Application of PBX1 in preparation of medicine for treating nuclear radiation injury

Abstract

The invention is applicable to the technical field of biological medicines, and provides application of PBX1 in preparation of a medicine for treating nuclear radiation injury, and a coding gene sequence of the PBX1 is as shown in SEQ ID No.1. The invention also provides a TAT-PBX1 recombinant protein, and an overexpression TAT-PBX1 virus, and also provides a TAT-PBX1 recombinant protein. The invention further provides a medicine for treating the nuclear radiation injury and a medical instrument for treating the nuclear radiation injury. According to the invention, the TAT-PBX1 recombinant protein is constructed, so that the PBX1 can enter cells by virtue of the transmembrane effect of TAT to play a corresponding role in resisting oxidative damage, and in addition, the PBX1 entering the cells has a strong capability of reducing DNA damage induced by nuclear radiation, especially an ROS-mediated DNA damage effect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine technology, and in particular relates to the application of PBX1 in preparing medicine for treating nuclear radiation damage. Background Art

[0002] Nuclear radiation damage can cause carcinogenicity, teratogenicity, mutagenicity, etc. by damaging human genetic material DNA, and even cause reproductive toxicity, seriously endangering the development of offspring. The damage caused by nuclear radiation to human tissue varies depending on the radiation dose, exposure level, radiation type and body exposure site. The damage can be local or systemic. The damage to human tissue may cause repeated wounds that are difficult to heal and difficult to treat for a long time, causing great pain to patients and a heavy economic burden to families and society. Therefore, actively treating nuclear radiation damage is a major scientific issue faced by every human society, as well as a major social issue.

[0003] At present, drugs for dealing with nuclear radiation and nuclear emergencies are mainly divided into two categories: one is drugs that prevent or reduce the absorption of radioactive elements and promote their excretion, and the other is drugs that repair radiation damage. These drugs mainly include: potassium iodide used to prevent or reduce the absorption of radioactive iodine by the thyroid gland, Prussian blue used to remove radioactive cesium and thallium from the human body, chelating agents used to promote the excretion of metals and radioactive elements, cytokines used to reduce damage to the bone marrow caused by acute radiation, and drugs used to treat vomiting, diarrhea and infection. Despite this, the above drugs still have shortcomings in the treatment of nuclear radiation damage: 1) The effect is limited, mainly preventing or promoting the excretion of radioactive nuclides, and there is a lack of effective drugs to repair nuclear radiation damage; 2) The above drugs are difficult to pass through the blood-brain barrier, and have little effect on the repair of brain damage caused by nuclear radiation; 3) The above drugs lack effective reduction of DNA damage caused by nuclear radiation, often leading to the occurrence of "carcinogenic, teratogenic, mutagenic" effects and reproductive toxicity related to DNA damage.

[0004] In recent years, with the continuous deepening of stem cell research and the widespread application of transplantation technology, the role of stem cells in regenerative medicine in tissue repair, organ reconstruction and gene therapy has attracted much attention. Mesenchymal stem cells (MSCs) are a type of cell with self-renewal, high proliferation and multidirectional differentiation capabilities. They show good repair effects in almost all human tissue damage. Studies have found that MSCs can have a good repair effect on tissue and organ damage caused by nuclear radiation through paracrine effects. However, MSCs have limited ability to repair nuclear radiation damage, especially in reducing DNA damage caused by nuclear radiation. Therefore, how to repair tissue and organ damage caused by nuclear radiation to humans, especially how to reduce DNA damage, has always been the primary problem that needs to be urgently solved in nuclear radiation emergency treatment. Summary of the invention

[0005] The purpose of the embodiments of the present invention is to provide the use of PBX1 in the preparation of a drug for treating nuclear radiation damage, aiming to solve the problems raised in the above-mentioned background technology.

[0006] The embodiment of the present invention is achieved by using PBX1 in the preparation of a drug for treating nuclear radiation damage, wherein the coding gene sequence of the PBX1 is shown in SEQ ID No.1.

[0007] Another object of the embodiment of the present invention is to provide a TAT-PBX1 recombinant protein, the preparation of which includes the following steps: amplifying the coding gene of the PBX1 by PCR, connecting the PCR product to the cloning vector by cloning, and picking the correctly connected white clones by blue-white screening; after bacterial liquid PCR, enzyme digestion identification and gene sequencing identification, the PBX1 coding gene fragment in the positive clone is inserted into the prokaryotic expression vector pTAT-HA by enzyme digestion and T4 connection, and then transferred into the prokaryotic expression strain, and the positive transformant is obtained by ampicillin resistance screening; activating in the expression culture medium, expanding the culture, and collecting the precipitate by centrifugation; placing the precipitate in the lysis solution for ultrasonic crushing and dissolution, centrifuging and collecting the supernatant, filtering, purifying, and then renaturing by pulse dilution, dialyzing and ultrafiltration of the renaturing liquid to obtain the TAT-PBX1 recombinant protein.

[0008] Another object of an embodiment of the present invention is to provide a TAT-PBX1 overexpressing virus, wherein the viral vector is one of a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

[0009] Another object of an embodiment of the present invention is to provide a drug for treating nuclear radiation damage, wherein the drug comprises the TAT-PBX1 recombinant protein or the TAT-PBX1 overexpressing virus.

[0010] Preferably, the medicine further comprises excipients.

[0011] Another purpose of an embodiment of the present invention is to provide a medical device for treating nuclear radiation damage, wherein the medical device is loaded with the above-mentioned medicine.

[0012] The embodiment of the present invention provides an application of PBX1 in the preparation of a drug for treating nuclear radiation damage. PBX1 belongs to the pre-B cell leukemia (PBC) transcription factor family, and plays an important role in maintaining the stemness and self-renewal of stem cells, resisting oxidative damage and DNA damage induced by endogenous reactive oxygen species (ROS), as well as anti-aging and maintaining the homeostasis of tissues and organs. Since PBX1, as a transcription factor, cannot enter the cell through the cell membrane due to its large molecular weight, the embodiment of the present invention constructs a TAT-PBX1 recombinant protein, so that PBX1 can enter the cell with the help of the transmembrane effect of TAT and exert a corresponding antioxidant damage effect. In addition, PBX1 that enters the cell has a strong ability to reduce nuclear radiation-induced DNA damage, especially ROS-mediated DNA damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG1a is a result diagram of the TAT-PBX1 recombinant protein prepared by the E. coli prokaryotic expression system provided in an embodiment of the present invention, FIG1b is a bright field observation result diagram and a Western Blot diagram of the control group and the umbilical cord mesenchymal stem cells overexpressing TAT-PBX1, Figure 1c This is a Western Blot image of TAT-PBX1 protein in the culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1;

[0014] FIG2 is an immunofluorescence and Western Blot diagram showing that the umbilical cord mesenchymal stem cell supernatant containing TAT-PBX1 can enter cells, and that the TAT-PBX1 recombinant protein can penetrate the membrane and enter cells, wherein a is the experimental result of the stem cell supernatant, and b is the experimental result of the TAT-PBX1 recombinant protein;

[0015] Figure 3 The results of the CCK-8 assay provided in the embodiments of the present invention for detecting the supernatant of umbilical cord mesenchymal stem cells containing TAT-PBX1 after irradiation and the inhibition of cell radiation damage by TAT-PBX1 recombinant protein;

[0016] Figure 4 A result diagram of the clone formation experiment provided in the embodiment of the present invention detecting the supernatant of umbilical cord mesenchymal stem cells containing TAT-PBX1 after irradiation and the promotion of cell clone formation by TAT-PBX1 recombinant protein;

[0017] 5 is a graph showing the results of reducing cell DNA damage by umbilical cord mesenchymal stem cell supernatant containing TAT-PBX1 and TAT-PBX1 recombinant protein provided in an embodiment of the present invention, wherein a and a' are graphs showing the results of alkaline comet assay, and b and b' are graphs showing the results of Western Blot.

[0018] Figure 6 A graph showing the results of flow cytometry detection of umbilical cord mesenchymal stem cell supernatant containing TAT-PBX1 after irradiation and apoptosis of cells intervened by TAT-PBX1 recombinant protein provided in an embodiment of the present invention;

[0019] Figure 7a and Figure 7b This is a graph showing the results of observing the changes in intracellular ROS levels after irradiation using the supernatant of umbilical cord mesenchymal stem cells containing TAT-PBX1 and intervention with TAT-PBX1 recombinant protein, as provided in an embodiment of the present invention.

[0020] Figure 8 HE staining of the tissue of mice intraperitoneally injected with TAT-PBX1 recombinant protein after abdominal irradiation provided in the embodiment of the present invention, wherein a is the result of the small intestine experiment and b is the result of the liver experiment;

[0021] Fig. 9 The plasmid maps of PBX1 and TAT-PBX1 provided in the embodiments of the present invention.

[0022] Fig.10 The results show that the TAT-PBX1 recombinant protein provided in the embodiments of the present invention can cross the blood-brain barrier and enter the mouse brain tissue after intraperitoneal injection, and that the TAT-PBX1 recombinant protein can reduce the radiation damage of HT22 cells. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0025] Example 1: Preparation of TAT-PBX1 recombinant protein, comprising the following steps:

[0026] The PBX1 gene (sequence shown in SEQ ID No.1), the PCR product was connected to the cloning vector pT-EASY by TA cloning, and then the correctly connected white clone was picked by blue-white screening; after bacterial liquid PCR, enzyme digestion identification and gene sequencing identification, the PBX1 coding gene fragment in the positive clone was inserted into the prokaryotic expression vector pTAT-HA by enzyme digestion and T4 connection, and then transferred into the prokaryotic expression strain Rosetta (DE3), and the positive transformant was obtained by ampicillin resistance screening; it was activated at 37°C overnight in the expression culture medium, expanded and cultured, and the precipitate was collected by centrifugation; the precipitate was placed in the lysis solution and ultrasonically crushed and dissolved, centrifuged at 12000g×30min, and the supernatant was collected; the supernatant was filtered with a 0.45μm filter, purified by nickel column affinity chromatography, and then renatured by pulse dilution, and the renatured liquid was dialyzed and ultrafiltered to obtain a high concentration of TAT-PBX1 recombinant protein, and the preparation of TAT-PBX1 recombinant protein was verified by SDS-PAGE, and the result was shown in Figure 1a;

[0027] The specific purification of TAT-PBX1 recombinant protein includes the following steps: using a Ni2+-NTA-agarose column, after equilibration with a binding buffer, loading the bacterial supernatant onto the chromatographic column, washing the chromatographic column with a washing buffer, and then eluting the TAT-PBX1 recombinant protein bound to the chromatographic column with an elution buffer;

[0028] The specific renaturation of TAT-PBX1 recombinant protein includes the following steps: renaturing the eluted sample in a renaturation buffer at a dilution ratio of 1:1-20; after the renaturation, introducing the supernatant into a dialysis bag and dialyzing it with phosphate buffer at 4°C overnight; thereafter, concentrating the denatured protein using a 30-kDa ultrafiltration tube, and determining the concentration of the recombinant protein using a BCA protein detection kit.

[0029] Example 2: Preparation of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1, comprising the following steps:

[0030] (1) Packaging empty vector TAT (Vector) into lentiviral particles: When the density of 293T cells reaches 70-75%, prepare for transfection. First, add 500 μL of serum-free culture medium (Opti-MEM), TAT plasmid, packaging plasmid and PMD2G into a 1.5 mL EP tube; at the same time, add LipoFiterTM transfection reagent into 500 μL of Opti-MEM, and both are incubated at room temperature for 5-100 min; add Opti-MEM containing plasmid to Opti-MEM containing lipofectamine 2000, incubate at room temperature for 15-20 min, then add to 293T, and culture at 37°C and 5% CO2; after culturing for 6-8 h, change to DMEM containing 10% FBS; after continuing to culture for 36 h, collect the DMEM supernatant containing viral particles, centrifuge at 3000 r / min for 5-10 min; collect the supernatant, filter it through a 0.45 μm filter, and set aside;

[0031] (2) Packaging TAT-PBX1 into lentiviral particles: When the density of 293T cells reaches 70-75%, prepare for transfection. First, add 500uL serum-free culture medium (Opti-MEM), TAT-PBX1 plasmid, packaging plasmid PAX2 and PMD2G into a 1.5mL EP tube; at the same time, add lipofectamine 2000 to 500μL Opti-MEM, and both are incubated at room temperature for 5-10min; After incubating Opti-MEM containing plasmid and Opti-MEM containing lipofectamine 2000 at room temperature for 15-20min, add them dropwise to 293T, culture at 37°C and 5% CO2 for 6-8h, and then replace with DMEM containing 10% FBS; continue to culture for 36h, collect the DMEM supernatant containing viral particles, and centrifuge at 3000r / min for 5-10min; collect the supernatant, filter it through a 0.45μm filter, and set it aside.

[0032] Example 3: Preparation of umbilical cord mesenchymal stem cells loaded with TAT-PBX1 protein, comprising the following steps:

[0033] Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached 70-75%, TAT-PBX1 recombinant protein was added to the medium to a final concentration of 1 ng / ml to 100 μg / ml. After the cells were cultured for 1 min to 120 h, the TAT-PBX1 recombinant protein penetrated the cell membrane and entered the cell nucleus, promoting cell growth.

[0034] Example 4: Preparation of culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1, comprising the following steps:

[0035] Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached 70-75%, the umbilical cord mesenchymal stem cell medium was discarded and a mixture of polybrene, bFGF and virus supernatant was added. The transduction was completed after 24 hours, and umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 were successfully established. The TAT-PBX1 protein penetrated the cell membrane and was secreted into the cell culture supernatant. The PBX1 transduction was observed using a fluorescence microscope. Umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 were obtained by screening with puromycin and verified by immunoblotting. The results are shown in Figure 1b.

[0036] Umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 protein were cultured with DMEM medium. When the cells reached a density of 70-75%, the medium was replaced with phenol red-free and serum-free DMEM medium. After culturing for 24 hours, the cell culture supernatant was collected, and the concentration was quantitatively determined using BCA protein. The culture supernatant was freeze-dried in a vacuum freezer, sealed, and stored at -80°C for later use. The expression of TAT-PBX1 protein in the cell culture supernatant was detected by immunoblotting, and the results were as follows: Figure 1c Immunofluorescence and immunoblotting were used to verify whether TAT-PBX1 and TAT-PBX1 recombinant protein in the umbilical cord supernatant entered the cells, and the results were as follows: Figure 2a , b, wherein Figure 2a For the TAT-PBX1 results in the supernatant, Figure 2b This is the result of TAT-PBX1 recombinant protein.

[0037] Example 5: Adenovirus packaging of TAT-PBX1, comprising the following steps:

[0038] When the 293A cells reach a confluence of 50-70%, they are ready for transfection, and TAT-PBX1 is recombined into the AdMAX system, which consists of a pHBAd series shuttle plasmid and an adenovirus backbone carrying a pBHGlox (delta) E13Cre double vector. After 6 hours of transfection, the medium is replaced with fresh culture medium. On the second day, the virus in the culture medium is added to fresh 293A cell culture medium for a small amount of virus amplification until the cells appear plaques again, and the cells and supernatant are collected. The virus is repeatedly frozen and thawed three times to collect the virus. This virus is the P1 generation virus, and the P1 generation adenovirus is used to infect 293A cells. The infection is carried out for three consecutive generations, and the adenovirus is amplified in large quantities to the P4 generation. After the plaques are formed, the virus is collected and purified and concentrated in vitro. Umbilical cord mesenchymal stem cells are cultured with DMEM culture medium, and when the cell density reaches 70-75%, adenovirus is added. The transduction is completed after 72 hours, and the successfully transduced TAT-PBX1 umbilical cord mesenchymal stem cells are screened using puromycin, and verified by immunoprotein blotting.

[0039] Example 6: Adeno-associated virus packaging of TAT-PBX1, comprising the following steps:

[0040] When the 293A cells reached 50-70% confluence, they were ready for transfection. Within 2 hours before transfection, the medium was replaced with serum-free medium, and Opti MEM was preheated in a 37°C water bath. The LipoFiterTM transfection reagent (Thermo Fisher) was restored to room temperature for use. TAT-PBX1 was recombined into the pAAV-IRES-PURO vector. The TAT-PBX1 plasmid of the AAV vector was constructed and transfected into 293A cells. After 6 hours, fresh complete medium was replaced. After 72 hours, the cells were centrifuged, and the supernatant was removed and the cell pellet was retained. The cell pellet was washed once with PBS and resuspended with 300 μL PBS. The centrifuge tube containing the cell suspension was repeatedly frozen and thawed in liquid nitrogen and a 37°C water bath for 3 times, and centrifuged at 4°C, 3000 rpm / min for 5 minutes. The lysed supernatant containing AAV particles was passed through 0.45 The virus was filtered through a μM filter and collected. To remove the residual plasmid DNA and cell genome in the crude virus extract, Benonase enzyme was added to the virus solution, incubated in a 37°C water bath for 1 hour, and then centrifuged at 4°C, 600 rpm / min for 10 minutes. The supernatant was taken and the virus was purified and concentrated. Umbilical cord mesenchymal stem cells were cultured with DMEM medium. When the cell density reached 70-75%, 0.5 ml of adeno-associated virus was added to every 1 ml. The transduction was completed after 72 hours. Puromycin was used to screen for successfully transduced TAT-PBX1 umbilical cord mesenchymal stem cells, which were verified using immunoblotting.

[0041] Example 7, Experiment on TAT-PBX1 promoting radiation damage repair:

[0042] In vitro experiments: X-RAD320 biological irradiator was used to model the cells, and the cells were divided into blank control group, irradiation group, wild-type hUC-MSCs supernatant group, empty hUC-MSCs supernatant group, TAT-PBX1 overexpressed hUC-MSCs supernatant group, and TAT-PBX1 recombinant protein group. CCK8 method was used to detect cell activity, cell clone formation experiment was used to detect cell proliferation ability, DCFH-DA probe was used to detect cell reactive oxygen species, flow cytometry was used to detect cell apoptosis, and alkaline comet assay and Western blot were used to detect the expression of DNA damage-related proteins.

[0043] In vivo experiment: 6-8 week old SPF male C57BL / 6 mice were randomly divided into 3 groups: blank solvent control group, irradiation control group, and TAT-PBX1 recombinant protein group. They were kept at a constant temperature of 22℃ and constant humidity in a quiet environment. X-RAD320 biological irradiator was used for local abdominal irradiation. The single irradiation dose was 8Gy. The voltage was 180kV, the current was 12.0mA, the source-target distance was 70cm, and the dose rate was 1.02Gy / min. TAT-PBX1 recombinant protein was injected intraperitoneally 2h after irradiation. Ten days later, the mice were killed by cervical dislocation. The required organs were taken and made into paraffin sections. After HE staining, they were observed under a microscope.

[0044] Statistical analysis: All data are expressed as mean ± standard deviation. Statistical analysis and statistical graph generation were performed using GraphPad Prism8 software. When comparing between groups, if the data conformed to normal distribution, one-way ANOVA test was used, and the least significant difference test (LSID) was used for multiple comparisons between groups; if the data variance was unequal, the Kruskal-Wallis test was used, and the test level was a-0.05. P<0.05 was considered statistically significant, and P<0.01 was considered statistically significant.

[0045] Result analysis:

[0046] Verification of the ability of culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 and TAT-PBX1 recombinant protein to inhibit irradiated cell damage and promote clone formation:

[0047] Compared with the irradiated group, TAT-PBX1 umbilical cord mesenchymal stem cell supernatant and TAT-PBX1 recombinant protein can significantly reduce the DNA damage caused by radiation (such as Figure 3 as shown) and promoting cell clone formation (as Figure 4 As shown in the figure, compared with the culture supernatant of wild-type umbilical cord mesenchymal stem cells and the culture supernatant of empty umbilical cord mesenchymal stem cells, the culture supernatant of umbilical cord mesenchymal stem cells overexpressing TAT-PBX1 has a more significant effect;

[0048] Verification that overexpression of TAT-PBX1 in umbilical cord mesenchymal stem cell culture supernatant and TAT-PBX1 recombinant protein reduces radiation-induced cell DNA damage, excessive generation of ROS, and cell apoptosis:

[0049] Compared with the irradiation group, TAT-PBX1 umbilical cord mesenchymal stem cell supernatant and TAT-PBX1 recombinant protein can significantly reduce the DNA damage of cells caused by radiation (as shown in Figure 5). At the same time, compared with the irradiation group, TAT-PBX1 umbilical cord mesenchymal stem cell supernatant and TAT-PBX1 recombinant protein can significantly reduce the cell apoptosis caused by radiation (as shown in Figure 5). Figure 6 As shown in Figure 7 ), in addition, compared with the irradiated group, TAT-PBX1 umbilical cord mesenchymal stem cell supernatant and TAT-PBX1 recombinant protein can also reduce the excessive generation of cellular ROS caused by irradiation (as shown in Figure 7 ). Compared with the wild-type umbilical cord mesenchymal stem cell culture supernatant and the empty umbilical cord mesenchymal stem cell culture supernatant, the umbilical cord mesenchymal stem cell culture supernatant overexpressing TAT-PBX1 has a more significant effect.

[0050] TAT-PBX1 recombinant protein can reduce radiation damage in mice:

[0051] Through the observation of HE staining, we explored the effect of PBX1 on the repair of mouse tissue after irradiation. Figure 8 The results of HE staining of liver tissue were observed under an optical microscope. The HE results after injection of TAT-PBX1 recombinant protein showed that the liver cell structure had a more obvious recovery, the liver lobule structure was relatively intact, and the liver cell inflammation and edema had recovered. The small intestine structure of mice injected with TAT-PBX1 recombinant protein was tighter and more complete than that of the irradiation group. At the same time, the mouse brain tissue was extracted for immunoglobulin imprinting to verify that the TAT-PBX1 recombinant protein can cross the blood-brain barrier to reach the brain tissue. Mouse hippocampal neuron cells were used for verification and it was found that the TAT-PBX1 recombinant protein can inhibit its radiation damage (such as Fig.10 shown).

[0052] In summary, PBX1 attenuates cell senescence and apoptosis by alleviating ROS-mediated DNA damage. Cell-penetrating peptides (CPPs) have been used to deliver a variety of molecules, especially large molecular weight active proteins from the extracellular to the intracellular, such as protamine, TAT peptide or R9, among which transcription transactivator (TAT) is a peptide derived from human immunodeficiency virus and is one of the most commonly used cell-penetrating peptides. When TAT binds to cells, it can enhance the cell's absorption of proteins.

[0053] The PBX1 provided in the embodiment of the present invention is present in the culture medium of cells (such as mesenchymal stem cells, neural crest stem cells, pigment stem cells, and human cells such as fibroblasts). In order to facilitate membrane permeation, PBX1 generally further includes a membrane-penetrating peptide, such as TAT;

[0054] The TAT-PBX1 overexpressing cells or culture supernatant provided in the embodiment of the present invention are cells infected with lentivirus to overexpress TAT-PBX1 protein, because the TAT membrane-penetrating peptide enables the PBX1 protein contained in the cell culture supernatant to enter the cell;

[0055] The TAT-PBX1 recombinant protein provided in the embodiment of the present invention is an exogenous protein expressed by Escherichia coli, yeast or CHO cells. Since the TAT cell-penetrating peptide enhances the absorption of proteins by cells such as neurons and mesenchymal stem cells, PBX1 can pass through the cell membrane and enter the cell nucleus. Experimental results show that the prokaryotically expressed TAT-PBX1 still has transcriptional activity after entering the cell nucleus.

[0056] The TAT-PBX1 recombinant protein provided in the embodiment of the present invention is packaged with lentivirus, adenovirus, adeno-associated virus and retrovirus, and then transduced into human cells, including but not limited to mesenchymal stem cells, neural crest stem cells, pigment stem cells and fibroblasts;

[0057] Upregulation of PBX1 can significantly reduce the excessive accumulation of intracellular ROS, DNA damage, and cell apoptosis caused by radiation. Infection of mesenchymal stem cells with lentivirus overexpressing TAT-PBX1 can upregulate the expression of PBX1 protein.

[0058] TAT-PBX1 recombinant protein can enter the brain tissue and other tissues of mice after intraperitoneal injection or gastrointestinal administration;

[0059] PBX1 or TAT-PBX1 can be used as an active ingredient to treat nuclear radiation damage. Specifically, PBX1 or TAT-PBX1 is mixed with other excipients to prepare a medicine (preparation) for treating nuclear radiation damage.

[0060] Pharmaceutical excipients may be those conventionally used in various preparations, including but not limited to isotonic agents, buffers, flavoring agents, excipients, fillers, adhesives, disintegrants and lubricants, etc.; they may also be selected for use in order to be compatible with the substance, such as emulsifiers, solubilizers, antibacterial agents, analgesics and antioxidants, etc. Such excipients can effectively improve the stability and solubility of the compounds contained in the composition or change the release rate and absorption rate of the compounds, thereby improving the metabolism of various compounds in the organism, thereby enhancing the administration effect of the composition;

[0061] In aqueous solution injections, excipients generally include isotonic agents and buffers, as well as necessary emulsifiers (such as Tweeen-80, Pluronic and Poloxamer), solubilizers and antibacterial agents, etc. In addition, other pharmaceutically acceptable excipients are also included, such as antioxidants, pH regulators and analgesics, etc.

[0062] The excipients used to prepare liquid preparations generally include solvents, water, oils (such as fatty acids), emulsifiers, and necessary preservatives;

[0063] Various excipients and the culture medium supernatant prepared in the embodiments of the present invention are made into dosage forms that are conducive to drug delivery, such as but not limited to aqueous solution injections, nasal drops, eye drops, powder injections, powders, patches, suppositories, emulsions, creams, gels, aerosols, sprays, powder aerosols, sustained-release agents and controlled-release agents. In addition, excipients such as but not limited to gelatin, albumin, chitosan, polyether and polyester polymer materials, such as but not limited to polyethylene glycol, polyurethane, polycarbonate and its copolymers, etc., which are "conducive to drug delivery" mainly include but not limited to improving therapeutic effects, improving bioavailability, reducing toxic side effects and improving patient compliance, etc.;

[0064] In addition, drugs and medical devices can also be combined to make drug-containing medical devices, such as dressings containing PBX1 or TAT-PBX1 of the embodiment of the present invention, PBX1 or TAT-PBX1 of the embodiment of the present invention is loaded or coated on the material as an active ingredient to make medical devices for treating radiation damage, common stent materials such as PLA, PLGA, GelMA and metals, etc., and mixed with biocompatible degradable materials to make microneedles and microneedle arrays, or loaded in metal microneedles to make microneedle chips. When the microneedle penetrates the skin, PBX1 or TAT-PBX1 is released in the epithelial tissue, enters the blood, reaches the damaged organ site, and treats radiation damage.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of PBX1 in the preparation of a drug for treating nuclear radiation damage, characterized in that: The coding gene sequence of PBX1 is shown in SEQ ID No.

1.

2. A TAT-PBX1 recombinant protein, characterized in that: The preparation of the recombinant protein comprises the following steps: The PBX1 coding gene as claimed in claim 1 is amplified by PCR, the PCR product is connected to the cloning vector by cloning, and the correctly connected white clone is picked by blue-white screening; after bacterial liquid PCR, enzyme digestion identification and gene sequencing identification, the PBX1 coding gene fragment in the positive clone is inserted into the prokaryotic expression vector pTAT-HA by enzyme digestion and T4 connection, and then transferred into the prokaryotic expression strain, and the positive transformant is obtained by ampicillin resistance screening; activation in expression culture medium, expansion culture, centrifugation to collect precipitation; placing the precipitation in a lysis solution for ultrasonic crushing and dissolution, centrifugation to collect the supernatant, filtering, purification, and then renaturation by pulse dilution, dialysis and ultrafiltration of the renaturation solution to obtain the TAT-PBX1 recombinant protein.

3. A virus overexpressing TAT-PBX1, characterized in that: The viral vector is one of a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.

4. A drug for treating nuclear radiation damage, characterized in that: The drug comprises the TAT-PBX1 recombinant protein as claimed in claim 2 or the TAT-PBX1 overexpressing virus as claimed in claim 3.

5. The drug according to claim 4, characterized in that The medicine also includes excipients.

6. A medical device for treating nuclear radiation damage, characterized in that: The medical device is loaded with the medicine as claimed in claim 4.

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