CN107312742B - Culture method and detection method of rat fibrotic lung cells - Google Patents
Culture method and detection method of rat fibrotic lung cells Download PDFInfo
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Abstract
The invention discloses a culture method and a detection method of rat fibrotic lung cells, which comprises the steps of preparing culture solution, perfusate and cleaning solution, taking materials and carrying out primary culture on rat type II alveolar epithelial cells, stimulating the type II alveolar epithelial cells by amiodarone to obtain fibrotic lung cells, and detecting the fibrotic lung cells.
Description
Technical Field
The invention relates to the field of medicine, in particular to a culture method and a detection method of rat fibrotic lung cells.
background
Amiodarone, also known as amiodarone, is an iodine-containing benzofuran derivative, is widely applied to clinic as a class III antiarrhythmic drug, and is mainly used for treating ventricular arrhythmia and atrial fibrillation. The side effects of the traditional Chinese medicine are mainly manifested as pulmonary toxicity, thyroid toxicity, cardiotoxicity, digestive system toxicity and the like, wherein the pulmonary toxicity reaction is the most serious, the incidence rate is 1% -10%, the pulmonary toxicity is mainly manifested as pulmonary fibrosis, the mortality rate is up to 33% due to unclear pathogenesis and clinical lack of effective treatment measures, and the life health of people is seriously influenced. But amiodarone has definite and stable curative effect in resisting arrhythmia, and has no other similar medicine substitute at present, thus still having wide clinical application. Therefore, the elucidation of the mechanism of the amiodarone induced pulmonary fibrosis has important clinical significance for reducing the toxic and side effects of the medicament, reversing or delaying the pulmonary fibrosis process, improving the prognosis of patients and improving the survival rate.
The pathophysiological process and mechanism of pulmonary fibrosis are very complex, the pathogenic process mainly comprises tissue injury, new tissue generation and tissue remodeling, and finally, the pulmonary fibrosis mainly characterized by fibroblast foci and abnormal deposition of extracellular matrix appears in the lung. The formation of fibroblast foci is an important pathological feature of pulmonary fibrosis, with myofibroblasts as the major effector cells. It is currently believed that EMT may be one of the major pathways for myofibroblast formation, and since the number of myofibroblasts in normal lung is not large, about 33% of myofibroblasts in pulmonary fibrosis are transformed from type ii alveolar epithelial cells through the EMT pathway. Therefore, the intensive study on the phenotype transformation regulation and mechanism of the II type alveolar epithelial cells is significant for delaying or even reversing the amiodarone-induced pulmonary fibrosis. Therefore, an in vitro cell model is prepared, the pathogenesis of the amiodarone induced pulmonary fibrosis mechanism is further explored from the cellular and molecular level, and the breakthrough of the treatment is very important.
Disclosure of Invention
aiming at the defects in the prior art, the invention aims to provide a culture method and a detection method of rat fibrotic lung cells, which are used for preparing amiodarone induced pulmonary fibrosis cells from a molecular level and can accurately reflect the condition of in vivo amiodarone induced pulmonary fibrosis.
In order to achieve the purpose, the invention provides the following technical scheme: a culture method and a detection method of rat fibrotic lung cells comprise the following steps:
Step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
Step two, material taking: injecting anesthetic into abdominal cavity of rat for anesthesia, injecting heparin sodium 2800-3Adding 3-6ml of inactivated calf serum enzyme into a culture dish, centrifuging the sample for 5-10min, removing the supernatant, re-suspending the precipitate with a cleaning solution, adding a red blood cell lysate preheated at 37 ℃, repeatedly blowing and uniformly mixing, standing at room temperature for 5-8min to completely lyse the red blood cells, centrifuging the sample for 5-10min, removing the supernatant, re-suspending the precipitate in a culture medium at 37 ℃ and counting;
step three, primary culture: inoculating the obtained cells in DMEM medium, standing at 37 deg.C with 5% CO by volume2Incubating in incubator for 40-60min, sucking out non-adherent cells, centrifuging sample for 5-10min, taking down layer precipitate, inoculating in DMEM culture medium, repeating for 3-4 times, sucking out non-adherent cells, inoculating suspension into culture dish coated with rat IgG, placing at 37 deg.C and 5% CO2Incubating in an incubator for 1-2h, collecting suspension without adhering free cells, centrifuging the sample for 5-10min, discarding supernatant, planting cells in a 24-well plate with 0.6-1ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study with primary cells after 24 h;
step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free culture medium DMEM to prepare a culture medium with the final amiodarone concentration of 10-180umolg/L, adding 1-sphingosine phosphate with the final concentration of 5-10 mu M into the culture medium containing amiodarone, wherein the 1-sphingosine phosphate has no toxic or side effect on lung tissues and is stored at 4-6 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the culture plate added with the CCK8 detection solution into a cell culture box at 37 ℃ again for culturing for 1-2 hours; the plate was removed and the absorbance was measured by a microplate reader at a wavelength of 450 nm.
As a further improvement of the invention, the SEW2871 agonist with the final concentration of 1-5 mu M is added into the culture medium containing amiodarone in the fourth step, and the SEW2871 agonist has no toxic or side effect on lung tissues.
As a further improvement of the invention, in the fourth step, dihydrocapsaicin with the final concentration of 0.1-0.5 mu M is added into the culture medium containing amiodarone, and the dihydrocapsaicin has no toxic or side effect on lung tissues.
As a further improvement of the invention, in the second step, rats are anesthetized by injecting 0.2-0.3ml/100g of 3% pentobarbital.
as a further improvement of the invention, the volume ratio of the resuspended precipitate and the erythrocyte lysate in the second step is 1: 8.
as a further improvement of the invention, in the second step, after the alveolar cavities are filled with the perfusate, the alveolar cavities are placed in a vibratory water bath at 37 ℃ for incubation for 10min, the perfusate is replaced with new perfusate, the alveolar cavities are incubated for 10min again, and the lungs are continuously massaged by hands in the incubation process.
the rat fibrotic pulmonary cells are separated, purified and subjected to primary culture, and then the rat type II alveolar epithelial cells are stimulated by amiodarone to obtain fibrotic pulmonary cells, so that the incidence rate of pulmonary fibers induced by the amiodarone can be increased, a large number of fibrotic pulmonary cells can be obtained in a short time, the condition of pulmonary fibrosis induced by the amiodarone in vivo can be accurately reflected, the phenotypic transformation regulation and mechanism of the type II alveolar epithelial cells are deeply researched, the significance is provided for delaying or even reversing the pulmonary fibrosis induced by the amiodarone, and a research object is provided for further exploring the pathogenesis of the mechanism of the pulmonary fibrosis induced by the amiodarone from the cellular and molecular level.
Detailed Description
the present invention will be described in further detail with reference to examples.
example 1
step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
step two, material taking: anesthetizing a rat by injecting 0.2ml/100g of 3% pentobarbital into the abdominal cavity of the rat for anesthesia, injecting 2800U heparin sodium into the abdominal cavity, opening the thoracic cavity of the rat after the rat is killed, washing blood by using a cleaning solution through the pulmonary artery of the right ventricle, then taking out the lung, perfusing the alveolar cavity with the perfusion solution, incubating in a vibration water bath at 37 ℃ for 10min, replacing the new perfusion solution, incubating for 10min again, continuously massaging the lung by hands in the incubating process, removing the trachea, the large airway and the heart, cutting the lung into 1mm in a culture dish containing DNase I3Adding 3ml of calf serum inactivated enzyme into a culture dish, centrifuging a sample for 5min, removing supernatant, resuspending a precipitate with a cleaning solution, adding a red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 5min to completely lyse red blood cells, centrifuging the sample for 5min, removing supernatant, resuspending the precipitate in a culture medium at 37 ℃ and counting;
Step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a culture box with a volume concentration of 5% CO2 at 37 deg.C for 40min, sucking out non-adherent cells, centrifuging the sample for 5min,collecting the lower layer, precipitating, inoculating into DMEM medium, repeating for 3 times, sucking out non-adherent cells, inoculating the suspension into culture dish coated with rat IgG, and standing at 37 deg.C and 5% CO2incubating in an incubator for 1h, collecting suspension without adhering free cells, centrifuging the sample for 5min, discarding supernatant, planting cells in a 24-well plate with 0.6ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
Step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free culture medium DMEM to prepare a culture medium with the amiodarone final concentration of 20umol g/L, adding sphingosine 1-phosphate with the final concentration of 5 mu M into the culture medium containing the amiodarone, and storing at 4 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 1 hour; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
Example 2
step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
Step two, material taking: anesthetizing a rat by injecting 0.3ml/100g of 3% pentobarbital into abdominal cavity of the rat, injecting 3000U of heparin sodium into the abdominal cavity, opening the thoracic cavity of the rat after the rat is killed, washing blood by using cleaning solution through pulmonary artery of right ventricle, taking out the lung, perfusing alveolar cavity by using perfusate, incubating in a 37 ℃ vibration water bath for 10min, replacing new perfusate, incubating again for 10min, replacing new perfusatethe perfusate was incubated for another 10min, the lungs were continuously massaged by hand during the incubation, the trachea, the large airways and the heart were removed, and the lungs were cut into 3mm pieces in a culture dish containing DNase I3Adding 6ml of inactivated calf serum enzyme into a culture dish, centrifuging the sample for 10min, removing the supernatant, resuspending the precipitate with a cleaning solution, adding a red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 8min to completely lyse the red blood cells, centrifuging the sample for 10min, removing the supernatant, resuspending the precipitate in a culture medium at 37 ℃ and counting;
Step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a culture box with 5% CO2 at 37 deg.C for 50min, sucking out non-adherent cells, centrifuging the sample for 10min, taking the lower layer precipitate, inoculating in DMEM culture medium, repeating the above steps for 4 times, sucking out non-adherent cells, inoculating the suspension in a culture dish coated with rat IgG, and placing at 37 deg.C with 5% CO2Incubating in an incubator for 2h, collecting suspension without adhering free cells, centrifuging the sample for 10min, discarding supernatant, planting cells in a 24-well plate with 0.8ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the final amiodarone concentration of 50umol g/L, adding sphingosine-1-phosphate with the final concentration of 8 mu M into the medium containing amiodarone, and storing at 6 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 1 hour; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
example 3
step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
step two, material taking: anesthetizing rat by injecting 3% pentobarbital 0.3ml/100g in abdominal cavity, injecting 3000U heparin sodium in abdominal cavity, opening thoracic cavity after rat death, washing blood with cleaning solution via right ventricle pulmonary artery, taking out lung, perfusing alveolar cavity with perfusate, incubating in 37 deg.C vibration water bath for 30min, removing trachea, airway and heart, cutting lung into 3mm pieces in culture dish containing DNase I3adding 5ml of inactivated calf serum enzyme into a culture dish, centrifuging the sample for 6min, removing supernatant, resuspending the precipitate with cleaning solution, adding red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 7min to completely lyse the red blood cells, centrifuging the sample for 5min, removing supernatant, resuspending the precipitate in a culture medium at 37 ℃ and counting;
Step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a culture box with 5% CO2 at 37 deg.C for 40min, sucking out non-adherent cells, centrifuging for 5min, collecting the lower layer precipitate, inoculating in DMEM culture medium, repeating the above steps for 4 times, sucking out non-adherent cells, inoculating the suspension in a culture dish coated with rat IgG, and standing at 37 deg.C with 5% CO2incubating in an incubator for 1h, collecting suspension without adhering free cells, centrifuging the sample for 10min, discarding supernatant, planting cells in a 24-well plate with 0.8ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
Step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the final amiodarone concentration of 80 umlg/L, adding sphingosine-1-phosphate with the final concentration of 8 mu M into the medium containing amiodarone, and storing at 6 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 2 hours; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
Example 4
Step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
Step two, material taking: anesthetizing a rat by injecting 0.2-0.3ml/100g of 3% pentobarbital into abdominal cavity of the rat, injecting 3000U of heparin sodium into the abdominal cavity, opening the thoracic cavity of the rat after the rat is killed, washing blood by using a cleaning solution through the pulmonary artery of the right ventricle, taking out the lung, perfusing the alveolar cavity by using a perfusion solution, incubating the rat in a 37 ℃ vibration water bath for 20min, removing the trachea, the large airway and the heart, cutting the lung into 2mm pieces in a culture dish containing DNase I3adding 6ml of inactivated calf serum enzyme into a culture dish, centrifuging the sample for 10min, removing the supernatant, resuspending the precipitate with a cleaning solution, adding a red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 8min to completely lyse the red blood cells, centrifuging the sample for 10min, removing the supernatant, resuspending the precipitate in a culture medium at 37 ℃ and counting;
step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a 37 deg.C 5% CO2 incubator for 60min, sucking out non-adherent cells, centrifuging the sample for 10min, taking down the lower layer precipitate, inoculating in DMEM culture medium, repeating the above steps for 4 times, sucking outNonadherent cells, the suspension was then seeded into rat IgG coated petri dishes at 37 ℃ with 5% CO2incubating in an incubator for 1h, collecting suspension without adhering free cells, centrifuging the sample for 5min, discarding supernatant, planting cells in a 24-well plate with 0.6ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
Step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the amiodarone final concentration of 110 umlg/L, adding sphingosine-1-phosphate with the final concentration of 9 mu M into the medium containing amiodarone, and storing at 6 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 1 hour; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
Example 5
step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
step two, material taking: anesthetizing a rat by injecting 0.2-0.3ml/100g of 3% pentobarbital into the abdominal cavity of the rat for anesthesia, injecting 2800U heparin sodium into the abdominal cavity, opening the thoracic cavity of the rat after the rat is killed, washing blood by using cleaning solution through the pulmonary artery of the right ventricle, then taking out the lung, perfusing the alveolar cavity with perfusate, incubating in a 37 ℃ vibration water bath for 10min, replacing with new perfusate, incubating again for 10min, continuously massaging the lung by hands in the incubating process, and removing the trachea, the large airway and the heartThe lungs were cut into 1mm pieces in a culture dish containing DNase I3Adding 3ml of calf serum inactivated enzyme into a culture dish, centrifuging a sample for 5min, removing supernatant, resuspending a precipitate with a cleaning solution, adding a red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 5min to completely lyse red blood cells, centrifuging the sample for 5min, removing supernatant, resuspending the precipitate in a culture medium at 37 ℃ and counting;
Step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a culture box with 5% CO2 at 37 deg.C for 45min, sucking out non-adherent cells, centrifuging for 5min, collecting the lower layer precipitate, inoculating in DMEM culture medium, repeating the above steps for 4 times, sucking out non-adherent cells, inoculating the suspension in a culture dish coated with rat IgG, and standing at 37 deg.C with 5% CO2incubating in an incubator for 2h, collecting suspension without adhering free cells, centrifuging the sample for 10min, discarding supernatant, planting cells in a 24-well plate with 0.8ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
Step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the final amiodarone concentration of 150 umlg/L, adding sphingosine-1-phosphate with the final concentration of 8 mu M into the medium containing amiodarone, and storing at 4 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 2 hours; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
Example 6
step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
step two, material taking: anesthetizing a rat by injecting 0.3ml/100g of 3% pentobarbital into the abdominal cavity of the rat, injecting 3000U of heparin sodium into the abdominal cavity, opening the thoracic cavity of the rat after the rat is killed, washing blood by using a cleaning solution through the pulmonary artery of the right ventricle, then taking out the lung, perfusing the alveolar cavity with the perfusion solution, placing the rat in a vibration water bath at 37 ℃ for incubation for 10min, replacing new perfusion solution, incubating for 10min again, continuously massaging the lung by hands in the incubation process, removing the trachea, the atmosphere and the heart, cutting the lung into 2mm pieces in a culture dish containing DNase I3Adding 4ml of inactivated calf serum enzyme into a culture dish, centrifuging the sample for 10min, removing supernatant, resuspending the precipitate with cleaning solution, adding red blood cell lysate preheated at 37 ℃ according to the volume ratio of 1: 8, repeatedly blowing and uniformly mixing, standing at room temperature for 8min to completely lyse the red blood cells, centrifuging the sample for 10min, removing supernatant, resuspending the precipitate in a 37 ℃ culture medium, and counting;
Step three, primary culture: inoculating the obtained cells in DMEM culture medium, incubating in a culture box with 5% CO2 at 37 deg.C for 55min, sucking out non-adherent cells, centrifuging for 5min, collecting the lower layer precipitate, inoculating in DMEM culture medium, repeating the above steps for 3 times, sucking out non-adherent cells, inoculating the suspension in a culture dish coated with rat IgG, and culturing at 37 deg.C with 5% CO2Incubating in an incubator for 2h, collecting suspension without adhering free cells, centrifuging the sample for 10min, discarding supernatant, planting cells in a 24-well plate with 1ml per well, culturing in a cell incubator at 37 ℃, and performing experimental study by using primary cells after 24 h;
Step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the amiodarone final concentration of 160 umlg/L, adding sphingosine-1-phosphate with the final concentration of 5 mu M into the medium containing amiodarone, and storing at 4 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the 24-hole plate added with the CCK8 detection solution into a 37 ℃ cell culture box again for culturing for 2 hours; the 24-well plate was taken out, and absorbance was measured using a microplate reader at a wavelength of 450 nm.
Example 7
The other experimental conditions were the same as in example 6, but in step four the agonist SEW2871 was added to the amiodarone containing medium to a final concentration of 1, 2, 3, 4 or 5. mu.M.
Example 8
The experimental conditions were the same as in example 6, but 3. mu.M of the agonist SEW2871 was added and in step four, 0.1, 0.2, 0.3, 0.4 or 0.5. mu.M dihydrocapsaicin was added to the amiodarone-containing medium.
CCK8 test results:
In example 7, the proliferation of fibroblasts obtained by adding different concentrations of the agonist SEW2871 is shown in the table, in which the agonist SEW2871 has an effect of promoting the proliferation of fibroblasts, and the proliferation of fibroblasts is accelerated as the concentration of the agonist SEW2871 is increased.
*P<0.05；**P<0.01
In example 8, the proliferation of fibroblasts obtained by adding different concentrations of dihydrocapsaicin is shown in the table, in which the dihydrocapsaicin concentration has an effect of promoting the proliferation of fibroblasts, and the proliferation of fibroblasts is accelerated as the dihydrocapsaicin concentration increases.
*P<0.05；**P<0.01
hydroxyproline content determination
Hydroxyproline is a specific amino acid in collagen fibers, the content of collagen can be converted by measuring the content of hydroxyproline in the lung so as to judge the degree of pulmonary fibrosis, the content of hydroxyproline in a culture medium is measured according to the method of a hydroxyproline detection kit specification, an A value is measured at 540nm of an enzyme labeling instrument, and the mass concentration (mg/L of the culture medium) of hydroxyproline in a sample is equal to the A value of a measuring tube multiplied by the concentration of a standard tube/the A value of a standard solution. The hydroxyproline content in the amiodarone fibrosis-causing lung cells is determined, and the result is as follows: the control group is type II alveolar epithelial cells obtained by primary culture
the above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may occur to those skilled in the art without departing from the principle of the invention, and are considered to be within the scope of the invention.
Claims (4)
1. A culture method and a detection method of rat fibrotic lung cells are characterized in that: the method comprises the following steps:
Step one, preparation of a culture medium, a perfusate and a cleaning solution: preparing a DMEM culture medium, and then adding fetal calf serum with the final concentration of 100ml/L, penicillin 100U/ml and streptomycin 100U/ml; preparing perfusate, preparing trypsin with the mass fraction of 0.1% by using PBS, adding penicillin with the final concentration of 100U/ml, streptomycin with the final concentration of 100U/ml and EDTA with the mass fraction of 0.02%, and filtering and sterilizing to obtain the perfusate; preparing PBS cleaning solution, and then adding penicillin and streptomycin with final concentration of 100U/ml;
Step two, material taking: injecting anesthetic into abdominal cavity of rat for anesthesia, injecting heparin sodium 2800-;
Step three, primary culture: inoculating the obtained cells in a DMEM culture medium, incubating in a 37 ℃ and 5% CO2 culture box for 40-60min, sucking out nonadherent cells, centrifuging the sample for 5-10min, taking the lower layer precipitate, inoculating in the DMEM culture medium, repeating the steps for 3-4 times, sucking out nonadherent cells, inoculating the suspension in a culture dish coated with rat IgG, incubating in a 37 ℃ and 5% CO2 culture box for 1-2h, collecting the suspension without adhering free cells, centrifuging the sample for 5-10min, discarding the supernatant, planting the cells in a cell culture plate with 0.6-1ml per hole, culturing in a 37 ℃ cell culture box, and carrying out experimental study on primary cells after 24 h;
step four, culturing the amiodarone-induced fibrotic lung cells: adding amiodarone into serum-free medium DMEM to prepare a medium with the final amiodarone concentration of 10-180umolg/L, adding sphingosine-1-phosphate with the final concentration of 5-10 mu M into the medium containing amiodarone, adding SEW2871 serving as an agonist with the final concentration of 1-5 mu M, adding dihydrocapsaicin with the final concentration of 0.1-0.5 mu M, and storing at 4-6 ℃; replacing the culture medium with a fresh culture medium containing amiodarone with different concentrations, and placing the cell culture plate in a cell culture box at 37 ℃ for culture;
Step five, detection: adding a CCK8 reagent into each hole of the culture plate, slightly knocking the culture plate after the reagent is added, and uniformly mixing a CCK8 detection solution; placing the culture plate added with the CCK8 detection solution into a cell culture box at 37 ℃ again for culturing for 1-2 hours; the plate was removed and the absorbance was measured by a microplate reader at a wavelength of 450 nm.
2. The method for culturing rat fibrotic lung cells and the method for detecting the same according to claim 1, wherein: in the second step, rats are anesthetized by injecting 0.2-0.3ml/100g of 3% pentobarbital.
3. The method for culturing rat fibrotic lung cells and the method for detecting the same according to claim 2, wherein the method comprises: and the volume ratio of the resuspended precipitate to the erythrocyte lysate in the step two is 1: 8.
4. the method for culturing rat fibrotic lung cells and the method for detecting the same according to claim 3, wherein the method comprises: and in the second step, after the alveolar cavities are filled with the perfusate, putting the alveolar cavities into a 37 ℃ vibration water bath for incubation for 10min, replacing with a new perfusate, incubating for 10min again, and continuously massaging the lungs with hands in the incubation process.
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