A method for promoting erythrocyte enucleation or improving in vitro erythropoiesis efficiency, and the application of HSF1 activators and / or HRI activators.

Abstract

This invention provides a method for promoting erythrocyte enucleation or improving in vitro erythropoiesis efficiency, and the application of HSF1 activator and / or HRI activator. This invention discovers that by applying HSF1 activator and / or HRI activator to cells undergoing erythroid differentiation, the enucleation efficiency of erythroid cells during the terminal differentiation stage can be significantly improved, increasing the enucleation rate.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a method for promoting erythrocyte denucleation or improving the efficiency of erythrocyte production in vitro, and the application of HSF1 activators and / or HRI activators. Background Technology

[0002] Blood transfusion therapy is currently the primary clinical treatment for anemia, blood loss, and other diseases, relying mainly on volunteer donations for blood supply. With the global blood transfusion volume increasing annually and the blood donor population growing slowly, blood shortages have become a widespread public health problem. Difficulties in collecting rare blood types, seasonal fluctuations in blood supply, and supply-demand mismatches caused by unforeseen events make blood shortages a persistent and complex challenge. Furthermore, volunteer blood donations have inherent drawbacks such as immune risks, pathogen transmission, and short storage periods. Therefore, developing in vitro red blood cell preparation technologies that can be produced independently of blood donation, are stable and controllable, and suitable for large-scale production is of great significance for clinical blood transfusions, cell therapy, and regenerative medicine.

[0003] Among numerous potential sources of initiating cells, CD34 + Hematopoietic stem and progenitor cells are the most commonly used cell type in in vitro erythroid differentiation studies. CD34 + Cells can proliferate in a suitable microenvironment and gradually enter the erythroid development stage, eventually generating oxygen-carrying erythrocytes. However, one of the key bottlenecks in current in vitro erythrocyte preparation is the low efficiency of enucleation. Mature erythrocytes must undergo enucleation to form an enucleated structure to acquire the physiologically required flexibility, sequential metabolic properties, and circulatory dynamics. In vitro culture systems often fail to effectively reproduce the in vivo extramedullary microenvironment, making CD34... + The enucleation efficiency of cells at the end of erythroid development is significantly lower than that in vivo, thus limiting the yield and maturation quality of artificially generated erythrocytes. Summary of the Invention

[0004] The purpose of this invention is to provide a method for promoting erythrocyte denucleation or improving the efficiency of in vitro erythrocyte production, and the application of HSF1 activator and / or HRI activator, to solve the problem of low denucleation efficiency in existing methods for in vitro erythrocyte production.

[0005] This invention provides the following technical solution: This invention provides the application of HSF1 activator and / or HRI activator in promoting erythrocyte denucleation or improving in vitro erythrocyte production efficiency.

[0006] Preferably, the HSF1 activator is HSF1A; the HRI activator is BtdCPU.

[0007] This invention provides the application of HSF1 activator and / or HRI activator in the preparation of blood products / cell materials.

[0008] Preferably, the HSF1 activator is HSF1A; the HRI activator is BtdCPU.

[0009] Preferably, the blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products.

[0010] Preferably, the cell material includes one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated erythrocytes, and immortalized erythroid cell lines.

[0011] This invention provides the use of an HSF1 activator and / or an HRI activator in the preparation of a medicament for the treatment or prevention of diseases related to abnormal erythrocyte enucleation.

[0012] Preferably, the HSF1 activator is HSF1A; the HRI activator is BtdCPU.

[0013] Preferably, the erythrocyte enucleation abnormality-related diseases include erythrocyte enucleation abnormality diseases caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress.

[0014] The present invention also provides a method for promoting erythrocyte denucleation or improving the efficiency of erythrocyte production in vitro, characterized by comprising the following steps: applying HSF1 activator and / or HRI activator.

[0015] Preferably, the method for promoting erythrocyte enucleation or improving in vitro erythrocyte production efficiency includes the following steps: adding HSF1 activator and / or HRI activator to the erythroid differentiation culture system.

[0016] Preferably, the method for promoting erythrocyte enucleation or improving in vitro erythropoiesis efficiency includes the following steps: taking CD34 + Cells or HUDEP2 cells are seeded in a culture medium and cultured with HSF1 activator and / or HRI activator added to the culture medium until red blood cells are generated.

[0017] Preferably, the HSF1 activator is HSF1A; the HRI activator is BtdCPU.

[0018] Preferably, the cultivation specifically includes: Phase 1: Obtaining CD34 + Cells were seeded in the first culture medium to achieve a cell density of 10-1. 5Cells / mL, cultured for 7 days; that is, the first stage is the 0th to 7th day of culture.

[0019] Phase 2: Remove cytokine IL-3 and culture for 4 days; that is, Phase 2 is from day 8 to day 11 of culture, and on day 9 of culture, add HSF1A and / or BtdCPU.

[0020] Phase 3: Remove cytokines IL-3 and SCF, and culture for 4 days; that is, phase 3 is from day 12 to day 15 of culture.

[0021] Preferably, the culture conditions are a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85%.

[0022] Preferably, in the second stage, HSF1A is added so that the concentration of HSF1A in the first culture medium is 10 mmol / L; Optionally, in the second stage, BtdCPU is added to make the concentration of BtdCPU in the first culture medium 1 mmol / L.

[0023] Preferably, the first culture medium comprises: IMDM medium, 8% (v / v) FBS, 2% (v / v) PB plasma, 10 µg / mL insulin, 3 IU / mL heparin, 10 μg / mL SCF, 1 μg / mL IL-3, 3 IU / mL EPO, 200 μg / mL TF, and 1% (v / v) PS.

[0024] Preferably, the cultivation specifically includes: Phase 1: HUDEP2 cells were harvested and seeded in the second culture medium to achieve a cell density of 102. 5 Cells / mL, cultured for 4 days; that is, the first stage is day 0-4 of culture.

[0025] Second stage: Continue culturing for 4 days; that is, the second stage is from day 5 to day 8 of culturing, and on day 6 of culturing, add HSF1A and / or BtdCPU.

[0026] Preferably, the culture conditions are a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85%.

[0027] Preferably, in the second stage, HSF1A is added so that the concentration of HSF1A in the second culture medium is 10 mmol / L; Optionally, in the second stage, BtdCPU is added to make the concentration of BtdCPU in the second culture medium 1 mmol / L.

[0028] Preferably, the second culture medium comprises: IMDM medium, 3% (v / v) FBS, 2% (v / v) PB plasma, 10 µg / mL insulin, 3 IU / mL heparin, 50 ng / mL SCF, 1 μg / mL DOX, 3 IU / mL EPO, 300 μg / mL TF, and 1% (v / v) PS.

[0029] In this invention, the IMDM medium refers to Iscove's Modified Dulbecco's Medium, and conventionally known IMDM media in the prior art can be used. FBS refers to Fetal Bovine Serum, and heat-inactivated fetal bovine serum can be used. PB plasma refers to Peripheral Blood Plasma; SCF refers to Stem Cell Factor. IL-3 refers to Interleukin-3. DOX refers to Doxycycline. EPO refers to Erythropoietin. TF refers to Transferrin. PS refers to a penicillin-streptomycin mixture.

[0030] In this invention, HSF1A is a cell-penetrating activator of heat shock transcription factor 1 (HSF1), with CAS number 1196723-93-9. BtdCPU is a heme-regulated eIF2α kinase (HRI) activator, with CAS number 1257423-87-2.

[0031] This invention utilizes CD34 cells undergoing erythroid differentiation. + Application of HSF1 activators and / or HRI activators to cells significantly increased CD34 activity. + Increase the efficiency of cell enucleation at the end of erythroid development and improve the enucleation rate.

[0032] Although previous studies have shown that HSF1 activators and / or HRI activators play a role in erythroid stress protection, no related studies have yet explored the role of HSF1 activators and / or HRI activators in erythroid differentiation, or their effect on CD34. + The effect of cell enucleation efficiency in vitro.

[0033] This invention reveals a close correlation between cellular proteostasis and erythroid cell enucleation efficiency. During erythroid differentiation, the synthesis of large amounts of hemoglobin, cytoskeleton reconstruction, and nuclear compression all increase protein folding pressure, making cells more dependent on the effective operation of the proteostasis regulation system. Imbalance in proteostasis can lead to the accumulation of misfolded proteins, overactivation of stress responses, or impaired cellular structural alterations, thus hindering erythroid maturation and enucleation. Key pathways regulating intracellular proteostasis include the heat shock response (HSF1-mediated) and the eIF2α-dependent stress-induced translation pathway (HRI-mediated). HSF1 (Heat Shock Factor 1) is a major transcription factor regulating molecular chaperones, maintaining folding stability by inducing HSP family proteins to participate in the recognition, repair, and clearance of misfolded proteins. HRI (Heme-regulated eIF2α kinase) is an important erythroid-specific kinase that regulates protein synthesis rates, enabling cells to maintain homeostasis under heme synthesis stress, oxidative stress, or folding stress.

[0034] This invention selectively activates the HSF1 pathway by applying an HSF1 activator, enhancing molecular chaperone expression and improving the cell's ability to maintain homeostasis under folding stress. By applying an HRI activator, it regulates the eIF2α stress translation response, enabling cells to maintain a more stable translational and metabolic environment under high protein synthesis load. HSF1 and / or HRI activators activate both the HSF1 and HRI pathways, allowing cells to achieve higher protein homeostasis adaptability at the end of erythroid development, thereby promoting enucleation through homeostasis regulation. This not only helps cells alleviate the folding stress caused by massive hemoglobin synthesis but also promotes cellular processes closely related to enucleation, such as nuclear membrane compression, chromatin remodeling, and cytoskeleton rearrangement.

[0035] The beneficial effects of this invention are as follows: (1) By enhancing protein homeostasis rather than exogenous mechanical drive to promote denucleation, the natural cell development process can be simulated to the greatest extent, which is more in line with the natural cell development law, reduces the damage caused by artificial intervention, and improves the safety of clinical application.

[0036] (2) HSF1 activators and / or HRI activators are used to act on key intrinsic pathways to enhance the cells' ability to cope with erythroid synthesis stress, thereby improving the quality of cytoplasmic maturation. This approach can increase in vitro erythrocyte yield, providing an important technological foundation for the future development of alternative transfusion products, rare blood type erythrocyte products, and personalized cell therapy. Furthermore, it exhibits good compatibility with existing erythroid culture systems, requiring no significant modifications to the culture system, and is highly feasible for industrial production. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0038] Figure 1 The bar chart shows the protein synthesis rate of Example 3 (BtdCPU group) and Comparative Example 1 (control group); Figure 2 Bar chart showing the chymotrypsin activity of Example 2 (HSF1A group) and Comparative Example 1 (control group); Figure 3 The bar chart shows the protein synthesis rate of Example 1 (combined drug administration group) and Comparative Example 1 (control group); Figure 4 The bar chart shows the chymotrypsin activity of Example 1 (combined drug administration group) and Comparative Example 1 (control group); Figure 5 These are flow cytometry images of enucleated cells from Examples 1-3 and Comparative Example 1; Figure 6 The bar chart shows the nucleation rate of Examples 1-3 and Comparative Example 1; Figure 7 Giemsa staining images of cells from Examples 1-3 and Comparative Example 1; Figure 8 The bar chart shows the protein synthesis rate of Example 6 (BtdCPU group) and Comparative Example 2 (control group); Figure 9 Bar chart showing the chymotrypsin activity of Example 5 (HSF1A group) and Comparative Example 2 (control group); Figure 10 The bar chart shows the protein synthesis rate of Example 4 (combined drug administration group) and Comparative Example 2 (control group); Figure 11 The bar chart shows the chymotrypsin activity of Example 4 (combined drug administration group) and Comparative Example 2 (control group); Figure 12 These are flow cytometry images of enucleated cells from Examples 4-6 and Comparative Example 2; Figure 13 Bar charts showing the nucleation rates of Examples 4-6 and Comparative Example 2; Figure 14 The images show Giemsa staining of cells from Examples 4-6 and Comparative Example 2. Detailed Implementation

[0039] The embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention. Unless otherwise specified in the embodiments of the present invention, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments used that do not specify the manufacturer are all commercially available conventional products; different manufacturers and models of raw materials do not affect the implementation of the technical solution or the achievement of the technical effects of the present invention. Example 1

[0040] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: applying HSF1A and BtdCPU. HSF1A is an activator of HSF1; BtdCPU is an activator of HRI.

[0041] In this embodiment, the specific steps include: taking CD34 + Cells were seeded in a first culture medium and cultured at 37°C, 5% carbon dioxide (v / v), and ≥85% humidity until red blood cells were produced. The culture specifically included: Phase 1: Obtaining CD34 + Cells were seeded in the first culture medium to achieve a cell density of 10-1. 5 Cells / mL, cultured for 7 days; that is, the first stage is the 0th to 7th day of culture.

[0042] Second stage: Remove cytokine IL-3 and culture for 4 days; that is, the second stage is from day 8 to day 11 of culture, and on day 9 of culture, add HSF1A and BtdCPU, so that the concentration of HSF1A in the first culture medium is 10 mmol / L and the concentration of BtdCPU in the first culture medium is 1 mmol / L.

[0043] Phase 3: Remove cytokines IL-3 and SCF, and culture for 4 days, i.e., phase 3 is from day 12 to day 15 of culture.

[0044] It should be noted that, in this embodiment, the CD34 + The cells are derived from human umbilical cord blood and are isolated using existing techniques, such as a magnetic bead sorting system. As an alternative implementation of this embodiment, the CD34... + The cells can also be one of the following: erythroid in vitro differentiation systems derived from fetal livers of animals such as mice, pigs, rabbits, and monkeys; erythroid in vitro differentiation systems derived from pluripotent stem cells; or erythroid in vitro differentiation systems derived from peripheral blood (the same applies below, and will not be repeated hereafter).

[0045] The first culture medium comprises: IMDM medium, 8% (v / v) FBS, 2% (v / v) PB plasma, 10 µg / mL insulin, 3 IU / mL heparin, 10 μg / mL SCF, 1 μg / mL IL-3, 3 IU / mL EPO, 200 μg / mL TF, and 1% (v / v) PS.

[0046] Wherein, IMDM medium refers to Iscove's Modified Durbecco's Medium, and conventionally known IMDM media in the prior art can be used. FBS refers to Fetal Bovine Serum, and heat-inactivated fetal bovine serum can be used. PB plasma refers to Peripheral Blood Plasma; SCF refers to Stem Cell Factor. IL-3 refers to Interleukin-3. EPO refers to Erythropoietin. TF refers to Transferrin. PS refers to a penicillin-streptomycin mixture.

[0047] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0048] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Example 2

[0049] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: administering HSF1A. HSF1A is an activator of HSF1.

[0050] In this embodiment, the specific steps include: taking CD34 + Cells were seeded in a first culture medium and cultured at 37°C, 5% carbon dioxide (v / v), and ≥85% humidity until red blood cells were produced. The first culture medium was the same as in Example 1.

[0051] The cultivation specifically includes: Phase 1: Obtaining CD34 + Cells were seeded in the first culture medium to achieve a cell density of 10-1. 5 Cells / mL, cultured for 7 days; that is, the first stage is the 0th to 7th day of culture.

[0052] Second stage: Remove cytokine IL-3 and culture for 4 days; that is, the second stage is from day 8 to day 11 of culture, and on day 9 of culture, add HSF1A to make the concentration of HSF1A in the first culture medium 10 mmol / L.

[0053] Phase 3: Remove cytokines IL-3 and SCF, and culture for 4 days, i.e., phase 3 is from day 12 to day 15 of culture.

[0054] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0055] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Example 3

[0056] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: applying BtdCPU. BtdCPU is an activator of HRI.

[0057] In this embodiment, the specific steps include: taking CD34 + Cells were seeded in a first culture medium and cultured at 37°C, 5% carbon dioxide (v / v), and ≥85% humidity until red blood cells were produced. The first culture medium was the same as in Example 1.

[0058] The cultivation specifically includes: Phase 1: Obtaining CD34 + Cells were seeded in the first culture medium to achieve a cell density of 10-1. 5 Cells / mL, cultured for 7 days; that is, the first stage is the 0th to 7th day of culture.

[0059] Second stage: Remove cytokine IL-3 and culture for 4 days; that is, the second stage is from day 8 to day 11 of culture, and on day 9 of culture, add BtdCPU so that the concentration of BtdCPU in the first culture medium is 1 mmol / L.

[0060] Phase 3: Remove cytokines IL-3 and SCF, and culture for 4 days, i.e., phase 3 is from day 12 to day 15 of culture.

[0061] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0062] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Example 4

[0063] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: applying HSF1A and BtdCPU. HSF1A is an activator of HSF1; BtdCPU is an activator of HRI.

[0064] In this embodiment, the specific steps include: taking HUDEP2 cells, seeding them in a second culture medium, and culturing them at a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85% until red blood cells are generated. The culture specifically includes: Phase 1: HUDEP2 cells were harvested and seeded in the second culture medium to achieve a cell density of 102. 5 Cells / mL, cultured for 4 days; that is, the first stage is day 0-4 of culture.

[0065] Second stage: Continue culturing for 4 days; that is, the second stage is from day 5 to day 8 of culturing, and on day 6 of culturing, add HSF1A and BtdCPU, so that the concentration of HSF1A in the second culture medium is 10 mmol / L and the concentration of BtdCPU in the second culture medium is 1 mmol / L.

[0066] It should be noted that, in this embodiment, the HUDEP2 cells are derived from a human erythrocyte progenitor cell line derived from umbilical cord blood cells, specifically from human umbilical cord blood CD34. + Hematopoietic stem cells can achieve immortalization by inducing the expression of HPV16 E6 / E7 oncogenes, which can be obtained through existing technologies (the same applies below, and will not be repeated hereafter).

[0067] The second culture medium comprises: IMDM medium, 3% (v / v) FBS, 2% (v / v) PB plasma, 10 µg / mL insulin, 3 IU / mL heparin, 50 ng / mL SCF, 1 μg / mL DOX, 3 IU / mL EPO, 300 μg / mL TF, and 1% (v / v) PS.

[0068] Wherein, IMDM medium refers to Iscove's Modified Durbecco's Medium, and conventionally known IMDM media in the prior art can be used. FBS refers to Fetal Bovine Serum, and heat-inactivated fetal bovine serum can be used. PB plasma refers to Peripheral Blood Plasma; SCF refers to Stem Cell Factor; DOX refers to Doxycycline; EPO refers to Erythropoietin; TF refers to Transferrin; and PS refers to a penicillin-streptomycin mixture.

[0069] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0070] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Example 5

[0071] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: administering HSF1A. HSF1A is an activator of HSF1.

[0072] In this embodiment, the specific steps include: taking HUDEP2 cells, inoculating them into a second culture medium, and culturing them at a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85% until red blood cells are generated. The second culture medium is the same as in Example 4.

[0073] The cultivation specifically includes: Phase 1: HUDEP2 cells were harvested and seeded in the second culture medium to achieve a cell density of 102. 5 Cells / mL, cultured for 4 days; that is, the first stage is day 0-4 of culture.

[0074] Second stage: Continue culturing for 4 days; that is, the second stage is from day 5 to day 8 of culturing, and on day 6 of culturing, add HSF1A to make the concentration of HSF1A in the second culture medium 10 mmol / L.

[0075] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0076] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Example 6

[0077] The method for promoting erythrocyte enucleation / in vitro erythrocyte generation in this embodiment includes the following steps: applying BtdCPU. BtdCPU is an activator of HRI.

[0078] In this embodiment, the specific steps include: taking HUDEP2 cells, inoculating them into a second culture medium, and culturing them at a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85% until red blood cells are generated. The second culture medium is the same as in Example 4.

[0079] The cultivation specifically includes: Phase 1: HUDEP2 cells were harvested and seeded in the second culture medium to achieve a cell density of 102. 5Cells / mL, cultured for 4 days; that is, the first stage is day 0-4 of culture.

[0080] Second stage: Continue culturing for 4 days; that is, the second stage is from day 5 to day 8 of culturing, and on day 6 of culturing, add BtdCPU to make the concentration of BtdCPU in the second culture medium 1 mmol / L.

[0081] The red blood cells obtained by the above method can be used to prepare blood products or cell materials. The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products. The cell materials include one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated red blood cells, and immortalized erythroid cell lines. Those skilled in the art can use existing technology to prepare the aforementioned blood products or cell materials from the red blood cells obtained in this embodiment.

[0082] Given the role of HSF1 activators and / or HRI activators in promoting erythrocyte enucleation and improving in vitro erythropoiesis efficiency, these activators can also be used to prepare drugs for the treatment or prevention of diseases related to abnormal erythrocyte enucleation. These diseases include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress. Those skilled in the art can add HSF1 activators and / or HRI activators to conventional pharmaceutical excipients to formulate clinically acceptable dosage forms, such as tablets, capsules, and injections, for the treatment or prevention of the aforementioned diseases related to abnormal erythrocyte enucleation. Comparative Example 1

[0083] The comparative method for generating erythrocytes in vitro includes the following steps: without the administration of HSF1A and BtdCPU.

[0084] This comparative example includes the following steps: Take CD34 + Cells were seeded in a first culture medium and cultured at 37°C, 5% carbon dioxide (v / v), and ≥85% humidity until red blood cells were produced. The first culture medium was the same as in Example 1. The culture specifically included: Phase 1: Obtaining CD34 + Cells were seeded in the first culture medium to achieve a cell density of 10-1. 5 Cells / mL, cultured for 7 days; that is, the first stage is the 0th to 7th day of culture.

[0085] Phase 2: Remove cytokine IL-3 and culture for 4 days; that is, phase 2 is from day 8 to day 11 of culture.

[0086] Phase 3: Remove cytokines IL-3 and SCF, and culture for 4 days, i.e., phase 3 is from day 12 to day 15 of culture. Comparative Example 2

[0087] The comparative method for generating erythrocytes in vitro includes the following steps: without the administration of HSF1A and BtdCPU.

[0088] This comparative example specifically includes the following steps: HUDEP2 cells are taken, seeded in a second culture medium, and cultured at a temperature of 37°C, a carbon dioxide volume fraction of 5%, and a humidity of ≥85% until red blood cells are generated. The second culture medium is the same as in Example 4. The culture specifically includes: Phase 1: HUDEP2 cells were harvested and seeded in the second culture medium to achieve a cell density of 102. 5 Cells / mL, cultured for 4 days; that is, the first stage is day 0-4 of culture.

[0089] Phase Two: Continue cultivation for 4 more days; that is, Phase Two is from day 5 to day 8 of cultivation. Effect Experiment Example

[0090] The following experiments were conducted to verify the technical effects of the present invention: I. CD34 + Flow cytometry detection of HUDEP2 cell enucleation Red blood cells were cultured according to the methods of Examples 1-6 and Comparative Examples 1-2, and CD34 was tested on day 13 of culture. + On day 8 of cell culture, HUDEP2 cells were counted, and 1×10⁻⁶ cells were collected. 5 Cells were centrifuged at 300×g for 5 min at 4℃, and the supernatant was discarded. Cells were washed with 1 mL of PBS and centrifuged again at 300×g for 5 min at 4℃, and the supernatant was discarded. The resulting precipitate was resuspended in 100 µL of PBS. 1 µL of Hoechst 33342 antibody was added to the cell suspension, and the mixture was thoroughly mixed and incubated on ice in the dark for 30 min. After antibody incubation, PBS buffer was added to an EP tube, and the sample was centrifuged at 300×g for 10 min. Unbound antibody in the supernatant was discarded. Cells were resuspended in PBS and transferred to flow cytometry tubes. 1 / 100 volume of 7AAD buffer was added to the tube, and the cells were analyzed using a flow cytometer. The results were processed using FlowJo software.

[0091] 2. CD34 + / HUDEP2 Cell Protein Synthesis Rate Flow Cytometry Detection Red blood cells were cultured according to the methods in Examples 1-6 and Comparative Examples 1-2. When the cells reached the Ortho phase, they were seeded at a density of 0.1 M / ml in culture dishes, followed by the addition of 1 μL of 20 μm OPP (O-propargyl-puromycin), and cultured for another 30 minutes. Cells were then harvested, the culture medium discarded, and the cells were centrifuged at 300 × g for 5 minutes, discarding the supernatant. Cells were washed with 1 mL of PBS and centrifuged at 300 × g for 5 minutes, discarding the supernatant. The resulting precipitate was fixed with 3.7% paraformaldehyde for 15 minutes, then centrifuged at 300 × g for 5 minutes, discarding the supernatant. The cells were permeabilized with 0.5% Triton X-100 for 20 minutes, centrifuged at 300 × g for 5 minutes, and the supernatant discarded. The prepared Click-iT reaction mixture (containing a fluorescent azide probe, copper catalyst, and buffer) was then added to the cells, mixed thoroughly, and incubated in the dark for 30 minutes. Wash cells 2–3 times with PBS to remove unbound dye. Finally, resuspend cells in PBS and transfer them to flow cytometry tubes. Add 1 / 100 volume of 7AAD buffer to the tube and analyze the cells using a flow cytometer. Analytical results are processed using FlowJo software.

[0092] III. CD34 + / HUDEP2 Cell Proteasome Activity Detection Red blood cells were cultured according to the methods of Examples 1-6 and Comparative Examples 1-2. When the cells reached the Ortho stage, they were counted, and 1×10⁶ cells were collected. 5 Centrifuge cells at 300×g for 5 min at 4℃, discard the supernatant, wash cells with 1 mL PBS, and centrifuge again at 300×g for 5 min at 4℃, discard the supernatant. Resuspend the cell pellet in 100 μL PBS and add it to a clean 96-well plate. Add 100 µL of Proteasome-Glo™ reagent to each well. Cover with a sealing film and shake on a plate shaker at 700 rpm for 2 min, then incubate at room temperature for 8 min. Finally, measure the fluorescence intensity (RLU) of each well using a microplate reader in luminescent mode.

[0093] IV. CD34 + / HUDEP2 cells Giemsa staining Red blood cells were cultured according to the methods of Examples 1-6 and Comparative Examples 1-2. After the culture was completed, 1×10⁻⁶ red blood cells were cultured. 5Cells were suspended in 200 μL of PBS and centrifuged at 280 rpm for 10 minutes to prepare cell slides. The slides were fixed with methanol for 2 minutes and then rinsed with distilled water. The cells were stained with diluted Giemsa stain at room temperature for 10–15 minutes, followed by rinsing with distilled water. The cells were then imaged using a microscope. Experimental results

[0094] Based on the above experiment, the results are as follows: In CD34 + Flow cytometry detection of cellular protein synthesis rate, CD34 + In the assay of cellular proteasome activity, the results of Examples 1-3 and Comparative Example 1 were compared to evaluate the effects of HSF1A and BtdCPU on protein homeostasis during erythroid differentiation. In the flow cytometry assay of HUDEP2 cell protein synthesis rate and the assay of HUDEP2 cell proteasome activity, the results of Examples 4-6 and Comparative Example 2 were compared to evaluate the effects of HSF1A and BtdCPU on protein homeostasis during erythroid differentiation.

[0095] like Figure 1 The figure shows a bar chart of protein synthesis rates in Example 3 (BtdCPU group) and Comparative Example 1 (control group). It is evident that the HRI activator BtdCPU significantly reduces the overall cellular protein synthesis rate, showing a marked decreasing trend compared to Comparative Example 1, suggesting that HRI activation can reduce translational load and alleviate protein folding pressure. In contrast, as... Figure 2 The image shows a bar chart of chymotrypsin activity in Example 2 (HSF1A group) and Comparative Example 1 (control group). Figure 8 The figure shows a bar chart of protein synthesis rates in Example 6 (BtdCPU group) and Comparative Example 2 (control group). It is evident that the HRI activator BtdCPU significantly reduces the overall cellular protein synthesis rate, exhibiting a marked decreasing trend compared to Comparative Example 2. Figure 9 The figure shows a bar chart of chymotrypsin activity in Example 5 (HSF1A group) and Comparative Example 2 (control group). It is evident that the HSF1 activator HSF1A significantly enhances proteasome activity, indicating that it can enhance protein degradation and quality control capabilities, thereby improving the efficiency of cellular degradation of abnormal proteins. Based on the above results, it can be concluded that the HRI activator BtdCPU and the HSF1 activator HSF1A can improve cellular protein homeostasis by simultaneously reducing protein production stress and enhancing degradation capabilities, respectively.

[0096] like Figure 3 The figure shows a bar chart of protein synthesis rates for Example 1 (combined drug administration group) and Comparative Example 1 (control group). It can be seen that the protein synthesis level in Example 1 also shows a decreasing trend. Figure 4The image shows a bar chart of chymotrypsin activity in Example 1 (combined drug administration group) and Comparative Example 1. The proteasome activity also increased synchronously, consistent with the direction observed in Example 2 (HSF1A group). Figure 10 The figure shows a bar chart of protein synthesis rates for Example 4 (combined drug administration group) and Comparative Example 2 (control group). It can be seen that the protein synthesis level in Example 4 also shows a decreasing trend. Figure 11 The bar chart shown is a comparison of chymotrypsin activity in Example 4 (combined drug administration group) and Comparative Example 2. The proteasome activity also increased synchronously, consistent with the direction observed in Example 5 (HSF1A group). This result indicates that simultaneous activation of the HSF1 and HRI pathways can jointly create a more balanced protein mass control system without producing any additional additive effects on any single indicator.

[0097] Therefore, it can be seen that drug activation of the HSF1 and HRI pathways can significantly improve protein homeostasis during erythroid differentiation, and bidirectional regulation to improve erythroid cell protein homeostasis can lay a more optimized cellular microenvironment for subsequent cell maturation and enucleation processes.

[0098] To further verify whether improved protein homeostasis affects cell enucleation, the enucleation efficiency of Examples 1-6 and Comparative Examples 1-2 was detected by flow cytometry. Figure 5-6 As shown, the enucleation rate of red blood cells generated using the method in Comparative Example 1 (control group) was approximately 41%, while the enucleation rate in Example 2 (HSF1A group) increased to approximately 46%, and the enucleation rate in Example 3 (BtdCPU group) increased to approximately 45.8%. The enucleation rate in Example 1 (combined drug administration group) was approximately 48%. Figure 12-13 As shown, the enucleation rate of red blood cells generated using the method in Comparative Example 2 (control group) was approximately 2.13%, while the enucleation rate in Example 5 (HSF1A group) increased to approximately 6.10%, and the enucleation rate in Example 6 (BtdCPU group) increased to approximately 6.42%. The enucleation rate in Example 1 (combined drug administration group) was approximately 6.30%.

[0099] It is evident that improving protein homeostasis can effectively promote cell enucleation, with the combined activation effect of the two drugs being slightly stronger, thus improving enucleation efficiency.

[0100] Cell morphology was observed using Giemsa staining, and the results were as follows: Figure 7 As shown, compared with Comparative Example 1 (control group), Examples 1-3 all showed more enucleated erythrocytes, which were smaller in size and more similar in morphology to mature erythrocytes, consistent with the trend observed by flow cytometry. Figure 14 As shown, compared with Comparative Example 2 (control group), more anucleate red blood cells were observed in Examples 4-6. These cells were smaller in size and more similar in morphology to mature red blood cells, consistent with the trend of flow cytometry detection.

[0101] In summary, regulating protein homeostasis by activating the HSF1 and HRI pathways not only improved protein homeostasis during erythroid differentiation but also further promoted enucleation at the terminal maturation stage, indicating that improved protein homeostasis is closely related to erythroid cell maturation.

[0102] As is known from common technical knowledge, this invention can be implemented through other embodiments that do not depart from its spirit or essential characteristics. Therefore, the disclosed embodiments described above are merely illustrative in all respects and are not the only ones. All modifications within the scope of this invention or its equivalents are encompassed by this invention.

Claims

1. The application of an HSF1 activator and / or HRI activator in promoting erythrocyte denucleation or improving in vitro erythropoiesis efficiency.

2. The application according to claim 1, characterized in that, The HSF1 activator is HSF1A; the HRI activator is BtdCPU.

3. The application of an HSF1 activator and / or HRI activator in the preparation of blood products / cell materials.

4. The application according to claim 3, characterized in that, The HSF1 activator is HSF1A; the HRI activator is BtdCPU.

5. The application according to claim 3, characterized in that, The blood products include one or more of the following: in vitro prepared red blood cell products, red blood cell suspensions, enucleated red blood cell preparations, cell products rich in erythroid cells, and artificial red blood cell products; Optionally, the cellular material includes one or more of the following: hematopoietic stem / progenitor cells, erythroid progenitor cells, erythroid precursor cells, orthochromatic erythrocytes, enucleated erythrocytes, and immortalized erythroid cell lines.

6. The use of an HSF1 activator and / or HRI activator in the preparation of a medicament for the treatment or prevention of diseases related to abnormal erythrocyte enucleation.

7. The application according to claim 6, characterized in that, The HSF1 activator is HSF1A; the HRI activator is BtdCPU.

8. The application according to claim 6, characterized in that, The diseases related to abnormal erythrocyte enucleation include those caused by erythroid differentiation disorders, protein homeostasis imbalances, or abnormal cellular stress.

9. A method for promoting erythrocyte denucleation or improving the efficiency of in vitro erythrocyte production, characterized in that, The steps include: applying HSF1 activator and / or HRI activator.

10. The method for facilitating red blood cell denucleation or improving the efficiency of red blood cell production in vitro according to claim 9, comprising the steps of: taking CD34 + cells or HUDEP2 cells, inoculating the cells in a culture medium, adding an HSF1 activator and / or an HRI activator to the culture medium, and culturing until red blood cells are produced.

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