Construction method and application of acute lung injury model induced by seawater drowning

Abstract

The present invention provides a method for constructing a seawater drowning-induced acute lung injury model and its application. When constructing the model, both pulmonary microvascular endothelial cells and type II alveolar epithelial cells are inoculated onto a transwell insert, the transwell insert is placed in a lung chip, and the lung chip is placed on a shaker for dynamic culture for 5 days to form a complete air-liquid interface, thus completing the construction of the lung chip; the lower channel of the lung chip is replaced with 1 mL of fresh mixed culture medium, and 500 μL of artificial seawater is slowly added to each of the two holes in the upper channel, and then it is placed on a shaker in an incubator at 37°C with 5% carbon dioxide for dynamic culture for 2 hours. The present invention applies the lung chip technology to the seawater drowning-induced acute lung injury model, and at the same time uses pulmonary microvascular endothelial cells and type II alveolar epithelial cells. The seawater is directly added to the prepared lung chip and then slowly penetrates into the lower channel. The model is closer to the real occurrence process of seawater drowning and has more clinical value in the related research of seawater drowning-induced acute lung injury.

Description

Technical Field

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

[0002] Since acute lung injury (ALI) was first described in 1967, it has been regarded as a common and fatal disease. Despite the use of lung-protective ventilation, neuromuscular blockers, and the prone position, its mortality rate still remains at about 40%, so it cannot be ignored [6, 12]. Seawater is a liquid with complex components, and its hyperosmolarity is three times that of plasma. After inhaling seawater, the hyperosmolar seawater will cause serious damage to the lungs, especially the function of alveoli, including insufficient pulmonary surfactant, destruction of the air-blood barrier, formation of pulmonary edema, inflammation, etc. Seawater drowning-induced lung injury is often more severe than that caused by fresh water [7]. An 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 seawater drowning-induced acute lung injury.

[0003] Common research models for acute lung injury caused by seawater drowning include ordinary cell culture models, which often use human normal lung epithelial cells (BEAS-2B). In the experimental group, the cells are stimulated with DMEM medium containing 20% seawater for 6 hours, while the control group is cultured with normal complete DMEM medium. Then, cell viability, growth inhibition, and cell death are determined by microscopic observation and CCK8 assay [1]. In some experimental groups, the target cells are also stimulated with DMEM medium containing 30% seawater for 6 hours [2]. The main problems in the above cell models of acute lung injury caused by drowning are as follows: 1) Usually, seawater is mixed with the medium before being used for cell culture, which does not conform to the actual process of seawater directly entering the airway and causing damage to cells during drowning; 2) There is usually only one type of cell in the model, lacking cell-cell interaction and unable to maintain the differentiation and expression of tissue-specific functions. The human body is a complex organism, and seawater drowning does not only act on one type of cell. Alveolar epithelial cells and microvascular endothelial cells play very important roles throughout the process; 3) In the model, the cells are in a static state without the influence of hemodynamics. Some studies have shown that hemodynamics has an impact on cell function. For example, when there is hemodynamic force, the absorption of drugs by cells is greatly reduced [3, 4], which is also the reason why some cell experiments are effective in drug research and development but ineffective in humans; 4) All cells in the model are cultured in the medium and isolated from the air, while alveolar epithelial cells participate in the gas exchange process of oxygen and carbon dioxide in the alveoli. Therefore, these cells can be in close contact with gases in the human body; 5) The cells commonly used in the model are BEAS-2B cells, which are actually human normal bronchial epithelial cells rather than alveolar epithelial cells. There are literature reports that dripping seawater into the trachea of rabbits can increase the permeability of the alveolar capillary membrane [5], and at the same time, the air-blood barrier is damaged [6]. Therefore, during seawater drowning, a large amount of seawater enters the alveolar cavity, affecting the function of the air-blood barrier and further affecting normal gas exchange. Alveolar epithelial cells are an important type of cell that constitutes the air-blood barrier and play a very important role during seawater drowning. Therefore, the cell models of acute lung injury caused by seawater drowning need to be improved. In addition to cell models, another common research model for acute lung injury caused by seawater drowning is the animal model. There are two drowning methods for the animal model. One is to precisely control the amount of seawater inhaled through tracheal intubation, such as injecting 4 ml / kg of seawater through tracheal intubation [1, 2, 7]. The other is to directly immerse the mice in water at 25 ± 2°C for 35 s [8 - 10].However, the problems with animal models are as follows: 1) Due to species differences, animal models cannot accurately simulate the physiological and pathological environments of the human body [3, 11]; 2) They often require a longer model manufacturing time; 3) Animal models have poor stability. Due to individual differences, the degree of seawater tolerance of each animal is inconsistent. During the process of seawater drowning, the mortality rate of animals is high, about 30% [1]; 4) There are controversies regarding 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 mainly focused on the cellular and animal levels, and various problems exist in both research methods. Summary of the Invention

[0005] The present invention aims to provide a method for constructing a model of acute lung injury caused by seawater drowning and its application, so as to overcome the defects of the commonly used cell models and animal models for acute lung injury caused by seawater drowning. The model of acute lung injury caused by seawater drowning in the present invention is more in line with the real process of human drowning and has greater clinical value in related research on acute lung injury caused by seawater drowning.

[0006] To solve the above technical problems, the technical solution of the present invention is as follows:

[0007] According to one aspect of the present invention, a method for constructing a model of acute lung injury caused by seawater drowning is provided, including the following steps: S1. Ultraviolet sterilization treatment of the lung chip; S2. Modifying the transwell insert with type I collagen colI; S3. Inoculation of pulmonary microvascular endothelial cells: Placing 50 μl of 1×10 6 / ml cell suspension at the center of the bottom of a 24-well plate well, placing the transwell insert into the well of the 24-well plate, inverting the 24-well plate with the transwell insert and placing it in a 37°C 5% carbon dioxide incubator for 2 h, and then placing it upright; S4. Inoculation of type II alveolar epithelial cells: Inoculating 100 μl of 1×10 5 / ml cell suspension on the upper surface of the transwell insert, placing it in a 37°C 5% carbon dioxide incubator overnight, and sucking out the liquid in the transwell insert; S5. Removing the transwell insert and placing it into the middle two holes of the lung chip, adding 2 ml of mixed culture medium to the liquid addition holes on both sides of the lung chip, and removing the bubbles under the transwell insert; S6. Dynamically culturing the lung chip on a shaker for 5 days, replacing the mixed culture medium every 1 - 2 days to form a complete air-liquid interface of the lung, and completing the construction of the lung chip; S7. Replacing the lower channel of the lung chip with 1 mL of fresh mixed culture medium, slowly adding 500 μL of artificial seawater to the two cell holes in the middle of the upper channel respectively, and dynamically culturing it on a shaker in a 37°C 5% carbon dioxide incubator for 1 - 4 hours.

[0008] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in step S2, type I collagen coll is diluted at a ratio of 1:100 in 0.02 M glacial acetic acid to obtain a modification solution. 100 μL of the modification solution is added into the Transwell insert, and 500 μL of the modification solution is added into the well, and left overnight.

[0009] 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 mixing RPMI 1640 and HULEC-5a medium at a volume ratio of 1:1.

[0010] 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°.

[0011] 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.

[0012] Optionally, in the above method for constructing a seawater drowning-induced acute lung injury model, in step S7, the components of the artificial seawater include: 26.518 g / L of NaCl, 3.305 g / L of MgSO4, 2.447 g / L of MgCl2, 0.725 g / L of KCl, 1.141 g / L of CaCl2, 0.202 g / L of NaHCO3, and 0.083 g / L of NaBr.

[0013] According to one aspect of the present invention, there is provided the use of the seawater drowning-induced acute lung injury model constructed by the above method in the study of the mechanism of seawater drowning-induced acute lung injury, screening for drugs for treating seawater drowning-induced acute lung injury, and studying the function of specific genes in seawater drowning-induced acute lung injury.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] The present invention applies the lung-on-a-chip technology to the model of acute lung injury caused by seawater drowning, making up for the shortcomings of traditional cell models and animal models. When constructing the model, both pulmonary microvascular endothelial cells and type II alveolar epithelial cells are used simultaneously, overcoming the defect that there is only one type of cell in the original cell culture model. Since the lung-on-a-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 possess the characteristics of fluid dynamics, which cannot be achieved by traditional cell models and animal models. Secondly, the present invention directly adds seawater to the prepared lung-on-a-chip and then slowly infiltrates it into the lower channels, breaking the traditional mode of adding seawater after mixing it with the culture medium, which conforms to the process of seawater entering the airway, flowing into the alveoli, and then slowly infiltrating into the blood vessels during seawater drowning. Therefore, the model of acute lung injury caused by seawater drowning in the present invention simulates the process of seawater slowly infiltrating into the blood vessels through the airway, being closer to the actual occurrence process of seawater drowning, and having more clinical value in the related research on acute lung injury caused by seawater drowning. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art.

[0017] Figure 1 is a schematic structural diagram of an existing lung-on-a-chip device;

[0018] Figure 2 is a schematic diagram of the lung-on-a-chip, where A is a picture of the prepared lung-on-a-chip being dynamically cultured on a shaker in a carbon dioxide incubator; B is a working unit of the lung-on-a-chip; C is a schematic side view of the lung-on-a-chip;

[0019] Figure 3 is a picture of immunofluorescence staining of the lung-on-a-chip;

[0020] Figure 4 is a fluorescence staining picture showing the effect of different seawater addition methods on E-cadherin in type II alveolar cells;

[0021] Figure 5 is a fluorescence staining picture showing the effect of different seawater addition methods on VE-cadherin in pulmonary microvascular endothelial cells;

[0022] Figure 6 is a fluorescence staining picture showing the expression of ZO1 in type II alveolar epithelial cells and the expression of VE-cadherin on pulmonary microvascular endothelial cells at different times after adding seawater;

[0023] Figure 7It is a fluorescence staining picture showing the effects of LCMR1 knockout and overexpression on type II alveolar epithelial cells in a seawater-induced acute lung injury model;

[0024] Figure 8 It is a fluorescence staining picture showing the effect of PFC on type II alveolar epithelial cells in seawater drowning-induced acute lung injury;

[0025] Figure 9 It is a fluorescence staining picture showing the effect of PFC on pulmonary microvascular endothelial cells in seawater drowning-induced acute lung injury. Detailed implementation manners

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0027] The present invention provides a method for constructing a seawater drowning-induced acute lung injury (ALI) model and its application. In the construction of the ALI model, a method of directly pouring seawater is adopted to simulate the process of seawater slowly penetrating into blood vessels through the airway, rather than mixing seawater with a culture medium in a certain proportion and then using it for cell culture; two types of human cells, namely human alveolar epithelial cells and human microvascular endothelial cells, are used, both of which are derived from humans and are incomparable to animal models. In addition, the cell types are more abundant, and the interaction between cells is increased compared with the culture of a single type of cell; a new technology of lung-on-a-chip is adopted to construct the gas-blood barrier structure of the lung. The lung-on-a-chip is a new technology reported in 2010 and has been widely used in the research of lung diseases [3, 15 - 17]. The lung-on-a-chip is realized through a microfluidic system, which contains two closely spaced microchannels separated by a specific membrane, and 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 and air is introduced into the upper chamber, a gas-liquid interface can be formed. Therefore, the lung-on-a-chip technology can more accurately simulate the gas-blood barrier, see Figure 1 . The lung-on-a-chip technology is particularly suitable for constructing the key functional alveolar-capillary interface of the lung. It increases liquid flow, and the alveolar epithelial cells are directly in contact with air, meeting the characteristics of the gas-liquid interface, solving the deficiencies of traditional cell culture. At present, there is no report on applying this technology to the construction of a seawater drowning-induced acute lung injury model. The successful construction of this model will bring rapid development to the research related to seawater drowning-induced acute lung injury. Constructing seawater drowning-induced acute lung injury on a lung-on-a-chip requires a short time and has good stability, and can overcome various problems brought by animal research, such as racial differences, animal ethics, time, stability, etc.

[0028] The following will detail the method for constructing a seawater drowning-induced acute lung injury model and its application of the present invention.

[0029] Construction of Acute Lung Injury Model Induced by Seawater Drowning

[0030] 1. Experimental Materials

[0031] Reagent Preparation: Artificial seawater was prepared according to the main components of seawater in the southeast coastal areas of China

[18] : 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, NaBr 0.083 g / L.

[0032] Human alveolar type II epithelial cells and human pulmonary microvascular endothelial cells were kindly provided by the Dalian Institute of Chemical Physics. The required instruments and reagents are shown in Tables 1 - 3.

[0033] Table 1 Primary Antibodies Used in the Experiment

[0034]

[0035] Table 2 Secondary Antibodies Used in the Experiment

[0036]

[0037] Table 3 Reagents, Instruments and Equipment Used in the Experiment

[0038]

[0039] 2. Construction of Acute Lung Injury Model Induced by Seawater Drowning

[0040] 2.1 Construction Steps of Acute Lung Injury Model Induced by Seawater Drowning, including:

[0041] S1. Ultraviolet sterilization treatment of the lung chip device: Ultraviolet the lung chip device overnight.

[0042] S2. Modify the transwell insert with type I collagen (colI): Dilute type I collagen (coll) in 0.02 M glacial acetic acid at a ratio of 1:100 to obtain the modification solution. Add 100 μL of the modification solution into the transwell insert and 500 μL of the modification solution into the well, and leave it overnight.

[0043] S3. Inoculation of pulmonary microvascular endothelial cells: Place 50 μl of 1×10 6 / ml cell suspension at the center of the bottom of the well in a 24 - well plate. Place the transwell insert into the well of the 24 - well plate. The cell suspension will quickly spread after contacting the transwell insert. Invert the 24 - well plate with the transwell insert and place it in a 37°C, 5% carbon dioxide incubator for 2 h, then place it upright.

[0044] Inoculation of type II alveolar epithelial cells of S4: 100 μl of 1×10 5 / ml cell suspension was inoculated on the upper surface of the transwell insert, placed in an incubator at 37°C with 5% carbon dioxide overnight, and the liquid in the transwell insert was blotted dry the next day.

[0045] S5. Take out the transwell insert and place it in the middle two holes of the lung chip. Add 2 ml of mixed medium (the volume ratio of RPMI 1640 to HULEC-5a is 1:1) to the liquid addition holes on both sides of the lung chip. Note: Remove the bubbles under the transwell insert. See Figure 2 .

[0046] S6. Place the prepared lung chip on a shaker for dynamic culture for 5 days (as Figure 2 shown), the shaker speed is 5 rpm, the rocking oscillation angle is 10°, and the mixed medium is replaced every 1 - 2 days for 5 days to form a complete air-liquid interface of the lung, and the construction of the lung chip is completed.

[0047] S7. Replace the lower channel of the above constructed lung chip with 1 mL of fresh mixed medium, and slowly add 500 μL of artificial seawater to the two cell holes in the middle of the upper channel respectively. Place it in an incubator at 37°C with 5% carbon dioxide and perform dynamic culture on a shaker for 1 - 4 hours, the shaker speed is 5 rpm, the rocking oscillation angle is 10°, preferably perform dynamic culture on a shaker for 2 hours, and the lung chip is used for subsequent experiments.

[0048] The present invention applies the lung chip technology to the seawater drowning-induced acute lung injury model, making up for the deficiencies of traditional cell models and animal models. Since the lung chip is constructed from human cells, it can simulate the air-liquid interface structure, and the cells on the chip can directly contact the air and possess the characteristics of liquid dynamics, which cannot be achieved by traditional cell models and animal models; secondly, the present invention directly adds seawater to the prepared lung chip and then slowly penetrates it into the lower channel, breaking the traditional mode of mixing seawater with the medium and then adding it, which conforms to the process of seawater entering the airway, flowing into the alveoli, and then slowly penetrating into the blood vessels during seawater drowning. Therefore, the seawater drowning-induced acute lung injury model of the present invention simulates the process of seawater slowly penetrating into the blood vessels through the airway, is closer to the real occurrence process of seawater drowning, and has more clinical value in the related research of seawater drowning-induced acute lung injury.

[0049] 2.2. Immunofluorescence staining on the lung chip

[0050] The steps of immunofluorescence staining on the lung chip include:

[0051] a. Rinse the upper and lower channels of the lung chip with PBS, 3 min × 2 times (place on a shaker during rinsing, slow speed);

[0052] b. Fix at room temperature with 4% paraformaldehyde for 20 minutes;

[0053] c. Rinse with PBS 5 min × 3 times (place on a shaker during rinsing, slow speed);

[0054] d. Incubate with the HC / IF special blocking solution for 10 minutes;

[0055] e. Incubate with the primary antibody (diluted 1:100 with the blocking solution): overnight at 4°C, protected from light;

[0056] f. Rinse with PBS 5 min × 3 times (place on a shaker);

[0057] g. Incubate with the secondary antibody (diluted 1:1000 with the blocking solution): 45 minutes at room temperature;

[0058] h. Under light protection, rinse with PBS 5 min × 3 times (place on a shaker during rinsing, slow speed);

[0059] i. Counterstain the nucleus: add DAPI and incubate protected from light for 5 min - 10 min;

[0060] j. Under light protection, rinse with PBS 3 min × 3 times to wash away excess DAPI and observe under fluorescence.

[0061] 2.3 Successfully constructed the lung chip

[0062] To detect the integrity of the lung chip, E-cadherin was used to label type II alveolar epithelial cells, and VE-cadherin was used to label lung microvascular endothelial cells for immunofluorescence staining. The results showed that the cells grew densely and the lung chip was successfully constructed. See Figure 3 , where A and C are planar pictures of the chip, and B and D are three-dimensional imaging pictures of the chip. Among them, the red color is the expression of E-cadherin in type II alveolar epithelial cells, the green color is the expression of VE-cadherin in lung microvascular endothelial cells, and the blue color is the nucleus labeled with DAPI.

[0063] 2.4 When constructing an acute lung injury model caused by seawater drowning on the lung chip, the choice of the method for adding seawater

[0064] In this study, seawater was added in two ways: For the first way of adding seawater, referring to the traditional cell research model, 1 mL of culture medium and 1 mL of seawater were first mixed and then added to the lower channel of the chip; for the second way of adding seawater, according to the actual occurrence process during seawater drowning, 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, the acting time was 6 h for both [1]. The test results showed that 6 h after the addition of seawater, in the second way of directly adding seawater, the expressions of E-cadherin and VE-cadherin in type II alveolar epithelial cells and pulmonary microvascular endothelial cells decreased significantly, as shown in Figure 4 and Figure 5 . Both E-cadherin and VE-cadherin are cell adhesion proteins expressed on the cell membrane. The decrease in their expression levels indicates that the connections between cells are disrupted. The results of this experiment fully demonstrate that the second way of directly adding seawater has a stronger destructive effect on type II alveolar cells and pulmonary microvascular endothelial cells, and the time to cause an acute lung injury model is shorter, which is closer to the animal model of acute lung injury [9]. Secondly, during the process of seawater drowning, seawater is directly inhaled into the lungs rather than being inhaled after being mixed with other liquids. Therefore, the second way of directly adding seawater is more in line with the actual process of seawater drowning.

[0065] 2.5. Selection of seawater acting time when constructing an acute lung injury model caused by seawater drowning on the lung chip

[0066] Add 1 mL of culture medium to the lower channel of the lung chip, and then slowly add 1 mL of seawater directly to the upper channel. At 1 h, 2 h, 3 h, and 4 h, ZO1 was stained in type II alveolar epithelial cells in the upper layer of the chip, and VE-cadherin was stained in pulmonary microvascular endothelial cells in the lower layer of the chip. The results are as shown in Figure 6 . It shows that at 1 h, the expressions of ZO1 and VE-cadherin began to decrease, but the structure of the cell membrane remained basically intact. At 2 h, the expressions of ZO1 and VE-cadherin further decreased, and the structure of some cell membranes was damaged. At 3 h, the expressions of ZO1 and VE-cadherin decreased significantly, the structure of the cell membrane of type II alveolar epithelial cells was basically damaged, and the structure of most of the cell membranes of pulmonary microvascular endothelial cells was damaged. Since seawater penetrated from the upper layer of alveolar epithelial cells into the pulmonary microvascular endothelial cells in the lower channel, the damage to alveolar epithelial cells was earlier, successfully simulating the process of seawater penetrating from the airway to the blood vessels during seawater drowning. At 4 h, the structures of both cell membranes were completely damaged. Thus, it can be seen that for this chip model of acute lung injury caused by seawater, 2 h is a suitable seawater acting time.

[0067] 3. Application of the acute lung injury model caused by seawater drowning

[0068] 3.1. Study on the function of specific genes in acute lung injury caused by seawater drowning

[0069] LCMR1 is a new gene discovered by the applicant's research group

[19] . 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 3 groups, namely the normal group, the knockdown group and the overexpression group. After the construction of the lung chips of the 3 groups was successful, 1 mL of culture medium was added to the lower channel of the lung chips, and 1 mL of seawater was slowly added on the upper layer. 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 decreased significantly, indicating that the knockdown of LCMR1 aggravated the acute lung injury caused by seawater, as shown in Figure 7 . The applicant's research group also reported previously that type II alveolar epithelial cell-specific conditional knockout mice of LCMR1 lacked a complete alveolar structure, and their lung permeability and compliance were both lower than those of the control group

[20] , which was consistent with the results obtained by 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 acute lung injury caused by seawater drowning.

[0070] 3.2. Screening drugs for the treatment of acute lung injury

[0071] The 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 3 groups, namely the normal group, the seawater group, and the seawater + PFC group. After the construction of the lung chips was successful, 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, E-cadherin and surfactant protein C (SPC) staining were performed on the upper layer of type II alveolar epithelial cells, and VE-cadherin staining was performed on the lower layer of pulmonary microvascular endothelial cells. 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, as shown in Figure 8 and Figure 9 . The previous experimental results of the applicant's research group also showed that the vaporization of perfluorocarbon can reduce the acute lung injury caused by seawater drowning in canines

[21] , which was consistent with the results obtained by 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.

[0072] The present invention applies the lung-on-a-chip technology to the model of acute lung injury caused by seawater drowning to overcome the defects existing in the commonly used cell models and animal models of acute lung injury caused by seawater drowning. In the model of acute lung injury caused by seawater drowning of the present invention, seawater is directly added to the lung-on-a-chip, which changes the traditional method and simulates the process of seawater slowly penetrating from the airway into the blood vessels, being closer to the real occurrence process of seawater drowning and having more clinical value in the related research of acute lung injury caused by seawater drowning.

[0073] The above embodiments are only specific implementation manners of the present invention, used to illustrate the technical solutions of the present invention rather than to limit it. The protection scope of the present invention is not limited thereto. Although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: any person skilled in the art within the technical scope disclosed by the present invention can still modify or improve the technical solutions described in the foregoing embodiments, or perform equivalent replacement on some of the technical features; 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 all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to 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. Perform UV sterilization on the lung chip; S2. Modify the transwell plug with type I collagen colI: dilute type I collagen colI in 0.02M glacial acetic acid at a ratio of 1:100 to obtain a modification solution, add 100uL of the modification solution to the transwell plug, add 500uL of the modification solution to the well, and leave overnight; 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 into the liquid addition holes on both sides of the lung chip, and remove the bubbles under the transwell plug; S6. The lung chip is placed on a shaker for dynamic culture for 5 days, and the mixed culture medium is replaced every 1-2 days to form a complete lung air-liquid interface to complete the construction of the lung chip, wherein the rotation speed of the shaker is 5 rpm and the rocking oscillation angle is 10°; 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% carbon dioxide incubator on a shaker for dynamic culture for 2 hours, wherein the shaker has a rotation speed of 5 rpm and a rocking oscillation angle of 10°, and the composition of the artificial seawater includes: 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, NaBr 0.083 g / L, Wherein, 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.

2. Application of the seawater drowning-induced acute lung injury model constructed according to the method described in claim 1 in the study of the mechanism of seawater drowning-induced acute lung injury.

3. Use of the seawater drowning-induced acute lung injury model constructed according to the method of claim 1 in screening drugs for treating seawater drowning-induced acute lung injury.

4. Application of the seawater drowning-induced acute lung injury model constructed according to the method of claim 1 in studying the function of specific genes in seawater drowning-induced acute lung injury.

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