild anemia, Manifested by elevated red blood cell count and hemoglobin level. And it was found that the spleen of β-thalassemia mice lacking expression of miR-144/451 was significantly reduced, thus confirming that reducing the expression and function of miR-144/451 can initiate a therapeutic effect on β-thalassemia.

Description of drawings

Figure 1 shows the overexpression of miR-144 and miR-451 in mouse embryonic liver, bone marrow and peripheral blood;

Figure 2 is the morphology of peripheral blood erythrocytes in mice of different genotypes;

Figure 3 is a comparison of spleen morphology and spleen weight in different genotype mice;

Figure 4 is the gel electrophoresis images of mice with different genotypes, the left image is the electrophoresis image of miR-144/451 genotype identification, and the right image is the electrophoresis image of β-het genotype identification.

detailed description

The present invention will be further described below in conjunction with the embodiments. The following descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention to other forms. Changes to equivalent embodiments with equivalent changes. Any simple modifications or equivalent changes made to the following embodiments according to the technical essence of the present invention without departing from the solution content of the present invention fall within the protection scope of the present invention.

The miR-144/451 mature sequence, precursor sequence and original sequence involved in this embodiment are as follows:

(1) The mature sequences (completely conserved) of miR-144 and miR-451 in mice and humans are as follows:

1.Mature sequence mmu-miR-144-5p(MIMAT0016988):

5'-GGAUAUCAUCAUAUACUGUAAGU-3';

2. Mature sequence mmu-miR-144-3p (MIMAT0000156):

5'-UACAGUAUAGAUGAUGUACU-3'

3.Mature sequence mmu-miR-451a(MIMAT0001632):

5'-AAACCGUUACCAUUACUGAGUU-3'

4.Mature sequence mmu-miR-451b(MIMAT0025178):

5'-UGGGAGCAGCAAGAGAACCGU-3'

(2) The antisense nucleic acid sequence of miR-144/451 mature sequence is as follows:

Antisense sequence of miR-144-5p mature sequence: 5'-ACTTACAGTATATGATGATATCC-3'

Antisense sequence of miR-144-3p mature sequence: 5'-AGTACATCATCTATACTGTA-3'

Antisense sequence of miR-451a mature sequence: 5'-AACTCAGTAATGGTAACGGTTT-3'

Antisense sequence of miR-451b mature sequence: 5'-ACGGTTCTCTTGCTGCTCCCA-3'

(3) The precursor sequences and antisense sequences of mouse miR-144 and miR-451 are as follows:

Pre-miR-144: >mmu-mir-144MI0000168

5'-GGCUGGGAUAUCAUCAUAUACUGUAAGUUUGUGAUGAGACACUACAGUAUAGAUGAUGUACUAGUC-3'

Pre-miR-144: >mmu-mir-144MI0000168 antisense sequence

5'-GACUAGUACAUCAUCUAUACUGUAGUGUCUCAUCACAAACUUACAGUAUAUGAUGAUAUCCCAGCC-3'

Pre-miR-451a:>mmu-mir-451a MI0001730

5'-CUUGGGAAUGGCGAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAACGGUUCUCUUGCUGCUCCCACA-3'

Pre-miR-451a: >mmu-mir-451a MI0001730 antisense sequence

5'-UGUGGGAGCAGCAAGAGAACCGUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUCGCCAUUCCCAAG-3'

Pre-miR-451b:>mmu-mir-451b MI0021960

5'-UGUGGGAGCAGCAAGAGAACCGUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUCGCCAUUCCCAAG-3'

Pre-miR-451b: >mmu-mir-451b MI0021960 antisense sequence

5'-CUUGGGAAUGGCGAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAACGGUUCUCUUGCUGCUCCCACA-3'

(4) The precursor sequences of human miR-144 and miR-451 are as follows:

Pre-miR-144: >hsa-mir-144MI0000460

5'-UGGGGCCCUGGCUGGGAUAUCAUCAUAUACUGUAAGUUUGCGAUGAGACACUACAGUAUAGAUGAUGUACUAGUCCGGGCACCCCC-3'

Pre-miR-451a:>hsa-mir-451a MI0001729

5'-CUUGGGAAUGGCAAGGAAACCGUUACCAUUACUGAGUUUAGUAAUGGUAAUGGUUCUCUUGCUAUACCCAGA-3'

Pre-miR-451b:>hsa-mir-451b MI0017360

5'-UGGGUAUAGCAAGAGAACCAUUACCAUUACUAAACUCAGUAAUGGUAACGGUUUCCUUGCCAUUCCCA-3'

(5) The original sequence (primary sequence) of mouse miR-144 and miR-451 is shown as SEQ ID NO.1.

(6) SEQ ID NO.2 of the reverse complementary chain sequence of the original sequence of mouse miR-144 and miR-451.

(7) The original sequence of human miR-144 and miR-451 is SEQ ID NO.3.

(8) The reverse complementary chain sequence of the original sequence of human miR-144 and miR-451 is SEQ ID NO.4.

(9) The promoter sequence of mouse miR-144 and miR-451 is SEQ ID NO.5.

(10) The promoter sequences of human miR-144 and miR-451 are SEQ ID NO.6.

(11) The gata-1 binding site sequence in the upstream sequence of miR-144 and miR-451 in mice is SEQ ID NO.7.

An experimental method for treating β-thalassemia according to the present method, comprising the following steps:

S1: Prepare relevant experimental reagents

(1) The experimental reagents involved in this embodiment are shown in the table below:

(2) The main self-prepared reagents involved in this embodiment are shown in the table below:

(3) PCR primers applied in this embodiment:

(3.1) Primers for real-time fluorescence quantitative PCR:

miR-144 upstream primer 5'-GGATATCATCATATACTGTAAGT-3

miR-451 upstream primer 5'-AAACCGTTACCATTACTGAGTT-3'

The downstream primers are the general primers in All-in-One miRNAqRT-PCRDetection Kit of GeneCopoeia Company.

3.2) Genotype identification primers:

miR451 upstream primer: 5’-TTCTGCCTGTAACTCTGGATCCCTAAGAGA

miR451 downstream primer-1: 5'-GGGTACCCAGACTAGTACCATCATCTATA

miR451 downstream primer-2: 5'-ATCCCCTCGAGGGACCTAATAACTTC

(3.3) Primer sequences for identification of β-thalassemia mice:

Upstream Thal-1:5'-GCTTTCCAGCAGGCACTAAC-3'

Downstream of Thal-1: 5'-AGGAGGAGGGGAAGCTGATA-3'

Upstream Thal-2:5'-GGCAAAGGATGTGATACGTGGAAG-3'

Downstream of Thal-2:5'-CCAGTTTCACTAATGACACAAACATG-3'

S2: Two β-globin-/+ and two miR-144/451-/- knockout male mice were introduced by the Children's Hospital of Philadelphia affiliated to the University of Pennsylvania from the Non-coding RNA Center of Yangzhou University, and two C57/BL6 females were introduced from the Comparison Center of Yangzhou University. There are several mice, and a large number of gene knockout β-thalassemia mice, miR-144/451 knockout mice and miR-144/451 knockout β-thalassemia mouse models have been bred. See: 1, Yu D, dos Santos C O, Zhao G, et al.miR-451 protects against erythroid oxidant stress by repressing 14-3-3ζ[J].Genes&development,2010,24(15):1620-1633.2, Yang B, Kirby S, Lewis J, et al.1995.A mousemodel for beta0-thalassemia.Proc Natl Acad Sci U S A.1995,92(25):11608-11612. The contents of the above two references are incorporated in the specification of this application in full and as a part of the specification The reagents used in the literature are also incorporated in full as the reagents used in this application, which can be used as the basis for the modification of the subsequent procedures.

S3: The mice were fed according to the SPF (Specific Pathogen Free) feeding standard. The humidity is controlled at 40%-70%, the temperature is controlled at 20-25°C, and the sterilized feed, litter and drinking water are replaced two to three times a week.

S4: The method of housing is 1 male knockout mouse and 1-5 female knockout mice for breeding. If the female mouse is found to be pregnant, such as a vaginal plug or a pear-shaped abdomen, she is immediately separated into cages and recorded in time. Mouse parental origin.

S5: mouse DNA extraction: take 3-week-old mouse tail end tissue with a size of about 0.5cm, put it into a 1.5ml EP tube, add 100μl lysate to each tube, and incubate at 95°C for 20min. After cooling, add 100 μl of neutralizing solution, mix well, and take the above supernatant containing mouse DNA for PCR to identify the mouse genotype.

S6: Polymerase chain reaction (PCR) of mouse DNA: primers were provided by Shanghai Sangon Bioengineering Co., Ltd. PCR reaction system: The following reactants constitute a 20 μl reaction system. Mouse tail DNA 2 μl, primer (20 μmol) μl, DNA polymerase mixture (Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer) 10 μl, double distilled water 7 μl. After mixing and centrifuging, set the conditions in a PCR instrument for cycle amplification: pre-denaturation, 95°C, 5 minutes; denaturation, 95°C, 40 seconds; annealing, 60.5°C, 40 seconds; extension, 72°C, 1 minute; cycle; 72°C, 5 minutes.

S7: Perform agarose gel electrophoresis: Take 0.75 g of agarose powder and add it to 50 ml of TAE electrophoresis buffer, heat it to boiling in a microwave oven, cool the dissolved and clarified agar to 60°C at room temperature, add 5 μl of Goldview or 3 μl of EB and mix well. Slowly pour it into the gel plate where the comb has been placed, and leave it at room temperature for 30 minutes until the gel is completely solidified, then gently pull out the comb. Put the prepared 1.5% agarose gel into the electrophoresis tank, add 1xTAE solution to the surface of the gel. Take 10 μl of the final product of the PCR reaction and inject it into the gel well. Voltage 100V electrophoresis, time is 30 minutes.

S8: Gel imaging: After the electrophoresis, take out the gel and place it under the gel imaging analyzer to take pictures. The electrophoresis genotype fragment of the mouse is: β-Thalassemia mouse identification is, a size of 567bp (base pair, base pair) ) bands are wild-type normal mice, one band with a size of 1000bp is homozygous β-thalassemia mice; the above two bands are β-thalassemia heterozygotes. miR-144/451 wild-type mice are 290bp; homozygous 190bp; heterozygous (-/+) 250bp and 190bp.

Example 1

As shown in Figure 1, A: The gestation period of mice is 20 days, and the embryonic liver is the hematopoietic organ in the embryonic stage of mammals. We found in the microarray data that miR-451 of β-het (heterozygous β-thalassemia. Note: homozygous β-thalassemia is embryonic lethal and unable to be born) was higher than that of wt (wild type , wild type) mice expressed 50-80% higher (p<0.01); B: Comparing the expression of miR-144 and miR-451 in the peripheral blood of β-het mice and wt mice, suggesting that the peripheral blood of β-het mice The expressions of miR-144 and miR-451 were significantly up-regulated (p<0.01); C: Comparing the expressions of miR-144 and miR-451 in reticulocytes and nucleated erythrocytes in the bone marrow of β-het mice, suggesting that β- The expressions of miR-144/451 in bone marrow Retic (reticulocyte) and NRBC (nucleated red blood cell) were significantly up-regulated (p<0.01) during het.

Example 2

The comparison of various indicators in the peripheral blood of mice of different genotypes is shown in the table below: index wt mko βhet mko/βhet WBC(*10^9/l) 7.28±2.86 6.25±1.66 36.69±13.50** 8.24±2.76** RBC(*10^12/l) 10.10±1.46 10.51±1.15 7.25±1.24** 11.2±0.65 Hb(g/dl) 130.18±20.61 117.11±14.37** 81.46±13.33** 102.25±8.42** HCT(%) 55.87±7.53 53.48±5.11 35.81±5.61** 48.52±3.30** PLT(*10^12/l) 1074.06±305.27 1013.67±398.53 1775.77±631.75** 1352.50±292.81** RDW(%) 17.01±0.77 23.24±1.31** 37.75±2.73** 30.93±3.23**

After taking the peripheral blood of the mice, the whole blood cell analyzer detected the indicators of the peripheral blood. Compared with the wt mice, it was found that the hemoglobin (Hb) in the peripheral blood of the mko mice was significantly reduced, and the red blood cell distribution width (RDW) was increased, indicating that the size of the red blood cells was different. Uniform, mild anemia. βhet mice are severely anemic, red blood cells (RBC), Hb, and hematocrit (HCT) are significantly decreased, and RDW is increased, indicating that the size of red blood cells is uneven. After removing miR-144/451 in βhet mice by genetic methods , the RBC, Hb and HCT indexes of the obtained mko/βhet mice were significantly increased, indicating that anemia was improved. Compared with βhet, RDW decreased, indicating that the red blood cells were uniform in size.

Example 3

As shown in Figure 2, the peripheral blood of mice with different genotypes was taken, and the blood smears were stained with Swiss Giemsa, and the slides were sealed with neutral gum and observed under a microscope. The peripheral blood red blood cells of wt mice were biconcave discs with uniform shape and size ; The peripheral morphology of β-het mice varies in size, and target-shaped red blood cells can be seen, and the number of reticulocytes increases; while the peripheral blood morphology of mko/βhet mice is compared with that of β-het, it is found that the central light-stained area is significantly reduced, and the number of target-shaped red blood cells is absent Reduced, red blood cell shape and size more uniform, suggesting that anemia improved.

Example 4

As shown in Figure 3, adult mammals are mainly hematopoietic from the bone marrow. In anemia, the extramedullary organs including the spleen can compensatory hematopoiesis, resulting in an increase in the size of the extramedullary organs. The spleens of mice with different genotypes in four groups of wt, mko, β-het and mko/βhet were collected at 8 weeks of adulthood, and the size of the spleens (Panel A) and weight (Panel B) were compared. The results showed that the spleens of miR-144/451 knockout mice (B) were enlarged by an average of 0.5 to 1 times compared with wt, suggesting mild anemia. However, the spleen of β-thalassemia mice (C) was particularly enlarged, with an average increase of 6 times, indicating severe anemia. However, after crossing with miR-144/451 knockout mice, that is, after removing miR-144/451 in β-thalassemia mice, the spleen of the hybrid β-thalassemia mice (D) only increased by 1.5 times, indicating compensatory hematopoiesis Significantly decreased, anemia was significantly reduced.

Example 5

As shown in Figure 4, the mouse genotype identification gel electrophoresis image, the left picture is the genotype identification band after hybridization of miR-144/451 gene knockout mice, 0 is the electrophoretic marker, and lanes 1, 2 and 10 are normal Mouse, size is 290 bases (base pair, bp), electrophoresis lanes 3, 8 and 9 are miR-144/451 heterozygous mice, electrophoresis lanes 4 to 7, size is 156bp, is miR-144/451 knockout Homozygous mice were excluded. The image on the right is the genotype-reduced electrophoresis of β-thalassemia mice, where 0 is a marker, electrophoresis lanes 1, 2, 3, 6, and 7 are β-thalassemia wild-type, and electrophoresis lanes 4 and 5 are heterozygotes.

The application of the application model of reducing miR-144/451 gene expression in the treatment of β-thalassemia described in the present invention to the research of anemia is also within the protection scope of the present invention.

The application of the gene knockout β-thalassemia mouse model for reducing the expression level of miR-144/451 in the screening of drugs for treating anemia is also within the protection scope of the present invention.

The miR-144/451 knockout β-thalassemia mouse model constructed by the method of the present invention is also within the protection scope of the present invention.

The above schematically describes the present invention and its implementation, which is not restrictive, and what is shown in the drawings is only one of the implementations of the present invention, and the actual structure is not limited thereto. Therefore, if a person of ordinary skill in the art is inspired by it, without departing from the inventive concept of the present invention, without creatively designing a structural mode and embodiment similar to the technical solution, it shall all belong to the protection scope of the present invention .

Claims (10)

1. treatment β-thalassemic reagent, it is characterised in that: described reagent can reduce miR-in erythrocyte The expression of 144/451 gene is or/and function.

Treatment β-thalassemic reagent the most according to claim 1, it is characterised in that: in described reduction erythrocyte The expression of miR-144/451 gene and/or the reagent of function include: be used for reducing miR-144 and miR-451 mature sequence Express or/and the method for function, regulation precursor or the method for original miR-144 and miR-451 rna expression, regulation miR-144/ 451 gene regulations are to reduce the method that ripe miR-144/451 expresses, the molecular biosciences changing miR-144 and miR-451 regulation and control Approach changes miR-144/451 to be expressed and the reagent of function.

Treatment β-thalassemic reagent the most according to claim 2, it is characterised in that: described reduction miR-144/ In 451 erythrocyte, the expression of gene and/or the reagent of function include: small nucleic acids medicine, chemicals, biogenetics side Method, gene therapy (include for hematopoietic stem cell, embryonic stem cell and multipotency induce stem cell) and/or miR-can be knocked out The reagent of 144/451 gene.

Treatment β-thalassemic reagent the most according to claim 2, it is characterised in that: described regulation miR-144 and The reagent that miR-451 gene regulation is expressed to reduce ripe miR-144/451 includes: can change promoter sequence and function, Change the reagent of the transcription factor activities such as Gata-1, KLF1.

Treatment β-thalassemic reagent the most according to claim 3, it is characterised in that: described reduction miR-144 and MiR-451 mature sequence express or/and the reagent of function be shRNA, siRNA, RNAi, antisense sequences, LNA technology (Locked Nucleic Acids) and Crispr-Cas9 combine miR-144/451 sequence suppression miR-144/451 mature sequence express and Function.

6. the treatment β described in claim 5-thalassemic reagent, it is characterised in that described reagent includes: miR-144 Forward primer 5 '-GGATATCATCATATACTGTAAGT-3

MiR-451 forward primer 5 '-AAACCGTTACCATTACTGAGTT-3 '

Downstream primer is that general in GeneCopoeia company All-in-One miRNA qRT-PCR Detection Kit draws Thing.

7. the application in preparation treatment β-thalassemia medicine of the reagent described in claim 5.

8. the expression reducing miR-144/451 gene in erythrocyte is or/and the method for function, it is characterised in that: bag Include following steps,

S1: get out related experiment reagent；

S2: if β is-globin-/+and each two of the male mice of miR-144/451-/-gene knockout, C57/BL6 female mice Dry, obtain substantial amounts of gene knockout β-thalassemia Mus, miR-144/451 clpp gene deratization and miR-144/ by breeding The lean mouse model in β-ground of 451 gene knockouts；

S3: raise mice, humid control according to SPF (Specefic pathogen Free no-special pathogen) level feeding standard At 40%-70%, temperature controls at 20-25 DEG C, within one week, changes two to three times with the feedstuff of sterilization treatment, bedding and padding and drinking-water；

S4: the mode of mating is the male Mus of 1 gene knockout and the female Mus copulation of 1-5 gene knockout is bred, and finds female Mus Conceived performance is had to divide cage to give birth to immediately, and record mice father's maternity in time；

S5: mouse DNA extracts: takes the mouse tail end flap of 3 week old, size about 0.5cm, puts in 1.5ml EP pipe, often Pipe adds l/95 ° of C lysate of l00 μ, stands 20 minutes, adds the mixing of l00 μ l neutralizer after cooling, takes above upper containing Mus DNA Clear liquid is used for PCR(polymerase chain reaction) carry out murine genes type qualification；

The PCR of S6: mouse DNA: following reactants constitutes the PCR reaction system of 20 μ l: rat-tail DNA 2 μ l, primer 1 μ 1, DNA gathers Synthase mixture 10 μ l, distilled water 7 μ l.

9. the centrifugal PCR instrument that is placed on of mixing imposes a condition cyclic amplification: denaturation, 95 DEG C, 5 minutes；Degeneration, 95 DEG C, 40 seconds；Move back Fire, 60.5 DEG C, 40 seconds；Extend, 72 DEG C, 1 minute, circulation；72 DEG C, 5 minutes；

S7: carry out agarose gel electrophoresis: take in the TAE electrophoretic buffer that agarose powder 0.75g adds 50ml, in microwave oven It is heated to boiling, the agar thing room temperature dissolving clarification is cooled to 60 DEG C and adds Goldview 5 μ l or EB 3 μ l mixing, slowly Pouring in the offset plate having put comb, room temperature is placed 30min and is extracted comb after gel solidifies completely gently；By prepare 1.5% agarose gel puts into electrophoresis tank, adds 1xTAE solution to exceeding gel surface；Take PCR reacting final product 10 μ l sample-adding In glue hole, voltage 100V electrophoresis, the time is 30 minutes；

S8: gel imaging: after electrophoresis terminates, taking-up gel is placed under gel image analyser takes pictures, mice electrophoresis gene matrix Duan Wei: β-thalassemia mice identify be that a size is 567bp(base pair, base) band be that wild type is normal Mus, size be the band of 1000bp be homozygote β-thalassemia mice；Occur above two band in β-ground Sea anemia heterozygote；MiR-144/451 wild-type mice is 290bp；Homozygote 190bp；heterozygous (-/+)250bp And 190bp.

In reduction erythrocyte the most according to claim 8, the expression of miR-144/451 gene is or/and the side of function Method, it is characterised in that: described lysate is 0.5M EDTA40 μ l, lON NaOH5 μ l and 100ml distilled water are mixed when PH is 8 The mixture closed；Described neutralizer is the mixed solution that 1M Tris-HCl4ml and distilled water are settled to 100 ml；Described polymerization Enzymatic mixture includes: Taq DNA polymerase, dNTPs, MgCl2 and reaction buffer.