Application of itaconic acid-related substances in prognostic assessment products and hematopoietic stem cell protective drugs after hematopoietic stem cell transplantation

Abstract

This invention provides the application of itaconic acid-related substances in prognostic assessment products and hematopoietic stem cell protective drugs after hematopoietic stem cell transplantation. It belongs to the interdisciplinary field of medicine and medical testing. This invention is the first to demonstrate that itaconic acid and cis-aconitine decarboxylase can serve as biomarkers for prognostic assessment after hematopoietic stem cell transplantation. It also demonstrates that itaconic acid, itaconic acid derivatives, and cis-aconitine decarboxylase expression promoters can be used as drugs to improve the maintenance rate of hematopoietic stem cells and promote their differentiation after hematopoietic stem cell transplantation with myeloablative pretreatment. This provides a safe and novel drug strategy with a clearly defined source for improving the efficiency of hematopoietic stem cell transplantation and shortening the post-transplant hematopoietic function failure period in clinical practice.

Description

Technical Field

[0001] This invention relates to the interdisciplinary field of medicine and medical testing, specifically to the application of itaconic acid-related substances in post-hematopoietic stem cell transplantation prognostic assessment products and hematopoietic stem cell protective drugs. Background Technology

[0002] Bone marrow hematopoietic stem cells (HSCs), as the seed cells of all hematopoietic and immune cells, possess the potential for self-renewal and multi-lineage differentiation. Currently, hematopoietic stem cell transplantation (HSCT) is an effective, and sometimes the only, treatment for diseases such as leukemia, severe aplastic anemia, Hodgkin's lymphoma, and myeloma. Clinically, prior to HSCT, recipients typically undergo chemotherapy or radiotherapy to ablate their bone marrow to create a "niche" for donor HSC implantation. However, chemotherapy or radiotherapy can severely damage the recipient's bone marrow microenvironment, leading to the death of a large number of donor HSCs after HSCT. This reduces the already limited implantation efficiency of donor HSCs and seriously affects the speed and quality of hematopoietic and immune function reconstruction. Therefore, how to protect HSCs and utilize the limited number of donor HSCs to achieve efficient and rapid reconstruction of the recipient's hematopoietic and immune systems has become a critical bottleneck problem that urgently needs to be solved in the field of HSCT.

[0003] Studies have shown that the nano-iron oxide drug Ferumoxytol has a good protective effect on donor HSCs in bone marrow transplantation for acute myeloid leukemia (AML). It can reduce ROS levels in donor HSCs by increasing ROS-induced AML cell killing, thereby protecting HSCs and improving the success rate of HSCT. However, the safety of exogenous inorganic nanomaterials still needs to be carefully considered in clinical practice. Furthermore, Ferumoxytol's protection of HSCs requires starting from the in vitro expansion phase, resulting in a long dosing period and high cost. Also, in the early post-HSCT period, Ferumoxytol cannot accurately and specifically assess the survival status of donor HSCs.

[0004] In summary, there is an urgent clinical need for a safe HSCs protection drug that can effectively prevent donor HSCs death caused by myeloablation. At the same time, there is also a need for a biomarker or method that can monitor the status of donor HSCs in the early postoperative period of HSCT. Summary of the Invention

[0005] To address the technical problem of the lack of effective monitoring of the donor HSC status in the recipient in the early post-HSCT period in existing technologies, the first aspect of this invention provides the application of an itaconic acid detection reagent in the preparation of a prognostic assessment product after hematopoietic stem cell transplantation.

[0006] The second aspect of this invention provides the application of a cis-aconitine decarboxylase detection reagent in the preparation of a product for prognostic assessment after hematopoietic stem cell transplantation.

[0007] A third aspect of the present invention provides a prognostic assessment product after hematopoietic stem cell transplantation, the prognostic assessment product after hematopoietic stem cell transplantation includes a donor hematopoietic stem cell hematopoietic capacity assessment reagent, wherein the donor hematopoietic stem cell hematopoietic capacity assessment reagent is an itaconic acid detection reagent and / or aconitine decarboxylase detection reagent.

[0008] The fourth aspect of this invention provides the use of itaconic acid, itaconic acid derivatives, or cis-aconitine decarboxylase expression promoters in the preparation of hematopoietic stem cell protective drugs.

[0009] Preferably, the itaconic acid derivative is 4-octyl itaconic acid ester.

[0010] Preferably, the hematopoietic stem cell protective drug includes pharmaceutically acceptable excipients.

[0011] Preferably, the hematopoietic stem cell protective drug is used to maintain the viability of recipient donor hematopoietic stem cells after hematopoietic stem cell transplantation with myeloablative pretreatment.

[0012] Preferably, the hematopoietic stem cell protective drug is used to reconstruct the hematopoietic function of the recipient hematopoietic stem cells after hematopoietic stem cell transplantation with myeloablative pretreatment.

[0013] In the early stages of donor HSCs reinfusion into the recipient body, donor HSCs are highly susceptible to large-scale death due to changes in the bone marrow microenvironment. The hematopoietic stem cell protection drug provided by this invention can exert a significant anti-damage effect by supplementing exogenous itaconic acid derivatives or promoting endogenous itaconic acid synthesis, thereby protecting donor HSCs and improving the early survival rate and implantation efficiency of donor HSCs in the recipient's bone marrow niche.

[0014] On the other hand, the hematopoietic stem cell protective drug provided by the present invention also provides long-term protection for donor HSCs, significantly improves the implantation rate of donor HSCs in the recipient's bone marrow and accelerates the comprehensive reconstruction of whole blood cells in the recipient's peripheral blood, ultimately effectively shortening the time of hematopoietic failure after HSCT and improving the patient's survival rate.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to demonstrate that itaconic acid deficiency exacerbates donor HSC damage caused by myeloablative pretreatment, thereby aggravating bone marrow hematopoietic failure and reducing HSC transplantation efficiency. Based on this, this invention develops itaconic acid and its rate-limiting enzyme, cis-aconitate decarboxylase, as biomarkers for prognostic assessment after HSCT. By detecting itaconic acid and cis-aconitate decarboxylase, the success of HSC implantation can be determined in the early postoperative period, and the hematopoietic function reconstitution can be accurately predicted. 2. This invention also found that the lack of itaconic acid after surgery directly reduces the success rate of HSCT, while supplementing itaconic acid, itaconic acid derivatives, and cis-aconitine decarboxylase expression promoters can effectively increase the number of donor HSCs in the recipient, thereby enabling HSCs to maintain long-term hematopoietic reconstitution capacity. This provides a safe and well-known new drug strategy for improving the efficiency of hematopoietic stem cell transplantation and shortening the period of hematopoietic failure after transplantation in clinical practice. Attached Figure Description

[0016] Figure 1 This is a statistical comparison chart of the number of donor HSCs and the content of donor HSCs in the underwear of mice before and 1 day after HSCT in Example 1 of the present invention. Figure 2 The results of the correlation analysis between the number of donor HSCs and the content of donor HSCs in their underwear 1 day after HSCT in Example 1 of the present invention; Figure 3 The statistical results of the expression of cis-aconitine decarboxylase in donor HSCs before and 1 day after HSCT in Example 2 of the present invention; Figure 4 The correlation analysis results of the number of donor HSCs and the expression of cis-aconitine decarboxylase in donor HSCs 1 day after HSCT in Example 2 of the present invention; Figure 5 The results show the statistical results of the number of HSCs and blood cell counts of the two groups of donor mice before HSCT and at the third week after HSCT in Example 3 of the present invention. Figure 6 This is the statistical result of the number of donor HSCs in the two groups of recipient mice 1 day after HSCT surgery in Example 4 of the present invention; Figure 7 This is the statistical result of the number of donor HSCs in the two groups of recipient mice 1 day after HSCT in Example 5 of the present invention; Figure 8 The results of the number of bone marrow cells and blood cells in the two groups of mice at week 3 after HSCT in Example 6 of this invention; Figure 9 The results of the number of bone marrow cells and blood cells in the two groups of mice at week 3 after HSCT in Example 7 of this invention; Figure 10 The results of this invention are as follows: the number of donor HSCs, the expression level of cis-aconitine decarboxylase in donor HSCs, and the content of cis-aconitine in donor HSCs in the inner lining of mice in two groups of mice 1 day after HSCT surgery. Detailed Implementation

[0017] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not intended to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the present invention.

[0018] It should be noted that the endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0019] As described in the background section, chemotherapy or radiotherapy for myeloablation before hematopoietic stem cell transplantation will lead to the death of the recipient's bone marrow hematopoietic stem cells after the procedure, thus causing HSCT failure.

[0020] In view of this, a new biomarker has been developed in specific embodiments of the present invention for early determination of whether HSCs have been successfully implanted and for predicting the hematopoietic function reconstitution of HSCs after HSCT.

[0021] In a specific embodiment of the present invention, the aforementioned biomarkers include itaconic acid and its rate-limiting enzyme, cis-aconitine decarboxylase (IRG1).

[0022] More specifically, the present invention provides the application of the above-mentioned itaconic acid detection reagent in the preparation of a prognostic assessment product after hematopoietic stem cell transplantation.

[0023] More specifically, in the above embodiments, the itaconic acid detection reagent includes a high-performance liquid chromatography (HPLC) detection reagent.

[0024] More specifically, the aforementioned liquid chromatography detection conditions include: the selected chromatographic column is a C18 column, the selected mobile phase is an aqueous solution of phosphoric acid containing 5% acetonitrile (v / v=0.05%), and the preferred high-performance liquid chromatography detection conditions are: injection volume: 20 μL; flow rate: 1.00 mL / min; column temperature: 40℃; detection wavelength: 205 nm.

[0025] More specifically, the present invention provides the application of the above-mentioned aconitine decarboxylase detection reagent in the preparation of a prognostic assessment product after hematopoietic stem cell transplantation.

[0026] More specifically, in the above embodiments, the aconitine decarboxylase detection reagent includes flow cytometry reagent.

[0027] More specifically, in the above embodiments, the flow cytometry reagent includes a primary antibody reagent and a secondary antibody reagent, wherein the primary antibody reagent includes an IRG1-specific antibody and the secondary antibody reagent includes a secondary antibody against an IRG1-specific antibody.

[0028] The present invention also provides a post-hematopoietic stem cell transplantation prognostic assessment product, which includes a donor hematopoietic stem cell hematopoietic capacity assessment reagent, wherein the donor hematopoietic stem cell hematopoietic capacity assessment reagent is an itaconic acid detection reagent and / or aconitine decarboxylase detection reagent.

[0029] More specifically, in the above embodiments, the postoperative prognostic assessment product for hematopoietic stem cell transplantation also includes calibrators, which include a number of itaconic acid solutions and / or a number of cis-aconitine decarboxylase solutions.

[0030] In the process of hematopoietic stem cell transplantation, the mass mortality period of HSCs is usually in the very early stage after hematopoietic stem cell transplantation (usually the first day after HSCT). Based on this, the present invention can effectively determine the viability and survival of donor HSCs by detecting the itaconic acid metabolism level of donor HSCs in the recipient in the early stage after HSCT, thereby predicting the ability to reconstruct hematopoietic function in the very early stage after HSCT.

[0031] More specifically, in the above embodiments, the postoperative prognostic assessment product for hematopoietic stem cells also includes quality control products, which include a certain concentration of itaconic acid solution and / or a certain concentration of aconitine decarboxylase solution.

[0032] Another embodiment of the present invention provides the application of itaconic acid, itaconic acid derivatives or cis-aconitine decarboxylase expression promoter in the preparation of hematopoietic stem cell protective drugs.

[0033] In the above embodiments, the itaconic acid derivative is 4-octyl itaconic acid ester.

[0034] As mentioned above, the very early post-HSCT period is the peak time for large-scale donor HSC mortality. Immediately after the recipient completes HSCT, administering the hematopoietic stem cell protective drug provided in this invention can significantly improve the maintenance of the number of donor HSCs in the recipient in the very early post-HSCT period and reduce the donor HSC mortality rate caused by myeloablation during HSCT. Furthermore, continuous administration of the hematopoietic stem cell protective drug provided in this invention after HSCT can significantly increase the number of donor HSCs in the recipient and the efficiency of peripheral blood cell regeneration, thereby significantly improving the transplantation efficiency of HSCT.

[0035] The technical solutions of the present invention are further described below through specific embodiments. Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. In some cases, terms with conventional meanings are limited herein for clarification or ease of reference, and such limitations should not be construed as indicating a significant difference from conventional understanding in the art. The technical methods described or referenced herein are generally well understood by those skilled in the art and have been employed by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments shall be performed according to the manufacturer's instructions and parameters.

[0036] In this embodiment of the invention, the normal wild-type C57BL / 6 mice used were purchased from the Institute of Zoology, Chinese Academy of Sciences (Beijing, China), C57BL / 6Smoc-Acod1. em1Smoc (Irg1 - / - Mice were purchased from the Shanghai Southern Model Organisms Research Center. All mice were carefully housed in SPF-grade animal rooms, ensuring a stable indoor temperature between 18 and 25°C. The mice had ample freedom to eat and drink at any time. To ensure the safety of the mice's drinking water, all drinking water was autoclaved; simultaneously, to maintain a clean and sterile environment, bedding and feed underwent strict sterilization. 60 Sterilization procedure using Co γ-ray irradiation. All mice involved in the experiment were male, with matching breeding background, breeding conditions, and age (8-10 weeks old), weighing 20-25g.

[0037] In embodiments of the present invention, unless otherwise specified, irradiation is carried out... 60 A single whole-body irradiation with gamma rays from a Co radioactive source (dose rate approximately 90 cGy / min) was performed. HSCs transplantation mouse models were established by either whole-body irradiation of recipient mice with 10 Gy or treatment with 40 mg / kg busulfan.

[0038] In an embodiment of the present invention, the specific procedures for preparing mouse bone marrow single-cell suspension are as follows: Mice are anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg / kg), and then quickly euthanized by cervical dislocation. The bilateral femurs and tibias of the mice are carefully separated and placed in 1640 culture medium (containing 2% FBS). The separated femurs and tibias are then gently lifted with ophthalmic forceps, and an appropriate amount of culture medium is drawn up with a 1 mL syringe to flush the cells into a 6-well plate. After flushing, the cell suspension in the culture medium is gently agitated with a pipette to mix it. Then, the cell suspension is collected into a 5 mL EP tube using a 1 mL syringe. The cell suspension is filtered through a 70 μm cell filter and quickly collected into a new 5 mL EP tube. The 5 mL EP tube is then placed in a centrifuge and centrifuged at 250 × g at room temperature for 5 minutes. After centrifugation, discard the supernatant, add 1 mL of erythrocyte lysis buffer, lyse at room temperature for 2 min, then add 3 mL of 1640 medium (containing 2% FBS) to an EP tube; centrifuge again at 250×g at room temperature for 5 min, then discard the supernatant; then add 1 mL of PBS and gently mix the cells, centrifuge again under the same conditions for 5 min, discard the supernatant, finally resuspend the cells in an appropriate amount of PBS, and count the cells using a Countess™ II FL automated cell counter to provide an accurate concentration of cell samples for subsequent experiments.

[0039] In an embodiment of the present invention, the steps for obtaining mouse peripheral blood single-cell suspension are as follows: 270 μL of EDTA (1% concentration) solution is added to a 5 mL EP tube in advance, and the groups are marked in advance according to the experimental design. The mice to be tested are selected, and after tail disinfection, a 2.5 mm tip of tissue is cut off. 30 μL of blood is quickly drawn using a capillary and added to the EDTA solution. 3 mL of erythrocyte lysis buffer is added to the 5 mL EP tube, and the mixture is blown up and down to ensure uniform mixing. Then, the cells are lysed at room temperature for 30 min. After lysis, there is no need to terminate the erythrocyte lysis process. The 5 mL EP tube is centrifuged at 250 × g at room temperature for 5 min. The supernatant is removed, and 2 mL of PBS is drawn for centrifugation and rinsing again. Finally, an appropriate amount of PBS is drawn to resuspend the cells, and the cells are counted using a Countess™ II FL automated cell counter to obtain peripheral blood single-cell suspension of the corresponding concentration for subsequent experiments.

[0040] In an embodiment of the present invention, the steps for detecting surface marker molecules of mouse bone marrow HSCs and donor hematopoietic cells by flow cytometry are as follows: obtaining a mouse bone marrow single-cell suspension and adjusting its cell concentration to 3 × 10⁻⁶ cells / mL. 7Cells were stained per mL. An appropriate amount of single-cell suspension was taken, and antibody staining solution was prepared according to specific experimental requirements. The dilution ratios of the flow cytometry antibodies used are shown in Table 1. After gently mixing the cells, they were incubated at 4°C in the dark for 35 min. After staining, 1 mL of PBS was added to a 1.5 mL EP tube, and then centrifuged at 250 × g for 5 min at room temperature. After centrifugation, the supernatant was carefully discarded, and the cells were rinsed twice with PBS. 200 μL of PBS was taken to resuspend the cells, and the cell samples were then tested as soon as possible. The raw flow cytometry data were processed and analyzed using FlowJo 10.8.1 software. The surface marker molecules of mouse bone marrow HSCs and donor hematopoietic cells are shown in Table 2.

[0041] Table 1 Table 2 In an embodiment of the present invention, the steps for sorting mouse bone marrow HSCs by flow cytometry are as follows: Before flow cytometry sorting, Lineage-negative cells are pre-enriched using Direct Lineage Cell Depletion magnetic beads from Miltenyi (Germany). Single-cell suspensions of bone marrow are obtained according to the aforementioned method, centrifuged at 250×g at room temperature for 5 min. After centrifugation, the supernatant is carefully discarded, and the cells are sorted at a ratio of 2×10⁻⁶ cells / cell. 7Resuspend cells in 90 μL of MACS Buffer (containing 10 μL of Miltenyi Direct Lineage Cell Depletion magnetic beads), mix well, and incubate at 4°C in the dark for 20 min. Place the Miltenyi LS column (pre-washed with MACS Buffer) in the VarioMACSSeparator magnet. Add an appropriate amount of MACS Buffer to the bone marrow single-cell suspension, and slowly add the cell suspension to the Miltenyi LS column. Collect Lineage-negative cells into a 5 mL sterile flow cytometry tube. Add an appropriate amount of MACS Buffer again to the Miltenyi LS column. The LS column was rinsed, and the remaining Lineage-negative cells were collected. The rinse fluid was also collected into a 5 mL flow cytometry tube. The 5 mL flow cytometry tube containing the Lineage-negative cell suspension was capped and placed in a centrifuge at 250 × g for 5 min at room temperature. After centrifugation, the supernatant was discarded, and the surface marker molecules of bone marrow HSCs were stained by flow cytometry according to the previously described method. After staining, the cells were resuspended in sterile PBS and centrifuged at 250 × g for 5 min at room temperature. After centrifugation, the supernatant was discarded again, and an appropriate amount of sterile PBS (containing 2% FBS) was taken to resuspend the cells. The target cells were accurately sorted using a FASCArial II or FASCArial III flow cytometer and received using sterile PBS (containing 4-8% FBS, the FBS content is adjusted according to the number of cells to be received) to provide high-quality cell samples for subsequent experiments.

[0042] In an embodiment of the present invention, the steps for detecting the expression of IRG1 protein in mouse bone marrow HSCs by flow cytometry are as follows: Bone marrow single-cell suspension was obtained according to the aforementioned method, and HSCs surface marker molecules were stained and washed three times with PBS. The cell pellet was set aside. The cell pellet was gently resuspended in 100 μL of Foxp3 fixative to ensure thorough mixing. Subsequently, the cells were incubated at room temperature in the dark for 30 min to ensure complete cell fixation. 1 mL of 1×Permeabilization Wash Buffer was added to an EP tube, followed by centrifugation at 280×g at room temperature for 5 min. After centrifugation, the supernatant was carefully discarded. This washing process was repeated twice. IRG1 primary antibody was prepared using 1×Permeabilization Wash Buffer at a dilution of 1:100. The prepared primary antibody staining solution was added to the EP tube and incubated at room temperature in the dark for 45 min. A sample was collected to set up an FMO control for subsequent analysis. 1 mL of 1×Permeabilization Wash Buffer was added to the EP tube. The cells were washed twice with 280×g at room temperature for 5 min. After centrifugation, the supernatant was carefully discarded. The washing process was repeated twice. 1×Permeabilization Wash Buffer was used to prepare Goat anti-Rabbit IgG (H+L) Alexa Fluor 594 secondary antibody staining solution (dilution ratio 1:2000). 100 μL of the prepared secondary antibody staining solution was added to an EP tube and incubated at room temperature in the dark for 30 min. Note that the FMO control should also be stained with secondary antibody. After staining, 1 mL of PBS was added to the EP tube and the cells were centrifuged at 280×g at room temperature. After centrifugation, the supernatant was discarded, and this washing step was repeated twice. 200 μL of PBS was used to resuspend the cells, and the cell samples were then tested as soon as possible.

[0043] In an embodiment of the present invention, the steps for detecting the protein concentration of mouse bone marrow HSCs are as follows: Bone marrow HSCs are sorted and obtained according to the aforementioned method, centrifuged to retain cell clusters, and an appropriate amount of PBS is added. Cells are disrupted using an ultrasonic device under ice bath conditions, centrifuged at 14000×g at 4℃ for 15 min, and the supernatant is collected and placed on ice. Based on the sample quantity, an appropriate amount of BCA working solution is prepared by adding 50 volumes of reagent A to 1 volume of reagent B, and thoroughly mixed. The pre-prepared protein standard (0.5 mg / ml) is added to the standard wells of a 96-well plate at concentrations of 0, 1, 2, 4, 8, 12, 16, and 20 μL, respectively. Standard diluent is added to bring the total volume to 20 μL. An appropriate volume of sample is added to the sample wells of the 96-well plate. If the sample volume is less than 20 μL, standard diluent is added to bring the total volume to 20 μL. 200 μL of sample is added to each well. BCA working solution, incubated at 37℃ for 20-30 min, absorbance value at 562 nm wavelength measured using an ELISA reader, protein concentration calculated based on standard curve and sample volume used.

[0044] In an embodiment of the present invention, the steps for detecting itaconic acid content in mouse bone marrow HSCs by HPLC are as follows: Bone marrow HSCs are sorted and obtained according to the aforementioned method, centrifuged to retain cell clusters for later use, an appropriate amount of PBS is added, and the cells are disrupted using an ultrasonic instrument under ice bath conditions. The cells are centrifuged at 14000×g at 4℃ for 15 min, and the supernatant is collected and stored on ice for later use. The itaconic acid standard is diluted to a series of concentrations and stored on ice. The mobile phase is prepared as follows: 5% acetonitrile and 95% phosphate aqueous solution (v / v=0.05%). The analytical method is set as follows: injection volume: 20 μL; flow rate: 1.00 mL / min; column temperature: 40℃; detection wavelength: 205 nm. The standard and the sample to be tested are detected by the HPLC. The itaconic acid concentration is calculated according to the standard curve and the sample peak area. The itaconic acid concentration is quantified according to the protein concentration.

[0045] In an embodiment of the present invention, the steps for routine blood testing in mice are as follows: Each group of experimental mice is tested every two days after continuous drug administration or before and after irradiation. 140 μL of blood cell analysis solution is added to a 1.5 mL EP tube beforehand. Each test is performed at a fixed time point. The mice to be tested are removed, the tail tissue is carefully cut off, and 10 μL of peripheral blood is quickly drawn using a capillary needle and added to the EP tube. The tube is gently blown to ensure thorough mixing of the blood cells and analysis solution. Before testing, the EP tube is gently inverted again to further mix the sample. Then, the XT-2000i fully automated blood analyzer is used to detect key indicators such as red blood cells, white blood cells, and platelets in the sample.

[0046] In the embodiments of this invention, all data in the experimental analysis were processed, statistically analyzed, and plotted using Graphpad Prism 9.5 software. Appropriate statistical methods were employed based on the Shapiro-Wilk normality test and the corresponding homogeneity of variance test results. Normally distributed data were expressed as mean ± standard deviation (SD), while non-normally distributed data were expressed as median ± interquartile range. For normally distributed data, paired or unpaired Student's t-tests were used for comparisons between two groups, and Welch-corrected or uncorrected t-tests were applied based on homogeneity of variance. For non-normally distributed data, non-parametric tests such as the Kolmogorov-Smirnov Z-test or the Mann-Whitney U-test were used for comparison. A significance level of 0.05 was used as the criterion; a p-value < 0.05 was considered statistically significant.

[0047] In the following examples, if 4-octylitaconate administration is involved, 4-octylitaconate is in powder form, which is dissolved in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% physiological saline and then filtered through a 0.22 μm filter membrane before use.

[0048] Example 1

[0049] Itaconic acid as a biomarker validation In this embodiment, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with 10 Gy ionizing radiation. After myeloablation, healthy WT C57BL / 6 mice were used as donors, and 1×10⁻⁶ mice were used as donors. 6 Donor bone marrow cells were injected into recipient mice via tail vein. The number of donor HSCs in the graft before HSCT, the number of donor HSCs implanted in the recipient mice 1 day after HSCT, the itaconic acid content in donor HSCs before HSCT, and the itaconic acid content in donor HSCs in the recipient mice 1 day after HSCT were recorded. The relevant results are as follows: Figure 1 As shown, Figure 1 A in the figure represents the statistical chart of the number of donor HSCs in the graft before HSCT and the number of donor HSCs implanted in the recipient mouse 1 day after HSCT. Figure 1 Figure B in the figure represents a statistical graph of itaconic acid content in donor HSCs implanted in HSCT grafts before HSCT and in donor HSCs implanted in recipient mice 1 day after HSCT. Data are expressed as Mean ± SD, n = 10. Figure 1 It is evident that 1 day after HSCT, due to changes in the bone marrow microenvironment, donor HSCs synthesize a large amount of itaconic acid, as shown in the correlation analysis. Figure 2 As shown, by Figure 1 and Figure 2 It is evident that a significant early increase in itaconic acid levels is positively correlated with good hematopoietic engraftment, demonstrating that itaconic acid can serve as a biomarker for assessing the prognosis of HSCT in the early postoperative period.

[0050] Example 2

[0051] Validation of aconitine decarboxylase as a biomarker In this embodiment, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with 10 Gy ionizing radiation. After myeloablation, healthy WT C57BL / 6 mice were used as donors, and 1×10⁻⁶ mice were used as donors. 6 Donor bone marrow cells were injected into recipient mice via tail vein. The relative expression levels of cis-aconitate decarboxylase (IRG1) in donor HSCs implanted before HSCT and in recipient HSCs implanted 1 day after HSCT were calculated. The relevant results are as follows: Figure 3 As shown, by Figure 3 It is evident that 1 day after HSCT, due to changes in the bone marrow microenvironment, donor HSCs express a large amount of aconitine decarboxylase. Correlation analysis is shown below. Figure 4 As shown, by Figure 3 and Figure 4 It is evident that the early and significant increase in the expression level of aconitine decarboxylase is positively correlated with good hematopoietic engraftment, which proves that aconitine decarboxylase can be used as a biomarker to assess the prognosis of HSCT in the early postoperative period.

[0052] Example 3

[0053] Verification of the impact of itaconic acid formation defects on the maintenance of donor HSCs In this embodiment, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with 10 Gy ionizing radiation. After myeloablation, itaconic acid production deficiency (Irg1) was identified. - / - Mice and healthy WT C57BL / 6 mice were used as donors, and 1×10⁻⁶ mice were used. 6 Donor bone marrow cells were injected into two groups of recipient mice via tail vein. The number of long-term hematopoietic reconstitution hematopoietic stem cells (LT-HSCs) and blood cell counts were recorded in both groups of donor mice before transplantation, and again 3 weeks after transplantation. The results are as follows: Figure 5 As shown, Figure 5 In this figure, A represents the statistical results of the number of LT-HSCs in the bone marrow of the two groups of donor mice under steady-state conditions; Figure 5 B in the table represents the statistical results of peripheral blood routine tests (white blood cells, red blood cells, and platelets) in the two groups of donor mice under steady-state conditions. Figure 5In the figure, C represents the statistical results of the number of donor bone marrow LT-HSCs and bone marrow cells in the two groups of recipient mice 3 weeks after transplantation. Figure 5 In the figure, D represents the statistical results of peripheral blood routine tests (white blood cells, red blood cells, and platelets) in the two groups of recipient mice 3 weeks after transplantation. Figure 5 It can be seen that Irg1 - / - The maintenance capacity and long-term hematopoietic reconstitution capacity of donor HSCs at steady state were not significantly different from those of WT donor HSCs, but their maintenance capacity and hematopoietic reconstitution capacity in recipient mice were significantly lower than those of WT donor HSCs. This can essentially rule out Irg1 from HSCs. - / - The effects of cells on HSC transplantation efficiency: The above results indicate that itaconic acid deficiency directly reduces HSC transplantation efficiency.

[0054] Example 4

[0055] 4-Octylitaconate administration promotes the maintenance of donor HSCs in the very early period after chemotherapy-induced myeloablation combined with HSCT. In this embodiment, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with busulfan. After myeloablation, healthy WT C57BL / 6 mice were used as donors, and 1×10⁻⁶ mice were used as donors. 6 Donor bone marrow cells were injected into recipient mice via tail vein. In the experimental group, 4-octyl itaconic acid derivative (OI) was injected intraperitoneally concurrently with hemodialysis (HSCT), while in the control group, an equal volume of 4-octyl itaconic acid solvent was injected intraperitoneally concurrently with HSCT. The number of donor HSCs in the mice of both groups was counted one day after HSCT. The results are as follows: Figure 6 As shown, by Figure 6 It is evident that OI can effectively resist toxic damage in the harsh bone marrow microenvironment caused by chemotherapy-induced myeloablation and significantly improve the maintenance rate of donor HSCs in the recipient.

[0056] Example 5

[0057] 4-Octylitaconate administration promotes the maintenance of donor HSCs in the very early postoperative period following radiotherapy-assisted myeloablation combined with HSCT. In this embodiment, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with 10 Gy ionizing radiation. After myeloablation, healthy WT C57BL / 6 mice were used as donors, and 1×10⁻⁶ mice were used as donors. 6 Donor bone marrow cells were injected into recipient mice via tail vein. In the experimental group, 4-octyl itaconic acid derivative (OI) was injected intraperitoneally concurrently with hemodialysis (HSCT), while in the control group, an equal volume of 4-octyl itaconic acid solvent was injected intraperitoneally concurrently with HSCT. The number of donor HSCs in the mice of both groups was counted one day after HSCT. The results are as follows: Figure 7 As shown, by Figure 7It is evident that OI can effectively resist toxic damage in the harsh bone marrow microenvironment caused by radiotherapy myeloablation and significantly improve the maintenance rate of donor HSCs in the recipient.

[0058] Example 6

[0059] 4-Octyl itaconic acid ester administration promotes long-term remodeling of receptor hematopoietic capacity after chemotherapy combined with HSCT. Based on Example 4, the number of bone marrow cells and blood cells in both groups of mice was counted at week 3, and the results are as follows: Figure 8 As shown, Figure 8 Figure A shows a statistical comparison of the number of donor LT-HSCs and donor bone marrow cells in the two groups of mice at week 3. Figure 8 Figure B shows a comparison of blood cell counts between the two groups of mice at week 3. Figure 8 It is evident that, in response to the deterioration of the bone marrow microenvironment caused by chemotherapy, OI can effectively enhance the activity of donor LT-HSCs, improve their long-term hematopoietic differentiation capacity in the recipient, promote hematopoietic function reconstruction, and effectively shorten the period of hematopoietic function failure after HSCT.

[0060] Example 7

[0061] 4-Octyl itaconic acid ester administration promotes long-term hematopoietic reconstitution of receptors after radiotherapy combined with HSCT. Based on Example 5, the number of bone marrow cells and blood cells in both groups of mice was counted at week 3, and the results are as follows: Figure 9 As shown, Figure 9 Figure A shows a statistical comparison of the number of donor LT-HSCs and donor bone marrow cells in the two groups of mice at week 3. Figure 9 Figure B shows a comparison of blood cell counts between the two groups of mice at week 3. Figure 9 It is evident that, in response to the deterioration of the bone marrow microenvironment caused by radiotherapy, OI can effectively improve the vitality of donor LT-HSCs, enhance their long-term hematopoietic differentiation capacity in the recipient, promote hematopoietic function reconstruction, and effectively shorten the period of hematopoietic function failure after HSCT.

[0062] Example 8

[0063] Administration of aconitine decarboxylase expression promoters promotes the maintenance of donor HSCs in the very early postoperative period following radiotherapy combined with HSCT. In this example, healthy WT C57BL / 6 mice were used as recipients. The recipient mice underwent myeloablation pretreatment with 10 Gy ionizing radiation. After myeloablation, healthy WT C57BL / 6 mice were used as donors. Donor bone marrow cells were harvested and pretreated with a recombinant lentiviral vector carrying the IRG1 gene as an IRG1 expression promoter to upregulate the expression of aconitine decarboxylase in the donor HSCs. The control group was transduced with an empty vector. Then, 1 × 10⁻⁶ HSCs were... 6 Modified donor bone marrow cells were injected into recipient mice via tail vein. The number of donor HSCs, the expression level of aconitine decarboxylase, and the itaconic acid content in HSCs were recorded in both groups of mice one day after HSCT surgery. The results are as follows: Figure 10 visible, Figure 10 In the figure, A is a statistical comparison of the number of donor HSCs in the two groups of recipient mice; Figure 10 Figure B in the figure shows the comparison of the expression levels of cis-aconitine decarboxylase in donor HSCs in the two groups of recipient mice. Figure 10 Figure C in the graph shows a comparison of the donor HSC uric acid content in the undercoatol of the two groups of recipient mice. Figure 10 It is evident that the aconitine decarboxylase expression promoter can effectively resist toxic damage in the harsh bone marrow microenvironment caused by radiotherapy by highly expressing aconitine decarboxylase and synthesizing itaconic acid, and significantly improve the maintenance rate of donor HSCs in the recipient.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. Application of itaconic acid detection reagent in the preparation of products for prognostic assessment after hematopoietic stem cell transplantation.

2. Application of aconitine decarboxylase detection reagent in the preparation of products for prognostic assessment after hematopoietic stem cell transplantation.

3. A product for assessing the prognosis after hematopoietic stem cell transplantation, characterized in that, The postoperative prognostic assessment product for hematopoietic stem cell transplantation includes a donor hematopoietic stem cell hematopoietic capacity assessment reagent, which is an itaconic acid detection reagent and / or aconitine decarboxylase detection reagent.

4. Application of itaconic acid, itaconic acid derivatives or cis-aconitine decarboxylase expression promoters in the preparation of hematopoietic stem cell protective drugs.

5. The application as described in claim 4, characterized in that, The itaconic acid derivative is 4-octyl itaconic acid ester.

6. The application as described in claim 4, characterized in that, The hematopoietic stem cell protection drug includes pharmaceutically acceptable excipients.

7. The application as described in claim 4, characterized in that, The hematopoietic stem cell protective drug is used to maintain the viability of recipient intracellular donor hematopoietic stem cells after hematopoietic stem cell transplantation with myeloablative pretreatment.

8. The application as described in claim 4, characterized in that, The hematopoietic stem cell protective drug is used to reconstruct the hematopoietic function of recipient hematopoietic stem cells after hematopoietic stem cell transplantation with myeloablative pretreatment.

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