Construction method of immortalized human alveolar type II epithelial cell silicosis model
By using the combination of immortalized human alveolar type II epithelial cells and SiO2 particle suspension in vitro, a stable and repetitive silicosis cell model was constructed, solving the difficulties in diagnosis and treatment of silicosis in the prior art, and providing a study on the mechanism of silicosis disease and important technical support for the development of drugs.
Patent Information
- Application Number
- CN202411417177.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2025-01-07
AI Technical Summary
The existing technology is difficult to effectively diagnose and treat silicosis, and traditional detection methods cannot directly reflect the degree of lung tissue damage. The experimental results of animal models are biased from the actual application effect, and there is a lack of an effective in vitro silicosis cell model.
By utilizing immortalized human alveolar type II epithelial cells, silicosis models were constructed using different concentrations of SiO2 particle suspensions to determine the optimal time and concentration of infection, and a stable and reproducible in vitro silicosis cell model was established.
The successful construction of a stable and repetitive in vitro silicosis cell model has provided a technical basis for studying the mechanism of silicosis and developing anti-injury drugs, and improved the possibility of early diagnosis and treatment.
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Figure CN119265102A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the field of medical technology, and in particular to a method for constructing an immortalized human alveolar type II epithelial cell silicosis model. Background Art
[0002] Silicosis is a lung disease caused by long-term exposure to large amounts of inhalable free silica dust particles (<5-10 μm). Its basic pathological changes are silicosis nodules and extensive pulmonary fibrosis, which are mainly collagenous fibrous tissue. Its pathogenesis is still unclear, and the mechanism of disease progression has not yet been elucidated.
[0003] However, due to the influence of traditional concepts, most patients do not realize that they have serious diseases, and some patients are not active in treatment, thus missing the best time for treatment, which brings a heavy burden to their families and society. Therefore, how to improve the diagnosis rate of early silicosis and reduce the mortality and disability rate of patients is an urgent problem that needs to be solved in my country. Although the current clinical detection methods can realize the pathological examination of lung interstitial and lesion tissue, they cannot directly reflect the degree of damage to human lung tissue and its functional changes; secondly, although there are many drugs for anti-silicosis, most of these drugs are slow to act and have certain side effects; furthermore, current research mostly uses animals as models to verify the effectiveness of new drugs, but there is often a large deviation between the experimental results and the actual application effects.
[0004] At present, most studies believe that when free SiO2 particles enter the alveoli, they will cause inflammatory cell infiltration in the lower respiratory tract, abnormal proliferation of fibroblasts, and expression and release of multiple cytokines by alveolar epithelial cells, which promotes fibroblast proliferation and collagen deposition. They can be transdifferentiated into fibroblasts and transformed into myofibroblasts under the stimulation of cytokines, leading to pulmonary fibrosis. As the main structural cells of the alveoli, alveolar type II epithelial cells have the functions of regulating the metabolism of epidermal active substances, maintaining the alveolar environment, and transforming into type I epithelial cells when lungs are injured. They are widely involved in various biological activities such as inflammatory response, immune response, extracellular matrix regulation, and injury repair in the lungs. Under the attack of SiO2 particles, the number of apoptosis or necrosis of alveolar type II epithelial cells directly determines the severity and prognosis of SiO2-induced lung injury. By preparing a cell model, SiO2 dust suspension can be used to stimulate alveolar type II epithelial cells in vitro to simulate the dusty environment in vivo, and explore the pathogenic mechanism of silicosis at the cellular and molecular levels, especially providing more intuitive and convenient conditions for studying the interaction between cells.
[0005] Alveolar type II epithelial cells are an important type of cell distributed in the alveoli. Their main function is to secrete pulmonary surfactant (PS). They have important physiological functions in maintaining alveolar function and regulating blood-gas exchange in the lungs. Pulmonary surfactant is distributed on the surface of alveolar epithelial cells. It is a complex composed of lipids and surfactant proteins secreted by alveolar type II epithelial cells, of which about 90% are phospholipids and about 10% are surfactant proteins. Surfactant proteins include four types: A, B, C, and D. Among them, SP-C is a protein that is highly specific to alveolar type II epithelial cells and is an important marker for identifying alveolar type II epithelial cells. Pulmonary surfactant has important physiological functions such as reducing alveolar surface tension, maintaining alveolar size, improving alveolar compliance, and preventing atelectasis and pulmonary edema. It plays a vital role in maintaining the normal physiological function of the lungs.
[0006] The A549 cell line is currently used in the alveolar epithelial cell model for silicosis. The A549 cell line is a widely used human lung adenocarcinoma cell line. Although it has some alveolar epithelial characteristics, it is derived from a primary lung tumor and cannot fully simulate the immediate reaction and subsequent effects of the normal lung microenvironment to free SiO2 particles. Primary cells isolated from animals or humans are difficult to obtain specimens and are not easy to amplify in large quantities in vitro, which is not conducive to repeated verification of experiments. At present, immortalized human alveolar type II epithelial cells have been prepared using immortalization technology, but there is still a lack of research data on the use of these cells to prepare silicosis cell models. Summary of the invention
[0007] To this end, the present invention provides a method for constructing an immortalized human alveolar type II epithelial cell silicosis model, and in particular provides a method for establishing an immortalized human alveolar type II epithelial cell silicosis model by inducing free SiO2 particles. The successfully established in vitro silicosis model is of great significance for studying the occurrence and development mechanism of silicosis, and provides a technical basis and theoretical reference for further research on cell-cell interactions and the development of related anti-injury drugs.
[0008] In order to achieve the above object, the present invention provides the following technical solutions:
[0009] According to the present invention, a method for constructing an immortalized human alveolar type II epithelial cell silicosis model is provided, the method comprising:
[0010] Step 1: Silica Dust Treatment
[0011] After grinding the silica crystal standard, use serum-free DMEM medium to prepare a mother solution, and use the mother solution and serum-free DMEM medium to prepare a SiO2 particle suspension;
[0012] Step 2: Cell treatment
[0013] After primary culture of immortalized human alveolar type II epithelial cells, subculture was performed. Trypsin was used for digestion in subculture. When the cell morphology became round and the corners disappeared, culture medium was added to stop digestion and then the cells were cultured to the logarithmic phase. After centrifugation, complete culture medium was used to prepare cell suspension. The cell density was adjusted, and the cells were inoculated in the culture plate. After culturing until they adhered to the wall, the original culture medium in the wells was discarded and washed with 1×PBS. Subsequently, SiO2 particle suspension was added to each well for poisoning culture to prepare a biochemical human alveolar type II epithelial cell silicosis model.
[0014] Furthermore, in step 2, the primary culture uses a DMEM medium containing 10% fetal bovine serum.
[0015] Furthermore, in step 2, the concentration of pancreatic enzyme used is 0.25%.
[0016] Furthermore, in step 2, the complete culture medium is 10% fetal bovine serum + 90% DMEM.
[0017] Furthermore, in step 2, the cell density is adjusted to 1×10 6 / mL.
[0018] Furthermore, in step 2, the concentration of the SiO2 particle suspension is 75-150 μg / mL, preferably 75 or 150 μg / mL.
[0019] Furthermore, in step 2, the infection culture time is 6-12 hours.
[0020] Furthermore, the method also includes staining the infected cells with CCK-8 reagent, performing cell activity detection, and observing the color change after incubation for 3 hours.
[0021] The present invention has the following advantages:
[0022] The present invention uses immortalized human alveolar type II epithelial cells as the research object, uses SiO2 particle suspensions of different concentrations to infect immortalized human alveolar type II epithelial cells, and determines the optimal intoxication time and concentration by detecting cell viability. A method for in vitro use of SiO2 particle suspensions to induce immortalized human alveolar type II epithelial cells to construct a silicosis model has been successfully established. The model has good stability and is easy to repeat, providing a technical basis and theoretical reference for subsequent research on the occurrence and development mechanism of silicosis, screening differential molecules, and developing related anti-injury drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.
[0024] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.
[0025] Figure 1 A process diagram for constructing an immortalized human alveolar type II epithelial cell silicosis model provided in Example 1 of the present invention;
[0026] Figure 2 This is an immunofluorescence image of immortalized human alveolar type II epithelial cells provided in Example 1 of the present invention;
[0027] Figure 3 This is a graph of cell viability after intervention after 6 hours of culture at different silica concentrations provided in Example 1 of the present invention;
[0028] Figure 4 This is a graph of cell viability after intervention at different silica concentrations after 12 hours of culture provided in Example 1 of the present invention. DETAILED DESCRIPTION
[0029] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are 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 ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0030] Example
[0031] In this example, a SiO2 particle suspension was used to induce immortalized human alveolar type II epithelial cells. The concentrations of the SiO2 particle suspension were 0, 50, 75, 125, 150, and 200 μg / mL. The induction time was 6 h and 12 h, respectively. An immortalized human alveolar type II epithelial cell silicosis model was successfully established.
[0032] Preparation process such as Figure 1The specific experimental steps are as follows:
[0033] (1) Dust treatment
[0034] The dust used was a silica crystal standard. Before use, it was ground using an agate mortar in a fume hood, weighed after grinding, and the weight was recorded. It was then sterilized by high pressure at 121°C for 30 min, and then dried. The serum-free DMEM culture medium was filtered through a 0.22 μm pore size microporous filter membrane to prepare a 4000 μg / mL stock solution, which was sealed and stored in a 4°C refrigerator for later use.
[0035] (2) Cell treatment
[0036] Immortalized human alveolar type II epithelial cells were purchased from a cell bank.
[0037] Immortalized human alveolar type II epithelial cells were cultured in T25 culture flasks using DMEM medium containing 10% fetal bovine serum and passaged every 2-3 days. In a 6-well plate, 10 μL of culture medium was added, and then a sterile coverslip was added to each well of the plate. The third generation cells were plated at 1x10 5 Density inoculated on the slide, shaken with the cross method, cultured for 24 hours to allow the cells to completely adhere to the wall. After the cells adhere, aspirate the culture supernatant, wash three times with 1X PBS, 5 minutes each time, and use a shaker at a low speed for washing. Then, the washing solution was aspirated, and 1 mL of 4% paraformaldehyde was added to each well for fixation for 20 min; 1X PBS was used to wash again for 3 times, 5 min each time; permeabilization: 500 μL of 0.5% triton-X100 was added to each well, and permeabilization was performed at room temperature for 20 min; 1X PBS was used to wash 3 times, 5 min each time, and goat serum was added to cover the cell slides after the washing solution was aspirated, and the cell slides were incubated at room temperature for 1 h, and the goat serum was aspirated, and SP-C antibody diluted according to the instruction manual was added to each well to cover the slides, and incubated at 4°C overnight; the primary antibody was aspirated the next day, and 1X PBS was used to wash 3 times, 5 min each time, and the excess PBS was aspirated, and the fluorescent secondary antibody diluted according to the instruction manual was added to cover the slides, and incubated at room temperature in the dark for 1 h; the secondary antibody was aspirated and 1X PBS was used to wash 3 times, 5 min each time, and the excess liquid was aspirated, DAPI was added to incubate in the dark for 10 min, and then 1X Wash with PBS at low speed for 4 times, 5 minutes each time; use a pipette to absorb the liquid on the slide, drip the sealing solution containing fluorescence quenching solution on the slide, then cover the slide on the slide, drip the fixative around it, and then use a fluorescence microscope to observe and collect images. Figure 2 As shown, the cells are irregular in morphology, angular, and grow in an adherent manner. When subcultured, they need to be digested with 0.25% trypsin. They can be placed in a 37°C, 5% CO2 incubator for digestion for 1 minute and 30 seconds, and then the digestion status can be observed under a microscope. When the cell morphology becomes round and the horns disappear, culture medium can be added to stop digestion and subsequent operations can be performed.
[0038] Observe the cells in the logarithmic growth phase. When the cell density in the culture flask is about 80%, the digestion operation can be performed. After centrifugation, use complete culture medium (10% fetal bovine serum + 90% DMEM) to prepare cell suspension and adjust the cell density to 1×10 6 / mL, the cells were inoculated into a 96-well plate, 100 μl per well, and 1× PBS was added to the periphery of the 96-well plate to prevent the evaporation of the internal culture medium.
[0039] (3) Cell poisoning
[0040] The mother solution was taken out from the 4°C refrigerator and ground with an ultrasonic grinder for 10 minutes. The mother solution and serum-free DMEM medium were used to prepare SiO2 particle suspensions of different concentrations according to 0μg / mL, 50μg / mL, 75μg / mL, 100μg / mL, 125μg / mL, 150μg / mL, and 200μg / mL. After 24 hours of cell culture in the 96-well plate, observe whether the cells are attached to the wall. After confirming that the cells are attached to the wall, discard the original culture medium in the wells and wash them with 1×PBS 1-2 times. Then, 100μL of the above-mentioned SiO2 particle suspensions of different concentrations were added to each well and cultured for 6h and 12h, respectively. The cell viability after intervention at different times and silica concentrations was as follows: Figure 3 and Figure 4 shown.
[0041] Results: A silicosis cell model was constructed based on immortalized human alveolar type II epithelial cells. The optimal concentrations of SiO2 particle suspension for induction in the in vitro construction of the above cell model were determined to be 75μg / mL and 150μg / mL, and the culture times were 12h and 6h, respectively.
[0042] (4) Cell activity detection
[0043] The viability of immortalized human alveolar type II epithelial cells induced by different concentrations of SiO2 particle suspensions and different time periods was tested according to the conventional CCK-8 reagent detection process. The cells were not easy to stain, and obvious color reaction could be observed after incubation for 3 hours. The results are as follows Figure 3 and Figure 4 As shown, it can be seen that the optimal concentrations of SiO2 particle suspension for induction when constructing the cell model in vitro are 75μg / mL and 150μg / mL, and the culture time is 12h and 6h, respectively.
[0044] Subsequently, intervention experiments can be carried out using appropriate concentrations and times to extract relevant proteins and RNA, or to separate the supernatant after culture to prepare conditioned medium for cell-cell interaction studies.
[0045] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.
Claims
1. A method for constructing an immortalized human alveolar type II epithelial cell silicosis model, characterized in that: The method comprises: Step 1: Silica Dust Treatment After grinding the silica crystal standard, use serum-free DMEM medium to prepare a mother solution, and use the mother solution and serum-free DMEM medium to prepare a SiO2 particle suspension; Step 2: Cell treatment After primary culture of immortalized human alveolar type II epithelial cells, subculture was performed. Trypsin was used for digestion in subculture. When the cell morphology became round and the corners disappeared, culture medium was added to stop digestion and then the cells were cultured to the logarithmic phase. After centrifugation, complete culture medium was used to prepare cell suspension. The cell density was adjusted, and the cells were inoculated in the culture plate. After culturing until they adhered to the wall, the original culture medium in the wells was discarded and washed with 1×PBS. Subsequently, SiO2 particle suspension was added to each well for poisoning culture to prepare a biochemical human alveolar type II epithelial cell silicosis model.
2. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In the step 2, the primary culture uses a DMEM medium containing 10% fetal bovine serum.
3. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In the step 2, the concentration of pancreatin used is 0.25%.
4. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In the step 2, the complete culture medium is 10% fetal bovine serum + 90% DMEM.
5. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In step 2, the cell density was adjusted to 1×10 6 / mL.
6. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In the step 2, the concentration of the SiO2 particle suspension is 75-150 μg / mL.
7. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: In the step 2, the infection culture time is 6-12 hours.
8. The method for constructing an immortalized human alveolar type II epithelial cell silicosis model according to claim 1, characterized in that: The method further comprises staining the cells after the poisoning culture with a CCK-8 reagent, performing a cell activity test, and observing the color change after incubation for 3 hours.