Method, system and equipment for predicting lung injury caused by radon exposure

Abstract

The invention provides a method, a system and equipment for predicting lung injury caused by radon exposure. The application finds that CCL5 shows significant up-regulation in a radon exposure model for the first time, the invention innovatively discloses a prediction method for the lung injury caused by radon exposure, and the prediction method can well predict whether a sample suffers from the lung injury caused by radon exposure; and meanwhile, the CCL5 mediated radon exposure induced DC migration and antigen presentation function enhancement are found, the CCL5 is neutralized to relieve the radon exposure induced lung injury and inflammation, and a new direction is provided for early intervention and early treatment of the radon exposure induced lung injury.

Description

Technical Field

[0001] The present invention belongs to the field of intelligent medical treatment, and specifically relates to a method, system and device for predicting lung damage caused by radon exposure. Background Art

[0002] Radon is a colorless, odorless, inert radioactive gas, mainly produced by the decay of uranium in soil and building materials. When radon decays, it releases high-energy alpha particles and produces a series of radon progeny. Radon and its progeny contribute the most to the natural radiation exposure to the public. Under poor ventilation conditions, radon and its progeny are widely present in human working and living environments. When inhaled by the human body, they are deposited in the lungs. The radiation they release can induce cytotoxic and genotoxic effects in lung tissue. Long-term inhalation can cause damage to the respiratory system and even cause lung cancer.

[0003] Immune response plays a vital role in the early stages of lung injury and lung cancer development. Radon exposure causes oxidative stress, leading to upregulation of inflammatory proteins, which affect the migration and activation of immune cells (such as B cells, dendritic cells, and T cells), and may induce immune activation or disorder. From autoimmune diseases to anticancer treatment, targeting immune factors has become an immunotherapy for many diseases. Immune molecular markers are a hot topic in immunopathology.

[0004] The results of studies on the immune mechanism of lung injury caused by radon exposure are inconsistent, and no relevant immune molecular markers have been found. In addition, radon exposure protection measures focus on radiation protection measures such as ventilation to reduce radon and shielding to prevent radon, and there are few basic studies on medical protection. Therefore, exploring markers of radon exposure-induced lung injury can provide a basis for medical protection research on radon exposure lung injury. Summary of the invention

[0005] To remedy the deficiencies of the prior art, the present invention provides a method, system and device for predicting lung damage caused by radon exposure.

[0006] To achieve the above purpose, the present invention adopts the following technical solution.

[0007] A first aspect of the present invention provides a method for predicting lung damage caused by radon exposure, the method comprising: Obtain gene / protein expression data of the sample to be tested; Extracting expression data of a target gene / protein from the gene / protein expression data, wherein the target gene / protein includes CCL5; Classification prediction is performed based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, a classification result is obtained that the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is low, a classification result is obtained that the sample to be tested does not suffer from lung damage caused by radon exposure.

[0008] A second aspect of the present invention provides a system for diagnosing lung damage caused by radon exposure, the system comprising: Extraction unit: used to extract the expression data of the target gene / protein in the sample to be tested, wherein the target gene / protein includes CCL5; Prediction unit: Perform classification prediction based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, a classification result is obtained that the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is low, a classification result is obtained that the sample to be tested does not suffer from lung damage caused by radon exposure.

[0009] A third aspect of the present invention provides a computer device, the device comprising: Memory: The memory is used to store program instructions; Processor: The processor is used to call program instructions, and when the program instructions are executed, it is used to execute the prediction method described in the first aspect of the present invention.

[0010] A fourth aspect of the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein when the computer program is executed by a processor, the steps of the method described in the first aspect of the present invention are implemented.

[0011] A fifth aspect of the present invention provides use of a reagent for detecting CCL5 expression levels in the preparation of a product for diagnosing lung damage caused by radon exposure.

[0012] Furthermore, the reagent is selected from a probe that specifically recognizes the CCL5 gene, a primer that specifically amplifies the CCL5 gene, or a binding agent that specifically binds to a protein encoded by the CCL5 gene.

[0013] Furthermore, the reagent also includes a detectable label.

[0014] Furthermore, the detectable labels include radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, and bioluminescent moieties.

[0015] Furthermore, the product includes a test kit, a chip or a test paper.

[0016] A sixth aspect of the present invention provides use of a CCL5 inhibitor in the preparation of a pharmaceutical composition for treating lung damage caused by radon exposure.

[0017] Furthermore, the inhibitors include nucleic acid inhibitors and protein inhibitors.

[0018] Furthermore, the inhibitor is selected from protein inhibitors.

[0019] Furthermore, the protein inhibitor is selected from antibodies that neutralize CCL5.

[0020] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0021] The seventh aspect of the present invention provides the use of CCL5 as a target in screening candidate drugs for treating lung injury caused by radon exposure.

[0022] Furthermore, the method for screening candidate drugs for treating lung injury caused by radon exposure includes: treating a culture system expressing or containing the CCL5 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CCL5 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CCL5 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating lung injury caused by radon exposure.

[0023] The eighth aspect of the present invention provides a method for screening candidate drugs for treating lung injury caused by radon exposure, the method comprising: treating a culture system expressing or containing the CCL5 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CCL5 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CCL5 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating lung injury caused by radon exposure.

[0024] The ninth aspect of the present invention provides the use of CCL5 in preparing a product for improving the antigen presentation function of DC cells.

[0025] Furthermore, improving the antigen presenting function of DC cells includes increasing the expression of MHC, CD80 and CD86 on the surface of DC.

[0026] Furthermore, the MHC includes MHC-I and MHC-II.

[0027] Furthermore, the CCL5 is a recombinant CCL5 cytokine.

[0028] The tenth aspect of the present invention provides a method for improving the antigen presenting function of DC cells, the method comprising administering CCL5.

[0029] Furthermore, improving the antigen presenting function of DC cells includes increasing the expression of MHC, CD80 and CD86 on the surface of DC.

[0030] Furthermore, the MHC includes MHC-I and MHC-II.

[0031] Furthermore, the CCL5 is a recombinant CCL5 cytokine.

[0032] Advantages and beneficial effects of the present invention: This application discovered for the first time that CCL5 was significantly upregulated in the radon exposure model, and innovatively disclosed a prediction method for lung injury caused by radon exposure, which can well predict whether a sample suffers from lung injury caused by radon exposure. At the same time, it was found that CCL5 mediated radon exposure-induced DC migration and enhanced antigen presentation function, and neutralization of CCL5 alleviated radon exposure-induced lung injury and inflammation, providing a new direction for early intervention and early treatment of lung injury caused by radon exposure. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a schematic flow chart of the method provided by the present invention; Figure 2 is a schematic flow chart of the system provided by the present invention; Figure 3 is a schematic diagram of a computer device provided by the present invention; Figure 4 It is a diagram of radon exposure and poisoning device and experimental animal poisoning, among which 4A is the poisoning device diagram, 4B is the radon concentration diagram of the external environment of the radon room, 4C is the poisoning condition parameter control diagram, and 4D is the animal poisoning room diagram; Figure 5 This is a graph showing the weight measurement results of mice after exposure to radon; Figure 6 This is a picture of pathological changes in the lungs of mice after radon exposure; Figure 7 Figure 7 is a graph of cytokine levels in lung lavage fluid of mice after radon exposure for 1, 3, and 7 days, wherein Figure 7A is a graph of cytokine levels in lung lavage fluid, and Figures 7B and 7C are graphs of fold changes between the radon-exposed group and the respective control groups; Figure 8 Figure 8 is a graph of cytokine levels in mouse lung tissue after radon exposure for 1, 3, and 7 days, wherein 8A is a graph of cytokine levels in lung tissue, and 8B and 8C are graphs of fold changes between the radon-exposed group and the respective control groups; Fig. 9 9A is a graph of peripheral serum cytokine levels in mice after radon exposure for 1, 3, and 7 days, wherein 9B and 9C are graphs of the fold change between the radon-exposed group and the respective control groups; Fig.10 10A is a graph of CCL5 levels in the lungs of mice exposed to radon detected by Elisa, and 10B is a graph of CCL5 levels in the lung lavage fluid of mice exposed to radon detected by Elisa; Fig.11 The figure is a graph of the expression level of CCL5 protein in the lung tissue of mice after radon exposure detected by WB, wherein 11A is a Western blot graph and 11B is a Western blot statistical graph; Fig.1212A is a graph showing the level of CCL5 in the lung lavage fluid and lung tissue homogenate supernatant of mice after neutralization of CCL5 by Elisa, wherein 12A is a graph showing the level of CCL5 in the lung lavage fluid, and 12B is a graph showing the level of CCL5 in the lung tissue homogenate; Fig.13 This is a graph showing the effect of neutralizing CCL5 on the body weight of mice induced by radon exposure; Fig.14 This is a graph showing that neutralization of CCL5 inhibits the increase in lung weight coefficient in mice induced by radon exposure; Fig.15 This is a graph showing the routine changes in peripheral blood of mice after neutralization of CCL5; Fig.16 Figure 16A is a 100× HE staining image, and Figure 16B is a 400× HE staining image. Fig.17 These are the proliferation and activation diagrams of DC cells in the lung draining lymph nodes of mice 1, 3, and 7 days after a single radon exposure, wherein 17A is a flow cytometric diagram of DC ratio changes, 17B is a statistical diagram of DC ratio changes, 17C is a statistical diagram of DC number changes, 17D is a diagram of changes in DC surface CD86 expression, and 17E is a diagram of changes in DC surface CD80 expression; Fig.18 Figure 18 is a graph showing the proliferation and activation of DC cells in mouse lung tissue 1, 3, and 7 days after a single radon exposure. Figure 18A is a graph showing the change in DC ratio, Figure 18B is a graph showing the change in DC number, Figure 18C is a graph showing the change in DC surface CD86 expression, Figure 18D is a graph showing the change in DC surface CD80 expression, and Figure 18E is a graph showing the change in DC surface PDL1 expression; Fig.19 The graphs are the proliferation and activation of DC and CCR5+DC in mouse lung tissue after cumulative radon exposure for 1, 3, and 7 days, wherein 19A is a graph showing the change in the ratio of DC and CCR5+DC, 19B is a graph showing the change in the number of DC, 19C is a graph showing the change in the expression of CD86 on the surface of DC, 19D is a graph showing the change in the expression of CD80 on the surface of DC, 19E is a graph showing the change in the expression of MHC-II on the surface of DC, and 19F is a graph showing the change in the expression of MHC-I on the surface of DC; Fig. 20 20A is a graph showing the chemotaxis of bone marrow-derived DCs by lung lavage fluid of radon-exposed mice and CCL5, wherein 20A is a graph showing the number of cells that migrated to the lower layer of the Transwell chamber by lung lavage fluid of radon-exposed mice and chemotactic DCs of different concentrations of CCL5, and 20B is a graph showing the regulation of chemotactic DCs by adding CCL5 or Anti-CCL5 neutralizing antibody to lung lavage fluid of radon-exposed mice; Fig.2121A is a graph showing the effect of neutralizing CCL5 in vitro on the immune response of DC in lung tissue induced by radon exposure, wherein 21A is a graph showing the proportion of cells positively expressing activation marker molecules on the surface of DC by flow cytometry, 21B is a statistical graph showing changes in the expression of MHC-I on the surface of DC, 21C is a statistical graph showing changes in the expression of MHC-II on the surface of DC, 21D is a statistical graph showing changes in the expression of CD80 on the surface of DC, and 21E is a statistical graph showing changes in the expression of CD86 on the surface of DC; Fig. 22 22A is a graph showing the effect of neutralizing CCL5 in vivo on the immune response of DC in lung tissue induced by radon exposure, wherein 22B is a graph showing the change in the proportion of CCR5+DC detected by flow cytometry, 22C is a graph showing the change in MHC-II expression on the surface of DC, 22D is a graph showing the change in CD86 expression on the surface of DC, and 22E is a graph showing the change in MHC-I expression on the surface of DC. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0035] In some of the processes described in the specification and claims of the present invention and the above-mentioned figures, multiple operations that appear in a specific order are included, but it should be clearly understood that these operations may not be executed in the order in which they appear in this article or executed in parallel. The serial numbers of the operations, such as 101, 102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions of "first", "second", etc. in this article are used to distinguish different messages, devices, modules, etc., do not represent the order of precedence, and do not limit the "first" and "second" to be different types.

[0036] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0037] Figure 1 The present invention provides a method for predicting lung damage caused by radon exposure. Specifically, the method comprises the following steps: 101: Obtain gene / protein expression data of the sample to be tested; Extract the expression data of the target gene / protein from the gene / protein expression data, wherein the target gene / protein includes CCL5 Classification prediction is performed based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, a classification result is obtained that the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is low, a classification result is obtained that the sample to be tested does not suffer from lung damage caused by radon exposure.

[0038] In some embodiments, the sample to be tested, the subject, or the person to be tested refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., which will be the recipient of a specific treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to human subjects.

[0039] The specific process is as follows: 1. CCL5 levels in lung tissues of radon-exposed mice increased significantly Experimental methods 1. Animal grouping and radon exposure: Vital River purchased SPF female BALB / c mice, all of which were 5-6 weeks old and weighed 16-18g. The mice were divided into a control group and a radon-exposed group. Sampling was immediately performed after the last radon exposure after 1, 3, 7, 15, 30, and 60 days of radon accumulation. Ten mice were sampled from each group at each time point. Mice were exposed to radon using an HD-3PLUS multifunctional automatic radon chamber: radon and its progeny were generated by a radium-226 source with a standard activity of 992 kBq, and gaseous radon-222 and its progeny were pumped into the radon chamber at a rate of 451 Bq / min. When the radon concentration in the exposure chamber stabilized at 1.2×10 5 Bq / m 3 Afterwards, the mice were exposed to radon for 16 hours every day.

[0040] 2. Specimen Collection and Processing After weighing, mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, and peripheral blood was collected from the eyeballs. After opening the chest cavity, the lungs, trachea, thymus and other organs were observed, the trachea of ​​the mice was exposed and intubated with a catheter, the lungs were washed three times with 0.5 ml pre-cooled PBS and the lung lavage fluid was collected, with a recovery rate of more than 90%. The lung lavage fluid was centrifuged at 3000 rpm / min at 4°C for 10 min to remove airway cells, and the supernatant was stored at -80°C for later use. The trachea was cut off and the lungs were removed. After removing excess tissue, the blood on the lung surface was cleaned with filter paper and stored at -80°C for later use.

[0041] 3. Lung tissue pathology sections The isolated lung tissue was fixed, dehydrated, permeabilized, and paraffin-embedded, and then cut into 5 μm slices. The slices were soaked in 90% alcohol, 80% alcohol, and 70% alcohol for 5 min, and finally washed with double distilled water for 5 min, and the washing was repeated 3 times. The water on the slices was dried, and finally the dewaxed slice samples were obtained. The slices were placed in hematoxylin staining solution for 5 min, and then slowly and gently rinsed with running water for 5 s; then differentiated with 1% hydrochloric acid ethanol for 3 s, rinsed with running water for 30 s, and then placed in eosin staining solution for 3 min, and slowly and gently rinsed with running water. The stained slices were soaked in 95% alcohol, 80% alcohol, and 70% alcohol for 5 min, and this operation was repeated once, and then soaked in anhydrous ethanol for 5 min, dehydrated after repeating once, and soaked in xylene for 5 min, and then repeated once again for transparent treatment. Finally, the slices were taken out of xylene, dried, and sealed with neutral gum. The prepared slides were examined under a microscope, image scanned, and pathologically analyzed.

[0042] 4. Cytokine chip technology Raybiotech mouse antibody chip (cat#GSM-CYT-1) was purchased from Huaying Biotechnology (Shanghai, China). The chip can detect 20 cytokines in mouse samples, and the operation process was carried out according to the product instructions.

[0043] 5. Enzyme-linked immunosorbent assay (ELISA) The levels of CCL5 in the lung tissue supernatant and lavage fluid were detected using the mouse CCL5 ELISA kit (SEKM-0043) from Solebio Biotech (Beijing, China) and compared with the results of the cytokine chip. The operation steps were carried out according to the instructions of the kit, and finally the absorbance (OD) values ​​were obtained at wavelengths of 450 nm and 630 nm using a microplate reader (Multiskan Go, Thermo Scientific, USA), and the calibration readings were obtained by OD 450 nm-OD630 nm. The standard curve was constructed using software, and the level of CCL5 in each well was determined according to the standard curve.

[0044] 6. Western Blot After washing with PBS, the tissue was ground with a high-speed low-temperature tissue homogenizer, and then lysed with M-PERVR mammalian protein extraction reagent (Thermo Scientific, USA) at 4 °C for 40 min to extract total protein. All protein samples were denatured by boiling at 100 °C for 10 min with SDS-PAGE loading buffer. Protein samples were separated according to molecular weight by SDS-PAGE electrophoresis, and then electrophoretically transferred to polyvinylidene difluoride membrane (BioRad, USA), blocked with 5% skim milk powder for 1 hour at room temperature, and then incubated with anti-CCL5 (cat#AF5151, Affinity, China) primary antibody at a ratio of 1:1000 at 4 °C overnight, washed, and incubated with secondary antibody (Cambridge, UK) at room temperature for 1.5 hours.

[0045] Experimental Results The results of mouse weight monitoring showed that the weight of mice in the radon exposure group was significantly lower than that in the control group, and this was most significant in the early stage of radon exposure (within 15 days) ( P <0.05), indicating that mice had a stress response in the early stage of radon exposure, resulting in a significant weight loss in the early stage and recovery in the later stage ( Figure 5 ).

[0046] HE staining results of lung tissue pathological sections showed that under low power microscope, some alveolar septa of mice in the radon-exposed group were widened, some alveoli collapsed and fused, and the alveolar cavity in the adjacent area was compensatory dilated; necrotic and sloughed epithelial cells were seen in some bronchi and bronchioles. Under high power microscope, alveolar septa were ruptured in the widened areas, and alveolar epithelial cells were necrotic and sloughed; interstitial venules and alveolar wall capillaries were dilated and congested; inflammatory cell infiltration such as lymphocytes was seen in the pulmonary interstitium, and a small amount of homogeneous powder-stained protein-like substances could be seen; some bronchial and bronchiolar epithelial cells were necrotic and sloughed, with incomplete folds, and inflammatory cell infiltration around bronchi and bronchioles; a small amount of neutrophils were also seen; infiltration of inflammatory cells such as lymphoid cells was seen in some alveolar cavities ( Figure 6 ).

[0047] The results of the cytokine chip detection of 20 cytokine levels in the lung lavage fluid of mice showed that the levels of different types of cytokines in the lung lavage fluid increased after radon exposure for 1, 3, and 7 days. Compared with the control group at each time point, the increase in CCL5 was most obvious after three days of radon exposure ( Figure 7 ).

[0048] The results of the cytokine chip detection of 20 cytokine levels in the supernatant of mouse lung tissue homogenate showed that the levels of different types of cytokines in the lung tissue increased after radon exposure for 1, 3, and 7 days. Among them, after comparing with the control group at each time point, the increase of CCL5 was the most obvious after three days of radon exposure ( Figure 8 ).

[0049] The results of cytokine chip detection of 20 cytokine levels in peripheral serum of mice after radon exposure showed that different types of cytokine levels increased in peripheral serum after radon exposure for 1, 3, and 7 days. Among them, CCL5 increased significantly after radon exposure for 7 days ( Fig. 9 ).

[0050] Elisa detection of CCL5 levels showed that after radon exposure, CCL5 levels in the lung tissue and lung lavage fluid of mice first increased and then tended to recover, but were still significantly higher than those in the control group ( Fig.10 ). Western blot showed that the expression of CCL5 protein in mouse lung tissue increased significantly after radon exposure for 3-15 days ( Fig.11 ).

[0051] In summary, the results of the study showed that radon exposure caused slow weight gain in mice, resulting in damage to the structure of the mouse lung tissue and infiltration of inflammatory cells in the lung interstitium. The levels of 20 cytokines in the lung tissue, BALF and peripheral serum of radon-exposed mice were further detected. The results showed that after 3 days of radon exposure, the level of CCL5 in the lung tissue and BALF of mice was significantly increased; after 7 days of radon exposure, the level of CCL5 in the serum of mice was significantly increased. The results suggest that CCL5 may be a key factor mediating the progression of lung injury in the model of lung injury caused by radon exposure.

[0052] 2. CCL5 mediates the progression of radon exposure-induced lung injury Experimental methods 1. Animal grouping and radon exposure Weitong Lihua purchased SPF female BALB / c mice, all of which were 5-6 weeks old and weighed 16-18g. The mice were divided into a control group (NC), a radon-exposed group (RN), a CCL5 neutralizing antibody (RN+anti-CCL5) and an antibody isotype control (RN+IgG) group during radon exposure. Sampling was immediately performed after the last radon exposure, which lasted for 7, 15, 30 and 60 days. Ten mice were sampled from each group at each time point. The mice were exposed to radon using an HD-3PLUS multifunctional automatic radon chamber, and the radon concentration was stabilized at 1.2×10 5 Bq / m 3 The mice were exposed to cumulative radon for 16 hours per day.

[0053] 2. Neutralization of CCL5 function in lung tissue of mice Within 2 hours before the mice were exposed to radon, the mice in the radon IgG group (RN+IgG) were intraperitoneally injected with 50 μg of isotype control antibody IgG2SA (A3176, Selleck) with an original concentration of 5.56 mg / ml, which was diluted with normal saline to a final volume of 0.1 ml for injection. The mice in the radon inhibition group (RN+anti-CCL5) were intraperitoneally injected with 50 μg of anti-CCL5 neutralizing antibody (A2571, Selleck), and the injection was repeated at the same time every week after radon exposure until the mice were sampled.

[0054] Detection indicators 1) Monitoring of mouse body weight, diet, and lung weight measurement 2) Elisa test to verify whether CCL5 in lung lavage fluid / lung tissue supernatant is neutralized 3) Blood tester to detect peripheral blood routine 4) Pathological sections of lung tissue: HE staining was used to observe inflammatory infiltration and structural changes; Masson staining was used to observe collagen deposition.

[0055] Experimental Results In the CCL5 inhibition mouse model, anti-CCL5 neutralizing antibodies effectively inhibited the function of CCL5 in lung lavage fluid and lung tissue induced by radon exposure ( Fig.12 ); Radon exposure caused slow weight gain in mice, and anti-CCL5 did not improve the slow weight gain caused by radon exposure ( Fig.13 ); Radon exposure causes an increase in the lung weight coefficient of mice, and anti-CCL5 inhibits the increase in the lung weight coefficient caused by radon exposure ( Fig.14 ); Routine blood tests found that radon exposure induced an increase in the number of white blood cells and lymphocytes in peripheral blood first and then a decrease, while neutralization of CCL5 inhibited the fluctuation of white blood cells and lymphocytes ( Fig.15 , Note: a means compared with the NC group, P <0.05; b means compared with RN+IgG group, P <0.05).

[0056] The results of lung tissue HE staining pathological analysis showed that no obvious lesions were found in the untreated group (NC group), and multiple lung tissue sections in the radon-exposed group (RN group) showed mild to mild proliferative lesions. The specific manifestations were: under low-power microscope, local alveolar cavity structure was destroyed, alveolar septa were broken and fused, and slightly widened and thickened; some blood vessels were dilated, congested, and congested, and the surrounding interstitial loose edema was observed, and bleeding was observed in local areas. Under high-power microscope, degenerated, necrotic, and detached alveolar epithelial cells were observed in the widened and thickened alveolar septa, and a small amount of cell hyperplasia was observed in local areas; a small amount of inflammatory cells such as lymphocytes and macrophages were observed in the interstitium; some blood vessels were dilated, and the lumen was filled with red blood cells, and a small amount of red blood cells were observed in the interstitium. Compared with the group injected with the isotype control antibody (RN+IgG group) after radon exposure, the group injected with the anti-CCL5 antibody (RN+anti-CCL5) after radon exposure had less damage to the alveolar structure and less infiltration of inflammatory cells in the lung interstitium. This suggests that neutralization of CCL5 alleviates radon exposure-induced lung injury and inflammation ( Fig.16 ).

[0057] 3. CCL5 may mediate radon exposure-induced lung injury by regulating the migration and function of dendritic cells (DCs) Experimental methods 1. Preparation of Single Cell Suspension from Mouse Lung Tissue The isolated lung tissue was placed in a 50 ml centrifuge tube and cut into pieces with ophthalmic scissors. 5 ml collagenase digestion solution (cat#5798821, Roche) was added to each tube, and the cells were digested in a 37 °C water bath shaker for 40 min. The digested cells were blown and the cells were passed through a 40 μm cell sieve into a 50 ml centrifuge tube. The cells were centrifuged at 4 °C, 2000 r / min for 3 min. The supernatant was removed, and 5 ml PBS was added to each tube. The cells were centrifuged at 4 °C, 3200 r / min for 5 min. 5 ml of 1× red blood cell lysis solution (cat#555899, BD Company) prepared in advance was added. After fully lysed on ice for 8 min, 5 ml PBS was added to wash once, and the cells were centrifuged at 4 °C, 2000 r / min for 3 min. The supernatant was discarded, and 500 μl PBS was added to each tube to resuspend the cells and count the cells. The cell concentration was diluted to 1×10 6 cells / 100 μl.

[0058] 2. Multicolor flow cytometry technology Cell surface markers were detected using an AttuneNxT flow cytometer (Thermo Corporation, USA) with 4 lasers and 14 colors. 6Lung tissue single cell suspension at a concentration of 1 cell / 100 μl was pre-incubated with 1 μl of anti-devitalization dye (cat#423101, Biolegend) and 1 μl of purified anti-mouse CD16 / CD32-FcBlock (cat#101301, Biolegend) at 4°C for 10 min. After washing with PBS, the cells were stained with a mixture of fluorescently conjugated antibodies at 4°C in the dark for 30 min, washed once with buffer, and cell surface markers were detected by flow cytometry. Antibodies used for DC staining included anti-CD45 (cat#157204), anti-CD11c (cat#550261), anti-CCR5 (cat#107006), anti-CD86 (cat#105116), anti-CD80 (cat#104722), anti-MHC-I (cat#116511) and anti-MHC-II (cat#107605). All of the above flow cytometry antibodies were purchased from Biolegend.

[0059] 3. In vitro Isolation, Induction and Co-culture of Mouse Bone Marrow-derived Dendritic Cells (BMDCs) Bone marrow cells were aseptically isolated from the tibia and femur of mice and resuspended in RPMI medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 10 ng / ml interleukin-4 (IL-4) and 25 ng / ml granulocyte-macrophage colony stimulating factor (GM-CSF) for in vitro induction culture for 7 days. On the third day of culture, the medium containing cytokines was renewed at half volume, and immature DCs were collected on the seventh day. The collected DC cells were cultured at 1×10 6 The concentrations of cells / mL were co-cultured with lung lavage fluid from mice exposed to radon, recombinant CCL5 cytokine (HEK293, MCE Company) or CCL5 neutralizing antibody.

[0060] 4. Transwell assay The ability of CCL5 to chemotactic DC cell migration was evaluated using a Transwell co-culture chamber with a filter diameter of 6.5 mm and a pore size of 0.4 μm (cat#3422, Corning). In this model, the Transwell chamber was placed in a 24-well plate. Lung lavage fluid extracted from radon-exposed mice or different concentrations of recombinant CCL5 cytokine (10 / 10 2 / 10 3 ng / ml), and 1×10 5After 24 h of co-culture, the filter was removed and fixed with 700 μl of 4% paraformaldehyde for 15 min, and then washed twice with 1× PBS. The filter was then stained with 700 μl of 0.1% crystal violet for 10 min, washed three times with 1× PBS, and the cells on the top of the filter were carefully wiped off with a cotton swab and photographed under a microscope.

[0061] Experimental results: Flow cytometry results showed that the number and activation of DCs in the lung draining lymph nodes and lung tissues of mice exposed to radon once increased ( Fig.17 , Fig.18 ); The proportion of DC and CCR5+DC in the lung tissue of mice exposed to cumulative radon increased, and the antigen presentation function of DC cells was enhanced, suggesting that CCL5 induced by radon exposure may induce the recruitment of DC cells to migrate to the lung tissue and promote the enhancement of DC cell antigen presentation function ( Fig.19 ).

[0062] Transwell migration experiments showed that mouse lung lavage fluid (RN-BALF) after radon exposure and different concentrations of CCL5 can chemotactic DC cell migration. After regulating the level of CCL5 in the lavage fluid, the effect of RN-BALF on DC cell migration was also effectively regulated, suggesting that CCL5 mediates the migration of DC cells induced by radon exposure ( Fig. 20 ).

[0063] After RN-BALF and DC cells were co-cultured in vitro, flow cytometry was used to detect the activation and antigen presentation function of DC. The results showed that the expression of activation molecules CD80 / 86 and antigen presentation molecules MHC-II on the surface of DC cells stimulated by RN-BALF increased, indicating that RN-BALF can induce DC cell activation and enhance antigen presentation in vitro. Further, recombinant CCL5 or anti-CCL5 neutralizing antibody was added to the co-culture system. CCL5 further promoted RN-BALF-induced DC cell activation and enhanced antigen presentation, while anti-CCL5 inhibited RN-BALF-induced DC cell activation and enhanced antigen presentation ( Fig.21 ). This suggests that CCL5 mediates the enhanced DC migration and antigen presentation function induced by radon exposure.

[0064] After neutralizing the function of CCL5 in mice, they were exposed to radon. After cumulative exposure to radon for 15, 30, and 60 days, flow cytometry was used to detect the number, activation, and antigen presentation function of DC in lung tissue. The results showed that anti-CCL5 inhibited the activation and enhanced antigen presentation function of CCR5+DC cells induced by radon exposure, suggesting that CCL5 mediates the recruitment of DC cells in lung tissue and the enhanced antigen presentation function induced by radon exposure ( Fig. 22 ).

[0065] The above results show that CCL5 mediates the recruitment of DC cells and the enhancement of antigen presentation function in lung tissue induced by radon exposure. As a powerful professional antigen presenting cell, DC plays an initial and core immune regulatory role in the process of innate immunity and adaptive immunity. In addition, studies have reported that DC also plays a key role in the immune mechanism of radiation-induced lung injury. This suggests that CCL5 may mediate radon exposure-induced lung injury by regulating the migration and function of dendritic cells (DC).

[0066] 102: Extracting expression data of a target gene / protein from the gene / protein expression data, wherein the target gene / protein includes CCL5.

[0067] 103: Perform classification prediction on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, the classification result of the sample to be tested suffering from lung damage caused by radon exposure is obtained; if the expression level of the CCL5 is low, the classification result of the sample to be tested not suffering from lung damage caused by radon exposure is obtained.

[0068] Figure 2 A system for diagnosing lung damage caused by radon exposure is provided in an embodiment of the present invention, and the system comprises: Extraction unit 201: used to extract the expression data of target genes / proteins in the sample to be tested, wherein the target genes / proteins include CCL5 Prediction unit 202: Perform classification prediction based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, the classification result of the sample to be tested suffering from lung damage caused by radon exposure is obtained; if the expression level of the CCL5 is low, the classification result of the sample to be tested not suffering from lung damage caused by radon exposure is obtained.

[0069] Figure 3 A computer device provided by an embodiment of the present invention includes: Memory: the memory is used to store program instructions; Processor: the processor is used to call program instructions, and when the program instructions are executed, it is used to execute the steps of the prediction method described in the first aspect of the present invention.

[0070] The present invention also discloses a computer-readable storage medium on which a computer program is stored. When the computer program is executed by a processor, the steps of the method described in the first aspect of the invention are implemented.

[0071] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working processes of the systems, devices and units described above can refer to the corresponding processes in the aforementioned method embodiments and will not be repeated here.

[0072] In the several embodiments provided in the present application, it should be understood that the disclosed systems, devices and methods can be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be an indirect coupling or communication connection through some interfaces, devices or units, which can be electrical, mechanical or other forms.

[0073] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed on multiple network units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0074] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0075] A person skilled in the art may understand that all or part of the steps in the various methods of the above embodiments may be completed by instructing related hardware through a program, and the program may be stored in a computer-readable storage medium, and the storage medium may include: a read-only memory (ROM), a random access memory (RAM), a disk or an optical disk, etc.

[0076] A person skilled in the art can understand that all or part of the steps in the above-mentioned embodiment method can be completed by instructing related hardware through a program, and the program can be stored in a computer-readable storage medium. The above-mentioned storage medium can be a read-only memory, a disk or an optical disk, etc.

[0077] The present invention provides application of a reagent for detecting the expression level of CCL5 in preparing a product for diagnosing lung damage caused by radon exposure.

[0078] In some embodiments, CCL5 includes wild-type, mutant, or fragments thereof. The term encompasses full-length, unprocessed CCL5, any form of CCL5 derived from processing in cells, and naturally occurring variants of CCL5 (e.g., splice variants or allelic variants). The term encompasses, for example, human CCL5 as well as CCL5 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), Gene ID: 6352.

[0079] In some embodiments, a probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing. Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. The probe can be directly or indirectly labeled, including primers. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays.

[0080] In some embodiments, a primer refers to a short nucleic acid sequence, which, as a nucleic acid sequence having a short free 3' terminal hydroxyl group (free 3' hydroxyl group), can form a base pair with a complementary template and serve as a starting point for replicating the template.

[0081] In some embodiments, a binding agent refers to a naturally occurring or non-naturally occurring molecule that specifically binds to a target. Examples of specific binding agents include, but are not limited to, proteins, peptides, nucleic acids, carbohydrates, and lipids. Specific examples of specific binding agents are peptides, peptide mimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies.

[0082] The reagent also includes a detectable label.

[0083] In some embodiments, detectable label refers to a composition that can produce a detectable signal indicating the presence of a target polynucleotide in a determination sample. Suitable labels include, but are not limited to, radioisotopes, nucleotide chromophores, enzymes, substrates, fluorescent molecules, chemiluminescent moieties, magnetic particles, bioluminescent moieties. Therefore, labeling is any composition that can be detected by a device or method, including but not limited to spectroscopy, photochemistry, biochemistry, immunochemistry, electricity, optics, chemical detection devices or any other suitable device. In some embodiments, labeling can be detected visually without the aid of a device.

[0084] Among them, radioactive isotopes include but are not limited to 3 H. 14 C. 35 S. 125 I.131 I.

[0085] Enzymes include, but are not limited to, horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, acetylcholinesterase.

[0086] Fluorescent molecules include, but are not limited to, FITC, rhodamine, and lanthanide phosphors.

[0087] The present invention provides the use of a CCL5 inhibitor in preparing a pharmaceutical composition for treating lung damage caused by radon exposure.

[0088] In some embodiments, the inhibitor refers to any substance that can reduce the activity of CCL5 protein, reduce the stability of CCL5 gene or protein, down-regulate the expression of CCL5 protein, reduce the effective action time of CCL5 protein, or inhibit the transcription and translation of CCL5 gene. These substances can be used in the present application as substances useful for down-regulating CCL5.

[0089] In some embodiments, the inhibitor includes a nucleic acid inhibitor and a protein inhibitor. The nucleic acid inhibitor is selected from: an interfering molecule that uses CCL5 or its transcript as a target sequence and can inhibit CCL5 gene expression or gene transcription, including but not limited to shRNA, siRNA, ribozyme, antisense oligonucleotide, or a construct that can express or form the shRNA, siRNA, ribozyme, antisense oligonucleotide. The protein inhibitor is selected from a substance that specifically binds to the CCL5 protein, such as an antibody or ligand that can inhibit the activity of the CCL5 protein.

[0090] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0091] Pharmaceutically acceptable carriers include, but are not limited to, diluents, binders, surfactants, wetting agents, adsorbent carriers, lubricants, fillers, and disintegrants.

[0092] Among them, diluents such as lactose, sodium chloride, glucose, urea, starch, water, etc.; adhesives such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginate, xanthan gum, hydroxypropyl cellulose and hydroxypropyl methyl cellulose, etc.; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, hexadecanol, etc.; humectants such as glycerol, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin and soap clay, etc.; lubricants such as zinc stearate, monostearate, etc. Glyceryl fatty acid esters, polyethylene glycol, talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolauric sucrose acid ester, sodium lauryl sulfate, magnesium lauryl sulfate, magnesium dodecyl sulfate, etc.; fillers such as mannitol (granular or powdered), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymerized sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, kelp polysaccharide powder, agar powder, calcium carbonate and sodium bicarbonate, etc.; disintegrants such as cross-linked vinyl pyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, cross-linked sodium carboxymethyl cellulose, soybean polysaccharides.

[0093] The pharmaceutical compositions described herein may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally, or via an implanted reservoir device.

[0094] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for predicting lung damage caused by radon exposure, characterized in that: The method comprises: Obtain gene / protein expression data of the sample to be tested; Extracting expression data of a target gene / protein from the gene / protein expression data, wherein the target gene / protein includes CCL5; Classification prediction is performed based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, a classification result is obtained that the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is low, a classification result is obtained that the sample to be tested does not suffer from lung damage caused by radon exposure.

2. A system for diagnosing lung damage caused by radon exposure, characterized in that: The system comprises: Extraction unit: used to extract the expression data of the target gene / protein in the sample to be tested, wherein the target gene / protein includes CCL5; Prediction unit: Perform classification prediction based on the expression data of the target gene / protein to obtain a classification result of whether the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is high, a classification result is obtained that the sample to be tested suffers from lung damage caused by radon exposure; if the expression level of the CCL5 is low, a classification result is obtained that the sample to be tested does not suffer from lung damage caused by radon exposure.

3. A computer device, characterized in that: The device comprises: Memory: The memory is used to store program instructions; Processor: The processor is used to call program instructions, and when the program instructions are executed, it is used to execute the prediction method described in claim 1.

4. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to claim 1 are implemented.

5. Application of reagents for detecting CCL5 expression levels in the preparation of products for diagnosing lung damage caused by radon exposure; Preferably, the reagent is selected from a probe that specifically recognizes the CCL5 gene, a primer that specifically amplifies the CCL5 gene, or a binding agent that specifically binds to a protein encoded by the CCL5 gene; Preferably, the reagent further comprises a detectable label; Preferably, the detectable label comprises a radioisotope, a nucleotide chromophore, an enzyme, a substrate, a fluorescent molecule, a chemiluminescent moiety, a magnetic particle, a bioluminescent moiety; Preferably, the product comprises a kit, a chip or a test paper.

6. Use of CCL5 inhibitors in the preparation of a pharmaceutical composition for treating lung damage caused by radon exposure; Preferably, the inhibitor includes a nucleic acid inhibitor and a protein inhibitor; Preferably, the inhibitor is selected from protein inhibitors; Preferably, the protein inhibitor is selected from antibodies that neutralize CCL5; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

7. Application of CCL5 as a target in screening candidate drugs for the treatment of lung injury caused by radon exposure; Preferably, the method for screening candidate drugs for treating lung injury caused by radon exposure comprises: Treating a culture system expressing or containing the CCL5 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CCL5 gene or the protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CCL5 gene or the protein encoded by it, the substance to be screened is a candidate drug for treating lung injury caused by radon exposure.

8. A method for screening candidate drugs for treating lung damage caused by radon exposure, characterized in that: The method comprises: treating a culture system expressing or containing a CCL5 gene or a protein encoded by it with a substance to be screened; and detecting the expression or activity of the CCL5 gene or a protein encoded by it in the system; wherein, when the substance to be screened inhibits the expression level or activity of the CCL5 gene or a protein encoded by it, the substance to be screened is a candidate drug for treating lung injury caused by radon exposure.

9. Application of CCL5 in the preparation of products for improving the antigen presentation function of DC cells; Preferably, improving the antigen presentation function of DC cells includes increasing the expression of MHC, CD80 and CD86 on the surface of DC; Preferably, the MHC includes MHC-I and MHC-II; Preferably, the CCL5 is a recombinant CCL5 cytokine.

10. A method for improving the antigen presentation function of DC cells, characterized in that: The method comprises administering CCL5; Preferably, improving the antigen presentation function of DC cells includes increasing the expression of MHC, CD80 and CD86 on the surface of DC; Preferably, the MHC includes MHC-I and MHC-II; Preferably, the CCL5 is a recombinant CCL5 cytokine.

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