Application of ikzf3 in peripheral blood leukocytes in calculating high-energy neutron radiation dose

Abstract

This invention discloses the application of Ikzf3 in peripheral blood leukocytes in calculating high-energy neutron radiation dose, belonging to the field of biodetection technology. This invention focuses on the biological effects and damage mechanisms of neutron radiation, screening potential biomarkers under dose gradients. The LD50 and LD100 of neutron radiation in mice were determined to be 8 Gy and 12 Gy, respectively. The degree of damage to radiation-sensitive organs such as the spleen and intestines increased with the dose gradient. The number of lymphocytes in the blood system decreased significantly with dose, which can serve as an indicator for assessing radiation damage. Cytokines such as IL-6 and IL-10 were significantly upregulated, suggesting inflammatory and immune regulatory mechanisms. Finally, Ikzf3 was screened as a potential key gene, and its expression level changed significantly before and after irradiation, exhibiting extremely high sensitivity. This method, by detecting the expression level of Ikzf3 in peripheral blood leukocytes and substituting it into a dose-response function, can achieve rapid and objective estimation of neutron radiation dose, possessing clinical translational value.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to the application of Ikzf3 in peripheral blood leukocytes in calculating high-energy neutron radiation dose. Background Technology

[0002] Neutron radiation has wide applications in nuclear power plants, radiotherapy, aerospace, and other fields. However, as a representative of high-LET (high-linear-energy-transfer) ionizing radiation, its biotoxicity is significantly higher than that of low-LET radiation such as gamma rays and X-rays. Neutrons induce repulsive protons through elastic scattering and generate heavy nuclear fragments through nuclear reactions, causing difficult-to-repair damage such as dense DNA double-strand breaks and base modifications in biological tissues, posing a serious threat to human health. With the expansion of nuclear technology applications, acute radiation syndrome (ARS) caused by acute neutron radiation exposure has become an important public health and occupational safety issue.

[0003] Currently, radiation dose assessment mainly relies on physical dosimeters and biodosimetry methods. Dicentric chromosome analysis (DCA) is considered the "gold standard" for radiation dose assessment, but its detection cycle is long, throughput is low, and it depends on professional personnel and laboratory conditions, making it difficult to meet the needs of rapid screening of large populations in emergencies such as nuclear accidents. In addition, existing dose assessment studies based on molecular markers such as microRNAs are mostly based on low-LET radiation models, and their dose response stability, tissue specificity, and discrimination ability under neutron radiation, especially high-energy neutron radiation, have not been fully verified, limiting their application in neutron-specific dose assessment.

[0004] Gene expression biomarkers have shown potential in radiation damage assessment due to their ability to be rapidly analyzed using techniques such as reverse transcription quantitative polymerase chain reaction (RT-qPCR) and their advantages of dose- and time-dependent analysis and high tissue specificity. Previous studies have shown a correlation between changes in the expression of genes related to DNA damage repair and cell cycle regulation and radiation dose. However, current research largely focuses on conventional radiation such as gamma rays or X-rays, and specific gene biomarkers for neutron radiation are still relatively scarce, lacking systematic validation across multiple tissues, dose sites, and different post-irradiation time points.

[0005] Neutron radiation exhibits unique microscopic energy deposition characteristics, and its relative biological effect (RBE) varies significantly with neutron energy, dose rate, and biological system, even exhibiting complex biological responses such as reverse dose extension. This limits the applicability of assessment systems based on conventional radiation models in neutron radiation scenarios. Furthermore, high-energy neutron radiation has strong penetrating power and causes severe damage, posing challenges to existing radiation protection materials and dose assessment methods.

[0006] Therefore, there is an urgent need in this field to develop a biomarker and detection method that can rapidly, accurately, and specifically assess neutron radiation exposure dose, in order to overcome the problems of long detection cycle, insufficient throughput, poor neutron specificity, and difficulty in field application in existing technologies, and to meet the urgent need for rapid assessment of neutron radiation dose in nuclear emergency response, occupational protection, and clinical treatment. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a novel strategy for neutron radiation dose determination based on the expression level of Ikzf3 in peripheral blood leukocytes, aiming to overcome key issues in current radiation dose assessment such as strong subjective dependence and complex operation procedures.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: One objective of this invention is to provide an application of a detection product for Ikzf3 in the preparation of products for calculating the neutron radiation dose suffered by a living organism.

[0009] Furthermore, the Ikzf3 is Ikzf3 found in peripheral blood leukocytes.

[0010] Furthermore, the peripheral blood is obtained 24 hours after the organism has been exposed to radiation.

[0011] Furthermore, the testing product includes testing reagents.

[0012] Furthermore, the detection reagents include next-generation sequencing detection reagents.

[0013] The method for detecting mRNA expression levels in peripheral blood leukocytes in this invention can employ various existing detection methods, including, in addition to the second-generation sequencing method, other existing detection methods such as qPCR.

[0014] Furthermore, the application includes using the following dose-response function: D = 13.74 + X Ikzf3 * (-21.502) / (0.5656 + X) Ikzf3 ), R 2 =0.9978; Where D is the irradiation dose and X is the fold change in mRNA expression level.

[0015] Furthermore, by detecting the expression level of Ikzf3 in peripheral blood leukocytes of an organism 24 hours after exposure to neutron radiation, calculating the relative change in its expression level, and substituting it into the dose-response function, the radiation dose can be calculated.

[0016] A second objective of this invention is to provide a product for calculating the neutron radiation dose suffered by an organism, the product comprising the aforementioned detection product for Ikzf3.

[0017] Furthermore, the product includes the following computing modules: D = 13.74 + X Ikzf3 * (-21.502) / (0.5656 + X) Ikzf3 ), R 2 =0.9978; Where D is the irradiation dose and X is the fold change in mRNA expression level.

[0018] The product described in this invention can be a miniature calculator, in which the functional relationship is embedded. Therefore, as long as the user inputs the relative change factor of each mRNA expression level, the neutron radiation dose can be calculated immediately.

[0019] A third objective of this invention is to provide a computer storage medium containing the computing module.

[0020] Compared with the prior art, the present invention has the following significant advantages and outstanding technological progress: This invention focuses on the biological effects and damage mechanisms of neutron radiation, with a particular emphasis on screening potential biomarkers at different dose gradients, and systematically analyzing the damage patterns of neutron radiation in mice. The study determined that 8 Gy and 12 Gy are the LD50 and LD100 of neutron radiation in mice, respectively, clarifying key dose nodes. At the histopathological level, the degree of damage to radiation-sensitive organs such as the spleen and intestines increased progressively with increasing dose, clearly revealing the quantitative relationship between radiation dose and tissue damage. In the hematologic system, the number of lymphocytes significantly decreased with increasing dose, effectively distinguishing radiation damage at different doses and demonstrating its potential as a biomarker for biological dose assessment; cytokines such as IL-6 and IL-10 were significantly upregulated with increasing dose, suggesting radiation-induced inflammatory and immune regulatory mechanisms.

[0021] At the molecular level, using multi-omics analysis, this invention focuses on B cell activation-related pathways and screens for Ikzf3 as a potential key gene. Ikzf3 was significantly downregulated in the spleen and intestine in a dose-dependent manner with increasing radiation dose. qPCR validation further solidified the feasibility of Ikzf3 as a potential biomarker.

[0022] In summary, this invention successfully screened Ikzf3 as a potential biomarker. Ikzf3 exhibits a gradient effect with increasing neutron radiation dose in different tissues, providing a powerful tool for early diagnosis and dose assessment of radiation damage, and possessing significant clinical translational value. Furthermore, the method described in this invention is convenient, rapid, and easy to operate, enabling quick estimation of the neutron radiation dose received by newborn organisms. Attached Figure Description

[0023] Figure 1 The results show the effects of neutron radiation dose on mouse survival and weight in Example 1.

[0024] Figure 2 This section presents the results of the effect of neutron radiation dose on intestinal pathological damage in Example 1, including: A. Changes in mouse intestinal tissue after treatment with different doses of neutron radiation. B. Pathological sections of mouse intestinal tissue after 0, 8, and 12 Gy neutron radiation (40X, 100X, and 200X). C. Effect of different doses of neutron radiation on the length of mouse intestinal villi. D. Effect of different doses of neutron radiation on the depth of mouse intestinal crypts. E. KI67 immunofluorescence staining. F. Statistical analysis.

[0025] Figure 3 The results show the effects of neutron radiation dose on the pathological damage of mouse spleen in Example 1, including: A. Changes in spleen size. B. Organ coefficient. C. Pathological sections of mouse spleen tissue after 0, 8, and 12 Gy neutron radiation (40X, 100X, and 200X). D. Statistical analysis of the number of spleen cells in mice irradiated with different doses of neutron during the same period. E. Ki67 immunofluorescence. F. Statistical analysis of the area of ​​Ki67 positive cells.

[0026] Figure 4 This is the result of the effect of neutron radiation dose on pathological damage of mouse femur in Example 1, including: A. Pathological sections of mouse femur tissue after 0, 8, and 12 Gy neutron radiation (40X, 100X, 200X). B. Statistical analysis of the number of nucleated cells in mouse femur after neutron radiation at different doses during the same period.

[0027] Figure 5 The results show the effect of neutron radiation dose on the blood routine indicators of mice in Comparative Example 1.

[0028] Figure 6 This is the result of the effect of neutron radiation dose on mouse cytokine levels in Example 1.

[0029] Figure 7 This is the result of differential gene analysis of the spleen after neutron radiation in Example 1, including: A. Heatmap of differential genes in the spleen. B. Statistical analysis of the number of differential genes. C. Venn diagram of differential genes.

[0030] Figure 8 The results are the pathway enrichment analysis results of differentially expressed genes in the spleen in Example 1, including: A. KEGG pathway classification analysis; B. GO enrichment analysis; C. Gene-pathway association chord diagram.

[0031] Figure 9The results of the differential gene analysis in the gut after neutron radiation in Example 1 include: A. Heatmap of differential genes in the gut. B. Statistical analysis of the number of differential genes. C. Venn diagram of differential genes.

[0032] Figure 10 The results are from the pathway enrichment analysis of differentially expressed genes in the gut in Example 1, including: A. KEGG pathway classification analysis; B. GO functional enrichment analysis (biological process dimension); C. Gene-pathway association chord diagram.

[0033] Figure 11 The results are the analysis results of spleen and intestinal B cell activation pathways in Example 1, including: A. Spleen GSEA. B. Spleen thermogram. C. Intestinal GSEA. D. Intestinal thermogram.

[0034] Figure 12 This is the result of screening and expression analysis of key genes downregulated in the spleen and intestine in a dose-dependent manner in Example 1, including: A. Venn diagram of the intersection of differentially downregulated genes. B. Gene expression. analyze.

[0035] Figure 13 These are the validation results of qPCR for potential biomarkers of neutron radiation in Example 1, including: A. qPCR detection of Ikzf3 in spleen tissue. B. qPCR detection of Ikzf3 in intestinal tissue. C. qPCR detection of Ikzf3 in blood.

[0036] Figure 14 This is a dose-response curve between the relative change in mRNA expression level in Example 1 and the calculated corresponding irradiation dose.

[0037] Figure 15 This section presents the results of the protective effect of the radiation shielding agent CPG against neutron radiation-induced pathological damage to the spleen, intestine, and femur of mice in Example 1. The results include: A. Histopathological sections of spleen tissue from mice receiving 0Gy, 12Gy, and 12Gy+CPG (100X and 200X). B. Statistical analysis of cell counts in mouse spleen. C. Histopathological sections of femur tissue from mice receiving 0Gy, 12Gy, and 12Gy+CPG (100X and 200X). D. Statistical analysis of nucleated cells in mouse femur. E. Histopathological sections of intestinal tissue from mice receiving 0Gy, 12Gy, and 12Gy+CPG (100X and 200X). F. Statistical analysis of intestinal villus length in mice. G. Statistical analysis of intestinal crypt depth in mice. Detailed Implementation

[0038] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0039] The main contents of the technical solution of this invention are as follows: 1) C57BL / 6 mice were divided into 3 groups (0, 8, 12 Gy) and received whole-body neutron irradiation. Peripheral blood was collected from the mice 24 hours after irradiation. Next-generation sequencing was used to detect changes in mRNA expression profiles in leukocytes and screen out mRNAs with stable and significant expression levels.

[0040] The selected genes should meet the following criteria: a) high radiation sensitivity, i.e., the up-regulation or down-regulation of expression levels before and after irradiation should be as large as possible; b) stable dependence between expression levels and irradiation dose, i.e., mRNA expression is clearly dose-dependent; c) mRNA exists in both mice and humans.

[0041] In addition, although the basal mRNA expression level of each mouse cannot be completely consistent, the difference is relatively small relative to the amount of change, so it will not affect the interpretation of the results.

[0042] 2) Using the unirradiated group as a control, calculate the relative change fold of gene expression and correlate it with the corresponding irradiation dose to construct a dose-response function model of irradiation dose-relative change fold of gene expression.

[0043] In practical applications, the radiation dose can be estimated by detecting the relative fold change in the expression level of this gene in the peripheral blood leukocytes of mice with unknown irradiation doses and then inputting it into the previously established dose-response function.

[0044] This invention ultimately selected a marker gene—Ikzf3—from peripheral blood leukocytes 24 hours after irradiation. Its expression level changed significantly before and after irradiation, exhibiting extremely high sensitivity. A dose-response function relationship was established based on the fold change in its expression level and the irradiation dose. D = 13.74 + X Ikzf3 * (-21.502) / (0.5656 + X) Ikzf3 ), R 2 =0.9978 Where D is the irradiation dose and X is the fold change in mRNA expression level.

[0045] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0046] Example 1 I. Experimental Materials and Methods (a) Chemicals and reagents Ki67 was supplied by Servicebio (Wuhan, China). Ki67 and DAPIFISH were supplied by Servicebio in Wuhan, China. The PCR kit was purchased from Yeasen in Shanghai, China. Primers are shown in Table 1.

[0047] Table 1 Primer Details

[0048] (ii) Animals and their handling Male C57BL / 6 mice were obtained from the Chinese Academy of Sciences (Shanghai). All mice were housed in the laboratory under standard conditions. This experiment was approved by the Laboratory Animal Center of the Naval Medical University of China and complied with the "Guidelines for the Care and Use of Laboratory Animals" (NIH Publication 85-23, National Academy of Sciences Press, Washington, D.C., revised in 1996). Age-, sex-, and weight-matched male C57BL / 6 mice were randomly divided into three groups: 0 Gy (no radiation), 8 Gy, and 12 Gy (all with 14.1 MeV neutron radiation). Each group contained at least three mice.

[0049] (III) Mouse irradiation model This experiment used the High Intensity DT Fusion Neutron Generator (HINEG) at the Institute of Nuclear Energy Safety Technology, Chinese Academy of Sciences, as the fast neutron radiation source. To meet experimental requirements, mice were exposed to different doses of radiation. Specifically, the mice were placed in a specially designed curved plastic box to ensure that each mouse maintained a consistent distance from the neutron source, thus precisely receiving a single dose of high-energy neutron radiation (HENR).

[0050] (iv) Peripheral blood cell count analysis Peripheral blood was collected from the corpus cavernosum / plexus posterior to the orbit and then analyzed using a blood cell analyzer (Sysmex, XN-1000V-1A). The experimental animals were euthanized under anesthesia overdose.

[0051] (v) H&E staining and immunohistochemistry (IHC) Three and a half days after irradiation, intestines, spleen, and femurs were fixed in 4% paraformaldehyde solution for 24 hours and repeatedly washed. Femurs were incubated with decalcification solution for 24 hours, dehydrated with ethanol, clarified with xylene I and II solutions, embedded, sectioned, and dewaxed using a 3µm thick microtome. Slides were stained with hematoxylin and eosin, dehydrated with anhydrous ethanol, and sealed. H&E staining was performed until hydrated, followed by antigen retrieval, rinsing, peroxidase blocking, and final blocking. The blocking solution was removed, and 50µL of primary antibody (Ki67, CD20) was added, and incubated overnight at 4°C. After rinsing, sections were incubated with secondary antibody at room temperature for 1 hour. Samples were incubated with 4′,6′-diamidinyl-2-phenylindole (DAPI) for 5–10 minutes before mounting. Rinsing with phosphate-buffered saline (PBS) was performed between steps. The degree of staining was evaluated under a light microscope.

[0052] (vi) Determination of biological characteristic parameters Each mouse was weighed every two days until death or euthanasia. Weight curves were generated based on the mean and standard error of the mean (SEM) for each group. To calculate the spleen coefficient, the spleen was removed after euthanasia, and after removing surface fat, connective tissue, and blood, the mice were weighed and weighted. The final result was obtained using the following formula: Spleen coefficient (vii) RT-qPCR of mouse tissues and peripheral blood leukocytes Twenty-four hours after irradiation, intestinal and spleen tissues and peripheral blood (200 μL, EDTA anticoagulated) were collected from mice. Tissue samples were used directly for RNA extraction; blood samples were used to separate leukocytes using erythrocyte lysis buffer, and RNA was extracted using the Trizol method, with RNA purity assessed. A PCR kit (asen, Shanghai, China) was used, and the reaction system was configured according to the manufacturer's protocol. PCR detection was performed on an applied biosystems (Thermo Fisher Scientific-Fic, USA) instrument.

[0053] (viii) Human peripheral blood leukocyte RT-qPCR Three healthy adult male volunteers (aged 23–27 years) with no history of acute or chronic diseases, smoking, alcohol consumption, or recent radiation exposure were recruited. Peripheral blood samples (12 mL) were collected via the antecubital vein and aliquoted into three EDTA-anticoagulated vacuum blood collection tubes (4 mL per tube). The samples were then irradiated with doses of 0, 8, and 12 Gy, respectively. The irradiated samples were mixed with an equal volume of RPMI-1640 medium containing 10% fetal bovine serum and transferred to a 25 cm² container. 2Incubation was performed in culture flasks at 37°C and 5% CO2 for 24 hours. Red blood cells were then removed using red blood cell lysis buffer, and the white blood cell pellet was collected by centrifugation. RNA was extracted using the Trizol method, and its purity was determined. A PCR kit (asen, Shanghai, China) was used, and the reaction system was configured according to the manufacturer's protocol. PCR detection was performed on an applied biosystems (Thermo Fisher Scientific-Fic, USA) instrument.

[0054] (ix) RNA sequencing and functional enrichment analysis Total RNA was extracted from mouse intestines and spleen 24 hours after irradiation using Trizol (Invitgen, USA). RNA purity was assessed using NanoVue (GE, USA). Each RNA sample had an A260-A280 ratio greater than 1.8 and an A260-A230 ratio greater than 2.0. Sequencing was performed at OeBiotech (Shanghai, China) using an Illumina HiSeq 2500 system. Raw data was filtered before sequencing to produce high-quality, clean data. All subsequent analyses were performed using this clean data.

[0055] (x) Statistical Analysis All statistical analyses and result graphs were performed using GraphPadPrism 9.5.1 (GraphPad Software, San Diego, California). When the data followed a normal distribution, t-tests and ANOVA were used appropriately. A value of 0.05 is considered statistically significant. These error bars represent the standard error (SEM) of the measurement mean.

[0056] II. Results and Discussion (I) Effects of neutron radiation on mouse survival 1.1 Effects of neutron radiation dose on mouse survival time and body weight changes, and determination of LD50 and LD100 This invention aims to clarify the effects of different doses of neutron radiation on the survival time and body weight of mice, and to determine the key dose effects. In the experiment, mice were treated with neutron radiation at doses of 0 Gy, 2 Gy, 8 Gy, and 12 Gy, respectively. During a 30-day observation period, the results showed ( Figure 1A) In the 0 Gy group, all mice survived, maintaining a survival rate of 100%; the survival rate in the 2 Gy group also remained at 100%; the survival rate in the 8 Gy group significantly decreased to 50%, with most mice dying within 5 to 12 days post-irradiation; and the survival rate in the 12 Gy group plummeted to 0%, with all mice dying within 7 days post-irradiation. Log-rank (Mantel-Cox) analysis showed a p-value of 0.0236 between the 0 Gy and 8 Gy groups, indicating a significant difference; and a p-value less than 0.0001 between the 0 Gy and 12 Gy groups, indicating a highly significant difference. These data clearly demonstrate that 8 Gy and 12 Gy represent the median lethal dose (LD50) and absolute lethal dose (LD100) of neutron radiation for mice, respectively.

[0057] Regarding weight change ( Figure 1 (B) Mice in the 0 Gy group showed stable weight gain during the experiment, increasing by approximately 20% from their initial weight by day 30. Mice in the 2 Gy group experienced a weight loss of approximately 10% in the first ten days after radiation exposure, followed by gradual recovery, reaching 110% of their initial weight by day 30, with no statistically significant difference compared to the 0 Gy group. Mice in the 8 Gy group experienced a weight loss of approximately 25%, with continued fluctuations and limited recovery during the observation period. Mice in the 12 Gy group showed a continuous weight loss, decreasing by approximately 40% by day 7. The dose-dependent trend in weight change was consistent with the survival results, further confirming that 8 Gy (LD50) and 12 Gy (LD100) are key dose points affecting mouse survival and weight.

[0058] Based on the preliminary experimental data above, 8 Gy and 12 Gy can be confirmed as the median lethal dose (LD50) and absolute lethal dose (LD100) of neutron radiation in mice. Both the survival curve and the weight change indicators show a highly consistent dose-response relationship. These findings not only provide a clear dose range for subsequent mechanistic studies and intervention exploration, but also provide reliable and reproducible experimental evidence for dose setting in radiation protection strategies.

[0059] (II) Effects of neutron radiation on pathological damage to mouse tissues and organs 2.1 Characteristics of intestinal pathological damage and dose relationship This invention aims to clarify the dose-response relationship of neutron radiation on pathological changes in mouse intestinal tissue. In the experiment, mice were treated with neutron radiation at doses of 0 Gy, 8 Gy, and 12 Gy, and tissue samples were collected on day 3.5 post-irradiation for observation. Observations revealed that with increasing radiation dose, the macroscopic manifestations of the mouse intestine changed: the intestinal surface became red and swollen, suggesting possible capillary dilation or hemorrhage; the intestinal wall thickness increased, possibly related to tissue edema or inflammatory response. Figure 2 A).

[0060] Histopathological examination of sections (magnifications of 40×, 100×, and 200×) revealed damage to intestinal villi and crypt structures with increasing radiation dose. At low doses, the villi were tightly packed and morphologically intact, and the crypt structures were clearly discernible; however, at high doses, the villi became shorter, fused, and even broke, the crypts became shallower and fewer in number, and the cell arrangement became disordered. Figure 2 B).

[0061] Statistical analysis of the intestinal villus length in mice after different doses of neutron radiation revealed that villus length decreased in a dose-dependent manner with increasing radiation dose. The adjusted p-value between the 8 Gy group and the 0 Gy group was 0.01, indicating a significant difference; however, at a dose of 12 Gy, the adjusted p-value was less than 0.001, indicating a highly significant difference. Figure 2 C).

[0062] Statistical analysis of crypt depth showed that crypt depth decreased significantly with increasing radiation dose. The adjusted p-values ​​between the 8 Gy group and the 0 Gy group were less than 0.001, and the adjusted p-values ​​between the 12 Gy group and the 0 Gy group were also less than 0.001, indicating highly significant differences. Figure 2 D).

[0063] Immunofluorescence staining results showed that neutron radiation significantly inhibited the proliferative activity of intestinal cells. In the 8 Gy and 12 Gy groups, the KI67-positive area was significantly reduced, indicating that intestinal stem cell function was suppressed. Statistical analysis of the cell proliferation index further confirmed this trend. Figure 2 EF).

[0064] In summary, the intestine exhibits significant sensitivity to neutron radiation. When the radiation dose reaches 8 Gy or higher, the damage to intestinal villi and crypt structures increases significantly with increasing radiation dose, while the functional inhibition of intestinal stem cells also shows a clear dose-dependent increasing trend.

[0065] 2.2 Pathological damage characteristics of the spleen and dose relationship To investigate the effects of neutron radiation on the spleen of mice, mice were treated with neutron radiation at doses of 0 Gy, 8 Gy, and 12 Gy, and the spleen was observed on day 3.5 post-irradiation. The results showed that the spleen volume gradually decreased with increasing radiation dose. Figure 3 A), and the decrease in organ coefficient further confirms the reduction in spleen volume ( Figure 3 B).

[0066] HE staining revealed that the damage to the red and white pulp structures of the spleen worsened with increasing radiation dose. Figure 3C). In the 0 Gy control group, the spleen tissue structure remained intact, with clear distribution of red and white pulp regions and abundant lymphocytes. When the radiation dose was increased to 8 Gy, the damage to the spleen tissue structure became obvious, the boundary between the red and white pulp became blurred, and the number of lymphocytes decreased significantly. When the radiation dose was further increased to 12 Gy, the damage to the spleen tissue was even more severe, with a sharp decrease in cell number and disordered tissue structure.

[0067] Cell count statistics revealed a dose-dependent decay in the number of spleen cells ( Figure 3 D). As the radiation dose increases, the number of spleen cells decreases significantly, indicating that radiation has a dose-dependent damaging effect on spleen cells.

[0068] Furthermore, statistical analysis of the Ki67 immunofluorescence-positive area showed that the Ki67-positive area decreased with increasing radiation dose, confirming that radiation has an inhibitory effect on spleen cell proliferation. Figure 3 EF).

[0069] In summary, neutron radiation has a significant dose-dependent effect on mouse spleen tissue. Radiation not only affects spleen volume and cell number but also inhibits spleen cell proliferation, thus causing comprehensive damage to the spleen tissue.

[0070] 2.3 Femoral pathological injury characteristics and dose relationship This invention aims to investigate the dose-response relationship of neutron radiation on the pathological effects of mouse femoral tissue. In the experiment, mice were treated with neutron radiation at doses of 0 Gy, 8 Gy, and 12 Gy, and tissue samples were collected on day 3.5 post-irradiation for observation. The results showed that the degree of damage to the femoral tissue structure gradually increased with increasing radiation dose. Figure 4 A). In the 0 Gy control group, the femoral tissue structure was intact, the trabeculae were arranged in an orderly manner, and the number of cells in the medullary cavity was abundant. Figure 4 A). However, when the radiation dose increased to 8 Gy, significant damage began to appear in the femoral tissue structure, the trabecular structure became blurred, and the number of cells in the medullary cavity decreased significantly.

[0071] Quantitative analysis results showed that with increasing radiation dose, the number of nucleated cells in the femur decreased significantly in a dose-dependent manner. Figure 4 (B) Specifically, the comparison between the 0 Gy and 8 Gy groups showed an adjusted p-value of less than 0.001, as did the comparison between the 0 Gy and 12 Gy groups, both indicating a significant difference between the two groups. Furthermore, the comparison between the 8 Gy and 12 Gy groups also showed an adjusted p-value of less than 0.001. These results all indicate a significant correlation between increased radiation dose and decreased femoral cell count.

[0072] In summary, the femur exhibits significant sensitivity to neutron radiation. At doses of 8 Gy and above, there is a clear positive correlation between increasing radiation dose and aggravated femoral tissue damage. With increasing radiation dose, the number of medullary canal cells in the femur significantly decreases, and the structural integrity of the trabecular bone is impaired.

[0073] (III) Effects of neutron radiation on blood physiological parameters in mice 3.1 Changes in blood routine indicators and their relationship with dosage This invention aims to investigate the effects of neutron radiation on physiological parameters of mouse blood. In the experiment, mice were treated with neutron radiation at doses of 0 Gy, 8 Gy, and 12 Gy, respectively. Blood samples were collected 24 hours after radiation, and their blood routine parameters were detected and analyzed to clarify the trends and patterns of changes in blood routine parameters with neutron radiation dose, providing a basis for assessing the biological effects of neutron radiation.

[0074] Regarding white blood cells (WBCs), the number of WBCs showed a significant decreasing trend with increasing neutron radiation dose. Figure 5 A) The white blood cell (WBC) levels in the 0 Gy group mice were relatively stable and at a high level, while the WBC counts in the 8 Gy and 12 Gy groups were significantly lower than those in the control group, with a more significant decrease in the 12 Gy group compared to the 8 Gy group. The difference between the groups was statistically significant (P < 0.001), indicating that neutron radiation has an inhibitory effect on white blood cells, and this inhibitory effect is positively correlated with the radiation dose. However, according to Tukey's multiple comparison analysis, the WBC difference between the 8 Gy and 12 Gy groups was not significant (P = 0.92). This suggests that although the white blood cell count was lower in the high-dose groups, the degree of dose-induced damage at 8 Gy and 12 Gy cannot be statistically distinguished from that at a statistically significant level. This indicates that while the white blood cell count can reflect the presence of radiation damage, it has certain limitations in accurately determining the specific range of neutron radiation dose.

[0075] In white blood cell differential count, the trend of neutrophil (NEUT) changes was similar to that of WBC. The NEUT content in the 0 Gy group remained stable. After neutron irradiation, the NEUT content in the 8 Gy and 12 Gy groups decreased significantly, with significant differences between groups (P < 0.001), indicating the killing or inhibitory effect of neutron irradiation on neutrophils. The inhibition was more severe in the 12 Gy group than in the 8 Gy group, reflecting that higher doses cause greater damage to neutrophils. However, according to Tukey's multiple comparison analysis, the difference in NEUT between the 8 Gy and 12 Gy groups was not significant (P = 0.39). This suggests that although the neutrophil count was lower in the high-dose groups, the degree of dose-induced damage at 8 Gy and 12 Gy cannot be statistically distinguished from that at a neutrophil count. This indicates that while neutrophil count can reflect the presence of radiation damage, it has limitations in accurately determining the specific range of neutron irradiation dose.

[0076] In contrast, changes in lymphocytes (LYMPH) showed a significant dose-dependent difference. LYMPH levels were normal in the 0 Gy group, while significantly decreased in the 8 Gy and 12 Gy groups, with a greater decrease in the 12 Gy group than in the 8 Gy group. According to Tukey's multiple comparison analysis, the differences between the 0 Gy and 8 Gy groups, the 0 Gy and 12 Gy groups, and the 8 Gy and 12 Gy groups were all statistically significant (P < 0.05). In particular, the comparison between the 8 Gy and 12 Gy groups showed a mean difference of 7.633, a 95% confidence interval of 0.2466 to 15.02, and an adjusted P value of 0.04, indicating that even at higher doses, lymphocyte markers can effectively distinguish radiation damage from different doses. This suggests that lymphocyte count not only reflects damage caused by neutron radiation but also allows for the inverse estimation of radiation dose through changes in lymphocyte count, thus possessing significant application value in neutron radiation biodosage assessment.

[0077] For macrophages (MONO), the experimental results showed no statistically significant difference between the dose groups (P>0.05). This indicates that under the experimental conditions, the change in macrophage number is insufficient to reflect the dose-response relationship of neutron radiation, and may require longer exposure times or higher doses of radiation to observe significant changes. Alternatively, the response mechanism of macrophages after neutron radiation may differ from that of other blood cells, and changes in their indicators cannot be used as an effective indicator for assessing neutron radiation damage. Figure 5 A).

[0078] Furthermore, there were no statistically significant differences in red blood cell count (RBC), hemoglobin (HGB), and hematocrit (HCT) among different dose groups (P>0.05). Figure 5A). This indicates that within the dose range of this experiment, the direct damaging effect of neutron radiation on erythrocytes is limited, possibly due to the relatively high radiation tolerance of erythrocytes, or the role of their damage repair mechanisms during the experimental period. Regarding reticulocytes (NRBCs), the differences between the 0 Gy group and the 8 Gy and 12 Gy groups were statistically significant (P < 0.05), indicating that neutron radiation inhibited the erythrocyte production process. However, the difference in NRBCs between the 8 Gy and 12 Gy groups was not statistically significant (P = 0.96), suggesting that while NRBCs can reflect radiation damage, they cannot distinguish between dose differences of 8 Gy and 12 Gy. Figure 5 B).

[0079] This experiment collected samples 24 hours after irradiation and focused on the acute damage effects induced by neutron radiation. The results showed that the number of lymphocytes decreased significantly and could effectively distinguish damage at different doses, serving as a key indicator for biological dose assessment. While leukocytes, neutrophils, and reticulocytes could reflect the presence of radiation damage, they could not accurately distinguish the high-dose range. Macrophages and erythrocyte-related indicators did not show significant changes.

[0080] 3.2 Changes in cytokine levels and dose relationship To further investigate the effects of neutron radiation on the hematopoietic system of mice, this experiment analyzed the changes in cytokine levels. Blood was collected 24 hours after irradiation, and the supernatant was separated by centrifugation. The expression levels of various cytokines were detected to clarify their trends and patterns of change with neutron radiation dose.

[0081] Experimental results showed that the expression levels of IL-6, IL-10, and IL-27 differed significantly among different dose groups. Figure 6 (B) Among them, the IL-6 level was lowest in the 0 Gy group, while the IL-6 levels in the 8 Gy and 12 Gy groups were significantly increased, with the 12 Gy group showing a higher level than the 8 Gy group, and the difference between the groups was significant (P<0.05). This indicates that IL-6 has a significant response to neutron radiation and may be involved in the early radiation-induced inflammatory response. IL-10 and IL-27 also showed a similar dose-dependent upregulation trend, suggesting that they may play an important role in post-radiation immune regulation. However, the significant changes in these factors were mainly observed between the 12 Gy and 0 Gy groups, while the difference between the 0 Gy and 8 Gy groups was not significant. This limits their application as biomarkers for low-dose radiation, but they still serve as potential biomarkers for acute injury from high-dose neutron radiation.

[0082] Meanwhile, the expression levels of IL-23, IL-1α, IFN-γ, TNF-α, MCP-1, and IL-12p70 also showed a significant dose-dependent upregulation. Figure 6(D) The expression levels of these factors were significantly higher in the 8 Gy and 12 Gy groups than in the 0 Gy group, and even higher in the 12 Gy group than in the 8 Gy group, with significant differences between groups (P < 0.05). This indicates that they are sensitive to neutron radiation and may be involved in radiation-induced immune and inflammatory responses. Similarly, the differences in these factors between the 0 Gy and 8 Gy groups were not significant, suggesting that their application in low-dose radiation assessment may be limited, but they have potential value in high-dose radiation damage assessment.

[0083] Furthermore, no significant differences were observed in the expression levels of cytokines such as IL-1β, IL-17A, IFN-β, and GM-CSF among different dose groups, suggesting that these factors may not be significantly affected in the early stages after neutron radiation, or their changes may be insignificant, thus failing to serve as effective indicators for assessing neutron radiation damage. Figure 6 AB).

[0084] Further analysis revealed that these significantly upregulated cytokines play crucial roles in inflammatory and immune responses. For example, IL-6 and TNF-α, classic pro-inflammatory cytokines, may reflect an early inflammatory response induced by neutron radiation. IFN-γ and IL-12p70 are closely related to immune regulation, and their increase may indicate that the body is attempting to respond to radiation damage by enhancing the immune response. Upregulation of MCP-1 may be associated with the recruitment of inflammatory cells, suggesting that neutron radiation may induce the migration of inflammatory cells to the site of injury. These changes in cytokine expression not only reveal the profound impact of neutron radiation on the mouse hematopoietic system but also provide important clues for understanding the mechanisms of neutron radiation-induced immune and inflammatory responses.

[0085] In summary, this experiment found that neutron radiation significantly upregulated the levels of multiple cytokines in mice, including IL-6, IL-10, IL-27, IL-23, IL-1α, IFN-γ, TNF-α, MCP-1, and IL-12p70. However, the significant changes in these factors were mainly observed between the 12 Gy and 0 Gy groups, while there was no significant difference between the 0 Gy and 8 Gy groups, limiting their application as biomarkers for low-dose radiation. Nevertheless, they could still serve as potential biomarkers for acute injury from high-dose neutron radiation. For cytokines such as IL-1β, IL-17A, IFN-β, and GM-CSF, since there were no significant differences in expression levels among the dose groups, it is speculated that their role in early neutron radiation injury is limited and they cannot be used as effective assessment indicators. These results suggest that the effects of neutron radiation on cytokines are complex, and further research is needed to explore their potential mechanisms and applications.

[0086] (iv) Study on the molecular mechanism of neutron radiation damage 4.1 Spleen sequencing analysis and differential gene screening Previous experiments have identified 8 Gy and 12 Gy as the LD50 and LD100 of neutron radiation in mice, respectively. To further investigate the molecular mechanisms of neutron radiation damage, this study sequenced and analyzed the gene expression profiles of mouse spleen tissue after 8 Gy and 12 Gy neutron radiation. The spleen, as a key organ of the immune system, is highly sensitive to radiation; therefore, this invention focuses on spleen tissue to reveal the gene expression changes induced by neutron radiation and their potential mechanisms. The screening criteria for differentially expressed genes were: a fold change (FC) greater than 1.5 or less than 1 / 1.5, and an adjusted p-value less than 0.05.

[0087] The differential gene heatmap visually illustrates the differences in gene expression levels among different dosage groups. Figure 7 A). The intensity of the color reflects the level of gene expression, helping to quickly identify genes whose expression changes significantly at different doses. The differential gene plot clearly shows the number of upregulated and downregulated genes in the comparisons of 0 Gy vs 8 Gy and 8 Gy vs 12 Gy. Figure 7 (B) Specifically, in the 0 Gy vs 8 Gy group, 2994 genes were upregulated and 1917 genes were downregulated; in the 8 Gy vs 12 Gy group, 947 genes were upregulated and 532 genes were downregulated. These data indicate that gene expression changes significantly with increasing radiation dose.

[0088] The Venn diagram shows the intersection and unique components of differentially expressed genes between the 0Gy vs 8Gy and 8Gy vs 12Gy groups. Figure 7 C). Further analysis identified common and unique genes across different radiation dose groups, revealing a common trend in gene expression changes with increasing radiation dose, as well as dose-specific gene responses. Specifically, 292 genes were upregulated in both comparisons, and 324 genes were downregulated, demonstrating the continuity of gene expression changes with increasing radiation dose. These common genes may play a central role in the response to radiation damage, providing important clues for further research into the molecular mechanisms of neutron radiation damage.

[0089] 4.2 Pathway enrichment analysis of differentially expressed genes from spleen sequencing To delve deeper into the molecular mechanisms of neutron radiation damage, this invention performed pathway enrichment analysis on differentially expressed genes obtained from spleen sequencing, aiming to reveal the functional enrichment of these genes in biological pathways. Through KEGG and GO enrichment analysis and chord diagram visualization of gene associations, the focus was placed on pathways related to the immune system and B cell activation.

[0090] KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in immune system-related pathways, especially immune system processes. Figure 8A). Among them, 59 genes related to the immune system were significantly enriched, and all of them were downregulated, suggesting that neutron radiation may have an inhibitory effect on the immune system. In addition, pathways related to infectious diseases (including viral, parasitic and bacterial) also showed significant gene enrichment, indicating that neutron radiation may weaken the body's immune defense against pathogens.

[0091] GO enrichment analysis further revealed the enrichment of differentially expressed genes in B cell activation-related biological processes. Figure 8 (B) Significant enrichment of B cell activation-related pathways and downregulation of their gene expression suggest that neutron radiation may weaken the immune response by inhibiting B cell function. Other related pathways, including B cell proliferation, positive regulation of type I interferon, and antigen processing and presentation, also showed significant gene enrichment and downregulation, further emphasizing the inhibitory effect of neutron radiation on B cell-mediated immune responses.

[0092] In addition, chord diagrams illustrate the distribution and interrelationships of differentially expressed genes in different pathways. Figure 8 C). The lines connecting genes represent genes participating in multiple pathways simultaneously, visually illustrating the complex interaction network between genes and pathways. These data indicate that neutron radiation-induced downregulation of gene expression involves multiple immune-related pathways, among which B cell activation may be a key link in the suppression of the immune system after neutron radiation damage.

[0093] 4.3 Intestinal sequencing analysis and differential gene screening Previous experiments have identified 8 Gy and 12 Gy as the LD50 and LD100 of neutron radiation in mice, respectively. To further investigate the molecular mechanisms of neutron radiation damage, this study sequenced and analyzed the gene expression profiles of mouse intestinal tissue after 8 Gy and 12 Gy neutron radiation. The intestine, as a vital organ of the digestive system, is highly sensitive to radiation; therefore, this invention focuses on intestinal tissue to reveal the gene expression changes induced by neutron radiation and their potential mechanisms. The screening criteria for differentially expressed genes were: a fold change (FC) greater than 1.5 or less than 1 / 1.5, and an adjusted p-value less than 0.05.

[0094] The differential gene heatmap visually illustrates the differences in gene expression levels among different dosage groups. Figure 9 A). The intensity of color reflects the level of gene expression, helping to quickly identify genes whose expression changes significantly at different doses. The differential gene statistics plot clearly shows the number of upregulated and downregulated genes in the comparisons of 0 Gy vs 8 Gy and 8 Gy vs 12 Gy. Figure 9(B) Specifically, in the 0 Gy vs 8 Gy group, 845 genes were upregulated and 1098 genes were downregulated; in the 8 Gy vs 12 Gy group, 558 genes were upregulated and 317 genes were downregulated. These data indicate that gene expression changes significantly with increasing radiation dose.

[0095] The Venn diagram shows the intersection and unique components of differentially expressed genes between the 0Gy vs 8Gy and 8Gy vs 12Gy groups. Figure 9 (C) Further analysis identified common and unique genes across different dose groups, revealing a common trend in gene expression changes with increasing radiation dose and dose-specific gene responses. Specifically, nine genes were upregulated in both comparisons, while ten genes were downregulated, demonstrating the continuity of gene expression changes with increasing radiation dose. These common genes may play a central role in the response to radiation damage, providing important clues for further research into the molecular mechanisms of neutron radiation damage.

[0096] 4.4 Pathway enrichment analysis of differentially expressed genes from intestinal sequencing To further explore the molecular mechanisms of neutron radiation-induced intestinal damage, this invention performed pathway enrichment analysis on differentially expressed genes obtained from intestinal sequencing, aiming to clarify the functional enrichment characteristics of these genes in biological pathways.

[0097] KEGG pathway classification analysis showed that the proportion of downregulated genes in immune system pathways was higher than in other categories such as infectious diseases and immune diseases. Figure 10 A) suggests that when neutron radiation induces downregulation of gut gene expression, immune system pathways may be one of the main targets. GO functional enrichment analysis further focused on the functional characteristics of downregulated genes; in the biological process (BP) dimension, "B cell activation" was a significantly enriched item. Figure 10 (B) Gene expression patterns showed a downregulated pattern in genes involved in B cell activation, suggesting that neutron radiation may inhibit the biological processes of intestinal B cell activation. Furthermore, pathways such as "B cell differentiation" and "cell surface receptor signaling pathway" also exhibited gene enrichment and downregulated expression, suggesting that radiation's impact on B cell function may involve multiple steps, thereby interfering with B cell-mediated intestinal mucosal immune responses (such as antibody production and pathogen neutralization).

[0098] Furthermore, chord diagrams show that a single gene can simultaneously participate in other immune pathways such as B cell activation pathway and antigen presentation, T cell co-activation, etc. Figure 10(C) This suggests that suppressed B cell activation may be related to synergistic changes in multiple pathways within the intestinal immune network. Synchronous gene expression changes in synergistic pathways involving B cells, antigen-presenting cells, and T cells may influence the "initiation-synergistic-effects" process of the intestinal immune response, exacerbating post-radiation intestinal immunosuppression. In summary, pathway enrichment analysis results indicate that neutron radiation-induced differentially expressed genes in the intestinal pathways related to B cell activation are significantly enriched, with the expression of relevant core genes predominantly downregulated.

[0099] 4.5 GSEA analysis and heatmap display of differentially expressed genes in spleen and intestine To investigate the regulatory mechanisms and tissue specificity of B cell activation pathways under neutron radiation, this invention conducted gene set enrichment analysis (GSEA) and differential gene heatmap visualization analysis on spleen and intestinal tissues, aiming to analyze the significant enrichment of B cell activation pathways and the expression of core genes among different radiation dose groups.

[0100] In spleen tissue, GSEA analysis was performed on the two radiation dose groups (0 Gy vs 8 Gy, 12 Gy vs 8 Gy). Figure 11 A). The results showed that in the spleen 0 Gy vs 8 Gy group, the normalized enrichment fraction (NES) of the B cell activation pathway (GO:0042113) was -2.22, the corrected p-value (p-adjust) was less than 0.001, and the false discovery rate (FDR) was 0.001, indicating that the pathway enrichment was statistically significant. In the spleen 12 Gy vs 8 Gy group, the NES was -2.05, the p-adjust was less than 0.001, and the FDR was 0.002, also indicating that the pathway enrichment was statistically significant. These results indicate that under 8 Gy and 12 Gy neutron radiation, the B cell activation pathway in spleen tissue exhibited significant gene set enrichment characteristics. Differential gene heatmap ( Figure 11 (B) This diagram illustrates the expression patterns of genes related to the B-cell activation pathway in the spleen, with color gradients reflecting relative changes in gene expression levels. The results showed a clear differentiation in gene expression between the 0 Gy group and the 8 Gy and 12 Gy groups, suggesting that increased radiation dose can significantly regulate the expression of genes related to the spleen's B-cell activation pathway.

[0101] In intestinal tissue, since there was no statistically significant difference between the 12Gy and 8Gy groups, a supplementary analysis was conducted on the 12Gy and 0Gy groups. Figure 11C). The results showed that in the 0Gy vs 8Gy group of the intestine, the NES of the B cell activation pathway was -2.05, p-adjust < 0.001, and FDR was 0.002, indicating statistically significant pathway enrichment. In the 12Gy vs 0Gy group of the intestine, the NES was -2.29, p-adjust < 0.001, also indicating statistically significant pathway enrichment. These results indicate that there is a significant enrichment of the B cell activation pathway in intestinal tissue between the 8Gy vs 0Gy and 12Gy vs 0Gy radiation groups; however, there is no statistically significant enrichment between the 12Gy vs 8Gy groups, suggesting that the enrichment pattern of the intestinal B cell activation pathway differs from that in the spleen in a dose-dependent manner. (Differential gene heatmap) Figure 11 D) The expression patterns of genes related to the B cell activation pathway in the gut were presented. The results showed that there were significant gene expression clusters between the 0 Gy group and the 8 Gy and 12 Gy groups, reflecting the regulatory effect of increased radiation dose on the expression of genes related to the B cell activation pathway in the gut.

[0102] In summary, this experiment demonstrates that B-cell activation pathways in both the spleen and intestinal tissues exhibit dose-dependent gene enrichment characteristics under neutron radiation. The differential gene heatmap further validates the regulatory effect of radiation dose on the expression of genes related to B-cell activation pathways, providing a basis for elucidating the tissue-specific mechanisms of neutron radiation-induced immune damage.

[0103] 4.6 Screening for potential biomarkers in the spleen and intestines that are downregulated in a dose-dependent manner To identify key genes that are downregulated in a dose-dependent manner with increasing neutron radiation and to clarify the association between their expression changes and radiation dose, this invention, based on previous differential gene analysis results, performed an intersection analysis on differentially downregulated genes in the spleen (8Gy vs 0Gy, 12Gy vs 8Gy) and intestine (8Gy vs 0Gy, 12Gy vs 8Gy). Figure 12 A).

[0104] The Venn diagram visually illustrates the intersection screening results of differentially downregulated genes in the spleen and intestines across different dose comparison groups, identifying Ms4a1, Cd79a, and Ikzf3 as key genes shared by both groups that are continuously downregulated with increasing dose. Figure 12 A). Figure 12B presents the log2 FoldChange values ​​of the above three genes at different dosage groups. Specifically, in the spleen, the log2 FoldChange values ​​for the 8 Gy vs 0 Gy groups were -1.69, -1.43, and -1.37, respectively, while those for the 12 Gy vs 8 Gy group were -0.87, -1.20, and -0.98; in the intestine, the values ​​for the 8 Gy vs 0 Gy group were -1.54, -0.97, and -0.48, while those for the 12 Gy vs 8 Gy group were -1.13, -0.62, and -0.66. These data clearly validate the dose-dependent downregulation characteristics of these genes.

[0105] In summary, Ms4a1, Cd79a, and Ikzf3 are dose-dependent downregulated genes shared by the spleen and intestine. Their screening results and expression patterns provide important targets for further investigation into the molecular mechanisms of neutron radiation-induced immune tissue damage.

[0106] 4.7 qPCR Validation for Potential Biomarker Screening Literature review revealed that Ikzf3 (Ikaros family zinc finger protein 3, also known as Aiolos), as an important transcription factor of the Ikaros family, plays a central role in B cell development, survival, and immune regulation. Existing research confirms that Ikzf3, through its N-terminal zinc finger domain binding to the DNA consensus sequence, regulates the B cell receptor (BCR) signaling pathway, the NF-κB transcriptional network, and cell cycle-related genes, directly determining B cell fate selection and functional maturation. During B cell development, Ikzf3 forms heterodimers with family members such as Ikzf1, regulating V(D)J rearrangements and immunoglobulin gene expression; its deficiency leads to B cell developmental arrest and a significantly increased risk of lymphoma. Although research on the specificity of neutron radiation is still in its early stages, the expression level of Ikzf3 has been established as a key indicator reflecting the integrity of the B cell lineage, and its transcriptional activity is closely related to lymphocyte homeostasis. In immune injury models, downregulation of Ikzf3 is directly associated with abnormal B cell development and immunosuppression, making it a reasonable candidate biomarker for monitoring radiation-induced immune injury. Based on this, this invention used qPCR to detect the relative expression levels of Ikzf3 in different neutron radiation dose groups (0 Gy, 8 Gy, 12 Gy).

[0107] The results showed that in the spleen tissue ( Figure 13 (A) Ikzf3 expression levels were highest in the 0 Gy group, but significantly downregulated in a dose-dependent manner in the 8 Gy and 12 Gy groups. In intestinal tissue ( Figure 13 (B) This gene also showed the highest expression level in the 0 Gy group, and its expression continuously decreased with increasing radiation dose, with statistically significant differences. Furthermore, this invention also examined the transcriptional level in the blood; the expression changes of Ikzf3 were consistent with the trends in the spleen and intestines. Figure 13 C), all decreased significantly with increasing radiation dose. In summary, the qPCR results were highly consistent with previous sequencing analyses, confirming that Ikzf3 was significantly downregulated in the spleen, intestine, and blood in a dependent manner with increasing neutron radiation dose, providing transcriptional evidence for further investigation into its role in radiation-induced immune tissue damage. Furthermore, changes in Ikzf3 expression in the blood can serve as a potential biomarker for monitoring the effects of neutron radiation on the immune system, possessing significant clinical and innovative value.

[0108] 4.8 Establish the dose-response function relationship Application of Ikzf3 in peripheral blood leukocytes in calculating neutron radiation dose Dose-response function relationship: D = 13.74 + X Ikzf3 * (-21.502) / (0.5656 + X) Ikzf3 ), R 2 =0.9978 Where D is the irradiation dose and X is the relative change fold of mRNA expression level, the radiation dose can be calculated.

[0109] The method for obtaining the relative expression level of mRNA is as follows: Step 1, Blood sample collection: Take 300-500 μL of fresh whole blood and add it to a 2 mL centrifuge tube containing 1 mL of Trizol. Mix thoroughly by pipetting.

[0110] Step 2, RNA Extraction: After standing for 5 minutes, add 0.2 mL of chloroform per 1 mL of Trizol, shake vigorously for 15 seconds, incubate at room temperature for 2-3 minutes, then centrifuge at 12000×g for 15 minutes at 4°C, resulting in three layers. Aspirate the upper aqueous phase (60% of the added Trizol volume), add 0.5 mL of isopropanol per 1 mL of Trizol to precipitate the RNA, incubate at room temperature for 10 minutes, then centrifuge at 12000×g for 10 minutes at 4°C to obtain the RNA precipitate. Wash the precipitate with 1 mL of 75% ethanol per 1 mL of Trizol, and centrifuge at 12000×g for 5 minutes at 4°C. Air dry inverted state at room temperature for 5-10 minutes (avoid over-drying), then dissolve the RNA in 25 μL of enzyme-free sterile water or TE buffer.

[0111] Step 3, RNA Concentration Determination: The total RNA extracted was determined using a Nano Drop nucleic acid quantification instrument. After starting the RNA quantification program, the upper and lower needle tips of the instrument were rinsed with RNase-free water and dried with absorbent paper. 2.5 μL of RNase-free water was loaded to detect background levels, followed by 2.5 μL of RNA sample for analysis. The sample concentration was recorded.

[0112] Step 4, mRNA reverse transcription: This experiment used the Yeasen Biotechnology reverse transcription kit. The reverse transcription reaction system is shown in Table 2 below: Table 2 Details of the reverse transcription reaction system

[0113] The reverse transcription program was set as follows: 25℃ for 5 min, 55℃ for 15 min, 85℃ for 5 min, and then incubated at 4℃. Step 5, mRNA quantification: Prepare the PCR reaction mixture according to the components in Table 3 below on an ice bath: Table 3 Component Configuration Details

[0114] Set the appropriate parameters according to the quantitative PCR instrument used. (1) Pre-denaturation (1 cycle) 95℃ 5min (2) PCR reaction (40 cycles) 95℃ for 10 seconds 60℃ for 30 seconds (3) Dissolution curve (1 cycle) Instrument default settings Record the Ct values ​​and obtain the relative expression level using the following formula: Relative expression level = 2 —［Ct（目的检测—管家检测）—Ct（目的对照—管家对照）］ Dosage estimation: Each sample was tested in triplicate. The relative expression level at each dose point was divided by the relative expression level at 0 Gy to obtain the relative fold change in the mRNA expression level. Substituting this into the equation yielded the estimated radiation dose. Specific experimental results are shown in Table 4 and... Figure 14 As shown in Table 5, the analysis results are as follows.

[0115] Table 4. Experimental Results of Dosage Estimation

[0116] Table 5. Results of dose estimation analysis

[0117] A smaller mean squared error indicates higher estimation accuracy, while a larger mean squared error indicates lower estimation accuracy. Table 5 shows that the dose-response function constructed in this invention is simpler, faster, and yields more objective results compared to existing methods.

[0118] 4.9 Verify the validity of the formula Mice were intraperitoneally injected with 50 μg of CPG7909 (a radiation protectant) 24 hours and 2 hours before whole-body irradiation. Pathological examination indicated a protective effect. Substituting the results into the formula, if the results indicated that the protectant did not affect the dosage assessment (Table 6), the radiation protectant was not affected. Figure 15 (Table 7).

[0119] Table 6. Experimental Results of Dose Estimation Values

[0120] Table 7. Results of Dose Estimation Analysis

[0121] 5.0 Validation of dose-response curves in human peripheral blood Peripheral blood samples from healthy individuals were anticoagulated and then irradiated with different dose groups (0, 8, 12 Gy). The samples were then cultured in 1640 culture medium containing 10% serum at 37°C for 24 hours. After separating leukocytes with erythrocyte lysis buffer, total RNA was extracted using the Trizol method. The extracted RNA was then analyzed by relative quantification of each selected mRNA using real-time quantitative PCR. The dose-response curve was then used, and the formula was valid (Tables 8 and 9).

[0122] Table 8. Experimental Results of Dose Estimation

[0123] Table 9. Results of Dose Estimation Analysis

[0124] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of a detection product for Ikzf3 in the preparation of a product for calculating the neutron radiation dose suffered by a living organism.

2. The application according to claim 1, characterized in that, The Ikzf3 mentioned is Ikzf3 in peripheral blood leukocytes.

3. The application according to claim 2, characterized in that, The peripheral blood was obtained 24 hours after the organism was exposed to radiation.

4. The application according to claim 3, characterized in that, The testing products include testing reagents.

5. The application according to claim 4, characterized in that, The detection reagents include next-generation sequencing detection reagents.

6. The application according to claim 5, characterized in that, The application includes using the following dose-response function: D=13.74+X Ikzf3 *(-21.502) / (0.5656+X Ikzf3 ),R 2 =0.9978; Where D is the irradiation dose and X is the fold change in mRNA expression level.

7. The application according to claim 6, characterized in that, The radiation dose can be calculated by detecting the expression level of Ikzf3 in peripheral blood leukocytes of an organism 24 hours after exposure to neutron radiation, calculating the relative change in expression level, and substituting it into the dose-response function.

8. A product for calculating the dose of neutron radiation received by an organism, characterized in that, The product includes the detection product for Ikzf3 in any of the applications described in claims 1 to 7.

9. The product according to claim 8, characterized in that, The product includes the following computing modules: D=13.74+X Ikzf3 *(-21.502) / (0.5656+X Ikzf3 ),R 2 =0.9978; Where D is the irradiation dose and X is the fold change in mRNA expression level.

10. A computer storage medium, characterized in that, The computer storage medium contains the computing module as described in claim 9.

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