Construction method and application of model of acute lung injury caused by sea water drowning

Abstract

The invention provides a construction method and application of an acute lung injury model caused by sea water drowning, during model construction, lung microvascular endothelial cells and type II alveolar epithelial cells are inoculated to a transwell insert, the transwell insert is arranged in a lung chip in parallel, the lung chip is arranged in a shaking table to be dynamically cultured for 5 days, a complete gas-liquid interface is formed, and construction of the lung chip is completed; replacing a lower channel of the lung chip with 1mL of a fresh mixed culture medium, slowly adding 500L of artificial seawater into two holes of an upper channel respectively, and placing the lung chip on a shaking table in a 37-DEG C 5% carbon dioxide incubator for dynamic culture for 2 hours. The lung chip technology is applied to the model of the acute lung injury caused by sea water drowning, meanwhile, lung microvascular endothelial cells and II-type alveolar epithelial cells are adopted, sea water is directly added to the prepared lung chip and then slowly permeates into a channel in the lower layer, the model is closer to the real occurrence process of sea water drowning, and the model has the advantages that the model is simple in structure and convenient to use. The method has the beneficial effect of higher clinical value in the related research of the acute lung injury caused by sea water drowning.

Description

Technical Field

[0001] The present invention relates to the field of disease model construction, and in particular to a method for constructing a seawater drowning-induced acute lung injury model and its application. Background Art

[0002] Since acute lung injury (ALI) was first described in 1967, it has been considered a common and fatal disease. Despite the use of lung protective ventilation, neuromuscular blockers and prone positioning, its mortality rate remains at around 40%, so it cannot be ignored [6,12]. Seawater is a complex liquid with a hyperosmotic pressure three times that of plasma. After inhalation of seawater, hyperosmotic seawater can cause serious damage to the lungs, especially the alveolar function, including insufficient pulmonary surfactant, destruction of the air-blood barrier, pulmonary edema formation, inflammation, etc. Seawater drowning lung injury is often more serious than freshwater-induced lung injury [7]. The important feature of ALI is the destruction of the alveolar-capillary barrier due to the death or dysfunction of alveolar epithelial cells and / or pulmonary capillary endothelial cells [13-14]. It can be seen that the construction of the air-blood barrier is the key to the study of acute lung injury caused by seawater drowning.

[0003] Commonly used research models for acute lung injury caused by seawater drowning include ordinary cell culture models, which often use normal human lung epithelial cells (BEAS-2B). The experimental group was stimulated with DMEM culture medium containing 20% ​​seawater for 6 hours, and the control group was cultured with normal complete DMEM culture medium. Then, cell viability, growth inhibition, and cell death were determined by microscopic observation and CCK8 detection [1]. Some experimental groups also stimulated the target cells with DMEM culture medium containing 30% seawater for 6 hours [2]. The main problems in the above-mentioned cell models of acute lung injury caused by drowning are: 1) Seawater is usually mixed with culture medium before being used for cell culture, which does not conform to the actual process of seawater directly entering the airway during drowning and causing damage to cells; 2) There is usually only one type of cell in the model, lacking cell-to-cell interaction, and it is even more impossible to maintain the differentiation and expression of tissue-specific functions. The human body is a complex organism. Seawater drowning does not only affect one type of cell. Alveolar epithelial cells and microvascular endothelial cells play a very important role in the whole process. 3) In the model, the cells are in a static state and there is no hemodynamic influence. Studies have shown that hemodynamics has an impact on cell function, such as cell absorption of drugs. When hemodynamics exists, cell absorption of drugs is greatly reduced [3, 4]. This is also the reason why cell experiments are effective in the development of some drugs, but ineffective in humans. 4) All cells in the model are cultured in culture medium and isolated from air, while alveolar epithelial cells participate in the gas exchange process of oxygen and carbon dioxide in the alveoli, so the cells can be in close contact with gas in the human body. 5) The cells commonly used in the model are BEAS-2B cells, which are actually normal human bronchial epithelial cells, not alveolar epithelial cells. Literature has reported that dripping seawater into the rabbit's trachea increases the permeability of the alveolar capillary membrane [5] and destroys the gas-blood barrier [6]. Therefore, during seawater drowning, a large amount of seawater enters the alveolar cavity, affecting the function of the gas-blood barrier and, in turn, affecting normal gas-blood exchange. Alveolar epithelial cells are a very important type of cell that constitutes the gas-blood barrier and play a very important role in the process of seawater drowning. Therefore, the cell model of acute lung injury caused by seawater drowning needs to be improved. In addition to the cell model, the animal model is also a commonly used research model for acute lung injury caused by seawater drowning. There are two types of drowning methods for animal models. One is to accurately control the amount of seawater inhaled through tracheal intubation, such as injecting 4ml / kg of seawater through tracheal intubation [1, 2, 7], and the other is to directly immerse mice in 25±2℃ water for 35s [8-10].However, there are some problems with animal models: 1) Due to species differences, animal models cannot accurately simulate the physiological and pathological environment of the human body [3, 11]; 2) It often takes a longer time to make the model; 3) Animal models have poor stability. Due to individual differences, each animal has a different tolerance to seawater. During seawater drowning, the mortality rate of animals is high, about 30% [1]; 4) There are disputes about animal ethics [3, 11]. Therefore, animal models are not ideal models for acute lung injury caused by seawater drowning.

[0004] In summary, previous studies on ALI have mainly focused on the cellular and animal levels, and both research methods have various problems. Summary of the invention

[0005] The present invention aims to provide a method for constructing and applying a seawater drowning-induced acute lung injury model, so as to overcome the defects of the commonly used cell models and animal models of existing seawater drowning-induced acute lung injury. The seawater drowning-induced acute lung injury model of the present invention is more consistent with the actual process of human drowning and has more clinical value in the relevant research on seawater drowning-induced acute lung injury.

[0006] In order to solve the above technical problems, the technical solution of the present invention is: According to one aspect of the present invention, a method for constructing a seawater drowning-induced acute lung injury model is provided, comprising the following steps: S1. sterilizing a lung chip with ultraviolet light; S2. modifying a transwell plug with type I collagen colI; S3. inoculating pulmonary microvascular endothelial cells: inoculating 50ul of 1×10 6 / ml cell suspension was placed in the center of the bottom of the 24-well plate, and the transwell insert was placed in the well of the 24-well plate. The 24-well plate with the transwell insert was inverted and placed in a 37℃ 5% carbon dioxide incubator for 2 hours, and then placed upright; S4. Inoculation of type II alveolar epithelial cells: 100ul 1×10 5 / ml cell suspension was inoculated on the upper surface of the transwell plug, placed in a 37℃ 5% carbon dioxide incubator overnight, and the liquid in the transwell plug was aspirated; S5. The transwell plug was removed and placed in the two middle holes of the lung chip, 2ml of mixed culture medium was added to the liquid addition holes on both sides of the lung chip, and the bubbles under the transwell plug were removed; S6. The lung chip was placed on a shaker for dynamic culture for 5 days, and the mixed culture medium was replaced every 1-2 days to form a complete lung air-liquid interface to complete the construction of the lung chip; S7. The lower channel of the lung chip was replaced with 1mL of fresh mixed culture medium, 500µL of artificial seawater was slowly added to the two cell holes in the middle of the upper channel, and the chips were placed in a 37℃ 5% carbon dioxide incubator for dynamic culture on a shaker for 1~4 hours.

[0007] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in step S2, type I collagen coll is diluted in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution, 100uL of the modification solution is added to the Transwell plug, and 500uL of the modification solution is added to the well overnight.

[0008] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, the mixed culture medium in steps S5, S6 and S7 is prepared by RPMI 1640 and HULEC-5a culture medium in a volume ratio of 1:1.

[0009] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in steps S6 and S7, the rotation speed of the shaker is 5 rpm, and the rocking oscillation angle is 10°.

[0010] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in step S7, dynamic culture is performed on a shaker for 2 hours.

[0011] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in step S7, the components of artificial seawater include: NaCl 26.518 g / L, MgSO 4 3.305 g / L, MgCl 2 2.447 g / L, KCl 0.725 g / L, CaCl 2 1.141 g / L, NaHCO 3 0.202 g / L, NaBr 0.083 g / L.

[0012] According to one aspect of the present invention, there is provided an application of the seawater drowning-induced acute lung injury model constructed by the above method in studying the mechanism of seawater drowning-induced acute lung injury, screening drugs for treating seawater drowning-induced acute lung injury, and studying the functions of specific genes in seawater drowning-induced acute lung injury.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention applies lung chip technology to the model of acute lung injury caused by seawater drowning, which makes up for the shortcomings of traditional cell models and animal models. When constructing the model, pulmonary microvascular endothelial cells and type II alveolar epithelial cells are used at the same time, which overcomes the defect of only one type of cell in the original cell culture model; because the lung chip is constructed by human cells, it can simulate the air-liquid interface structure of the lung, and the cells on the chip can directly contact the air and have the characteristics of liquid dynamics, which is impossible for traditional cell models and animal models to achieve; secondly, the present invention adopts the method of adding seawater directly to the prepared lung chip, and then slowly infiltrating into the channel of the lower layer, breaking the traditional mode of mixing seawater with culture medium and then adding it, which is in line with the process of seawater drowning, seawater enters the airway, flows into the alveoli, and then slowly infiltrates into the blood vessels. Therefore, the model of acute lung injury caused by seawater drowning of the present invention simulates the process of seawater slowly infiltrating into the blood vessels through the airway, which is closer to the actual occurrence process of seawater drowning, and has more clinically valuable beneficial effects in the relevant research on acute lung injury caused by seawater drowning. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific implementation of the present invention or the technical solution in the prior art, the drawings required for use in the specific implementation or the description of the prior art are briefly introduced below.

[0015] Figure 1 is a schematic diagram of the structure of an existing lung chip device; Figure 2 This is a schematic diagram of the lung chip, where A is a picture of the prepared lung chip being dynamically cultured on a shaker in a carbon dioxide incubator; B is a working unit of the lung chip; C is a schematic diagram of the structure of the lung chip on the side; Figure 3 This is a picture of immunofluorescence staining of the lung chip; Figure 4 It is a fluorescent staining picture showing the effect of different seawater addition methods on E-cadherin in type II alveolar cells; Figure 5 It is a fluorescence staining picture showing the effect of different seawater addition methods on VE-cadherin in pulmonary microvascular endothelial cells; Figure 6 The fluorescence staining images show the expression of ZO1 in type II alveolar epithelium and VE-cadherin in pulmonary microvascular endothelial cells at different times after adding seawater; Figure 7 It is a fluorescent staining picture showing the effect of LCMR1 knockout and overexpression on type II alveolar epithelium in the seawater-induced acute lung injury model; Figure 8is a fluorescent staining picture showing the effect of PFC on type II alveolar epithelial cells in acute lung injury caused by seawater drowning; Fig. 9 The fluorescence staining images show the effect of PFC on pulmonary microvascular endothelial cells in acute lung injury caused by seawater drowning. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution 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.

[0017] The present invention provides a method for constructing and applying a seawater drowning-induced acute lung injury (ALI) model. In the construction of the ALI model, seawater is directly infused to simulate the process of seawater slowly penetrating into blood vessels through the airways, rather than mixing seawater with culture medium in a certain proportion and then using it for cell culture. Two types of human cells, human alveolar epithelial cells and human microvascular endothelial cells, are used. Both are derived from humans and are incomparable to animal models. In addition, the cell types are richer, and the interaction between cells is increased compared to the culture of one type of cell. The new technology of lung chip is used to construct the air-blood barrier structure of the lung. Lung chip is a new technology reported in 2010 and has been widely used in the research of lung diseases [3, 15-17]. The lung chip is realized through a microfluidic system, which contains two tight microchannels separated by a specific membrane. The middle membrane is coated with collagen. Human alveolar epithelial cells and human pulmonary microvascular endothelial cells are cultured on opposite sides of the membrane. Once the cells grow to confluence, air is introduced into the upper chamber to form an air-liquid interface. Therefore, the lung chip technology can more accurately simulate the air-blood barrier. Figure 1 . Lung chip technology is particularly suitable for constructing the key functional alveolar-capillary interface of the lungs, which increases liquid flow, and the alveolar epithelial cells are in direct contact with the air, which conforms to the characteristics of the air-liquid interface and solves the shortcomings of traditional cell culture. There are currently no reports on the application of this technology to the construction of a seawater drowning-induced acute lung injury model. The successful construction of this model will bring about rapid development in research related to seawater drowning-induced acute lung injury. The construction of seawater drowning-induced acute lung injury on the lung chip takes a short time and has good stability, which can overcome various problems brought about by animal research, such as racial differences, animal ethics, time issues, stability issues, etc.

[0018] The construction method and application of the seawater drowning-induced acute lung injury model of the present invention are described in detail below.

[0019] Construction of a model of acute lung injury induced by seawater drowning 1. Experimental Materials Reagent preparation: Artificial seawater is prepared according to the main components of seawater from the southeastern coastal areas of my country.

[18] :NaCl 26.518 g / L, MgSO 4 3.305 g / L, MgCl 2 2.447 g / L, KCl 0.725 g / L, CaCl 2 1.141 g / L, NaHCO 3 0.202 g / L, NaBr 0.083 g / L.

[0020] Human type II alveolar epithelial cells and human pulmonary microvascular endothelial cells were kindly provided by Dalian Institute of Chemical Physics. The required instruments and reagents are shown in Tables 1 to 3.

[0021] Table 1 Primary antibodies used in the experiments Primary Antibody Production Company Part Number source Dilution concentration ZO-1 monoclonal antibody (ZO1-1A12), Alexa Fluor™ 555 Thermo Fisher Scientific MA3-39100-A555 mouse 1：100 Rabbit anti-pro-Surfactant Protein C (proSP-C) polyclonal antibodyAnti-Surfactant Protein C (proSP-C) Merck AB3786 rabbit 1：100 CD144 (VE-Cadherin) Monoclonal Antibody (16B1), eBioscience™ Thermo Fisher Scientific 14-1449-82 mouse 1：100 E-cadherin monoclonal antibody Wuhan Mitaka Company 60335-1-Ig mouse 1：100 VWF polyclonal antibody Wuhan Mitaka Company 27186-1-AP rabbit 1：100 Table 2 Secondary antibodies used in the experiments Secondary Antibodies Production Company Part Number Dilution concentration HYPERLINK "https: / / www.thermofisher.cn / antibody / product / Goat-anti-Mouse-IgG-HL-Cross-Adsorbed-Secondary-Antibody-Polyclonal / A-11001" Goat anti-Mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor™ 488 Thermo Fisher Scientific A-11001 1:1000 Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, AlexaFluor™ 647 Thermo Fisher Scientific A-21235 1:1000 Goat anti-rabbit IgG (H+L) Highly cross-adsorbed secondary antibody, Alexa Fluor™ 488 Thermo Fisher Scientific A-11034 1:1000 Goat anti-rabbit IgG (H+L) Highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus 647 Thermo Fisher Scientific A-32733 1:1000 Table 3 Reagents and instruments used in the experiment Reagents and instruments company IHC / IF dedicated blocking solution Beijing Sunshine Yingrui Biotechnology Co., Ltd. Cell culture chamber (transwell), containing 24-well plate (PET membrane 6.5mm, pore size 3.0um) Beijing Sunshine Yingrui Biotechnology Co., Ltd. HULEC-5a Cell-Specific Culture Medium Wuhan Pronocell Company Domestic culture medium RPMI 1640 Beijing Sunshine Yingrui Biotechnology Co., Ltd. Carbon dioxide incubator Thermo Scientific, USA Confocal fluorescence microscopy Leica, Germany chip Dalian Institute of Chemical Physics TECAN Multi-function Fluorescence Microplate Reader TECAN, Switzerland 2. Construction of seawater drowning-induced acute lung injury model 2.1. The steps for constructing the seawater drowning-induced acute lung injury model include: S1. UV sterilization of the Lung Chip device: UV sterilize the Lung Chip device overnight.

[0022] S2. Modify the transwell insert with type I collagen (colI): dilute type I collagen (coll) in 0.02M glacial acetic acid at a ratio of 1:100 to obtain the modification solution. Add 100uL of the modification solution to the Transwell insert and 500uL of the modification solution to the wells overnight.

[0023] S3. Pulmonary microvascular endothelial cells seeding: 50ul 1×10 6 / ml cell suspension was placed in the center of the bottom of a 24-well plate, and a transwell plug was placed in the well of the 24-well plate. The cell suspension quickly dispersed after contacting the transwell plug; the 24-well plate with the transwell plug was inverted and placed in a 37°C 5% carbon dioxide incubator for 2 hours, and then placed upright.

[0024] S4. Inoculation of type II alveolar epithelial cells: 100ul 1×10 5 / ml cell suspension was inoculated on the upper surface of the transwell plug and placed in a 37℃ 5% carbon dioxide incubator overnight. The next day, the liquid in the transwell plug was aspirated.

[0025] S5. Take out the transwell insert and place it in the two middle wells of the lung chip. Add 2 ml of mixed culture medium (RPMI 1640 and HULEC-5a volume ratio is 1:1) to the wells on both sides of the lung chip. Note: remove the bubbles under the transwell insert. Figure 2 .

[0026] S6. Place the prepared lung chip on a shaker for dynamic culture for 5 days (eg Figure 2 As shown in the figure), the shaker speed was 5 rpm, the rocking oscillation angle was 10°, and the mixed culture medium was replaced every 1-2 days for 5 days to form a complete lung air-liquid interface and complete the construction of the lung chip.

[0027] S7. Replace the lower channel of the lung chip constructed above with 1 mL of fresh mixed culture medium, slowly add 500 µL of artificial seawater into the two cell wells in the middle of the upper channel respectively, and place it in a 37°C 5% carbon dioxide incubator for dynamic culture on a shaker for 1 to 4 hours. The shaker speed is 5 rpm and the rocking angle is 10°. Preferably, the dynamic culture is carried out on the shaker for 2 hours, and the lung chip is used for subsequent experiments.

[0028] The present invention applies lung chip technology to the model of acute lung injury caused by seawater drowning, which makes up for the shortcomings of traditional cell models and animal models. Since the lung chip is constructed from human cells, it can simulate the air-liquid interface structure of the lung, and the cells on the chip can directly contact the air and have the characteristics of liquid dynamics, which is impossible for traditional cell models and animal models to achieve; secondly, the present invention adopts the method of adding seawater directly to the prepared lung chip, and then slowly infiltrating into the channel of the lower layer, breaking the traditional mode of mixing seawater with culture medium and then adding it, which is consistent with the process of seawater entering the airway, flowing into the alveoli, and then slowly infiltrating into the blood vessels when drowning in seawater. Therefore, the model of acute lung injury caused by seawater drowning of the present invention simulates the process of seawater slowly infiltrating into the blood vessels through the airway, which is closer to the actual occurrence process of seawater drowning, and has more clinically valuable beneficial effects in related research on acute lung injury caused by seawater drowning.

[0029] Please draft the claims of the invention patent application document based on the above content.

[0030] 2.2. Immunofluorescence staining of lung chip The steps of immunofluorescence staining on the lung chip include: a. Rinse the upper and lower channels of the lung chip with PBS for 3 min × 2 times (put on a shaker at a slow speed during rinsing); b. Fix with 4% paraformaldehyde at room temperature for 20 minutes; c. Rinse with PBS for 5 min × 3 times (put on a shaker at a slow speed during rinsing); d. HC / IF special blocking solution for 10 minutes; e. Incubate with primary antibody (diluted 1:100 with blocking solution): overnight at 4°C, protected from light; f. Rinse with PBS for 5 min × 3 times (on a shaker); g. Incubate with secondary antibody (diluted 1:1000 with blocking solution): room temperature for 45 minutes; h. Rinse with PBS for 5 min × 3 times in a dark environment (put on a shaker at a slow speed during rinsing); i. Counterstaining nuclei: add DAPI and incubate in dark for 5-10 minutes; j. In the dark, rinse with PBS for 3 min × 3 times to remove excess DAPI and observe under fluorescence.

[0031] 2.3 Successful construction of lung chip In order to detect the integrity of the lung chip, type II alveolar epithelial cells were labeled with E-cadherin and pulmonary microvascular endothelial cells were labeled with VE-cadherin. Immunofluorescence staining was performed, and the results showed that the cells grew densely and the lung chip was successfully constructed. Figure 3 , where A and C are planar images of the chip, and B and D are three-dimensional imaging images of the chip, where red represents the expression of E-cadherin in type II alveolar epithelial cells, green represents the expression of VE-cadherin in pulmonary microvascular endothelial cells, and blue represents the cell nucleus labeled with DAPI.

[0032] 2.4 Choice of method for adding seawater when constructing a seawater drowning-induced acute lung injury model on a lung chip This study added seawater in two ways: the first method of adding seawater was based on the traditional cell research model, where 1 mL of culture medium and 1 mL of seawater were mixed and then added to the lower channel of the chip; the second method of adding seawater was based on the actual process of seawater drowning, where 1 mL of culture medium was first added to the lower channel of the chip, and then 1 mL of seawater was directly added to the upper channel, referring to the traditional cell research model, and the action time was 6 hours[1]. The test results showed that 6 hours after the addition of seawater, the expression of E-cadherin and VE-cadherin in type II alveolar epithelial cells and pulmonary microvascular endothelial cells in the second method of directly adding seawater decreased significantly, as shown in Figure 1. Figure 4 and Figure 5Both E-cadherin and VE-cadherin are cell junction proteins expressed on the cell membrane. The decrease in their expression levels indicates that the connection between cells is destroyed. The results of this experiment fully demonstrate that the second method of directly adding seawater has a stronger destructive effect on type II alveolar cells and pulmonary microvascular endothelial cells, and the acute lung injury model is caused in a shorter time, which is closer to the animal model of acute lung injury [9]. Secondly, during seawater drowning, seawater is directly inhaled into the lungs instead of being mixed with other liquids before inhalation. Therefore, the second method of directly adding seawater is more consistent with the actual process of seawater drowning.

[0033] 2.5. Selection of seawater exposure time when constructing a seawater drowning-induced acute lung injury model on a lung chip 1 mL of culture medium was added to the lower channel of the lung chip, and then 1 mL of seawater was directly and slowly added to the upper channel. At 1 h, 2 h, 3 h, and 4 h, the type II alveolar epithelial cells on the upper layer of the chip were stained with ZO1, and the pulmonary microvascular endothelial cells on the lower layer of the chip were stained with VE-cadherin. The results are shown in Figure 6 As shown, at 1 hour, the expression of ZO1 and VE-cadherin began to decrease, but the structure of the cell membrane remained basically intact. At 2 hours, the expression of ZO1 and VE-cadherin further decreased, and the structure of some cell membranes was destroyed. At 3 hours, the expression of ZO1 and VE-cadherin decreased significantly, the structure of the cell membrane of type II alveolar epithelial cells was basically destroyed, and the structure of the pulmonary microvascular endothelial cell membrane was mostly destroyed. Since seawater infiltrates into the pulmonary microvascular endothelial cells of the lower channel through the upper alveolar epithelial cells, the damage to the alveolar epithelial cells is earlier, successfully simulating the process of seawater infiltration into the blood vessels through the airway during seawater drowning. At 4 hours, the structure of the two cell membranes was completely destroyed. It can be seen that 2 hours is a suitable seawater action time for this chip model of seawater-induced acute lung injury.

[0034] 3. Application of seawater drowning-induced acute lung injury model 3.1. Studying the function of specific genes in seawater drowning-induced acute lung injury LCMR1 is a new gene discovered by the applicant's research group

[19] . In order to further study its function, the seawater-induced acute lung injury model of the present invention was used. In the experiment, LCMR1 in type II alveolar epithelial cells was knocked down and overexpressed, and divided into three groups, namely normal group, knockdown group and overexpression group. After the three groups of lung chips were successfully constructed, 1 mL of culture medium was added to the lower channel of the lung chip, and 1 mL of seawater was slowly added to the top of the channel. After 2 hours, E-cadherin staining was performed on the upper layer. The results showed that the expression of E-cadherin in the knockdown group was significantly decreased, indicating that the knockdown of LCMR1 aggravated the acute lung injury caused by seawater, see Figure 7The applicant's research group has previously reported that type II alveolar epithelial cell-specific LCMR1 conditional knockout mice lack complete alveolar structure and have lower lung permeability and compliance than the control group

[20] . This is consistent with the results obtained using the seawater-induced acute lung injury model of the present invention, indicating that the seawater-induced acute lung injury model of the present invention can be used to study the function of specific genes in seawater drowning-induced acute lung injury.

[0035] 3.2. Screening of drugs for the treatment of acute lung injury A seawater drowning-induced acute lung injury model was used to explore the efficacy of perfluorocarbon (PFC) in seawater-induced acute lung injury. The experiment was divided into three groups, namely the normal group, the seawater group, and the seawater + PFC group. After the lung chip was successfully constructed, in the seawater group, 1 mL of culture medium was added to the lower channel, and 1 mL of seawater was added to the upper channel; in the seawater + PFC group, 1 mL of culture medium was added to the lower channel, and 1 mL of seawater and 0.2 mL of PFC were added to the upper channel. After 2 hours, the upper type II alveolar epithelial cells were stained for E-cadherin and pulmonary surfactant-associated protein C (SPC), and the lower pulmonary microvascular endothelial cells were stained for VE-cadherin. The results showed that after the addition of PFC, the damage of seawater to the pulmonary air-blood barrier was reduced, indicating that PFC has a protective effect on the pulmonary air-blood barrier, see Figure 8 and Fig. 9 The previous experimental results of the applicant's research group also showed that the vaporization of perfluorocarbons can alleviate the acute lung injury caused by seawater drowning in canines

[21] . This is consistent with the results obtained using the seawater-induced acute lung injury model of the present invention, indicating that the seawater-induced acute lung injury model of the present invention can be used to screen drugs for the treatment of acute lung injury.

[0036] The present invention applies lung chip technology to the acute lung injury model caused by seawater drowning to overcome the defects of the commonly used cell models and animal models of acute lung injury caused by seawater drowning. In the acute lung injury model caused by seawater drowning of the present invention, seawater is directly added to the lung chip, which changes the traditional method and simulates the process of seawater slowly penetrating into the blood vessels through the airway, which is closer to the actual occurrence process of seawater drowning and has more clinical value in the relevant research on acute lung injury caused by seawater drowning.

[0037] The above embodiments are only specific implementation methods of the present invention, which are used to illustrate the technical solutions of the present invention, rather than to limit them. The protection scope of the present invention is not limited thereto. Although the present invention is described in detail with reference to the above embodiments, ordinary technicians in the field should understand that any technician familiar with the technical field can still modify or improve the technical solutions recorded in the above embodiments within the technical scope disclosed by the present invention, or replace some of the technical features therein by equivalents; and these modifications, changes or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the protection scope of the claims.

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Claims

1. A method for constructing a seawater drowning-induced acute lung injury model, characterized in that: The following steps are involved: S1. UV sterilization of the lung chip; S2. Modification of transwell inserts with type I collagen colI; S3. Pulmonary microvascular endothelial cells seeding: 50ul 1×10 6 / ml cell suspension is placed in the center of the bottom of a 24-well plate well, the transwell insert is placed in the well of the 24-well plate, the 24-well plate with the transwell insert is inverted, placed in a 37°C 5% carbon dioxide incubator for 2 hours, and then placed upright; S4. Inoculation of type II alveolar epithelial cells: 100ul 1×10 5 / ml cell suspension was inoculated on the upper surface of the transwell plug, placed in a 37°C 5% carbon dioxide incubator overnight, and the liquid in the transwell plug was dried; S5. Take out the transwell plug and place it in the two middle holes of the lung chip, add 2 ml of mixed culture medium to the liquid addition holes on both sides of the lung chip, and remove the bubbles under the transwell plug; S6. The lung chip was placed in a shaker for dynamic culture for 5 days, and the mixed culture medium was replaced every 1-2 days to form a complete lung air-liquid interface, thereby completing the construction of the lung chip; S7. Replace the lower channel of the lung chip with 1 mL of fresh mixed culture medium, slowly add 500 µL of artificial seawater to the two cell wells in the middle of the upper channel, and place the cells in a 37°C 5% CO2 incubator on a shaker for dynamic culture for 1 to 4 hours.

2. The method for constructing a seawater drowning-induced acute lung injury model according to claim 1, characterized in that: In step S2, type I collagen coll is diluted in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution, 100uL of the modification solution is added to the Transwell plug, and 500uL of the modification solution is added to the well overnight.

3. The method for constructing a seawater drowning-induced acute lung injury model according to claim 1, characterized in that: The mixed culture medium in steps S5, S6 and S7 is prepared by mixing RPMI 1640 and HULEC-5a culture medium in a volume ratio of 1:

1.

4. The method for constructing a seawater drowning-induced acute lung injury model according to claim 1, characterized in that: In steps S6 and S7, the rotation speed of the shaking table is 5 rpm, and the rocking oscillation angle is 10°.

5. The method for constructing a seawater drowning-induced acute lung injury model according to claim 1, characterized in that: In step S7, dynamic culture was performed on the shaker for 2 hours.

6. The method for constructing a seawater drowning-induced acute lung injury model according to claim 1, characterized in that: In step S7, the components of the artificial seawater include: NaCl 26.518 g / L, MgSO4 3.305 g / L, MgCl2 2.447 g / L, KCl 0.725 g / L, CaCl2 1.141 g / L, NaHCO3 0.202 g / L, and NaBr 0.083 g / L.

7. Application of the seawater drowning-induced acute lung injury model constructed according to the method according to any one of claims 1 to 6 in the study of the mechanism of seawater drowning-induced acute lung injury.

8. Use of the seawater drowning-induced acute lung injury model constructed according to the method according to any one of claims 1 to 6 in screening drugs for treating seawater drowning-induced acute lung injury.

9. Use of the seawater drowning-induced acute lung injury model constructed according to the method according to any one of claims 1 to 6 in studying the function of specific genes in seawater drowning-induced acute lung injury.

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