Method for constructing alveolar epithelial cell in-vitro aging model by using tobacco tar

Abstract

The invention provides a method for constructing an alveolar epithelial cell in-vitro aging model from tobacco tar, and belongs to the technical field of cigarette extracts. The method comprises the following steps: firstly, adding fetal calf serum and penicillin-streptomycin double antibodies into a culture medium to obtain a culture solution, then culturing alveolar epithelial cells in a 5% CO2 cell incubator at 37 DEG C, and inducing by adopting tobacco tar to form an in-vitro aging model. According to the method, the culture mode of the alveolar epithelial cells is adjusted, the tobacco tar is added to stimulate aging, the obtained in-vitro aging model of the alveolar epithelial cells is good in stability and easy to repeat, and the tobacco tar is adopted for induction, so that the in-vitro aging model is closer to an in-vivo real environment.

Description

Technical Field

[0001] The present invention relates to the technical field of cigarette extracts, and particularly to a method for constructing an in vitro senescence model of alveolar epithelial cells with tobacco tar. Background Art

[0002] Chronic obstructive pulmonary disease (COPD) is a serious public health problem, characterized by progressive and irreversible decline in lung function caused by airflow obstruction, lung parenchymal destruction and emphysema. This disease has become the third leading cause of death globally, and cigarette smoke (CS) exposure is the main risk factor for COPD. Cigarette smoke contains a large number of oxidizing substances, tobacco tar, nicotine and other components, which trigger local and systemic inflammatory responses through oxidative stress mechanisms driven by free radicals and other oxidants.

[0003] In in vitro experiments studying the pathology of COPD, researchers have used a variety of stimulants to simulate the effects of cigarette smoke and establish a standardized in vitro system to more accurately simulate the effects of cigarette smoke on the single-cell characteristics of the human alveolar microenvironment under long-term exposure and to deeply reveal the pathogenesis of COPD.

[0004] Although in vitro experiments can only simulate some cell characteristics of COPD, it is still necessary to follow and be as close as possible to the objective environment in the human body. In experiments, 1% CSE is considered to be the tobacco smoke burned by 12.5 - 17.5 cigarettes per day. Therefore, it can be considered that the severe cell damage caused by stimulating cells with a high concentration (>2%) of CSE, including overly severe apoptosis, makes it difficult for such cytotoxicity experiments to simulate the real situation of the alveolar microenvironment in the human body, and there are often differences from the protein molecule changes and signal pathways in the body.

[0005] Tobacco smoke extract can cause various systemic diseases, and the occurrence of these systemic diseases is not due to direct exposure to the tobacco smoke environment. However, most current researchers still widely use traditional tobacco smoke exposure models in basic research.

[0006] Tobacco smoke extract CSE is obtained by dissolving the water-soluble gas and particulate phase of tobacco smoke. Tobacco smoke extract can be divided into the gas phase (CSGE) and the tar phase (CSTE). After tobacco smoke is inhaled into the lungs, the gaseous substances and nicotine can be rapidly absorbed into the blood through the alveoli in 5 - 7 seconds, inducing damage in tissues far from the lungs, while the tar phase accumulates continuously in the alveolar microenvironment with the increase of smoking age and acts for a long time. Therefore, only using CSE cannot simulate the real environment in the body and is not convenient for exploring which components cause the above diseases.

[0007] Therefore, it is necessary to explore the stimulation of alveolar cells by tar in order to more realistically simulate the real environment in the body. Summary of the Invention

[0008] In view of this, the present invention provides a method for constructing an in vitro senescence model of alveolar epithelial cells with tar, which adjusts the culture method of alveolar epithelial cells and adds tar stimulation for senescence. The obtained in vitro senescence model of alveolar epithelial cells has good stability and is easy to repeat.

[0009] The method for constructing an in vitro senescence model of alveolar epithelial cells with tar according to the present invention includes the following steps:

[0010] (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to the culture medium to obtain the culture solution;

[0011] (2) Place the alveolar epithelial cells in the culture solution described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture solution, and culture in a 37 °C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; replace the culture solution every 24 h during the culture process;

[0012] (3) Place the centrifuged cells in the culture solution described in step (1), adjust the cell density, and after culturing for 6-10 h, add tar to induce the cells to obtain the in vitro senescence model of alveolar epithelial cells constructed with tar.

[0013] Preferably, the culture medium described in step (1) is RPMI1640 medium or DMEM medium.

[0014] Preferably, the concentration of fetal bovine serum in the culture solution described in step (1) is 10 wt%.

[0015] Preferably, the concentration of penicillin in the penicillin-streptomycin double antibody described in step (1) is 10,000 U / mL, and the concentration of streptomycin is 10,000 U / mL.

[0016] Preferably, the concentration of penicillin-streptomycin double antibody in the culture solution described in step (1) is 1 wt%.

[0017] Preferably, the cell density described in step (3) is 50-60%.

[0018] Preferably, the preparation method of tar in step (3) is as follows:

[0019] Remove the filter tip of the cigarette, light the cigarette, place it in a sealed container, and use a syringe to extract tobacco smoke through a filter material. Tobacco smoke tar remains on the filter material. Then dissolve the tobacco smoke tar in a culture medium, filter it through a 0.22 μm filter membrane, and store it at a low temperature to obtain cigarette tar.

[0020] More preferably, the filter material is a Cambridge filter disc; the culture medium is RPMI 1640 culture medium.

[0021] Preferably, in step (3), the induction time of cigarette tar in the culture solution is 6 - 72 h, and the temperature is 37 °C.

[0022] Preferably, the content of cigarette tar in the culture solution in step (3) is 0.5 - 2 wt%.

[0023] The present invention provides a method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar. By adjusting the culture method of alveolar epithelial cells and adding cigarette tar to stimulate senescence, the obtained in vitro senescence model of alveolar epithelial cells has good stability, is easy to repeat, and is induced with cigarette tar, which is closer to the real in vivo environment. Description of the Drawings

[0024] Figure 1 Results of detecting the cell viability after 48 h of stimulation in Examples 1 - 4 and Comparative Examples 1 - 6;

[0025] Figure 2 Western blot images of mitochondrial autophagy and senescence-related proteins in A549 cells after 48 h of stimulation in Example 2, Comparative Example 2, and Comparative Example 6. Detailed Embodiments

[0026] The present invention provides a method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar, comprising the following steps:

[0027] (1) Preparation of the culture solution: Add fetal bovine serum and penicillin-streptomycin double antibody to the culture medium to obtain the culture solution;

[0028] (2) Place the alveolar epithelial cells in the culture solution prepared in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture solution, and culture them in a cell culture incubator at 37 °C and 5% CO2 until the logarithmic phase, then centrifuge and discard the supernatant; replace the culture solution every 24 h during the culture process;

[0029] (3) Place the centrifuged cells in the culture solution prepared in step (1), adjust the cell density, after culturing for 6 h, add cigarette tar to induce the cells to obtain the in vitro senescence model of alveolar epithelial cells constructed with cigarette tar.

[0030] Preferably, the culture medium in step (1) is RPMI1640 medium or DMEM medium.

[0031] Preferably, the concentration of fetal bovine serum in the culture solution in step (1) is 10 wt%.

[0032] Preferably, the concentration of penicillin-streptomycin double antibody in the culture solution in step (1) is 1 wt%. In a specific embodiment of the present invention, the concentration of penicillin in the penicillin-streptomycin double antibody in step (1) is 10,000 U / mL, and the concentration of streptomycin is 10,000 U / mL.

[0033] Preferably, the alveolar epithelial cells in step (2) are A549 cells, purchased from the National Experimental Cell Resource Sharing Platform.

[0034] Preferably, the cell density in step (3) is 50-60%.

[0035] Preferably, the induction time of tar in the culture solution in step (3) is 6-72 h, and the temperature is 37 °C.

[0036] Preferably, the content of tar in the culture solution in step (3) is 0.5-2 wt%.

[0037] Preferably, the preparation method of tar in step (3) is as follows:

[0038] Remove the filter tip of the cigarette and light the cigarette, place it in a sealed container, and use a syringe to extract tobacco smoke through a filter material. The tobacco smoke tar remains on the filter material; then dissolve the tobacco smoke tar in the culture medium, filter it through a 0.22 μm filter membrane and store it at a low temperature to obtain tar. Or use other methods to remove the gas phase components in the tobacco smoke extract to obtain tar.

[0039] More preferably, the filter material in the tar preparation process is a Cambridge filter; the culture medium is RPMI1640 medium; in a specific embodiment of the present invention, the Cambridge filter is purchased from Shanghai Yinda Scientific Instruments Co., Ltd., model 97039654.

[0040] In a specific embodiment of the present invention, the preparation method of the tar is as follows:

[0041] Take a cigarette, remove the filter tip and light it, place it in a sealed container, and use a syringe to extract tobacco smoke through the filter material. There is residual tobacco smoke tar on the filter material. Then dissolve the tobacco smoke tar in 10 mL of RPMI 1640 medium, filter it through a 0.22 μm filter membrane, and store it at -80 °C to obtain cigarette tar, denoted as CSTar (or CSTE). In a specific embodiment of the present invention, the cigarette is the Red Rose brand cigarette produced by China Tobacco Guangdong Industrial Co., Ltd. (carbon monoxide content in cigarette smoke: 1.1 mg, tar content: 9 mg, nicotine content in cigarette smoke: 0.9 mg).

[0042] The present invention will be further described below in conjunction with embodiments.

[0043] Example 1

[0044] A method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar, the steps are as follows:

[0045] (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to RPMI 1640 medium to obtain a culture medium; the concentration of fetal bovine serum in the culture medium is 10 wt%, and the concentration of penicillin-streptomycin double antibody in the culture medium is 1 wt%;

[0046] (2) Place the alveolar epithelial cells in the culture medium described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture medium, and culture them in a 37 °C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; replace the culture medium every 24 h during the culture process;

[0047] (3) Place the centrifuged cells in the culture medium described in step (1), adjust the cell density to 50%, culture for 6 h at 37 °C, and then add 0.5 wt% cigarette tar to stimulate the cells for 48 h to obtain the in vitro senescence model of alveolar epithelial cells constructed with cigarette tar.

[0048] Example 2

[0049] A method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar, the steps are as follows:

[0050] (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to RPMI 1640 medium to obtain a culture medium; the concentration of fetal bovine serum in the culture medium is 10 wt%, and the concentration of penicillin-streptomycin double antibody in the culture medium is 1 wt%;

[0051] (2) Place the alveolar epithelial cells in the culture medium described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture medium, and culture them in a 37°C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; change the culture medium every 24 h during the culture process;

[0052] (3) Place the centrifuged cells in the culture medium described in step (1), adjust the cell density to 50%, culture at 37°C for 6 h, then add 1 wt% tar to stimulate the cells for 48 h to obtain the in vitro aging model of alveolar epithelial cells constructed by tar.

[0053] Example 3

[0054] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are as follows:

[0055] (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to RPMI1640 medium to obtain the culture medium; the concentration of fetal bovine serum in the culture medium is 10 wt%, and the concentration of penicillin-streptomycin double antibody in the culture medium is 1 wt%;

[0056] (2) Place the alveolar epithelial cells in the culture medium described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture medium, and culture them in a 37°C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; change the culture medium every 24 h during the culture process;

[0057] (3) Place the centrifuged cells in the culture medium described in step (1), adjust the cell density to 50%, culture at 37°C for 6 h, then add 1.5 wt% tar to stimulate the cells for 48 h to obtain the in vitro aging model of alveolar epithelial cells constructed by tar.

[0058] Example 4

[0059] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are as follows:

[0060] (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to RPMI1640 medium to obtain the culture medium; the concentration of fetal bovine serum in the culture medium is 10 wt%, and the concentration of penicillin-streptomycin double antibody in the culture medium is 1 wt%;

[0061] (2) Place the alveolar epithelial cells in the culture medium described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture medium, and culture them in a 37°C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; change the culture medium every 24 h during the culture process;

[0062] (3) The centrifuged cells were placed in the culture medium described in step (1), the cell density was adjusted to 50%, and after culturing at 37 °C for 6 h, 2 wt% of tar was added to stimulate the cells for 48 h to obtain the in vitro aging model of alveolar epithelial cells constructed with tar.

[0063] Comparative Example 1

[0064] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are the same as those in Example 1, the difference is that the method for preparing tar used in Comparative Example 1 is as follows:

[0065] A cigarette was taken, the filter tip was removed and lit, placed in a sealed container, and tobacco smoke was extracted by a syringe through a filter material, and then the tobacco smoke was dissolved in 10 mL of RPMI 1640 culture medium, the pH value was adjusted to 7.0, and after filtration through a 0.22 μm filter membrane, it was used within 30 minutes.

[0066] Comparative Example 2

[0067] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are the same as those in Comparative Example 1, the difference is that the concentration of tar added in step (3) of Comparative Example 2 is 1 wt%.

[0068] Comparative Example 3

[0069] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are the same as those in Comparative Example 1, the difference is that the concentration of tar added in step (3) of Comparative Example 3 is 1.5 wt%.

[0070] Comparative Example 4

[0071] A method for constructing an in vitro aging model of alveolar epithelial cells with tar, the steps are the same as those in Comparative Example 1, the difference is that the concentration of tar added in step (3) of Comparative Example 4 is 2 wt%.

[0072] Comparative Example 5

[0073] A method for constructing an in vitro aging model of alveolar epithelial cells, the steps are the same as those in Example 1, the difference is that no tar is added in step (3) of Comparative Example 5, denoted as the Control group.

[0074] Comparative Example 6

[0075] A method for constructing an in vitro aging model of alveolar epithelial cells with nicotine, the steps are the same as those in Example 1, the difference is that the tar in step (3) of Comparative Example 6 is replaced with 100 μM nicotine, denoted as the nicotine group.

[0076] The cell viability after 48 h of stimulation in Examples 1 to 4 and Comparative Examples 1-6 was detected, and the results are as follows Figure 1 ; in the figure, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

[0077] The immunoblot images of mitophagy and senescence-related proteins (including mitochondrial membrane protein TOM20, LC3B protein, Pink1 protein, P21 protein, β-actin) in A549 cells after 48 h of stimulation in Example 2, Comparative Example 2 and Comparative Example 6 are as follows Figure 2 . It can be seen from Figure 2 that compared with CSE, CSTE did not cause obvious cytotoxic damage and apoptosis while causing mitophagy damage (decrease in Pink1, increase in TOM20, decrease in LC3BII / LC3B) and cell senescence (increase in P21), which is closer to the real environment in vivo.

[0078] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar, characterized in that, It includes the following steps: (1) Culture medium preparation: Add fetal bovine serum and penicillin-streptomycin double antibody to the culture medium to obtain the culture solution; (2) Place the alveolar epithelial cells in the culture solution described in step (1), centrifuge at 500 rpm for 3 min, discard the supernatant, place the cells in a new culture solution, and culture them in a 37°C, 5% CO2 cell culture incubator until the logarithmic phase, then centrifuge and discard the supernatant; Replace the culture solution every 24 h during the culture process; (3) Place the centrifuged cells in the culture solution described in step (1), adjust the cell density, after culturing for 6-10 h, add cigarette tar to induce the cells to obtain the cigarette tar-induced in vitro senescence model of alveolar epithelial cells.

2. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, wherein The culture medium described in step (1) is RPMI1640 medium or DMEM medium.

3. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, characterized in that, The concentration of fetal bovine serum in the culture solution described in step (1) is 10 wt%.

4. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, wherein, The concentration of penicillin in the penicillin-streptomycin double antibody described in step (1) is 10,000 U / mL, and the concentration of streptomycin is 10,000 U / mL.

5. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, characterized in that, The concentration of penicillin-streptomycin double antibody in the culture solution described in step (1) is 1 wt%.

6. The method for constructing an in vitro aging model of alveolar epithelial cells using cigarette tar according to claim 1, characterized in that, The cell density described in step (3) is 50-60%.

7. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, characterized in that The preparation method of cigarette tar in step (3) is as follows: Remove the filter tip of the cigarette and light the cigarette, place it in a sealed container, and use a syringe to extract tobacco smoke through a filter material. There is tobacco smoke tar remaining on the filter material; Then dissolve the tobacco smoke tar in the culture medium, filter it through a 0.22 μm filter membrane and store it at low temperature to obtain cigarette tar.

8. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 7, wherein, The filter material is a Cambridge filter disc; The culture medium is RPMI1640 medium.

9. The method for constructing an in vitro senescence model of alveolar epithelial cells with cigarette tar according to claim 1, wherein The induction time of cigarette tar in the culture solution described in step (3) is 6-72 h, and the temperature is 37°C.

10. The method for constructing an in vitro senescence model of alveolar epithelial cells using cigarette tar according to claim 1, characterized in that, The content of cigarette tar in the culture solution described in step (3) is 0.5-2 wt%.

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