DDX1 overexpression radiation resistance model and establishment method and application thereof

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a DDX1 overexpression radiation resistance model and an establishment method and application thereof. The invention firstly provides an establishment method and application of a DDX1 overexpression radiation resistance animal model, and the establishment method comprises the following steps: constructing a sequence carrying LOXP-DDX1 in a mouse embryonic stem cell, and further hybridizing the mouse with a mouse strain specifically expressed by Cre recombinase to obtain a double transgenic mouse, finally, the DDX1 gene in the double transgenic mouse is subjected to overexpression, and the DDX1 overexpression radiation resistance animal model is obtained. According to the scheme, a safer and more effective protection strategy is provided by optimizing an endogenous regulation and control path, and the defects of the prior art in organism radiation protection specificity, side effect management and cost are overcome.

Description

[0001] Priority Application This application claims the priority of the Chinese invention patent application [2025101742327] "A DDX1 Overexpression Radiation Resistance Model and Its Establishment Method and Application" filed on February 17, 2025, and the entire text of the priority invention patent application is incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of biomedical technology, and specifically relates to a DDX1 overexpression radiation resistance model and its establishment method and application. Background Art

[0003] Radiation protection is a key topic in the fields of medicine and biology, especially important in scenarios such as nuclear energy utilization, radiotherapy, and space exploration. Radiation causes great damage to organisms, mainly manifested in the destruction of cellular DNA, especially to rapidly dividing cells such as hematopoietic stem cells (HSCs). HSCs are located in the bone marrow and are responsible for generating all types of blood cells, which is crucial for maintaining the health of the body. Protecting HSCs from radiation damage is essential for maintaining the normal function of the blood system and preventing long-term health problems caused by radiation. However, with the progress of technology and the development of society, the risk of radiation exposure is also increasing (such as medical radiotherapy, industrial ray applications, cosmic ray exposure during air travel, etc.), so the development of effective radiation protection measures has become increasingly important.

[0004] In the prior art, the radiation protection measures for hematopoietic stem cells (HSCs) mainly rely on the application of physical shielding, chemical protectants, or biological agents. However, these methods have many limitations. For example, physical shielding is difficult to comprehensively protect the human body, especially ineffective for individuals who have already been exposed to radiation; chemical protectants may be accompanied by side effects and have limited effects, unable to provide safe long-term protection; although biological agents such as cytokines and growth factors can promote hematopoietic recovery, their mechanisms of action are complex, take a long time to take effect, and are also costly. In addition, most of the existing radiation protection strategies are broad-spectrum and do not specifically protect this specific cell population of hematopoietic stem cells. On the other hand, most of the existing radiation protection research focuses on reducing radiation dose or enhancing the overall cell survival rate, and more are some exogenous means. Currently, there is no endogenous regulatory means to directly improve the radiation resistance of HSCs themselves. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method and strategy for specifically and endogenously regulating the radiation resistance of HSCs, specifically involving a DDX1 overexpression radiation resistance model and its establishment method and application, which can partially solve or alleviate the above deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] On the one hand, the present invention provides an animal model resistant to ionizing radiation and its application.

[0007] A method for establishing a DDX1 overexpressing radiation-resistant animal model, comprising the following steps: S01: Design and synthesize a plasmid vector containing LOXP sites, the plasmid vector contains the DDX1 gene driven by CMV or EF1α, and LOXP sites are inserted in front of the DDX1 gene; the gRNA sequence of the DDX1 gene is as shown in SEQ ID NO.1; S02: Introduce the plasmid vector into mouse embryonic stem cells to construct transgenic mice carrying the LOXP-DDX1 sequence (in the present invention, embryonic stem cells are used to construct transgenic mice, and then Cre tool enzyme is used to specifically overexpress DDX1 in the hematopoietic system); S03: Hybridize a mouse strain with specific expression of Cre recombinase with the transgenic mouse carrying the LOXP-DDX1 sequence to obtain a double transgenic mouse model; inject polyinosinic acid into the double transgenic mouse model to trigger overexpression of the DDX1 gene, and construct the DDX1 overexpressing radiation-resistant animal model.

[0008] Furthermore, gene detection is performed on the constructed double transgenic mouse model to ensure that it carries the correct LOXP-DDX1 sequence; the primer pairs used for detection are as shown in SEQ ID NO.2 and SEQ ID NO.4, SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.6.

[0009] Furthermore, the expression level of the DDX1 gene in the constructed DDX1 overexpressing radiation-resistant animal model is detected.

[0010] In some preferred embodiments, the expression level of the DDX1 gene in the constructed DDX1 overexpressing radiation-resistant animal model is 10 times that of wild-type mice.

[0011] Furthermore, the mouse strains with specific expression of Cre recombinase include Vav1-Cre mouse strain or Mx1-Cre mouse strain.

[0012] The DDX1 overexpressing radiation-resistant animal model constructed by the above establishment method.

[0013] Application of the above DDX1 overexpressing radiation-resistant animal model in the preparation of a screening model for anti-radiation drugs.

[0014] And, application of the above DDX1 overexpressing radiation-resistant animal model in the preparation of a screening model for radioprotective agents.

[0015] The present invention further provides a cell model with radiation resistance by overexpressing DDX1.

[0016] A method for constructing a cell model with radiation resistance by overexpressing DDX1, comprising the following steps: S01: Design and synthesize a lentiviral expression vector containing the DDX1 gene, and the lentiviral expression vector includes a strong promoter (CMV) to ensure high-level expression of DDX1; the gRNA sequence of the DDX1 gene is as shown in SEQ ID NO.1; S02: Isolate CD34 + HSC from umbilical cord blood or bone marrow; S03: Infect the CD34 + HSC with the lentiviral expression vector constructed in S01, and detect and screen out GFP-positive cells by flow cytometry.

[0017] Furthermore, the lentiviral expression vector further contains enhanced green fluorescent protein (EGFP).

[0018] Furthermore, the lentiviral expression vector further contains a selection marker (such as neomycin resistance gene).

[0019] The present invention also provides a novel molecular target for evaluating the anti-radiation effect.

[0020] Application of DDX1 as a molecular marker in the preparation of a kit for evaluating the anti-radiation effect of hematopoietic stem cells.

[0021] Furthermore, the kit is used to detect the expression level of the DDX1 gene in hematopoietic stem cells to evaluate whether the hematopoietic stem cells have specific anti-radiation ability.

[0022] Furthermore, when the DDX1 gene is highly expressed in hematopoietic stem cells, the hematopoietic stem cells have specific anti-radiation ability.

[0023] Beneficial technical effects: The present invention provides a method and strategy for overexpressing DDX1 in hematopoietic stem cells (HSCs) to endow the hematopoietic stem cells with specific anti-ionizing radiation ability. Compared with the prior art, the present invention mainly has the following advantages.

[0024] (1) Most of the prior art are broad-spectrum protection measures. The present invention realizes conditional overexpression of DDX1 through the Cre-LOXP conditional overexpression system, ensuring activation only in target cells, and achieving the specificity and efficiency of protection.

[0025] (2) Compared with chemical protection measures, the method of directly enhancing the radiation resistance of HSCs through the endogenous regulation pathway in the present invention avoids the side effects brought by external drugs and improves safety.

[0026] (3) Compared with the treatment with high-cost biological agents, the present invention realizes the efficient and economical overexpression of DDX1 through means such as gene editing and lentiviral vectors, reduces the treatment cost, and improves the economic benefit.

[0027] (4) The radiation resistance model with overexpressed DDX1 constructed in the present invention has broad application prospects. The model constructed in the present invention can be used to improve the efficiency and accuracy of drug screening and the research and development of radiation protection strategies. Since ordinary mice generally die quickly under high-dose radiation, it is difficult to distinguish the mechanism of action of drugs, and only rough indicators such as "prolonged survival time" can be relied on. However, the radiation-resistant mice of the present invention can be accurately evaluated through more specific biological processes such as delaying damage and repairing DNA, and can be effectively used for the screening and research of drugs and radioactive protectants.

[0028] Generally speaking, the present invention not only alleviates the deficiencies of the prior art in terms of specificity, side effect management and cost, but also provides a safer and more effective protection strategy by optimizing the endogenous regulation pathway. The technical solution of the present invention not only improves the protection effect of HSCs, but also opens up a new research path for the health maintenance after radiation exposure in the medical and scientific research fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to actual scale. Obviously, the following-described drawings are some embodiments of the present invention, and those of ordinary skill in the art can obtain other drawings according to these drawings without creative efforts.

[0030] Figure 1 It is a synthetic identification diagram of the DDX1 gene; Figure 2 It is the result verification of the overexpression of DDX1 protecting the hematopoietic system of mice from radiation damage; Figure 3 It is the statistical chart of the overexpression level of DDX1; Figure 4 It is the result of exploring genes sensitive to ionizing radiation in hematopoietic stem cells; Figure 5 It is for constructing an in vitro model and verifying the effect of overexpressed DDX1 in vitro on the radiation resistance of CD34 + of HSC; Figure 6 It is a plasmid map for overexpression of DDX1 cells. Specific implementation manners

[0031] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

[0032] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0033] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0034] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0035] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this kind of description "within a certain range" is only for convenience and brevity, and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within this range. For example, the description of the range 1-6 should be regarded as having specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and the individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0036] Main experimental method steps: 1. Isolation of human hematopoietic stem cells Healthy adult mobilized spinal cord hematopoietic stem cells are obtained from the First Affiliated Hospital of Chongqing Medical University, and CD34 is selected using a human CD34 positive selection kit (Stem Cell Technologies). +Enrich the cells, and the steps refer to the kit instruction manual. Culture them in StemSpan SFEM II medium supplemented with complement reagents, and perform subsequent experimental steps after continuous culture for 24 h. All hematopoietic stem cell donors from the First Affiliated Hospital of Chongqing Medical University have signed the informed consent form. All research involving human hematopoietic stem cells has obtained the approval of the Ethics Committee of Chongqing Medical University.

[0037] 2. Hematopoietic Stem Cell Culture For mouse experiments, freshly sorted hematopoietic stem cells (HSCs) or lentivirally transfected lymphoid-myeloid progenitors (LSKs) are cultured in StemSpan SFEM medium (Stem Cell Technologies). Additionally, 10 ng / mL recombinant mouse stem cell factor (SCF), 20 ng / mL recombinant mouse thrombopoietin (TPO), 10 µg / mL heparin, and 1× concentration of penicillin / streptomycin solution are added. For human hematopoietic stem cell culture, CD34 + umbilical cord blood cells are cultured in StemSpan SFEM II medium, and 100 ng / mL recombinant human stem cell factor (SCF), 100 ng / mL recombinant human Flt3 ligand (Flt3L), 20 ng / mL recombinant human thrombopoietin (TPO), 20 ng / mL recombinant human interleukin-6 (IL-6), and 1× concentration of penicillin / streptomycin mixture are also added. All cells are cultured in a cell incubator at 37°C and 5% CO 2 . After 7 or 10 days of culture, harvest the cells for the next analysis.

[0038] 3. Western Blot Take the sorted mouse bone marrow LSK cells, wash them once with PBS, add 5× SDS loading buffer containing protease inhibitors on ice, boil at 100°C for 10 minutes to denature and linearize the proteins, and centrifuge at 4°C and 15,000g for 5 minutes. Then electrophorese the lysed protein samples on a 4%-10% gradient gel for 1 hour, transfer the separated proteins to a PVDF membrane, block the transferred PVDF membrane with TBST containing 5% non-fat milk powder. After that, incubate the membrane with the primary antibody diluted to 1:1000 at 4°C overnight, incubate with the horseradish peroxidase (HRP)-labeled secondary antibody diluted to 1:5000 at room temperature for 1 hour, finally develop the color with ECL chemiluminescence reagent, and image the membrane in a Biorad gel imaging system.

[0039] 4. Immunofluorescence After sorting mouse bone marrow HSC cells, they were placed on poly-L-lysine-coated slides for cell spreading for 5 minutes. Mitotacker-Green (MTG) and Lysotracker-Red (LTR) were used to stain for 30 minutes at 37°C, and then DAPI staining was performed to label the cell nuclei. For other immunofluorescence experiments, mouse bone marrow HSC cells were also sorted and placed on poly-L-lysine-coated slides for spreading for 5 minutes, fixed with 4% paraformaldehyde at room temperature for 25 minutes, and rinsed twice with PBS. Permeabilized with 0.1% Triton X-100 at room temperature for 10 minutes, washed 3 times with PBS for 5 minutes each time. Blocked with 10% goat serum at room temperature for 1 hour. After permeabilization and fixation, the samples were incubated overnight at 4°C with γ-H2AX S139, TOMM20, TIMM23 or LAMP1 respectively. Finally, the cells were stained with fluorescein-labeled secondary antibodies and DAPI, and photographed using a confocal microscope. The Pearson correlation coefficient was calculated using ImageJ software to quantitatively analyze the degree of colocalization between different markers.

[0040] 5. Single-molecule DNA replication stress assay After sorting and culturing HSC for 36 hours, pulsed labeling was performed with 50 µM CldU for 38 minutes, washed twice with PBS, then pulsed labeling was performed with 250 µM IdU for 38 minutes, washed once with PBS, and then 10,000 cells were dropped onto the slide and lysed with 10 μL of cell lysis solution (0.5% SDS in 200 mM Tris-HCl (pH 7.4) and 50 mM EDTA) for 5 minutes. The slide was tilted at a 15° angle to unfold the DNA, fixed with a 3:1 volume of methanol: glacial acetic acid fixative for 2 minutes, and then air-dried. The DNA was denatured with 2.5 M hydrochloric acid at room temperature for 30 minutes, the slide was rinsed three times with PBS, and blocked in PBS / 0.1% Triton X-100 / 1% BSA at room temperature for 1 hour. The cells were incubated with rat anti-BrdU antibody and mouse anti-BrdU antibody at room temperature for 1 hour to detect CldU and IdU respectively, and at the same time, mouse anti-single-stranded DNA antibody was used to check the integrity of the DNA fiber. The slide was washed three times with PBS, and then incubated with Alexa Fluor 488-labeled goat anti-mouse antibody and Alexa Fluor 594-labeled goat anti-rat antibody at room temperature for 30 minutes. After washing three times with PBS again, mounting was performed. Microscopic imaging was used, and the replication length, replication symmetry, and replication origin distance were calculated using ImageJ software based on the length of the IdU track.

[0041] 6. Flow cytometry analysis and sorting According to the conventional methods in the art, bone marrow, peripheral blood, spleen or thymus are obtained from an animal model, prepared into a single-cell suspension, and immunostained with a fluorescent dye-conjugated antibody; the cells are analyzed using a spectral flow cytometer, CantoII or other flow cytometers, and sorted using Aria III; the flow cytometry data is analyzed using FlowJo software.

[0042] The present invention proposes a method for obtaining a specific and endogenous anti-radiation means by conditional overexpression of DDX1. The technical solution of the present invention mainly includes two parts. The first part is to enhance DDX1 using the Cre-LOXP conditional overexpression system in mice; the second part is to also enhance CD34 using the DDX1 overexpression system in vitro. + The radiation resistance of HSC.

[0043] Example 1 This example provides an example of constructing a Cre-LOXP conditional overexpression system in mice.

[0044] Construct transgenic mice carrying the LOXP-DDX1 system and introduce a mouse strain with specific expression of Cre recombinase (such as Vav1-Cre or Mx1-Cre) to ensure that DDX1 is only expressed in specific tissues or conditions. Genotype identification is performed on newborn mice to ensure that they carry the correct gene editing sequence. The specific operation process is as follows.

[0045] 1.1 Design the gRNA for DDX1 overexpression mice through http: / / crispor.tefor.net / .

[0046] 1.2 Construct a plasmid vector by conventional molecular biology and cell biology means. This plasmid vector contains LOXP sites, specifically the 6*SV40 Poly sequence containing LOXP sites.

[0047] (1) Fragment amplification (5’arm, cKO, 3’arm): Use Novoprotein P515 high-fidelity enzyme to prepare a 50 μL system for PCR amplification with 30 cycles.

[0048] (2) Gel recovery: Electrophorese the PCR product and recover the gel at the position of the target product using the Qiagen gel recovery kit (product number: 28706).

[0049] (3) Ligation (backbone + fragment) / transformation: Use Novoprotein C115 ligase to ligate the recovered fragments, and transform the ligated DNA fragments into Escherichia coli using Takara's Stellar competent cells, and culture overnight at 37°C.

[0050] (4) Bacterial examination: Pick 16 well-shaped dental plaques, use Novoprotein P222 Taq enzyme, and prepare a 25 μL system for PCR amplification; pick the dental plaque with the correct band and inoculate it into a 4 mL broth medium for small-scale shaking culture.

[0051] (5) Plasmid extraction from positive clones: Extract plasmids using the alkaline lysis method.

[0052] (6) Enzyme digestion identification and sequencing: Select appropriate restriction enzymes from NEB, and prepare a 20 μL enzyme digestion system with 600 ng plasmid for enzyme digestion, and sequence the plasmid with correct enzyme digestion.

[0053] (7) Preparation of plasmid for injection: For the clones with correct sequencing results, inoculate them into 22.5 mL broth medium for overnight culture, and use the Qiagen 27106 plasmid extraction kit to extract the plasmids for injection.

[0054] 1.3 Synthesis of gRNA for DDX1 overexpressing mice.

[0055] Artificially synthesize the CrRNA (CRISPR RNA) sequence and tracrRNA (trans-activating crRNA) sequence. CrRNA binds to tracrRNA to form the gRNA sequence for DDX1 overexpression described in 1.1, and the gRNA sequence is shown as SEQ ID NO.1.

[0056] 1.4 Preparation of RNP complex.

[0057] (1) Solution in Tube 1: Add 0.8 μL of 100 pmol / μL CrRNA to 5.2 μL of RNase-free water, then add 0.6 μL of 100 pmol / μL tracrRNA, mix well and incubate for 5 min, and then add 0.2 μL of Cas9 protein (NEB, product number: M0646M), mix well and incubate for 10 min to obtain the solution in Tube 1.

[0058] (2) Solution in Tube 2: Prepare a plasmid vector with a final concentration of 15 ng / μL.

[0059] (3) Mix the solution in Tube 1 and the solution in Tube 2 to obtain the RNP injection complex.

[0060] 1.5 Preparation of fertilized eggs.

[0061] (1) Select 3 - 4 week-old C57BL / 6 female mice, and inject pregnant mare serum (PMSG) and human chorionic gonadotropin (HCG) respectively, with a time interval of 46 - 48 h between the two.

[0062] (2) After injecting HCG, mate the female mice with adult fertile male mice to fertilize the female mice.

[0063] (3)On the next day, after euthanizing the female mice, collect the fertilized eggs from the fallopian tubes and place them in a 37°C constant temperature incubator with 5% CO 2 for standby.

[0064] 1.6 Pronuclear microinjection.

[0065] (1)Dilute and mix the prepared RNP complex and plasmid DNA to form an injection solution, and then load the injection solution into the microinjection needle.

[0066] (2)Select fertilized eggs with normal morphology and place them in an injection dish. Under an inverted microscope with a magnification of 200 - 400 times, inject the foreign gene injection solution into the nucleus of the fertilized eggs by microinjection.

[0067] (3)Transfer the injected fertilized eggs to M16 medium and place them in a 37°C constant temperature incubator with 5% CO 2 for 0.5 - 1 h. Then perform transplantation; or culture until the two - cell stage during embryonic development and perform transplantation the next day.

[0068] 1.7 Preparation of surrogate mice and embryo transfer (1)Prepare pseudopregnant female mice: Select fertile female mice of appropriate age to mate with vasectomized male mice. Stimulate the female mice to undergo a series of pregnancy - like changes to obtain pseudopregnant female mice, which will be used as surrogate mice after the fertilized eggs are transgenic.

[0069] (2)Transfer the fertilized eggs that have been injected with foreign genes into the fallopian tubes of the surrogate female mice on the day of detecting vaginal plugs.

[0070] (3)After transplantation, place the surrogate female mice in a clean cage box and keep them warm. After they wake up, return them to the cage rack for breeding.

[0071] (4)After successful tubal transplantation, the female mice usually give birth 19 - 20 days after the operation.

[0072] (5)One week after the mice are born, the mice can be numbered by clipping their claws and at the same time, PCR identification can be performed; three weeks after the mice are born, they can be separately caged for independent breeding.

[0073] 1.8 Identification of F0 newborn mice Collect tissues of 1 - 2 - week - old young mice (tail or toe tissues), lyse and extract the genomic DNA of the tissues. Perform PCR amplification and electrophoresis detection using specific primers for the target gene to screen for offspring with integrated foreign genes. Perform genotype identification on F0 generation mice to ensure that they carry the correct LOXP - DDX1 sequence.

[0074] 1.9 Introduction of Cre recombinase.

[0075] The Cre recombinase-expressing mouse strain Mx1-Cre was selected and crossed with mice carrying the correct LOXP-DDX1 sequence to generate double transgenic offspring. MX1-Cre can specifically express Cre in the hematopoietic system and express Cre under the stimulation of IFN-γ signal.

[0076] The sequence information involved in the above experiments is shown in Table 1.

[0077] Table 1 The experimental results are as Figure 1 shown. Figure 1 Among them, mouse No. 1 is an Mx1 Cre mouse, mouse No. 2 and No. 4 are wild-type mice, mouse No. 5 is a Ddx1 flox / + mouse, and mouse No. 3 is a Ddx1 flox / + -Mx1 Cre mouse. After the experimental group mice were injected with pIpC, overexpression of Ddx1 in mice could be induced. The results showed that mice with F3-R3, F4-R3 bands and Cre bands would induce overexpression of Ddx1 after injection of pIPC and become Ddx1 overexpressing mice. 2. Induced overexpression Experimental mice (Ddx1 flox / + -MX1-Cre mice) carrying the LOXP-DDX1 sequence and Cre recombinase were injected with polyinosinic acid (Poly I:C, pIpC) to activate Cre recombinase, thereby triggering overexpression of the DDX1 gene, labeled as TgDdx1 fl / + . At the same time, a control group was taken and the inducer was injected simultaneously to evaluate the change of background level.

[0078] One month after injection of pIpC, Ddx1 overexpressing mice and control group mice were sacrificed by cervical dislocation, and bone marrow cells were taken. After enriching and sorting HSCs, total RNA was extracted, and cDNA was reverse transcribed from total RNA using the PrimeScript™ RT kit (Takara). Real-time qPCR was performed on a CFX96 Touch real-time PCR detection system (Bio-Rad) using the QuantiNova SYBR Green PCR kit (QIAGEN). The primer list is shown in Table 1. Statistical analysis was performed on the overexpression level of the DDX1 gene.

[0079] 5. Radiation treatment The experimental group and control group mice were subjected to the same dose of radiation treatment. Specifically, mice were irradiated with a sublethal radiation dose of 7.5 Gy and the survival rate of mice was detected; mice were irradiated with a sub-lethal dose of 5 Gy and the hematopoietic system phenotype of mice was detected.

[0080] 6. Data analysis and results Compare the differences between the experimental group and the control group to clarify the effect of DDX1 on radiation protection of HSCs in vivo. The results are as Figure 2 shown.

[0081] Figure 2 A is the construction pattern diagram of conditional overexpression mice of Ddx1 flox / + -MX1-Cre mice. This result shows that after inducing the expression of MXI-cre enzyme by pIpC, the specific overexpression of DDX1 gene in the hematopoietic system was achieved.

[0082] Figure 2 B is the survival curve of Ddx1 flox / + -MX1-Cre mice (Ddx1 fl / + ) after injection of pIpC for one month (labeled as TgDdx1 fl / + or TgDdx1) irradiated with a sublethal dose. This experimental result shows that one month after 3 times of pIpC induction, the mice were irradiated with a sublethal dose of 7.5 Gy, and it was found that the number of dead Ddx1 overexpression mice (TgDdx1) was significantly reduced. Among them, Ddx1 fl / + represents the control group mice without radiation treatment; Ddx1 fl / + +IR represents the control group mice treated with radiation; TgDdx1 represents the experimental group mice with overexpression of Ddx1 without radiation treatment; TgDdx1 +IR represents the experimental group mice with overexpression of Ddx1 treated with radiation Figure 2 C is the pattern diagram of Ddx1 overexpression mice injected with pIpC and irradiated with 5 Gy.

[0083] Figure 2 D and 2E are flow cytometry analyses of the number of hematopoietic stem and progenitor cells in the bone marrow of Ddx1 overexpression mice and their control group mice after irradiation. It shows that there is no significant difference in the hematopoietic system between Ddx1 overexpression mice and normal mice without irradiation. However, after irradiation, Ddx1 overexpression mice have stronger anti-radiation ability than control group mice, and there are more hematopoietic stem and progenitor cells in irradiated Ddx1 overexpression mice, including LSK, CD34-LSK, HSC, HPC, CMP, GMP, MEP.

[0084] Figure 2 F is the analysis of apoptosis of hematopoietic progenitor cells HPC and LSK in Ddx1 overexpression mice by AnneixV-DAPI staining. It shows that the apoptosis ratio of HPC and LSK in overexpression mice after irradiation is significantly lower than that of control mice, where the ordinate represents the early apoptosis events of cells.

[0085] Figure 2 G was to detect the mitochondrial membrane potential of hematopoietic stem cells (HSCs) in the bone marrow of mice in each group using JC-1 staining. It was shown that the mitochondrial membrane potential of mice overexpressing Ddx1 was higher than that of the control group of mice after irradiation. A decrease in mitochondrial membrane potential is a marker of early apoptosis of cells. The lower the membrane potential, the more cells die; the higher the membrane potential, the better the cell condition and the stronger the anti-radiation ability.

[0086] Figure 2 H was to detect the mitochondrial number of HSCs in the bone marrow of mice in each group using Mito-Tracker Green staining. It was shown that the number of mitochondria in mice overexpressing Ddx1 was more than that of the control group of mice after irradiation.

[0087] Figure 2 I was to detect the overall ROS content of HSCs in the bone marrow of mice in each group using DCFH-DA staining. It was shown that the overall ROS content in the cells of mice overexpressing Ddx1 was less than that of the control group of mice after irradiation.

[0088] Figure 2 J was to detect the mitochondrial ROS content of HSCs in the bone marrow of mice in each group using Mito-SOX staining. It was shown that the mitochondrial ROS in mice overexpressing Ddx1 was also less than that of the control group of mice after irradiation.

[0089] The above results proved that the mouse model with overexpression of Ddx1 had better anti-radiation ability, and the hematopoietic stem cells of mice with overexpression of Ddx1 also had better anti-radiation ability.

[0090] In addition, the relative expression level of the DDX1 gene in mice with overexpression of Ddx1 was quantified, as shown in Figure 3 . It was shown that the expression level of the DDX1 gene in mice overexpressing Ddx1 was increased by about 10 times compared with that of the control group of mice.

[0091] Example 2 Exploration of genes sensitive to ionizing radiation in hematopoietic stem cells.

[0092] To identify genes sensitive to ionizing radiation in the mitochondria of hematopoietic stem cells (HSCs), mitochondrial microproteomics analysis was performed on CD34 + HSCs cells before and after irradiation ( Figure 4 A). Subcellular localization analysis identified 1,129 mitochondrial proteins, covering 70.5% and 66.7% of the MitoCarta 3.0 and Mito-CoP datasets respectively ( Figure 4 B). KEGG, BP and pathway enrichment analysis showed that ionizing radiation significantly affected signal pathways such as mitochondrial energy metabolism and oxidative phosphorylation in CD34 + HSCs (Figure 4 C).

[0093] Furthermore, by performing an intersection analysis on the mitochondrial differential proteins and DNA damage-related gene sets in CD34 + HSCs before and after irradiation, 16 radiation-sensitive genes related to DNA damage were identified ( Figure 4 D). Among them, DDX1, as a significant gene, is closely related to DNA damage repair. Further, the new mitochondrial protein DDX1 determined by mitochondrial microproteomics was used as the focus of ionizing radiation response research for verification. After 24 hours of treatment with 5 Gy irradiation, immunofluorescence experiments showed that DDX1 in the mitochondria of CD34 + HSCs increased significantly ( Figure 4 E), thus proving that the DDX1 gene in hematopoietic stem cells is sensitive to ionizing radiation.

[0094] Example 3 This example provides an example of constructing an in vitro DDX1 overexpression system.

[0095] 1. Vector construction Design and synthesize a lentiviral expression vector containing the DDX1 gene to ensure high-efficiency expression in HSCs; the gRNA sequence of the DDX1 gene is shown in SEQ ID NO.1. This lentiviral expression vector includes a strong promoter (CMV) to ensure high-level expression of DDX1. In addition, enhanced green fluorescent protein (EGFP) is added to track the infection efficiency and screen positive cells. In addition, the plasmid also contains appropriate selection markers (such as neomycin resistance gene) for subsequent screening of successfully transfected cells. The overexpression plasmid map of the DDX1 cell line is shown in Figure 6 .

[0096] 2. Cell isolation and transfection Isolate CD34 + HSCs from umbilical cord blood or bone marrow, and use flow cytometry or magnetic bead sorting technology to ensure high purity. Infect these cells with the lentiviral expression vector particles obtained in step 1 to stably express DDX1. Then, detect the proportion of GFP-positive cells by flow cytometry to confirm the infection efficiency.

[0097] 3. Radiation treatment Perform the same dose of radiation treatment (5 Gy γ-ray) on the HSCs in the DDX1 gene overexpression group and the control group with empty vector, and then detect indicators such as cell viability, apoptosis, and DNA repair ability. Specifically, perform DNA damage detection 2 hours after 5 Gy radiation treatment, and use γ-H2AX immunofluorescence method to evaluate the DNA damage repair efficiency. Then, detect the relative cell viability at 24 h, 48 h, 72 h, and 96 h after radiation respectively.

[0098] 4. Data Analysis and Results Through the statistical analysis of experimental data, the actual effect of DDX1 overexpression on the radiation protection of HSCs was confirmed, and the results are as Figure 5 shown.

[0099] Figure 5 A shows the detection of the viability of hematopoietic stem cells after 5 Gy irradiation treatment following the overexpression of DDX1 in CD34 + HSCs. It shows that overexpression of DDX1 in CD34 + HSCs can improve their tolerance to irradiation. Among them, DDX1-OE represents the experimental group with overexpression of Ddx1 in CD34+HSCs. The cell survival rate of the DDX1-OE+5Gy group at 96 h was significantly higher than that of the NC+5Gy group.

[0100] Figure 5 B shows the detection of DNA damage in the above-mentioned identically treated CD34 + HSCs using immunofluorescence γ-H2AX foci. It shows that overexpression of DDX1 in CD34 + HSCs can reduce the DNA damage occurring after irradiation. The green fluorescence in the figure represents the DNA damage sites. The above experiments have proved that overexpression of the DDX1 gene in hematopoietic stem cells can improve their ability to resist ionizing radiation.

[0101] It should be noted that in this article, the terms "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitations, an element defined by the statement "including a..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0102] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the spirit and scope protected by the claims of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A method for establishing a DDX1 overexpression radiation-resistant animal model, characterized in that: The following steps are involved: S01: Design and synthesize a plasmid vector containing a LOXP site, wherein the plasmid vector contains a DDX1 gene driven by CMV or EF1α, and a LOXP sequence is inserted in front of the DDX1 gene, wherein the LOXP sequence is a 6*SV40 Poly sequence containing a LOXP site; the gRNA sequence of the DDX1 gene is shown in SEQ ID NO.1; S02: introducing the plasmid vector into mouse embryonic stem cells to construct transgenic mice carrying the LOXP-DDX1 sequence; S03: hybridizing a mouse strain that specifically expresses Cre recombinase with the transgenic mouse carrying the LOXP-DDX1 sequence to obtain a double transgenic mouse model; injecting polyinosinic-polycytidylic acid into the double transgenic mouse model to trigger overexpression of the DDX1 gene, thereby constructing the DDX1 overexpression radiation-resistant animal model.

2. The method of establishing as claimed in claim 1, characterized in that: The constructed double transgenic mouse model is subjected to gene detection to ensure that it carries the correct LOXP-DDX1 sequence; the primer pairs used for detection are shown in SEQ ID NO.2 and SEQ ID NO.4, SEQ ID NO.3 and SEQ ID NO.4, or SEQ ID NO.5 and SEQ ID NO.

6.

3. The establishment method according to claim 1, characterized in that: The DDX1 gene expression level of the constructed DDX1 overexpression radiation-resistant animal model was detected.

4. The establishment method according to claim 1, characterized in that: The mouse strains specifically expressing Cre recombinase include Vav1-Cre mouse strains or Mx1-Cre mouse strains.

5. A DDX1 overexpression radiation-resistant animal model constructed by the establishment method according to any one of claims 1 to 4.

6. Use of the DDX1 overexpression radiation-resistant animal model of claim 5 in preparing a radiation-resistant drug screening model; or use of the DDX1 overexpression radiation-resistant animal model of claim 5 in preparing a radiation protective agent screening model.

7. A method for constructing a DDX1 overexpression radiation-resistant cell model, comprising the following steps: S01: Design and synthesize a lentiviral expression vector containing the DDX1 gene, wherein the lentiviral expression vector includes at least one strong promoter to ensure high-level DDX1 expression; the gRNA sequence of the DDX1 gene is shown in SEQ ID NO.1; S02: Isolation of CD34 from umbilical cord blood or bone marrow + HSC cells; S03: Use the lentiviral expression vector constructed in S01 to infect the CD34 + HSC cells were detected by flow cytometry and GFP-positive cells were screened.

8. Application of DDX1 as a molecular marker in the preparation of a kit for evaluating the radiation protection effect of hematopoietic stem cells.

9. The use according to claim 8, characterized in that The kit is used to detect the expression level of the DDX1 gene in hematopoietic stem cells to evaluate whether the hematopoietic stem cells have specific radiation protection ability.

10. The use according to claim 9, characterized in that When the DDX1 gene is highly expressed in hematopoietic stem cells, the hematopoietic stem cells have specific radiation protection ability.

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