CN113337453A - Method for detecting pulmonary toxicity of compounds - Google Patents

Method for detecting pulmonary toxicity of compounds Download PDF

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CN113337453A
CN113337453A CN202110598316.5A CN202110598316A CN113337453A CN 113337453 A CN113337453 A CN 113337453A CN 202110598316 A CN202110598316 A CN 202110598316A CN 113337453 A CN113337453 A CN 113337453A
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alveolar
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杨仁君
刘抒羽
殷诺雅
费凡
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Research Center for Eco Environmental Sciences of CAS
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Abstract

The invention provides a method for detecting the toxicity of a compound to human lungs, which uses a human pluripotent stem cell lung differentiation model to simulate the real situation in a human body and can efficiently and accurately evaluate the toxicity of the compound to the human lungs. The method can be used as an effective means for evaluating the pulmonary toxicity of PM2.5 and other environmental pollutants, and can also be used for evaluating the pulmonary toxicity of preclinical medicines, so that the risk of clinical experiment failure is reduced.

Description

Method for detecting pulmonary toxicity of compounds
Technical Field
The invention belongs to the cross field of cell biology and environmental toxicology, and relates to a method for detecting the lung toxicity of a compound.
Background
With the development of world science and technology and the improvement of industry, a large amount of substances with unknown toxicity are continuously discharged into the environment, wherein a part of substances can be dissociated in the air or form haze, and further transmitted through the air and inhaled by a human body to cause adverse effects on the health of the human body. Therefore, there is a need for an effective research means for evaluating the effect of compounds on human lung, preventing and preventing the toxic air pollutants from harming human health.
Most of the previous lung toxicity researches depend on animal models or lung cancer cell models, however, animal experiment operation is difficult, experiment amount is large, and cross-species differences exist when results are extrapolated to human beings; cancer cell test since the test material is a cell that has become cancerous, it is impossible to objectively respond to the toxicity of the compound to normal cells. In addition, the method of directly isolating alveolar cells from human body as research material is also difficult, and although some experimental procedures for isolating alveolar epithelial precursor cells from human fetus have been reported in the prior art, alveolar epithelial precursor cells usually appear from 26 weeks of pregnancy, where no sample can be obtained unless spontaneous abortion occurs, and neither purification nor extensive culture techniques of alveolar epithelial cells are mature.
Therefore, there is a need for a method of testing compounds for pulmonary toxicity that at least partially addresses the above-mentioned problems.
Disclosure of Invention
The human pluripotent stem cells are sub-totipotent stem cells which can be proliferated almost indefinitely and keep normal karyotypes, a large number of human lung precursors or lung epithelial cells can be obtained by expanding and culturing the human pluripotent stem cells and then inducing the human pluripotent stem cells to differentiate, and the cells not only can meet the requirement of high-throughput toxicology screening in quantity, but also are normal human cells, so that the cells are very suitable for researching the human lung toxicity of compounds.
Therefore, an object of the present invention is to provide a highly efficient and accurate in vitro method for detecting pulmonary toxicity in human, wherein the detection is performed by using human pluripotent stem cell-induced lung precursor cells or alveolar cells.
The invention provides the following technical scheme:
in one aspect, the invention provides lung precursor cells induced by induced human pluripotent stem cells.
In some embodiments, the lung precursor cells are induced from induced human pluripotent stem cells by the following induction phase:
a. inducing the human pluripotent stem cells to differentiate into primitive streak cells;
b. inducing the primitive streak cells to differentiate into definitive endoderm cells;
c. inducing definitive endoderm cells to differentiate into head foregut cells;
d. inducing differentiation of the head foregut cells into lung precursor cells.
In some embodiments, the basal medium used to induce differentiation is DMEM/F12 medium containing 1 XGlutaMAX, 1 Xpenicillin-streptomycin, 1 XN 2 supplement, 1 XB 27 supplement, and ascorbic acid at 40-60 μ g/mL, preferably 40, 50, 60 μ g/mL, more preferably 50 μ g/mL.
In some embodiments, the medium used to induce differentiation of human pluripotent stem cells into primitive streak cells is the above-described basal medium to which Activin A (20-500ng/mL, preferably 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500ng/mL, more preferably 100ng/mL) and CHIR99021 (1-12. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. mu.M, more preferably 3. mu.M) are added.
In some embodiments, the induction time to induce differentiation of human pluripotent stem cells into primitive streak cells is 1-3 days, preferably 1, 2, 3 days, more preferably 1 day.
In some embodiments, the medium used to induce differentiation of primitive streak cells into definitive endoderm cells is the basal medium described above to which Activin A (20-500ng/mL, preferably 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500ng/mL, more preferably 100ng/mL) is added.
In some embodiments, the medium used to induce the differentiation of definitive endoderm cells into head foregut cells is the basal medium described above to which SB431542 (1-20. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. mu.M, more preferably 10. mu.M) and dorsomorphin (1-10. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. mu.M, more preferably 2. mu.M) are added.
In some embodiments, the induction time to induce definitive endoderm cells to differentiate into head foregut cells is 2-4 days, preferably 2, 3, 4 days, and more preferably 3 days.
In some embodiments, the medium used to induce differentiation of the head foregut cells into lung precursor cells is the basal medium described above to which CHIR99021 (1-12. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. mu.M, more preferably 3. mu.M), retinoic acid (10-100nM, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100nM, more preferably 50nM), BMP4(1-30ng/mL, preferably 1, 5, 8, 15, 20, 25, 30ng/mL, more preferably 10ng/mL), FGF7 (10-50/mL, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50ng/mL, more preferably 10ng/mL), FGF 26 (10-50ng/mL, preferably 10, 15, 20, 25, 30, 40, 45, 50ng/mL, more preferably 10ng/mL), FGF10(10-50ng/mL, preferably 10, 15, 20, 25, 30, 35. 40, 45, 50ng/mL, more preferably 10 ng/mL).
In some embodiments, the induction time to induce differentiation of the head foregut cells into lung precursor cells is 8-10 days, preferably 8, 9, 10 days, more preferably 9 days.
In another aspect, the present invention provides an alveolar cell induced by an induced human pluripotent stem cell.
In some embodiments, the alveolar cells are induced by inducing human pluripotent stem cells through the following induction stages:
a. inducing the human pluripotent stem cells to differentiate into primitive streak cells;
b. inducing the primitive streak cells to differentiate into definitive endoderm cells;
c. inducing definitive endoderm cells to differentiate into head foregut cells;
d. inducing differentiation of the head foregut cells into lung precursor cells.
e. Inducing lung precursor cells to differentiate into alveolar cells.
In some embodiments, the basal medium used to induce differentiation is DMEM/F12 medium containing 1 XGlutaMAX, 1 Xpenicillin-streptomycin, 1 XN 2 supplement, 1 XB 27 supplement, and ascorbic acid at 40-60 μ g/mL, preferably 40, 50, 60 μ g/mL, more preferably 50 μ g/mL.
In some embodiments, the medium used to induce differentiation of human pluripotent stem cells into primitive streak cells is the above-described basal medium to which Activin A (20-500ng/mL, preferably 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500ng/mL, more preferably 100ng/mL) and CHIR99021 (1-12. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. mu.M, more preferably 3. mu.M) are added.
In some embodiments, the induction time to induce differentiation of human pluripotent stem cells into primitive streak cells is 1-3 days, preferably 1, 2, 3 days, more preferably 1 day.
In some embodiments, the medium used to induce differentiation of primitive streak cells into definitive endoderm cells is the basal medium described above to which Activin A (20-500ng/mL, preferably 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500ng/mL, more preferably 100ng/mL) is added.
In some embodiments, the medium used to induce the differentiation of definitive endoderm cells into head foregut cells is the basal medium described above to which SB431542 (1-20. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20. mu.M, more preferably 10. mu.M) and dorsomorphin (1-10. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. mu.M, more preferably 2. mu.M) are added.
In some embodiments, the induction time to induce definitive endoderm cells to differentiate into head foregut cells is 2-4 days, preferably 2, 3, 4 days, and more preferably 3 days.
In some embodiments, the medium used to induce differentiation of the head foregut cells into lung precursor cells is the basal medium described above to which CHIR99021 (1-12. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. mu.M, more preferably 3. mu.M), retinoic acid (10-100nM, preferably 10, 20, 30, 40, 50, 60, 70, 80, 90, 100nM, more preferably 50nM), BMP4(1-30ng/mL, preferably 1, 5, 8, 15, 20, 25, 30ng/mL, more preferably 10ng/mL), FGF7 (10-50/mL, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50ng/mL, more preferably 10ng/mL), FGF 26 (10-50ng/mL, preferably 10, 15, 20, 25, 30, 40, 45, 50ng/mL, more preferably 10ng/mL), FGF10(10-50ng/mL, preferably 10, 15, 20, 25, 30, 35. 40, 45, 50ng/mL, more preferably 10 ng/mL).
In some embodiments, the induction time to induce differentiation of the head foregut cells into lung precursor cells is 8-10 days, preferably 8, 9, 10 days, more preferably 9 days.
In some embodiments, the medium used to induce differentiation of lung precursor cells into alveolar cells is the above-described basal medium supplemented with CHIR99021 (1-12. mu.M, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12. mu.M, more preferably 3. mu.M), FGF7(10-50ng/mL, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50ng/mL, more preferably 10ng/mL), dexamethasone (10-50nM, preferably 10, 15, 20, 25, 30, 35, 40, 45, 50nM, more preferably 50nM), cAMP (0.1-2mM, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 0.9, 1.0, 1.1.1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.1.8, 1.9, 1.2, 1.3, 1.4, 1.3, 1.9, 1.8, 1.0.0.0.0.9, 1.9, 1.0.0.0.0.0.9, 1.1.1.2, 1, 1.9, 1.1.2, 1.3, 1.4, 1.9, 1.4, 1.9, or more preferably 1.4, or 1.0.4, or 1.0.1.1.1.1.1.1.4, or more preferably 1.1.1.1.1.1.2 mM, or more preferably 1.2 mM, or more preferably 1.0.1.1.1.0.0.0.0 mM, or 1mM, or more preferably 1.0.0.0.0 mM, or 1.3, or more preferably 1mM, or 1.0.0.0.0.0.3, or more preferably 1mM, or 1.3, or more preferably 1mM, or more preferably 1.1.3, or more preferably 1.3, or more preferably 1mM, or more preferably 1.0.0.0.0.0.0.0 mM, or more preferably 1.1.1.1.1.1.1 mM, or more preferably 1.0.0.0.0.3, or more preferably 1.3, or more preferably 1.1.0.0.0.0.3, or more preferably 1.3, or more preferably 1.1.0.0.0.0.0.0.0.0.0.3, or more preferably 1.3, or more preferably 1.1.3, or more, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0mM, more preferably 0.1 mM).
In some embodiments, the induction time to induce differentiation of lung precursor cells into alveolar cells is 13-15 days, preferably 13, 14, 15 days, more preferably 14 days.
In another aspect, the invention provides the use of a lung precursor cell or an alveolar cell as described above for detecting pulmonary toxicity of a compound.
In another aspect, the present invention provides a method of detecting pulmonary toxicity of a compound, the method comprising the steps of:
s1, inducing human pluripotent stem cells to differentiate into lung precursor cells or alveolar cells;
s2. treating the lung precursor cells or alveolar cells with a compound at a concentration of 1nM to 1mM, preferably 10nM, 50nM, 100nM, 500nM, 1. mu.M, 5. mu.M and 10. mu.M for 1 to 8 days, preferably for 1, 2, 3, 4, 5, 6, 7, 8 days, more preferably for 2 days, 8 days;
s3, detecting the concentration of the compound which can affect the basic toxicity index of lung precursor cells or alveolar cells;
s4, detecting the change of the lung precursor cell or alveolar cell biomarker under non-lethal concentration.
In some embodiments, the basal toxicity index is selected from one or more of the group consisting of cell viability, mitochondrial membrane integrity, active oxygen production, calcium flux changes, and lactate dehydrogenase leakage, preferably cell viability and mitochondrial membrane integrity.
In some embodiments, the method of detecting cell viability is selected from the group consisting of resazurin, MTT, live/dead cell counting assays, and preferably resazurin.
In some embodiments, the method of detecting mitochondrial membrane integrity is selected from the group consisting of JC-1 staining, tetramethyl rhodamine methyl ester staining, acridine orange 10-bromononane staining, preferably JC-1 staining.
In some embodiments, the biomarker of lung precursor cells or alveolar cells is selected from the group consisting of NKX2.1, ID2, SFTPC, ABCA3, and NAPSA genes.
In some embodiments, the biomarker for lung precursor cells is selected from the group consisting of NKX2.1, ID2 and SFTPC.
In some embodiments, the biomarker of alveolar cells is selected from the group consisting of NKX2.1, ID2, SFTPC, ABCA3, and NAPSA genes.
In some embodiments, the method of detecting a change in a lung precursor cell or alveolar cell biomarker comprises fluorescent quantitative PCR.
Compared with the prior art, the invention has the following advantages:
1. the invention provides a feasible and reliable human lung toxicity research method.
2. Normal human lung cells are extremely difficult to obtain and therefore cannot be used for toxicology studies. The invention is based on the induced human pluripotent stem cell lung differentiation model, can easily obtain a large amount of normal human lung cells, can solve the source problem of the human lung cells, and lays a foundation for high-throughput accurate detection of pulmonary toxicity.
3. Compared with cancer cell experiments, the invention uses normal cells, which is closer to the real situation of healthy human body.
4. Compared with animal experiments, the method has the advantages of low cost, short time consumption, real-time monitoring and easy control of external factor influence.
Drawings
FIG. 1 is a lung precursor cell morphology according to an embodiment of the invention;
FIG. 2 is an alveolar cell morphology according to an embodiment of the present invention;
FIG. 3 is a graph of the effect of benzopyrene on lung precursor cell viability, p <0.05, p <0.01, t-test analysis, according to an embodiment of the present invention;
FIG. 4 is a graph of the effect of benzopyrene on alveolar cell viability, p <0.05, p <0.01, t-test analysis, according to an embodiment of the present invention;
FIG. 5 is a graph of the effect of benzopyrene on mitochondrial membrane integrity of lung precursor cells according to an embodiment of the present invention, p <0.05, p <0.01, t-test analysis;
FIG. 6 is a graph of the effect of benzopyrene on alveolar cell mitochondrial membrane integrity, p <0.05, p <0.01, t-test analysis, according to an embodiment of the present invention;
FIG. 7 is a graph of the effect of benzopyrene on the lung precursor cell biomarker NKX2.1, according to an embodiment of the present invention;
FIG. 8 is a graph of the effect of benzopyrene on the lung precursor cell biomarker ID2, according to an embodiment of the present invention;
FIG. 9 is a graph of the effect of benzopyrene on the lung precursor cell biomarker SFTPC according to an embodiment of the present invention;
FIG. 10 is a graph of the effect of benzopyrene on the alveolar cell biomarker NKX2.1, in accordance with an embodiment of the present invention;
FIG. 11 is a graph of the effect of benzopyrene on alveolar cell biomarker ID2, according to an embodiment of the present invention;
FIG. 12 is a graph of the effect of benzopyrene on the alveolar cell biomarker SFTPC, in accordance with an embodiment of the present invention;
FIG. 13 is a graph of the effect of benzopyrene on the alveolar cell biomarker ABCA3, according to an embodiment of the present invention;
FIG. 14 is a graph of the effect of benzopyrene on the alveolar cell biomarker NAPSA, according to an embodiment of the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
Example 1 Induction of differentiation of human pluripotent Stem cells into definitive endoderm cells
All cells of the present invention were incubated at 37 ℃ in a cell incubator containing 5% carbon dioxide.
The initial cell used in the present invention is induced human pluripotent stem cell DYR0100 (Chinese academy of sciences cell bank/stem cell technology platform, SCSP-1301). DYR0100 was maintained in matrigel (Corning, 356231) -coated 24-well plates using 10mL of mTesR1(stem cell Technologies, 85850) medium. When the confluency of DYR0100 clone reached 90%, differentiation was induced.
The basal medium used for inducing differentiation in the present invention was 100mL of DMEM/F12(Gibco, C1410500BT330500BT) medium supplemented with 1mL of 100 XGlutaMAX (Gibco, 35050061), 1mL of 100 Xpenicillin-streptomycin (Gibco, 15140163), 1mL of 100 XN 2 supplement (Gibco, 17502001), 2mL of 50 XB 27 supplement (Gibco, 17504044), and 0.1mL of 50mg/mL ascorbic acid (original leaf, S24742).
On day 0 of differentiation, 100ng/mL Activin A (Peprotech, 120-14E) and 3. mu.M CHIR99021(Selleck, S2924) were added to the basal medium to induce differentiation of the cells into primitive streak cells.
On day 1 of differentiation, 100ng/mL Activin A was added to the basal medium to induce primitive streak cells to differentiate for 3 days, and definitive endoderm cells were obtained.
The definitive endoderm obtained by the invention is a dense monolayer of cells.
EXAMPLE 2 Induction of definitive endoderm differentiation into Lung precursor cells
Definitive endoderm cells finally obtained in example 1 were used.
The definitive endoderm cells obtained in example 1 were induced to differentiate for 3 days into foregut cells by adding 10. mu.M of SB431542(Selleck, S2924) and 2. mu.M of dorsomorphin (Selleck, S7306) to the basal medium.
mu.M CHIR99021, 50nM retinoic acid (Sigma, R2625), 10ng/mL BMP4(Gibco, PHC9531), 10ng/mL FGF7(R & D, 251-KG-050), and 10ng/mL FGF10(R & D, 345-FG-025) were added to the basal medium, and the above-obtained anterior enterocytes were induced to differentiate for 9 days into lung precursor cells (FIG. 1).
Example 3 Induction of differentiation of Lung precursor cells into alveolar cells
The lung precursor cells finally obtained in example 2 were used.
mu.M CHIR99021, 10ng/mL FGF7, 50nM dexamethasone, 0.1mM cAMP (Sigma-Aldrich, B5386) and 0.1mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, I5879-250MG) were added to the basal medium to induce the lung precursor cells obtained in example 2 to differentiate for about 16 days, and finally become alveolar cells (FIG. 2).
Example 4 treatment of Lung precursor cells with Compounds
The lung precursor cells finally obtained in example 2 were used.
The lung precursor cells obtained in example 2 were digested with TryplE, and the cells were seeded in a matrigel-coated 96-well plate in an amount of 15,000 cells per square centimeter. Lung precursor cells were cultured in a basal medium supplemented with 3. mu.M CHIR99021, 50nM retinoic acid, 10ng/mL BMP4, 10ng/mL FGF7, 10ng/mL FGF10 and the compound.
The compound used is benzopyrene (BaP) which is the main component of the atmospheric pollutants.
The assay was performed using 96-well plates, with BaP concentrations in the medium of 1, 5, 10, 50, 100, 500 and 1000nM, respectively, and controls treated with DMSO.
Example 5 treatment of Lung precursor cells with Compounds
Example 5 was the same as example 4 except that 24-well plates were used for the culture and the BaP concentration in the medium was 10, 50, 100nM, and the control was also treated with DMSO.
Example 6 treatment of alveolar cells with Compounds
The alveolar cells finally obtained in example 3 were used.
Alveolar cells obtained in example 3 were digested with TryplE, and the cells were seeded in matrigel-coated 96-well plates in an amount of 15,000 cells per square centimeter. Alveolar cells were cultured in basal medium supplemented with 3. mu.M CHIR99021, 10ng/mL FGF7, 50nM dexamethasone, 0.1mM cAMP, 0.1mM 3-isobutyl-1-methylxanthine and compound.
The compound used was BaP.
The assay was performed using 96-well plates, with BaP concentrations in the medium of 1, 5, 10, 50, 100, 500 and 1000nM, respectively, and controls treated with DMSO.
Example 7 treatment of alveolar cells with Compounds
Example 7 was the same as example 6 except that 24-well plates were used for the culture and the BaP concentration in the medium was 10nM, 50nM, 100nM, and the control was also treated with DMSO.
Example 8 cell viability assay of Lung precursor cells and alveolar cells
Since normal human alveoli generally contain lung precursor cells and alveolar cells, lung precursor cells and alveolar cells obtained by induced differentiation are preferred as a test model for detecting the pulmonary toxicity of a compound. The cytotoxicity of the compound generally causes the reduction of the cell number or the functional damage, and the health condition of the cell (whether the cell activity is damaged) and the existence of the apoptosis precursor (whether the mitochondrial membrane potential is reduced) can be simply, conveniently and quickly known by detecting the cell activity and the mitochondrial membrane potential. In this example, the compound was selected to be the main component of air pollutants, BaP, and the lung precursor cells of example 4 and alveolar cells of example 6 were washed once with DPBS after 2 days of BaP treatment. To the DPBS-washed cell culture plate, 100. mu.L of resazurin cell viability assay working solution (basal medium containing 10. mu.M resazurin) was added, and the mixture was incubated in a carbon dioxide incubator at 37 ℃ for 1 hour. The fluorescence of the resazurin-treated cells was measured using a Varioskan LUX multi-functional microplate reader. The fluorescence is 590nm fluorescence emitted by each well under excitation of 530nm light. Fluorescence detection results show that more than 1 μ M benzopyrene can inhibit cell viability of lung precursor cells (as shown in FIG. 3), and more than 5 μ M benzopyrene can inhibit cell viability of alveolar cells (as shown in FIG. 4). The above results indicate that lung precursor cells are more sensitive to benzopyran treatment than terminally differentiated alveolar cells. Since current stem cell toxicology studies show that precursor cells of the same lineage tend to respond more strongly to environmental signals than terminally differentiated cells, the phenomenon in figure 4 is more reasonable.
Example 9 mitochondrial Membrane integrity assays of Lung precursor cells and alveolar cells
Since the compound concentration at μ M level tends to show toxicity at 0.5-2 days, the lung precursor cells of example 4 and the alveolar cells of example 6 were treated with benzopyrene for 0.5-2 days, preferably 0.5, 1, 1.5, 2 days, and for 2 days in this example. After treatment, cells were washed once with DPBS. To the DPBS-washed cell culture plates, the same volume of basal medium containing 2. mu.g/mL JC-1 dye was added per well and incubated in a carbon dioxide incubator at 37 ℃ for 30 min. The supernatant of the JC-1 stained cells was discarded and an equal volume of basal medium was added again. The JC-1 stained cells were examined for green fluorescence at 490/530nm and red fluorescence at 525/590nm using a Varioskan LUX multifunctional microplate reader. The green fluorescence shows free JC-1 in the cytoplasm and the red fluorescence shows the aggregated JC-1 in intact mitochondria. JC-1 staining results show that the benzopyrene treatment of more than 1 μ M of lung precursor cells can reduce the mitochondrial membrane potential of the lung precursor cells, the benzopyrene treatment of more than 5 μ M can reduce the mitochondrial membrane potential of the alveolar cells, which suggests that 1 μ M of benzopyrene can cause mitochondrial damage of the lung precursor cells, 5 μ M of benzopyrene can cause mitochondrial damage of the alveolar cells, and the results are similar to the results obtained by cell viability detection, and the lung precursor cells with stem cell characteristics are more sensitive to the benzopyrene treatment.
Example 10 detection of Lung precursor cell biomarkers
The lung precursor cells of example 5 and the alveolar cells of example 7 are treated with benzopyrene for 1, 2, 3, 4, 5, 6, 7 and 8 days, and the supernatant is discarded after 8 days in this example to amplify the toxic effect and ensure a short experimental period.
The RNA of the compound-treated sample was extracted using TRNzol Total RNA extraction reagent (DP 424, Tiangen Biochemical technology, Inc., Beijing).
Genomic DNA contamination and reverse transcription in the samples were removed using the FastKing kit (Tiangen Biochemical technology (Beijing) Ltd., KR 118).
The samples were subjected to fluorescent quantitative PCR using FastFire qPCR PreMix (tiangen biochemistry technologies (beijing) ltd., FP 207).
The primers used in the fluorescent quantitative PCR experiment are designed according to the human genome transcript information published by NCBI, the product length is about 80-120 bp, and the PCR primer information is as follows:
GAPDH-Fw：5′-GGTCACCAGGGCTGCTTTTA-3′
GAPDH-Rv：5′-GGATCTCGCTCCTGGAAGATG-3′
NKX2.1-Fw：5′-CTCATGTTCATGCCGCTC-3′
NKX2.1-Rv：5′-GACACCATGAGGAACAGCG-3′
ID2-Fw：5′-GACAGCAAAGCACTGTGTGG-3′
ID2-Rv：5′-TCAGCACTTAAAAGATTCCGTG-3′
SFTPC-Fw：5′-AGCAAAGAGGTCCTGATGGA-3′
SFTPC-Rv：5′-CGATAAGAAGGCGTTTCAGG-3′
ABCA3-Fw：5′-AAGATGTAGCGGACGAGAGG-3′
ABCA3-Rv：5′-CCCCGGTCAGCATTTTGAAA-3′
NAPSA-Fw：5′-ACGCCTCCACAAAACTTCAC-3′
NAPSA-Rv：5′-TGGCAAACTTGGTCCCATTG-3′
the reaction conditions of the fluorescent quantitative PCR are as follows: pre-denaturation at 95 ℃ for 30 seconds; 95 deg.C, 5 seconds, 60 deg.C, 30 seconds, 50 cycles.
The expression levels of marker genes NKX2.1, ID2 and SFTPC were detected in lung precursor cells using fluorescent quantitative PCR. The results of the correlation measurements are shown in FIGS. 7-9. The experimental results show that BaP treatment can inhibit the expression of transcription factors NKX2.1, ID2 and a lung surfactant protein coding gene SFTPC in lung precursor cells, and the inhibition effect shows a dose-dependent enhancement trend. The inhibition of the key transcription factors NKX2.1, ID2 and important lung surfactant protein coding gene SFTPC by BaP shows that the BaP has great influence on the function of lung precursor cells.
Since alveolar cells are differentiated from lung precursor cells, NKX2.1, ID2 and SFTPC genes are also expressed in alveolar cells, and thus, they are also used as markers of alveolar cells under study, and furthermore, alveolar cells have unique markers ABCA3 and NAPSA. Thus, this example uses fluorescent quantitative PCR to detect the expression levels of alveolar cell marker genes NKX2.1, ID2, SFTPC, ABCA3, and NAPSA. The results of the correlation measurements are shown in FIGS. 10-14. From the results, it can be seen that the expression levels of NKX2.1, ID2, SFTPC, ABCA3 and NAPSA all showed a dose-dependent decrease trend with an increase in BaP concentration, suggesting that BaP inhibits the expression of the above genes in which NAPSA encodes a protease required for lung surfactant protein maturation and ABCA3 encodes an important transporter associated with lung active protein secretion. Changes in the expression levels of the relevant genes suggest that BaP exposure inhibits the synthesis and transport of lung surfactant proteins, thereby impairing alveolar cell function.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The lung precursor cell is characterized in that the lung precursor cell is induced by inducing human pluripotent stem cells through the following induction stages:
a. inducing the human pluripotent stem cells to differentiate into primitive streak cells;
b. inducing the primitive streak cells to differentiate into definitive endoderm cells;
c. inducing definitive endoderm cells to differentiate into head foregut cells;
d. inducing differentiation of the head foregut cells into lung precursor cells.
2. The alveolar cell is characterized in that the alveolar cell is formed by inducing human pluripotent stem cells through the following induction stages:
a. inducing the human pluripotent stem cells to differentiate into primitive streak cells;
b. inducing the primitive streak cells to differentiate into definitive endoderm cells;
c. inducing definitive endoderm cells to differentiate into head foregut cells;
d. inducing differentiation of the head foregut cells into lung precursor cells.
e. Inducing lung precursor cells to differentiate into alveolar cells.
3. Use of a lung precursor cell according to claim 1 or an alveolar cell according to claim 2 for detecting pulmonary toxicity of a compound.
4. A method for detecting pulmonary toxicity of a compound, comprising the steps of:
s1, inducing human pluripotent stem cells to differentiate into lung precursor cells or alveolar cells;
s2. treating the lung precursor cells or alveolar cells with a compound at a concentration of 1nM to 1mM, preferably 10nM, 50nM, 100nM, 500nM, 1. mu.M, 5. mu.M and 10. mu.M for 1 to 8 days, preferably for 1, 2, 3, 4, 5, 6, 7, 8 days, more preferably for 2 or 8 days;
s3, detecting the concentration of the compound which can affect the basic toxicity index of lung precursor cells or alveolar cells;
s4, detecting the change of the lung precursor cell or alveolar cell biomarker under non-lethal concentration.
5. The method according to claim 4, wherein the basal toxicity index is selected from one or more of the group consisting of cell viability, mitochondrial membrane integrity, active oxygen production, calcium flux changes and lactate dehydrogenase leakage, preferably cell viability and mitochondrial membrane integrity.
6. The method according to claim 5, wherein the method for detecting the viability of the cells is selected from the group consisting of Resazurin, MTT, live/dead cell counting assay, preferably Resazurin.
7. The method according to claim 5, wherein the mitochondrial membrane integrity is detected by a method selected from the group consisting of JC-1 staining, tetramethyl rhodamine methyl ester staining, acridine orange 10-bromononane staining, preferably JC-1 staining.
8. The method of claim 4, wherein the biomarker for lung precursor cells or alveolar cells is selected from the group consisting of NKX2.1, ID2, SFTPC, ABCA3 and NAPSA genes.
9. The method of claim 8, wherein the biomarker for lung precursor cells is selected from the group consisting of NKX2.1, ID2 and SFTPC.
10. The method of claim 4, wherein the method of detecting a change in a biomarker of a lung precursor cell or an alveolar cell comprises fluorescent quantitative PCR.
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