Application of helichthrin in preparation of medicine for relieving and treating erythroid differentiation disorder of cells of subjects

Abstract

The invention discloses application of helichthrin in preparation of a medicine for relieving and treating erythroid differentiation disorder of cells of a subject, and belongs to the field of medical preparations. The chemical structure of the wax chrysanthemum element disclosed by the invention is as shown in formula 1, and experiments prove that the wax chrysanthemum element can effectively correct cell erythroid differentiation disorder caused by ribosomal protein gene mutation; specifically, hematopoietic stem cells with the erythroid differentiation disorder are promoted to be differentiated into pluripotent progenitor cells, common myeloid progenitor cells with the erythroid differentiation disorder are promoted to be differentiated into megakaryocyte-erythroid progenitor cells, and the problems that in the erythroid differentiation disorder, due to ribosome protein gene mutation, the proliferation capacity of erythroid phenotype cells is reduced, and cell apoptosis is increased are solved. The erythroid phenotype cells are relieved or recovered on the whole. Therefore, the invention can provide a thought for treating erythroid differentiation disorder diseases, such as DBA.

Description

Technical Field

[0001] The invention relates to the technical field of pharmaceutical preparations, and in particular to the application of helichrysanthemin in the preparation of a medicine for alleviating and treating erythroid differentiation disorders of test subjects. Background Art

[0002] Diamond Blackfan anemia (DBA) is a rare congenital erythrocyte aplasia disease with no difference in incidence between males and females. It usually manifests in infancy and is a ribosomal disease. Common clinical features of DBA include anemia, macrocytosis, and increased erythrocyte adenosine deaminase activity, and are often associated with short stature, physical abnormalities, and cancer susceptibility. Erythrocyte dysplasia is the main hematological feature of DBA patients, mainly manifested as macrocytic anemia, decreased erythroid progenitor cells, and decreased reticulocytes. Studies have shown that DBA patients have a significant lack of erythrocytes, mainly due to the lack of erythrocyte progenitor cells in the bone marrow (BM) at the time of initial diagnosis, such as burst forming unit erythroid (BFU-E) cells, but all other hematopoietic lines are minimally affected.

[0003] The genetic basis of DBA has been extensively characterized, with approximately 60–70% of DBA patients carrying lesions in genes encoding small 40s ribosomal protein subunits (RPS) or large 60s ribosomal protein subunits (RPL). Among RP genes, RPS19 is the most commonly mutated gene, accounting for approximately 25% of DBA patients, followed by RPL5 (approximately 7% of DBA patients), RPS26 (approximately 7% of DBA patients), and RPL11 (approximately 5% of DBA patients). In addition, a small number of non-RP genes have been found to have mutations, such as the erythroid transcription factor GATA1 and the RPS26-interacting protein TSR2. Possible pathological mechanisms include abnormal ribosome biogenesis in DBA patient cells due to mutations in the RPs genes, resulting in the release of RPL5 and RPL11 and their binding to MDM2, which activates p53 activity as MDM2 is a negative regulator of p53. P53 is a key cell cycle regulatory protein that plays an important role in cellular stress conditions such as DNA damage. Since erythroid progenitor cells are highly sensitive to p53 activation, cell cycle arrest and apoptosis occur, and ultimately lead to erythroid hematopoiesis failure. Another triggering factor is believed to be due to the overall reduction in ribosome supply, and the imbalance between globin protein synthesis and heme production leads to the accumulation of cytotoxic free heme, thereby impairing the growth of erythrocytes. Studies have also pointed out that in bone marrow tissue, compared with other lineages of hematopoietic stem and progenitor cells in the process of erythropoiesis, in the pre-erythroid stage, due to the activation of the cell cycle, a large number of ribosomes are required to synthesize proteins, but due to RPs mutations, ribosome haploid deficiency leads to erythroid-specific defects. Recent studies have shown that due to the excessive cell cycle in the BFU-E stage, replication stress stress is caused, thereby activating the expression of TP53, leading to a large number of apoptosis of cells in the BFU-E stage, affecting erythroid development. In summary, the pathological mechanism of erythroid-specific defects reported in previous DBA mainly focuses on the study of the erythroid progenitor (EP) stage, which may explain the cause of erythroid-specific defects in DBA to a certain extent. However, in the context of the universal requirement for ribosome biosynthesis in all cell types, especially in the context of the erythroid system (nucleated red blood cells) in the bone marrow, which does not account for the largest proportion, accounting for about 20% of nuclear cells, while the granulocytic system accounts for the largest proportion, accounting for about 40-60% of nuclear cells, the reason for the failure of erythropoiesis due to abnormal ribosome synthesis caused by RPs mutations that cannot meet the demand for large-scale protein synthesis required in erythroid development still appears to be a paradox.

[0004] In addition, some transcription factors play an important role in the differentiation of erythrocytes, including GATA1 and KLF1. The abnormal expression of these transcription factors may be related to the occurrence of DBA. Recent studies have shown that the inflammation, immunity, oxidative phosphorylation, lipogenesis and other pathways in DBA bone marrow are significantly abnormal, and the transcriptome profiles caused by different RPs mutations are quite different, which may indicate that in addition to erythroid hematopoietic defects, there are other abnormal cell components with unknown functions in the bone marrow. It is worth thinking that through the single cell data of DBA bone marrow, it was found that immune cells also have cell cycle activation, but the fate of immune cells in clinical manifestations is indeed very different from that of erythroid cells. This shows that there are other reasons besides the erythroid defect mechanism caused by ribosomal haploinsufficiency. Interestingly, in the single cell data of normal controls, it was observed that compared with cells in the later stages of development, more primitive hematopoietic stem / progenitor cells seem to have a greater demand for ribosomal protein synthesis. Therefore, it is speculated that the cell defects caused by RPs mutations in DBA may not only start at the erythroid progenitor (EP) stage, but may have appeared at the more primitive hematopoietic stem / progenitor cell (HSPC) stage.

[0005] At present, the mechanism of DBA erythrocyte failure has not been fully determined, and there are few reports on the effects of RP haploinsufficiency on HSPCs and the entire bone marrow microenvironment. The hematopoietic system can be traced back to the seed cells hematopoietic stem cells (HSCs), which are a type of cells that can self-renew and differentiate into various blood cells, including red blood cells, white blood cells and platelets. HSCs are mainly found in the bone marrow and placenta and are key cells required for maintaining the normal function of the blood system. Few studies have reported that some genes in hematopoietic stem / progenitor cells are abnormally expressed. In addition, DBA patients are susceptible to age-related pancytopenia, myelodysplastic syndrome and myeloid leukemia. Although Rps19+ / - mice do not show hematopoietic abnormalities, further reduction of Rps19 by in vivo expression of short hairpin RNA leads to trp53-dependent anemia and HSC failure. HSPCs from DBA patients have intrinsic quality defects and show reduced clonogenic output in long-term culture initiation experiments. Two studies examined HSPCs from DBA patients after xenotransplantation into immunodeficient mice. One of the studies showed that transducing CD34+ mutant HSPCs from DBA patients with a lentiviral vector (LV) expressing rps19 improved their engraftment, but healthy donor HSPCs were not tested as controls. In another report, HSPCs from DBA patients were normally engrafted in NOD / SCID mice, but erythropoiesis was reduced. The engraftment of both DBA and control HSPCs was less than 1%. A recent paper reported that DBA HSPCs are damaged, and RPS19 editing of HSPCs in a cell model resulted in erythroid lineage defects, indicating that HSPCs are damaged, but no abnormalities within hematopoietic stem / progenitor cells were given. Currently, research on Diamond-Blackfan anemia hematopoietic stem / progenitor cells is still in its early stages.

[0006] Although the research on the pathogenesis has been gradually deepened, the treatment of DBA is still mainly based on glucocorticoids, suspended red blood cell transfusion and allogeneic hematopoietic stem cell transplantation. These three treatment methods have different degrees of side effects and need to be considered in clinical application. Oral glucocorticoids are still the first-line treatment for DBA. Although the dose of glucocorticoids is small, the patients are young and take them for a long time during treatment. Some patients even need to take medication for life, and the side effects of hormones cannot be ignored. In addition, the most important clinical problem now is that different DBA patients have very different sensitivities to glucocorticoids (such as prednisone, whose mechanism of action can drive the expansion of red blood cell progenitor cells). Although about 80% of DBA children respond to glucocorticoids, only 20% of patients can achieve remission in the end, 40% of patients rely on drugs to maintain, and the other 40% of patients need blood transfusions to maintain, but the impact of iron overload caused by long-term blood transfusions on the growth and physical condition of children should not be underestimated. Hematopoietic stem cell transplantation is currently the only treatment that can cure DBA. Studies have shown that the 5-year overall survival rate is about 74%, and chronic graft-versus-host disease after transplantation is the main factor affecting its overall survival rate. In addition, cyclosporine A, lenalidomide, and L-leucine have also been used in the treatment of DBA, but the mechanism of action is still unclear and the efficacy varies. Therefore, finding other pathways to further explore the genetic abnormalities of DBA and precise medication is the current research focus to alleviate clinical problems. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a drug that can correct cell erythroid differentiation disorders, thereby alleviating and treating erythroid differentiation disorder-related diseases.

[0008] In a first aspect, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for alleviating and / or treating erythroid differentiation disorder in a subject; .

[0009] According to the application described above, the compound shown in Formula 1 is named Helichrysetin.

[0010] In the above application, the pharmaceutically acceptable salt refers to a salt formed by the compound represented by Formula 1 and a pharmaceutically acceptable acid or base. The "pharmaceutically acceptable" refers to a substance that is suitable for human use without excessive adverse reactions and side effects.

[0011] For the applications described above, the subject can be a mammal, which can be selected from bovines, equines, felines, canines, lagomorphs, porcines, camelids, rodents and primates, etc., including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, gorillas) and humans, preferably cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys and humans.

[0012] In the application as described above, the cell erythroid differentiation disorder includes the cell erythroid differentiation disorder caused by the cell ribosomal protein gene mutation and / or the cell erythroid differentiation disorder caused by the non-ribosomal protein gene mutation. Further, the cell erythroid differentiation disorder is caused by the cell ribosomal protein gene mutation. Further, the cell ribosomal protein gene mutation includes at least one of RPS19 mutation, RPL5 mutation, RPS26 mutation and RPL11 mutation. Further, the ribosomal protein gene mutation includes RPS19 mutation, and the RPS19 mutation is a mutation that causes a decrease in the expression of the RPS19 gene.

[0013] The use as described above, wherein the subject suffers from congenital pure red cell aplasia.

[0014] As described above, the relief and / or treatment of the subject's erythroid differentiation disorder is manifested as increasing the number and / or proportion of the subject's erythroid cells. Further, the erythroid differentiation disorder at least includes erythroid differentiation disorder of hematopoietic stem progenitor cells (HSPC) that can differentiate into the hematopoietic lineage and produce all types of blood cells. Furthermore, the relief and / or treatment of the subject's erythroid differentiation disorder is specifically embodied in at least one of the following: 1) Promote the differentiation of hematopoietic stem cells with erythroid differentiation disorders into multipotent progenitor cells; 2) Promote the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryotic-erythroid progenitor cells; 3) Increase the number and / or proportion of CD71-positive and CD235a-positive erythroid cells.

[0015] For the above-mentioned application, the dosage form of the drug can be determined according to actual needs, for example, it can be selected from one of injection, tablet, capsule, lozenge, oral liquid, granule, granule, pill, powder, ointment, syrup, mixture, dew, effervescent, paste, emulsion, tea, pill, suspension, powder, implant, ointment, plaster, cream, spray, drop, patch. Further, the dosage form of the drug is selected from one of injection, tablet, capsule, lozenge, oral liquid, suspension, implant, patch.

[0016] Furthermore, the dosage form of the drug is an injection, such as liquid injection, powder injection, injection tablets, etc. The injection form includes but is not limited to original drug form, drug-loaded nanoparticles, etc.; the injection site includes but is not limited to intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection and spinal cavity injection, etc.

[0017] Furthermore, the dosage form of the drug is an implant, and the implant is selected from one of a cavity-controlled implant, a skeleton-type implant, and an osmotic pressure-driven implant.

[0018] In the above application, the drug further comprises a pharmaceutically acceptable carrier. Further, the pharmaceutically acceptable carrier comprises at least one of an excipient, a lubricant, a pH regulator, a stabilizer, an encapsulating agent, a spray, an adhesive, a filler, a disintegrant, a wetting agent, a transdermal absorbent, an absorption enhancer, a surfactant, a flavoring agent, a colorant, a preservative, a sweetener, an osmotic pressure regulator, and an adsorption carrier.

[0019] In a second aspect, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for correcting and / or alleviating an in vitro cell erythroid differentiation disorder; .

[0020] In a third aspect, the present invention provides a method for correcting and / or alleviating erythroid differentiation disorder of ex vivo cells, comprising contacting the ex vivo cells with a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, so as to correct and / or alleviating erythroid differentiation disorder of the ex vivo cells; .

[0021] In the above-mentioned method, the compound represented by Formula 1 can be contacted with cells having erythroid differentiation disorder in a cell culture system or in other forms of carriers.

[0022] In the method described above, the concentration of the compound represented by Formula 1 in the cell culture system is 5-20 μM, and the culture time is 24-72 hours.

[0023] In a fourth aspect, the present invention provides a method for alleviating and / or treating erythroid differentiation disorder in a subject, the method comprising administering to the subject an alleviating and / or treating effective amount of a compound shown in Formula 1 or a pharmaceutically acceptable salt thereof, or a drug comprising the compound shown in the above Formula 1 or a pharmaceutically acceptable salt thereof.

[0024] The method as described above, wherein the subject suffers from congenital pure red cell aplasia.

[0025] The dosage of the drug used to alleviate and / or treat the erythroid differentiation disorder of the subject in the method described above is variable, depending on the mode of administration, route of administration, age and / or weight of the individual, and the condition of the individual being treated, and is ultimately determined by the attending physician. The dosage administered to the individual, in the case of the present invention, should be sufficient to induce a beneficial response in the individual over a period of time.

[0026] The chemical structure of helichrysanthemin disclosed in the present invention is shown in Formula 1. It has been verified by experiments that helichrysanthemin can effectively correct cell erythroid differentiation disorders caused by ribosomal protein gene mutations, specifically promoting the differentiation of hematopoietic stem cells with erythroid differentiation disorders into multipotent progenitor cells, promoting the differentiation of common myeloid progenitor cells with erythroid differentiation disorders into megakaryotic-erythroid progenitor cells, and alleviating the problems of reduced proliferation ability and increased cell apoptosis of erythroid phenotype cells in erythroid differentiation disorders caused by ribosomal protein gene mutations, so that erythroid phenotype cells are alleviated or restored as a whole. Therefore, the present invention can provide ideas for erythroid differentiation disorder diseases, such as DBA therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The HSPC cell analysis results of DBA patients; A is the clustering result of CD34+ HSPC cells; B is the analysis result of CD34+ HSPC cell markers; C is the result of GSVA verification of the clustering results against the known clustering gene set; D is the evaluation result of the CD34+ HSPC cell proportion in normal healthy people (Ctrl) and DBA patients (Patient); E is the statistical result of the significant difference in the number of cells in the CD34+ HSPC cell population between normal healthy people (Ctrl) and DBA patients (Patient); F is the result of pseudo-time differentiation trajectory analysis of four cell subsets (HSC / MPP, CMP, MEP, CLP) of normal healthy people (Ctrl) and DBA patients (Patient); Figure 2 The analysis results of the relative expression of the RPS19 gene in the constructed DBA HSPC cell model (sh-RPS19) and the control cells (sh-Random); Figure 3The expression of CD235a and CD71 antigens on the cell surface of the constructed DBA HSPC cell model (sh-RPS19) and the control cell (sh-Random) after 7 days of directed erythroid differentiation culture; A is the result of flow cytometry detection, and B is the statistical result of the proportion of CD235a and CD71 double-positive cells; Figure 4 The analysis results of the relative expression of TP53 gene after 7 days of directed erythroid differentiation culture in the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); Figure 5 Giemsa staining of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random) after 10 days of directed erythroid differentiation culture; Figure 6 The expression of CD11b antigen on the cell surface of the constructed DBA HSPC cell model (sh-RPS19) and the control cell (sh-Random) after 5 days of monocytic differentiation; A is the result of flow cytometry detection, and B is the statistical result of the proportion of CD11b positive cells; Figure 7 The expression of CD11b antigen on the cell surface of the constructed DBA HSPC cell model (sh-RPS19) and the control cell (sh-Random) after 36 days of monocytic differentiation; A is the result of flow cytometry detection, and B is the statistical result of the proportion of CD11b positive cells; Figure 8 The expression of CD15 and CD68 antigens on the cell surface of the constructed DBA HSPC cell model (sh-RPS19) and the control cells (sh-Random) after 7 days of granulocyte differentiation culture; A is the result of flow cytometry detection, and B is the statistical result of the proportion of CD15 and CD68 double-positive cells; Fig. 9 The expression of CD15 on the cell surface of the constructed DBA HSPC cell model (sh-RPS19) and the control cell (sh-Random) after 22 days of granulocyte differentiation culture; A is the result of flow cytometry detection, and B is the statistical result of the proportion of CD15 positive cells; Fig.10 Flow cytometry results of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); Fig.11 The clustering statistics of the constructed DBA HSPC cell model (sh-RPS19) and the control cells (sh-Random); Fig.12 The statistical results of the proportion of hematopoietic stem and progenitor cell subpopulations after adding different concentrations of helichrysanthemin during the proliferation of DBA HSPC cell model (sh-RPS19); Fig.13 The figure shows the erythrocyte differentiation after adding different concentrations of helichrysin during the differentiation process of the DBA HSPC cell model (sh-RPS19); A is the result of flow cytometry detection; B is the statistical result of the erythrocyte differentiation ratio.

[0028] In the figure above, * indicates p -value≤0.05, there is a statistical difference; ** indicates p -value≤0.01, there is a statistically significant difference; *** indicates p -value≤0.001, there is an extremely significant statistical difference; ns means there is no statistical difference. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments, and they should not be understood as limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0030] In the following examples, unless otherwise defined, the terms should be understood according to the conventional usage of ordinary technicians in the relevant fields. The experimental methods involved in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0031] The culture medium StemSpan SFEM II used in the following examples was purchased from StemCell, USA; the cytokines SCF, TPO, Flt-3 Ligand, and IL-3 were purchased from PeproTech, USA; streptomycin and penicillin were purchased from Yantai Saiers Biotechnology Co., Ltd.; Helichrysetin (Helichrysetin) with a chemical structure as shown in Formula 1 was purchased from MedChemExpress, USA, with a catalog number of HY-N4058 and a CAS number of 62014-87-3; the flow cytometry antibodies PE conjugated anti-CD34, APC conjugated anti-CD38, BV421 conjugated anti-CD90, AF700 conjugated anti-CD45RA, BV510 conjugated anti-CD10, PE-Cy7 conjugated anti-CD135, APC conjugated anti-CD235a, and PE conjugated Anti-CD71 was purchased from BioLegend, USA; flow cytometry antibodies APC-CD11b, APC-CD68, and PE-CD15 were purchased from Elabscience / Elarerite, China.

[0032] Example 1: Single-cell transcriptome analysis of HSPC (hematopoietic stem cell) in patients with DBA like Figure 1 As shown in A, a cell atlas of CD34+ HSPC cells was drawn using single-cell transcriptome sequencing data, and CD34+ HSPC cells were divided into 16 groups: HSC / MPP (hematopoietic stem cells / multipotent progenitor cells), CMP (common myeloid progenitor cells), CLP (common lymphoid progenitor cells), GMP (granulocyte-monocyte progenitor cells), MEP (megakaryocyte-erythroid progenitor cells), ERR-Early (early erythroid progenitor cells), ERP (erythroid progenitor cells), Erythroblast (erythroblast), MKP (megakaryocyte progenitor cells), MDP-1 (monocyte-dendritic progenitor cells-1 type), MDP-2 (monocyte-dendritic progenitor cells-2 type), Eo / B / Mast (eosinophilic / basophilic / mast cell progenitor cells), Lymphoid-UNK (NK cell progenitor cells), pre_PC (pre-plasma cell, plasma cell precursor cells), pre_B (B cell precursor cells), and pre_T (T cell precursor cells). right Figure 1 The biomarkers of the 16 cell subtypes shown in A were analyzed. The analysis results are shown in Figure 1As shown in Figure B, it can be seen that the specific biomarkers of 16 types of cells are significant and the cells are effectively distinguished. The clustering results are compared with the known clustering gene set for GSVA verification, as shown in Figure 2. Figure 1 As shown in C, the clustering results provided in this embodiment can be well matched with the cell populations known in public data, reflecting that the clustering results of this embodiment are reliable.

[0033] Based on the above clustering results, the clustering ratios of CD34+ HSPC cell subtypes in normal healthy people (Ctrl) and DBA patients (Patient) were evaluated. The evaluation results are as follows: Figure 1 As shown in D, the proportion of progenitor cell stages related to erythroid differentiation (MEP, ERP-early, EPR) in DBA patients is downregulated, while the proportion of lymphoid progenitor cells related to lymphoid differentiation (CLP, Lymphoid-UNK, pre_T) is upregulated. Afterwards, the number of cells in the CD34+HSPC cell population between normal healthy people (Ctrl) and DBA patients (Patient) was statistically analyzed for significant differences. The statistical results are shown in Figure 2. Figure 1 As shown in E, it can be seen that the erythroid lineage MEP and ERP-Early in the HSPC cell population are significantly reduced, and other lineages such as lymphoid lineage CLP are significantly increased. The early HSC / MPP is reduced to a certain extent, while the GMP early myeloid progenitor cells related to myeloid differentiation are increased to a certain extent, indicating that the proportions of each progenitor cell subset of hematopoietic stem / progenitor cells have changed. The four cell subsets (HSC / MPP, CMP, MEP, CLP) of normal healthy people (Ctrl) and DBA patients (Patient) were analyzed by pseudo-time differentiation trajectory. The analysis results are shown in Figure 1 As shown in F, it can be seen that the seed cells in DBA patients (Patient) are reduced to the early erythroid-megakaryocyte progenitor MEP stage.

[0034] The above results show that the proportion of HSPC cells in DBA patients is abnormal, indicating that there is an abnormality in hematopoietic stem progenitors and that the proportion of early erythroid-related cells is specifically downregulated.

[0035] Example 2: Construction of RPS19-deficient HSPC cell differentiation model 2.1. Extraction of model cells: CD34+ HSPC cells were isolated from human umbilical cord blood obtained from Beijing Maternity Hospital. The specific steps are as follows: 1.1. Cut the blood bag catheter containing human umbilical cord blood with sterilized scissors and disinfect the cut again with the outer flame of an alcohol lamp. 1.2. Transfer the umbilical cord blood to a 50 mL centrifuge tube, with each tube containing about 15 mL of umbilical cord blood. 1.3. Add RosetteSep at 5 μL / mL. TMCocktail, i.e. about 80 μL per tube, was placed at room temperature for 20 min. 1.4. Take 50 mL of SepMate TM Density gradient centrifuge tubes (the number is the same as that of the 50 mL centrifuge tubes containing umbilical cord blood), add 15 mL of lymphocyte separation solution to each tube, and avoid light. 1.5. Add 1×PBS solution containing 2% serum and 1 mM EDTA in the same volume as the umbilical cord blood (about 15 mL) to the 50 mL centrifuge tube containing umbilical cord blood, mix it upside down, and then gently add it to the density gradient centrifuge tube containing lymphocyte analysis solution. 1.6. Adjust the break of the centrifuge to the lowest setting, and centrifuge the mixture in step 1.5 at 1200 g for 20 min. 1.7. Discard the supernatant 1 cm away from the cloudy cell layer with a 10 mL pipette, quickly pour the remaining liquid containing the cloudy cell layer into a new 50 mL centrifuge tube, and finally add PBS to make up to 50 mL. 1.8. Adjust the break to 3300 g and centrifuge for 10 min. 1.9. Remove all supernatant, resuspend the pellet with 750 μL 1× PBS, and transfer to a 14 mL round-bottom test tube with a polypropylene lock cap. Add 75 μL EasySep TM Positive Selection Cocktail, let stand at room temperature for 10 min. 1.10, add 50 μL EasySep TM Dextran Rapid Spheres, after standing at room temperature for 3 min, fill up to 3 mL with 1×PBS and mix with a rubber-tipped dropper. 1.11. Insert the test tube into the EasySep™ Magnet, pour out the liquid in the tube after 3 min, add 3 mL of 1×PBS and mix, discard the liquid after 3 min, and repeat this washing 4 times. The round-bottom test tube cannot be removed during the whole process. 1.12. After washing, remove the test tube, add a certain amount of proliferation medium, resuspend the cells adsorbed on the tube wall, count and culture the cells according to the density in a 6-well plate to obtain HSPC (CD34+) cells.

[0036] 2.2. Cultivation of model cells: The extracted HSPC (CD34+) cells were cultured in proliferation medium for 7 days to expand a certain number of cells to meet the needs of subsequent experiments. The proliferation medium was SFEM II medium containing 50 ng / mL SCF, 50 ng / mL Flt3 Ligand, 50 ng / mL TPO and 1% double antibody. The cell density was 5×10 4 / mL and placed in a cell culture incubator (37°C, 5% (V / V) CO2) to prepare for the subsequent construction of a cell model.

[0037] 2.3. Construction of HSPC cells infected with lentivirus (shRPS19): 2.3.1. Design shRNA for knocking down RPS19 gene online through the website (URL: http: / / rnaidesigner.thermofisher.com / rnaiexpress / sort.do), synthesize oligonucleotide chains required for expressing sh-RPS19, and the nucleotide sequences of the oligonucleotide chains are shown in SEQ ID NO: 1-2 respectively.

[0038] SEQ ID NO: 1: 5'-GATCC gagatctggacagaatcgc cttcctgtcaga gcgattctgtccagatctc tttttg-3'; SEQ ID NO:2: 5'-AATTCAAAAA gagatctggacagaatcgc TCTGACAGGAAG gcgattctgtccagatctc G-3'.

[0039] 2.3.2. Mix 1 μl of 20 μM single-stranded DNA molecule with nucleotide sequence SEQ ID NO:1, 1 μl of 20 μM single-stranded DNA molecule with nucleotide sequence SEQ ID NO:2, 2 μl of 10×T4 Polynucleotide Kinase Buffer, 2 μl of 10 mM ATP and 1 μl of T4 Polynucleotide Kinase (10 U / μl), make up the total volume to 20 μl with ddH2O, heat the mixed system at 95°C for 2 min, and then reduce the temperature at a rate of 1°C every 30 s until the temperature drops to 25°C to obtain double-stranded DNA.

[0040] 2.3.3 Use BamH1 and EcoR1 The knockdown plasmid pSHI-H1-copGFP-T2A-Puro (#SI501A-1, System Biosciences) was digested with restriction endonucleases to obtain the digested plasmid; 1 μl 10× DNA Ligase buffer, 1 μl digested plasmid, 7 μl double-stranded DNA and 3 μl T4 DNA Ligase were mixed, and the digested plasmid and double-stranded DNA were ligated to obtain a ligation product.

[0041] 2.3.4. Take 5 μl of the ligation product and add it to 50 μl of E. coli stbl3 competent cells (#CD521-01, All-Gold), place on ice for 30 min, heat shock at 42°C for 1 min, and place on ice again for 3 min. Inoculate the transformed competent cells onto solid LB medium containing ampicillin (AMP) (#CA0006-10ml, LEAGENE), and culture at 37°C overnight.

[0042] 2.3.5. Select colonies with good growth status, put them into 5 mL LB medium containing Amp, and culture them in a shaking incubator at 37°C for 16 h. Extract the plasmid, sequence it, and after identification, the fragment was successfully inserted into the vector to obtain a vector expressing sh-RPS19, named PSIHI-H1-shRNA.

[0043] 2.3.6 Lentivirus packaging PSIHI-H1-shRNA and its auxiliary packaging plasmid pMDLg (#12251, Addgene), pRSV-Rev (#12253, Addgene) and pVSV-G (#138479, Addgene) were co-transfected into 293T cells. The culture medium was replaced with complete culture medium 6-8 hours after transfection. After culturing for 48 and 72 hours, the supernatant culture medium rich in lentiviral particles was collected, mixed and concentrated to obtain lentivirus.

[0044] 2.3.7. Infection of HSPC cells The concentrated lentivirus was used to infect HSPC cells for 24-48 hours, and the proliferation medium was replaced with new one, and the cells were expanded to the 7th day. The obtained cells were the constructed RPS19-deficient HSPC cell model, named sh-RPS19, to simulate the HSPC cells of DBA patients.

[0045] At the same time, a random sequence was used to construct a lentivirus using the same method as above, and HSPC cells were infected, named sh-Random, as the control cells for sh-RPS19.

[0046] Wherein: the nucleotide sequences of the oligonucleotide chains corresponding to sh-Random are shown in SEQ ID NOs: 3-4 respectively.

[0047] SEQ ID NO:3: 5'-GATCC GACTCCAGTGGTAATCTAC cttcctgtcaga GTAGATTACCACTGGAGTC tttttg-3'; SEQ ID NO:4: 5'-AATTCAAAAA CTGAGGTCACCATTAGATG TCTGACAGGAAG CATCTAATGGTGACCTCAG G-3'.

[0048] qPCR detection was performed on sh-RPS19 cells and sh-Random cells, and the test results were as follows Figure 2 As shown, the relative expression level of RPS19 in sh-RPS19 cells was significantly decreased, indicating that RPS19 was successfully knocked down.

[0049] 2.4. Verification of erythroid differentiation disorder in the RPS19-deficient HSPC cell model, as follows: a. The RPS19-deficient HSPC cell model constructed above was subjected to directed erythroid differentiation induction culture in a differentiation medium containing 10 ng / ml SCF, 10 ng / ml IL3, 3 U / ml EPO and 1% double antibody in SFEM II medium. The cell density was 5×10 4 The cells were placed in a cell culture incubator (37°C, 5% (V / V) CO2) and cultured for differentiation until the 7th day. They were incubated with APC conjugated anti-CD235a and PE conjugated anti-CD71 antibodies, and then the fluorescence was detected by flow cytometry. The degree of erythroid differentiation of the cells was determined by detecting the expression of CD235a and CD71 antigens on the cell surface.

[0050] The results are as follows Figure 3 As shown in the figure, CD71 cell surface antigen begins to express in the early stage of erythroid differentiation, while CD235a begins to express in the late stage of erythroid differentiation. Generally, the double positive expression of the two antigens represents the maturity of erythroid differentiation. Compared with the control group (sh-Random), after RPS19 knockdown, the erythroid phenotype (CD71+CD235a+) was significantly inhibited, indicating that RPS19 knockdown caused erythroid differentiation disorder.

[0051] b. TP53 is a molecule that is commonly abnormally activated in DBA. The expression of TP53 in the RPS19-deficient HSPC cell model was further detected by qPCR. The results are shown in Figure 4 As shown, after RPS19 knockdown, TP53 was abnormally activated, which is consistent with the DBA phenotype.

[0052] c. Check the cell morphology of erythroid differentiation by Giemsa staining, such as Figure 5 As shown, it can be seen that after RPS19 knockdown, most of them lost their differentiation ability and remained in the HPCs stage (large nucleus).

[0053] 2.5. Verification of the ability of the RPS19-deficient HSPC cell model to differentiate into myeloid lineage-monocytes was performed as follows: a. The constructed RPS19-deficient HSPC cell model was inoculated in a monocyte differentiation medium containing 50 ng / ml M-CSF and 1% double antibody in SFEM II medium. The cell density was 5×10 4 / mL, placed in a cell culture incubator (37°C, 5% (V / V) CO2), the liquid was changed every 4 days, and the cells cultured to the 5th day (early) and 36th day (late) were collected and incubated with APC-CD11b antibody, and then the fluorescence was detected by flow cytometry, where SSC-A: A is area, SSC-A represents the area of ​​the detected pulse signal wave curve, and its signal intensity is proportional to the complexity of the cell. By measuring the expression of CD11b antigen on the cell surface, the degree of differentiation of monocytes in the RPS19-deficient HSPC cell model can be determined.

[0054] The results are as follows Figure 6-7 As shown, compared with the control group, the early myeloid differentiation ability (CD11b+) was significantly enhanced after RPS19 knockdown, but as the culture time increased, the control group and the knockdown group tended to be consistent, indicating that RPS19 knockdown will stimulate myeloid differentiation in the short term, but has no significant effect on the myeloid lineage in the long term.

[0055] 2.6. Verification of the ability of the RPS19-deficient HSPC cell model to differentiate into myeloid lineage-granulocytes, as follows: a. The constructed RPS19-deficient HSPC cell model was inoculated in a granulocyte differentiation medium containing 50 ng / ml FLT3, 100 ng / ml G-CSF and 1% double antibody in SFEM II medium. The cell density was 5×10 4 The cells were placed in a cell culture incubator (37°C, 5% (V / V) CO2), the medium was changed every 4 days, and the cells cultured at the 7th day (early stage) and the 22nd day (late stage) were collected and incubated with APC-CD68 and PE-CD15 antibodies. The fluorescence was then detected by flow cytometry. The degree of granulocytic differentiation of the cells was determined by detecting the expression of CD68 and CD15 antigens on the cell surface.

[0056] The results are as follows Figure 8-9 As shown, compared with the control group, the early myeloid differentiation ability (CD68+CD15+) was significantly enhanced after RPS19 knockdown, but with the increase of culture time (CD15+), the control group and the knockdown group tended to be consistent, indicating that RPS19 knockdown will stimulate myeloid differentiation in the short term, but has no significant effect on the myeloid in the long term.

[0057] In summary, by further inducing differentiation of the constructed RPS19-deficient HSPC cell model, it was found that this cell model specifically inhibited the erythroid differentiation ability, resulting in abnormal activation of TP53, and had no long-term effect on the myeloid differentiation ability, which is consistent with the DBA phenotype.

[0058] Example 3 RPS19-deficient HSPC cell model shows lineage differentiation deviation of hematopoietic stem / progenitor cell components a. Collect the control (sh-Random) and RPS19 knockdown HSPC cell samples (sh-RPS19) in 1.5 mL centrifuge tubes, centrifuge at 300 g for 5 min, discard the supernatant completely, resuspend the precipitate with 1 ml of DPBS buffer containing FBS, and centrifuge at 300 g for 5 min.

[0059] b. After completely discarding the supernatant, add 60 μL of DPBS buffer to each tube to resuspend the precipitate.

[0060] c. Leave 10 μL of cell suspension unstained as a blank control. For the remaining 50 μL of cell suspension, when using multi-color flow cytometry dyes to detect HSPC cell components, add 2.5 μL of PE conjugated anti-CD34, APC conjugated anti-CD38, BV421 conjugated anti-CD90, BV510 conjugated anti-CD10, PE-Cy7 conjugated anti-CD135 and 1.5 μL of AF700 conjugated anti-CD45RA fluorescent dye to the same 1.5 mL EP tube and incubate at 4°C in the dark for 15 min.

[0061] d. Add 1 ml of DPBS buffer containing FBS to the cell suspension after staining and incubation, mix well by pipetting, centrifuge at 300 g for 5 min, and repeat the washing twice.

[0062] e. After completely discarding the supernatant of the staining group, resuspend the precipitate with 200 μL of DPBS buffer containing FBS. For the blank control group, add DPBS buffer containing FBS to make up to 200 μL. Detect the expression of different antigens on the cell surface by flow cytometry fluorescence detection to determine the various cell components in HSPC.

[0063] The results are as follows Figure 10-11 As shown, it can be seen that compared with the control group, the cell components in the HSPCs of the RPS19 knockdown group changed, including a significant decrease in the proportion of MPP and MEP, and a significant increase in the proportion of GMP, indicating that the knockdown of RPS19 has an effect on HSPCs.

[0064] Example 4 Helichrysanthemin alleviates the differentiation deviation of hematopoietic stem / progenitor cells caused by RPS19 deficiency The constructed RPS19-deficient HSPC cell model was inoculated in a proliferation medium containing 50 ng / ml SCF, 50 ng / ml Flt3 Ligand, 50 ng / ml TPO and 1% double antibody SFEM II medium, with a cell density of 5×10 4 / mL, placed in a cell culture incubator (37 ℃, 5% (V / V) CO2) for culture. On day 0, 5μM, 10μM, 20μM, and 40μM helichrysanthemin solutions (solvent: DMSO) were added to the proliferation medium, respectively, and placed in a cell culture incubator (37℃, 5% (V / V) CO2), and proliferation culture was continued for 72h, and flow cytometry was used for detection. The results are shown in Fig.12 shown.

[0065] according to Fig.12 It can be seen that compared with the control group (sh-Random), the production of MPP and MEP in the sh-RPS19 group cells was significantly reduced, while treatment with helichrysin can promote the production of multipotent progenitor cells (MPP) and megakaryocyte-erythroid progenitor cells (MEP) and significantly inhibit the expansion of myeloid progenitor cells GMP, indicating that helichrysin can alleviate the abnormal expansion of stem / progenitor cells caused by the reduction of RPS19 to a certain extent, and alleviate the loss of early erythroid-related cells.

[0066] Example 5 Helichrysin alleviates erythroid differentiation block caused by RPS19 deficiency The constructed RPS19-deficient HSPC cells were inoculated in a differentiation medium for directed erythroid differentiation culture. The differentiation medium was SFEM II medium containing 10 ng / ml SCF, 10 ng / ml IL3, 3 U / ml EPO and 1% double antibody. The cell density was 5×10 4 / mL. From day 0 of differentiation, helichrysanthemin solution (DMSO as solvent) with a final concentration of 5μM, 10μM, 20μM, and 40μM was added, and the cells were placed in a cell culture incubator (37 ℃, 5% (V / V) CO2) and cultured until day 7. The cells were collected and tested by flow cytometry.

[0067] The results are as follows Fig.13 As shown, compared with the control group (sh-Random), the production of red blood cells (CD71+CD235a+) in the sh-PRS19 group cells was significantly inhibited, while the red blood cell phenotype (CD71+CD235a+) was restored after treatment with helichrysin, indicating that helichrysin can alleviate the red blood cell defect caused by the decrease of RPS19 to a certain extent.

[0068] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for alleviating and / or treating erythroid differentiation disorder in a subject; 。 2. The use according to claim 1, characterized in that: The subject suffers from congenital pure red cell aplasia.

3. The use according to claim 1, characterized in that: The cell erythroid differentiation disorder is caused by a mutation in the cell ribosomal protein gene.

4. The use according to claim 3, characterized in that: The cell ribosomal protein gene mutation includes at least one of RPS19 mutation, RPL5 mutation, RPS26 mutation and RPL11 mutation.

5. The use according to claim 3, characterized in that: The cell ribosomal protein gene mutation includes an RPS19 mutation, and the RPS19 mutation is a mutation that causes a decrease in the expression level of the RPS19 gene.

6. The use according to any one of claims 1 to 5, characterized in that: The alleviation and / or treatment of the subject's erythroid differentiation disorder is manifested in increasing the number and / or proportion of the subject's erythroid cells.

7. The use according to any one of claims 1 to 5, characterized in that: The cell erythroid differentiation disorder at least includes erythroid differentiation disorder of hematopoietic stem and progenitor cells.

8. The use according to any one of claims 1 to 5, characterized in that: The alleviation and / or treatment of the subject's erythroid differentiation disorder is manifested by at least one of the following: 1) Promote the differentiation of hematopoietic stem cells with erythroid differentiation disorders into multipotent progenitor cells; 2) Promote the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryotic-erythroid progenitor cells; 3) Increase the number and / or proportion of CD71-positive and CD235a-positive erythroid cells.

9. Use of the compound represented by formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for correcting and / or alleviating erythroid differentiation disorders in vitro; 。 10. A method for correcting and / or alleviating erythroid differentiation disorder in vitro, characterized in that: The method comprises contacting the ex vivo cells with the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof, so as to correct and / or alleviate the erythroid differentiation disorder of the ex vivo cells; 。

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