CN116949096A - Construction method of mouse lung adenocarcinoma model with Nrf1 alveolus II type epithelial cells specifically knocked out - Google Patents

Construction method of mouse lung adenocarcinoma model with Nrf1 alveolus II type epithelial cells specifically knocked out Download PDF

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CN116949096A
CN116949096A CN202310968710.2A CN202310968710A CN116949096A CN 116949096 A CN116949096 A CN 116949096A CN 202310968710 A CN202310968710 A CN 202310968710A CN 116949096 A CN116949096 A CN 116949096A
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房昕
徐苑苑
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China Medical University
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Abstract

The invention relates to a specific knockout of alveolar type II epithelial cellsNrf1A method for constructing a lung adenocarcinoma model of a mouse belongs to the technical field of medicine. Specifically comprises the induction by using the carbamic acid ethyl esterNrf1Pulmonary adenocarcinoma occurs in alveolar type II epithelial cell-specific knockout mice. On the basis of the traditional model of the lung adenocarcinoma of the mice caused by the urethane, the tumor load of the lung tumor of the mice of the C57BL/6 strain is improved, the total molding time is shortened, the cost benefit is reduced, a reliable model is provided for the research of complex carcinogenesis mechanisms involving genes and exogenous environmental factors in the development of the lung adenocarcinoma, and the method has important research significance for the establishment of intervention strategies and prevention measures of the lung adenocarcinoma. The method is simple to operate, high in repeatability, easy to master and convenient to popularize.

Description

Construction method of mouse lung adenocarcinoma model with Nrf1 alveolus II type epithelial cells specifically knocked out
Technical Field
The invention belongs to the technical field of medicine, and in particular relates to a method for constructing a specific knockout of alveolar II type epithelial cellsNrf1A method for constructing a mouse lung adenocarcinoma model by combining a mouse with a classical mutagen, namely carbamic acid ester.
Background
Cancer is a "first killer" threatening human health, and lung cancer has become one of the most interesting types of cancer in recent years due to its extremely high morbidity and mortality, and poor prognosis. According to the latest world cancer statistics published by the international cancer research institute (International Agency for Research on Cancer, IARC) in 2020, about 1930 ten thousand new cases and about 1000 ten thousand death cases are worldwide in 2020, and although female breast cancer is the most common cancer beyond lung cancer, lung cancer is the cancer with the highest female mortality rate, and seriously threatens the life health of human beings. Lung cancer can be classified into small cell lung cancer and non-small cell lung cancer according to the degree of differentiation and morphological characteristics of lung cancer, the latter mainly including lung adenocarcinoma, lung squamous carcinoma and large cell carcinoma. The most common risk factor for lung adenocarcinoma is smoking, and in recent years, diagnosis of non-smoking patients has increased dramatically, and the age of onset is relatively small, with women relatively frequent. The lung adenocarcinoma treatment scheme comprises operation, radiotherapy, chemotherapy, targeted therapy, immunotherapy or combined therapy, but has high local recurrence and distant metastasis risks, poor prognosis and insufficient survival rate of 20% in 5 years, so that the development of a tumor-susceptible mouse model has important significance for the molecular mechanism of lung-heat-clearing adenocarcinoma and the development of an effective lung cancer treatment method.
Animal models are one of the indispensable tools in biomedical research, and mice are one of the most widely used experimental animals. The genetically engineered mouse model is a key model for analyzing the function of cancer-related genes in lung cancer. Because the C57BL/6 strain mice have the advantages of high stability on genetic background and consistency of experimental data, the genetic engineering mice mostly use the C57BL/6 strain mice, but the strain mice have strong resistance to exogenous chemical substances, and the construction of a cancer model by using classical chemical cancer model drugs is difficult.
Urethane is a recognized genotoxic carcinogen, widely found in fermented foods, alcoholic beverages and tobacco, and IARC in 2010 listed it as a class 2A carcinogen. Urethane-induced lung adenocarcinoma is a common experimental model today. The metabolism of urethane in vivo by CYP2E1 produces ethylene oxide carbamate, which forms adducts with DNA (including 1, N6-ethylene deoxyadenosine and 1, N4-ethyldeoxycytidine), which covalently bind to DNA and can lead to base mismatches during DNA replication, resulting in point mutations in Kras, with the gene at the Q61 position having a substitution L or R (Q61L is common in A/J mouse strains and Q61R is common in C57BL/6 mouse strains). Compared with human lung adenocarcinoma, the mouse-urethane model shows similar histological appearance and molecular change, and can be used as a valuable tool for not only knowing basic lung tumor biology, but also developing and verifying new tumor intervention measures. But inbred mouse strains have different susceptibility to urethane-induced lung tumor formation. The A/J strain mice are a lung cancer susceptible strain, spontaneous lung tumors appear in the life, the biological activation ability of the urethane is stronger, a higher level of DNA adducts can be formed, a plurality of lung adenomas can develop 25 weeks after a single intraperitoneal urethane injection, and the occurrence of lung adenocarcinomas can be observed from 40 weeks to the extension of the observation time. In contrast, C57BL/6 strain mice are resistant to both carcinogen induction and spontaneous lung tumor formation, mainly because of the low oxidative capacity of urethane in this strain of mice. To increase the incidence of lung tumors in mice of the C57BL/6 strain, a procedure of continuous multiple intraperitoneal injections of urethane was generally employed to overcome genetic resistance and induce lung tumorigenesis in mice of the C57BL/6 strain in a reproducible manner, which resulted in nearly 100% lung tumor incidence, but with relatively low tumor diversity.
The transcription factor NF-E2 related factor 1 (Nuclear factor erythroid-related factor 1, NFE2L1, also abbreviated as NRF 1) and the transcription factor NF-E2 related factor 2 (Nuclear factor erythroid-related factor 2, NFE2L2, also abbreviated as NRF 2) belong to the CNC basic leucine family (Cap 'n' collar basicleucine zipper, CNC-bZIP) and play the role of a hinge molecule in the organism environmental stress network. When external stress source exists, the two can be quickly combined with an antioxidant reaction element (Antioxidant response elements, ARE) of a downstream gene promoter region to play a role of a transcription factor, further regulate a series of response reactions in cells and participate in the determination of cell fate, and recent researches find that NRF1 plays an important role in the occurrence and development of cancers.
Disclosure of Invention
The invention aims at solving the problem that the existing urethane induces C57BL/6 strain mice to form lung tumor resistance, and provides a specific deletion of alveolar type II epithelial cellsNrf1A method for constructing lung adenocarcinoma of a mouse by combining the mouse with urethane.
In order to achieve the above object, the present invention adopts the following technical scheme.
The invention provides an animal model, which is characterized in that the model is used for preparing alveolar II type epithelial cell specific deletion by adopting a mouse with a C57BL/6 genetic backgroundNrf1Gene mice [ ]Nrf1(ATII)-KO）。
Further, the mouse isNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- Triple transgenic mice.
Further, the saidTeto-Cre +/- The gene should be inherited by female mice.
The invention also provides an application of the animal model in preparing a mouse lung adenocarcinoma model, which is characterized in that the mouse model is combined with the carbamic acid ethyl ester.
The invention also provides a construction method of the animal model, which is characterized by comprising the following steps:
(1) Preparation of F1 mice: mice with a C57BL/6 genetic background,Nrf1 f/f (female) andSPC-rtTA +/- (Male) mating to obtain F1 filial generation, and after genotyping, retaining genotype of F1 generation asNrf1 f/- /SPC-rtTA +/- Is a mouse of (2);Nrf1 f/f (Male) andTeto-Cre +/- (female) mating to obtain F1 filial generation, and after genotyping, retaining genotype of F1 generation asNrf1 f/- /Teto-Cre +/- Is a mouse of (2);
(2) F2 generation mice preparation: f1-generation mice prepared in (1)Nrf1 f/- /SPC-rtTA +/- (Male) and then toNrf1 f/f Mating (female) to obtain F2 filial generation, and after genotyping, retaining genotype of F2 generation asNrf1 f/f /SPC-rtTA +/- Is a mouse of (2); f1-generation mice prepared in (1)Nrf1 f/- /Teto-Cre +/- (female) andNrf1 f/f (Male) mating to obtain F2 filial generation, and after genotyping, retaining genotype of F2 generation asNrf1 f/f /Teto-Cre +/- Is a mouse of (2);
(3) Preparation of F3 mice: (2) F2-substituted mice obtained in the aboveNrf1 f/f /SPC-rtTA +/- (Male) andNrf1 f/f /Teto-Cre +/- (female) mating to obtain F3 filial generation, and after genotyping, retaining genotype of F3 generationNrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- is a mouse of (2);
(4) Alveolar type II epithelial cell specific deletionNrf1Preparation of mice: f3-substituted mice obtained in (3)Nrf1 f /f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- mice of several genotypes were given drinking water containing doxycycline 2mg/mL and sucrose 5% at 6 weeks of age for 2 weeks to give genotypeNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- A kind of electronic deviceNrf1(ATII) -KO mice.
Further, the feeding conditions of all mice were: SPF-level environment feeding is carried out, the feeding temperature is controlled to be 23+/-1 ℃, the humidity is controlled to be 55-70%, and the circadian rhythms are alternated.
The invention also provides a lung adenocarcinoma mouse model, which is characterized in that the animal model and the ethyl carbamate are combined to prepare the lung adenocarcinoma mouse model.
Further, the method specifically comprises the following steps:
selecting a weight within the range of 18-22 gNrf1(ATII) -KO and control mice were initially subjected to lung adenocarcinoma modeling: the urethane was injected intraperitoneally at a dose of 1g/kg, 1 time per week for 10 weeks continuously, and the pulmonary adenocarcinoma mouse model was obtained by further observation for 17 weeks after stopping the injection.
The invention also provides an application of the lung adenocarcinoma mouse model, which is characterized by comprising application in research on pathogenesis of lung adenocarcinoma diseases.
The invention also provides application of the lung adenocarcinoma mouse model, which is characterized by comprising application in screening lung adenocarcinoma medicaments.
Compared with the prior art, the invention has the following advantages and beneficial effects.
(1) The invention constructs the strain of C57BL/6Nrf1The model of the lung adenocarcinoma of the mice caused by combining (ATII) -KO mice with the urethane has the advantages of improving the lung tumor load of the mice of the strain on the basis of the traditional model, shortening the total molding time and reducing the cost benefit, along with simple operation, strong repeatability and easy mastering. Provides a reliable model for the research of complex mechanisms related to genes and environmental factors in the occurrence and development of lung cancer, can strengthen the basic research and clinical research of the occurrence and development of lung cancer, searches for prevention and treatment measures of lung cancer, and has important research significance.
(2) By adopting the breeding strategy of the invention, the strain can be stably obtained in the F3 generationNrf1(ATII) -KO genotype mice and control groupNrf1-Flox genotype mice. After DOX drinking water feeding is carried out at 6-8 weeks of age, KO mice and Flox mice have no difference in appearance and good growth state. Through the RT-qPCR verification of lung tissue immunohistochemistry and mouse ATII primary cells, the construction can be successfully carried outNrf1(ATII) -KO mice.
Drawings
FIG. 1 is a schematic diagram of the activation principle of Cre recombinase provided by the embodiment of the inventionNrf1Schematic of the breeding strategy for (ATII) -KO mice. Wherein A isNrf1Schematic representation of the principle of (ATII) -KO gene knockout, B isNrf1Schematic of the breeding strategy for (ATII) -KO mice.
FIG. 2 shows specific knockout of mouse ATII cells according to an embodiment of the present inventionNrf1Results graphs of mouse genotypes.
FIG. 3 shows the isolation and identification of primary cells of mouse ATII according to the present invention. Wherein A is a phase contrast microscope result diagram of the form of the mouse ATII primary cells separated by an immunomagnetic bead method. Wherein B is a result diagram of detecting the expression of the mouse ATII primary cell specific marker protein SPC by an immunofluorescence method.
FIG. 4 shows ATII cell-specific knockouts according to an embodiment of the present inventionNrf1Is a knock-out effect identification result graph. Wherein A is extraction by immunomagnetic bead separationNrf1-Flox、Nrf1Detection by qPCR in ATII primary cells and other lung cells of (ATII) -KO miceNrf1Is a comparison of mRNA expression level of (3). B is a graph of NRF1 staining in histopathological sections detected by immunohistochemical staining.
FIG. 5 shows an embodiment of the present inventionNrf1(ATII) -KO mice were combined with urethane-based mice lung adenocarcinoma model base index. Wherein A is dynamic monitoring of males and females during urethane moldingNrf1-FloxNrf1Body weight of (ATII) -KO mice. Wherein B is a photograph of the lungs of each group of mice at the end of molding.
FIG. 6 shows an embodiment of the present inventionNrf1(ATII) -KO mice were evaluated in combination with urethane-induced mouse lung adenocarcinoma model tumor burden. Wherein A is lung organ coefficient, lung surface tumor number and lung surface tumor diameter measured at the end point of the male mouse lung adenoma molding to evaluate the lung tumor load of the urethane molding. B is lung organ coefficient, lung surface tumor number and lung surface tumor diameter measured at the end point of lung adenoma molding of female mice to evaluate the lung tumor load of the urethane molding.
FIG. 7 shows an embodiment of the present inventionNrf1(ATII) -KO mice combined with urethane to cause lung tumor pathology and immunohistochemical results. Wherein A is a lung tissue pathological section adenoma area HE staining chart, and B is an adenoma area quantitative analysis chart. C is an immunohistochemical staining chart of the marker protein PCNA of the cell proliferation index, and D is a PCNA positive staining area quantitative analysis chart of the adenoma part. Immunohistochemical staining pattern of macrophage with E being marker protein F4/80 and F being quantitative analysis pattern of F4/80 cation stained cell area.
Detailed Description
The invention is described in further detail below with reference to specific examples and figures. The following examples are merely illustrative of the present invention and should not be construed as limiting the invention.
The invention is aimed at by constructing C57BL/6 strainNrf1The model of the lung adenocarcinoma of the mice caused by combining (ATII) -KO mice with the urethane has the advantages of improving the lung tumor load of the mice of the strain on the basis of the traditional model, shortening the total modeling time and reducing the cost benefit, and the construction method has simple operation and strong repeatability and provides a reliable model for the mechanism research of the occurrence and the development of lung cancer.
Example 1.
Alveolar type II epithelial cell specific deletionNrf1The construction method of the mice comprises the following steps.
1.Nrf1The breeding strategy for (ATII) -KO mice was as follows: mice with C57BL/6 genetic background were genotyped as followsNrf1 f/f 、SPC-rtTA +/- AndTeto-Cre +/- . Note thatTeto-Cre +/- The gene should be inherited by female mice in case of loss of Teto-Cre inheritance.
（1）Nrf1 f/f (female) andSPC-rtTA +/- f1 filial generation is obtained by (male) mating, numbering is carried out when the mice are 3 weeks old, a proper amount of rat tail is cut by disinfection operation for genotyping, and male is reservedNrf1 f/- /SPC-rtTA +/- Genotype mice for subsequent use;Nrf1 f/f (Male) andTeto-Cre +/- (female) mating to obtain F1 offspringNumbering is carried out when the mice are 3 weeks old, and a proper amount of rat tails are cut by a sterilizing operation for genotyping.
(1) Extracting rat tail DNA: a small amount of rat tail is sheared by using a sterile surgical scissors into a 1.5mL centrifuge tube, 100 mu L of alkaline lysate is added, heating is carried out for 45min at 95 ℃, 100 mu L of neutralization solution is added after cooling, and the rat tail can be stored at minus 20 ℃.
(2) And (3) PCR reaction: each genotype pool was prepared according to the PCR pool formula shown in Table 1, and the eight-strand tube with the reagents was placed in a PCR apparatus to perform PCR reactions, each genotype primer sequence was shown in Table 2, and each genotype PCR reaction procedure was shown in tables 3, 4 and 5.
TABLE 1 PCR reaction mixing pool
。
TABLE 2 PCR genotype primers
。
TABLE 3 Table 3Nrf1PCR reaction procedure
。
TABLE 4 Table 4SPC-rtTAPCR reaction procedure
。
TABLE 5Teto-CrePCR reaction procedure
。
(3) Horizontal electrophoresis and imaging: preparing 3% agarose gel, placing in a horizontal electrophoresis tank, adding 1 xTAE buffer solution, adding 8 μl of amplification product per channel, performing gel electrophoresis for 45min, and photographing.
Retaining femaleNrf1 f/- /Teto-Cre +/- Genotype mice were used later.
(2) F1 progeny of the foregoing reservationsNrf1 f/- /SPC-rtTA +/- (Male) andNrf1 f/f mating (female) to homozygouslyNrf1Numbering at 3 weeks of age, sterilizing, surgical cutting, genotyping, and retaining maleNrf1 f/f /SPC-rtTA +/- For subsequent use;Nrf1 f/- /Teto-Cre +/- (female) andNrf1 f/f (Male) mating for homozygosityNrf1Numbering the mice at 3 weeks of age, performing genotyping by performing a surgical operation to remove a proper amount of rat tail, and reserving femalesNrf1 f/f /Teto-Cre +/- For subsequent use.
(3) F2 progeny of the foregoing reservationsNrf1 f/f /SPC-rtTA +/- (Male) andNrf1 f/f /Teto-Cre +/- mating (female) to obtain F3 filial generation, numbering at 3 weeks of age of mice, sterilizing, surgical cutting, and collecting appropriate amount of rat tail for genotyping, and preserving genotype of F3 generationNrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- is a mouse of (2). The genotyping results are shown in FIG. 2. As can be seen from the results of gel electrophoresis, the Flox band (360 bp) alone was found in the mouseNrf1 f/f The method comprises the steps of carrying out a first treatment on the surface of the When the mice have Flox bands (360 bp) andSPC-rtTA +/- the bands (450 and 397 bp) areNrf1 f/f /SPC-rtTA +/- The method comprises the steps of carrying out a first treatment on the surface of the When the mice have Flox bands (360 bp) andTeto-Cre +/- the bands (324 and 100 bp) areNrf1 f/f /Teto-Cre +/- The method comprises the steps of carrying out a first treatment on the surface of the When the mice have Flox bands (360 bp) at the same time,SPC-rtTA +/- bands (450 and 397 bp) andTeto-Cre +/- the bands (324 and 100 bp) areNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- . Wherein the method comprises the steps ofNrf1 f/f ，Nrf1 f/f /SPC-rtTA +/- ，Nrf1 f/f /Teto-Cre +/- Is thatNrf1-a Flox control mouse, which was selected from the group consisting of,Nrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- is thatNrf1(ATII) -KO mice.
(4) Feeding DOX-primer water to induce knockout in ATIINrf1: obtained according to the aforementioned mouse breeding hybridization strategyNrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- several genotype mice were given drinking water containing 2mg/mL DOX at 6 weeks of age, DOX powder was kept in 4 ℃ protected from light, and since it was easy to degrade after preparation, it was required to prepare and pay attention to protected from light every day, while 5% sucrose was added to prevent the mice from reduced drinking water due to bitter taste, drinking time was 2 weeks. Wherein the method comprises the steps ofNrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- AndNrf1 f/f /Teto-Cre +/- is that (1)Nrf1-Flox mice, genotypeNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- Is thatNrf1(ATII) -KO group mice.
(5) The method for sorting the immunomagnetic beads extracts the primary cells of the ATII of the mice: to identify ATII primary cell-specific knockoutsNrf1After the DOX drinking water is finished, respectively selectingNrf1-Flox and FloxNrf1(ATII) -KO group mice, excess CO 2 Euthanized mice were euthanized, supine position was taken, whole body sterilized with 70% alcohol, the chest was exposed by surgical scissors, the free trachea was perfused through the right ventricle with sterile PBS until the whole lung was whitened, 5U/mL of dispese II was prepared, pre-warmed in a 37℃ water bath, 1mL of dispese II was perfused into the lung through the bronchi, then 0.3mL of 1% low melting point agarose was perfused, crushed ice was covered to allow rapid solidification of agarose to prevent the dispese II from leaking out, and the trachea was fastened with surgical threads followed by tying the trachea withThe lung tissue is cut off by a surgical scissors and placed in a 50mL centrifuge tube containing 1mL of dispese II, the lung tissue is placed in a horizontal shaking table at 37 ℃ for digestion for 30min at 200rpm, the digested lung tissue is transferred into a 10cm cell culture dish, 10mL of DMEM/F12 culture medium is added, the lung tissue is gently combed to the far end by holding the bent forceps with two hands until the center part of the lung tissue is dispersed into a cell suspension, then the cell suspension is obtained by filtering with a 70 mu m screen and a 40 mu m screen respectively, 300g is removed after 10min centrifugation, and the supernatant is removed (note: if the blood cells are too much after centrifugation, the cells can be resuspended with 2mL of erythrocyte lysate, after standing and lysing for 10min, 300g is removed after centrifugation for later use). The ATII cells were then purified and resuspended in pre-chilled buffer (4 ℃) and counted in a hemocytometer. Every 2×10 7 Cells were resuspended after 80. Mu.L of buffer and 20. Mu. L Lineage cell depletion (Lin) and incubated at 4℃for 10min. The MS separation column was placed in the magnetic field of a suitable MACS separator and a 30 μm filter was placed over the separation column. The column was washed with 3mL of buffer, the cell suspension was passed through the column, and unlabeled cells, lin, were collected - Is a cell of (a) a cell of (b). The column was washed with 3mL X3 times buffer and the residual Lin in the column was collected - Cells were centrifuged at 300g for 10min and kept for further use. Cells were resuspended in 90. Mu.L buffer and 10. Mu.L Ep-CAM microblades and incubated for 10min at 4 ℃. The MS separation column was placed in the magnetic field of a suitable MACS separator and a 30 μm filter was placed over the separation column. Washing the column with 3mL of buffer solution, allowing the cell suspension to pass through the column, and collecting unlabeled cells to obtain Ep-CAM - Is a cell of (a) a cell of (b). The column was washed with 3mL×3 buffer and the residual Ep-CAM in the column was collected + And (3) cells. And replacing a new centrifuge tube, adding 3mL of buffer solution into the separation column, and throwing the cell liquid in the separation column into the centrifuge tube to obtain the cell which is the ATII cell. The prepared culture solution (Small Airway Cell Basal Medium +5% C-FBS+10ng/mL rhKGF) is used for resuspending cells, the cells are placed in a cell culture box, the cell adherence time is longer than 24h, 1mL of culture solution is added after 24h, and the culture solution is replaced after 48h, so that the adherent cells are the mice ATII primary cells. The cell morphology was observed by inverted microscopy and the results are shown in figure 3A (scale = 200 or 100 μm).
(6) Immunofluorescence method for identifying mouse ATII primary cells: fixing: after the culture solution is discarded, the mice ATII primary cells cultured for 3 days are washed for 3 times for 1 XPBS for 10 min/time, 4% paraformaldehyde is added for 15 min/time at 4 ℃,1 XPBS for 10 min/time and 3 times for washing, pre-cooled methanol at minus 20 ℃ is added for 15min at minus 20 ℃ and 1 XPBS for 10 min/time and 3 times for washing; closing: goat serum working solution, incubating for 1h at room temperature; antigen-antibody reaction: SPC antibodies (1:200 goat serum working solution formulation) were used overnight at 4 ℃. Recovering primary antibody, 1 XPBS for 10 min/time, cleaning for 3 times, incubating for 1h at room temperature with red fluorescent secondary antibody (1:500 goat serum working solution configuration), 1 XPBS for 10 min/time, and cleaning for 3 times; DAPI counterstained nuclei, incubated for 5min at room temperature, 1 XPBS for 10 min/time, and washed 3 times. And shooting by an inverted fluorescent microscope. The results are shown in fig. 3B (scale=100 μm).
(7) RT-qPCR method for identifying ATII cellsNrf1Knocking out effect: after the primary cells of the mice ATII are cultured for 3 days, the culture solution is discarded, 1 XPBS is used for 10 min/time, the primary cells are washed for 3 times, 0.5mL/3.5-dish Trizol reagent is added, a pipetting gun repeatedly blows the bottom surface of a culture dish, the Trizol suspension containing the cells is transferred to a 1.5mL enzyme-free centrifuge tube, 100 mu L of chloroform is added, the primary cells are oscillated for 15s, and the primary cells are stood for 10min at room temperature; centrifuging at 12000rcf,15min and 4 ℃, sucking the upper transparent liquid into a new 1.5mL enzyme-free centrifuge tube by a pipetting gun, adding 250 mu L of isopropanol, oscillating for 5s, standing at room temperature for 10min, centrifuging at 12000g,15min and 4 ℃, wherein white precipitate at the bottom of the centrifuge tube is RNA, adding 75% ethanol prepared by enzyme-free water, cleaning the white precipitate, repeating for 2 times, and centrifuging at 7500g,15min and 4 ℃. The supernatant was discarded, the precipitate was dried at room temperature, and after complete drying, 10. Mu.L of enzyme-free water was added to dissolve RNA, and RNA concentration was measured by Nano-drop, and the sample concentration was uniformly adjusted to 50 ng/. Mu.L. Reverse transcription of RNA into cDNA was performed using the Takara reverse transcription kit. Next, RT-qPCR reaction is performed using cDNA as template, and data is analyzed by 2-DeltaCT method. The results are shown in FIG. 4A, andNrf1in comparison with the group of mice of the Flox,Nrf1in the (ATII) -KO group mice ATII primary cellsNrf1The expression quantity is obviously reducedP<0.05 While in other lung cells)Nrf1The expression level is not different.
(8) Identification of ATII cells by immunohistochemical methodNrf1Knockout effectThe method comprises the following steps: parallel validation of ATII cells using immunohistochemistryNrf1The knocking-out effect is that after DOX drinking water is ended, respectively selectingNrf1-Flox and FloxNrf1(ATII) -KO group mice, excess CO 2 The mice were euthanized, placed in a supine position, the chest cavity was exposed with surgical scissors, the free trachea was perfused through the right ventricle with sterile PBS until the whole lung was whitened, the lungs were separated with surgical scissors, 4% paraformaldehyde was slowly injected through the trachea with a 1mL syringe, and placed in a 50mL centrifuge tube containing 4% paraformaldehyde for fixation for 24h. The fixed lung tissue is dehydrated, transparent and waxed, then is embedded in normal paraffin, sliced by a slicing machine, the thickness of the slice is 5 mu m, and the glass slide is placed at 37 ℃ for overnight drying for standby. The sections were dewaxed and hydrated in xylene and alcohol, 1 XPBS for 5 min/time and washed 3 times. After antigen retrieval, appropriate amount of endogenous peroxidase blocking agent blocks endogenous peroxidase, 1 XPBS for 5 min/time, and washing for 3 times. An appropriate amount of goat serum working solution was added dropwise for blocking, incubated at room temperature for 30min, and NRF1 antibody (1:100 goat serum working solution preparation) was used overnight at 4 ℃. The primary antibody was recovered, 1 XPBS for 5 min/time and washed 3 times. The goat anti-rabbit IgG labeled with biotin was added dropwise, and washed 3 times at room temperature for 15min and 1 XPBS for 5 min/time. Dripping working solution of horseradish enzyme labeled chain enzyme ovalbumin at room temperature for 15min,1 XPBS for 5 min/time, and cleaning for 3 times. DAB was developed while being observed under a microscope. After hematoxylin, the hematoxylin is dehydrated, transparent and sealed conventionally. Microscope pictures were taken after complete drying of the gum. The result is shown in FIG. 4B, andNrf1the ratio of the Flox group to the,Nrf1NRF1 staining was significantly reduced in lung histopathological sections of (ATII) -KO group mice (figure 4B,P<0.05）。
in conclusion, we succeeded in constructingNrf1(ATII) -KO mouse model.
Example 2.
A construction method of mouse lung adenocarcinoma caused by combining an alveolar type II epithelial cell specific deletion Nrf1 mouse with urethane comprises the following steps.
(1) Experimental grouping: the breeding obtained in example 1Nrf1-Flox and FloxNrf1(ATII) -KO female and male mice were randomly assigned to pairs of urethane-injected experimental groups and physiological saline-injected groupsAnd (5) group illumination.
(2) Building a lung cancer gland model: after the drinking water of DOX is over, the mice with the weight of 18-22 g can start to model lung adenocarcinoma, the ethyl carbamate is injected intraperitoneally at the dosage of 1g/kg of the body weight (the ethyl carbamate is dissolved in sterile physiological saline), the injection is carried out continuously for 10 weeks every week, and the tumor formation is observed after 17 weeks of injection. During the molding period, the health status of the mice was monitored daily and body weight was measured weekly. The results are shown in FIG. 5, in which the weight gain of the model mice in the urethane group was suppressed during the injection period of the first 10 weeks, and the weight of the model mice was gradually recovered after the end of the urethane group, as compared with the control group injected with physiological saline, in the same treatment groupNrf1Flox andNrf1there was no significant difference between (ATII) -KO mice (fig. 5A). At the end of the molding, a photograph was taken of the lungs of each group of mice, and it can be seen whether male or female, as compared to the urethane treated groupNrf1Disease conditions of Flox mice, same treatment groupNrf1(ATII) -KO mice were more ill (FIG. 5B).
(3) Other viscera collection: at the end of the 27 th week experiment, mice were anesthetized with sodium pentobarbital anesthetic by intraperitoneal injection; the method comprises the steps that a mouse is placed on a pre-cooling operation table in a supine position, 75% alcohol is used for disinfecting the body surface of the mouse, surgical scissors are used for fully exposing the chest and the abdominal cavity of the mouse, after the abdominal aorta is cut off, a perfusion needle is inserted into the right atrium of the mouse, and PBS pre-cooled at 4 ℃ is uniformly injected until lung tissues are fully whitened; carefully cutting lung tissues by surgical scissors, simultaneously cutting other viscera (liver, spleen, heart and kidney), removing redundant connective tissues, cleaning impurities such as surface blood by using 4 ℃ precooled PBS (phosphate buffered saline), sucking redundant water by filter paper, and weighing so as to correct the viscera weight by using the weight later; the lung tissue was slowly injected with 4% paraformaldehyde fixative through the trachea using a 1mL syringe, and then placed in 15mL centrifuge tubes with 4% paraformaldehyde fixative for subsequent pathology detection. As shown in FIG. 6, the lung organ coefficients, the number of lung surface tumors and the lung surface tumor diameters measured at the end point of the lung adenoma molding of the mice were evaluated for the lung tumor load of the urethane molding, regardless of the maleSex or female, urethane treated groupNrf1The lung tissue weight, the lung surface tumor number and the lung surface tumor volume of the (ATII) -KO mice are all larger than those of the same treatment groupNrf1Flox mice, and the differences were statistically significant (fig. 6A&B，P<0.05). Furthermore, analysis of lung surface tumor diameter found that it was found to be compatible with the urethane treatment groupNrf1In contrast to the Flox mice,Nrf1the average tumor diameter of (ATII) -KO mice was larger.
(4) Lung tissue harvesting: at the end of the 27 th week experiment, by excess CO 2 Euthanasia the mice, open the chest cavity and expose the lungs, and the camera takes a rough picture of the lungs; PBS perfused to the lungs to whiten; the lung is separated by the surgical scissors, and the analytical balance is weighed; counting the number of lung surface tumors, and measuring the lung tumor diameter by a vernier caliper; a1 mL syringe was slowly injected with 4% paraformaldehyde through the trachea and placed in a 50mL centrifuge tube for 24h fixation. The fixed lung tissue is dehydrated, transparent and waxed, then is embedded in normal paraffin, sliced by a slicing machine, the thickness of the slice is 5 mu m, and the glass slide is placed at 37 ℃ for overnight drying for standby.
(5) HE staining: dewaxing with xylene and gradient alcohol to water, hematoxylin for 30s, fully turning blue with color separation liquid and blue turning liquid, and eosin for 5min, dewatering with gradient alcohol and xylene, sealing with neutral glue, completely drying gum, and microscopic photographing. The results are shown in FIG. 7A&B, group with urethane treatmentNrf1In contrast to the Flox mice,Nrf1the adenoma Area of (ATII) -KO mice was larger and the differences were statistically significant [ ]P<0.05）。
(6) Immunohistochemical staining identified cell proliferation index and macrophage marker protein: immunohistochemical procedure was as described in example 1 (8), using PCNA (1:100 goat serum working solution formulation) antibodies to identify whether tumor cells were in proliferative state, and F4/80 (1:100 goat serum working solution formulation) antibodies to identify macrophage infiltration. The correlation results are shown in FIGS. 7C-F. Wherein, FIG. 7C&D can be seen in comparison to the urethane treated groupNrf1Area of PCNA positive staining in Flox mice, same treatment groupNrf1The PCNA of (ATII) -KO mice had more cationic dye area and was differentStatistical significance%P<0.05 Description of this timeNrf1The adenoma cells of (ATII) -KO mice were more proliferative. Graph E&F it can be seen that the urethane treatment groupNrf1F4/80 cationic cell Area of (ATII) -KO mice compared to the same groupNrf1More Flox mice and statistically significant differencesP<0.05 Indicating ATII cell-specific knockoutNrf1More macrophages can be recruited to promote the development of tumors.
The above examples show that the method of the invention can be successfully used for breedingNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- And can be successfully obtained by DOX inductionNrf1The (ATII) -KO mice are combined with the carbamic acid ethyl ester to construct a mouse lung adenocarcinoma model, the lung tumor load of the mice is improved on the basis of the traditional model, the total modeling time is shortened, the cost benefit is reduced, the construction method is simple to operate and high in repeatability, and a reliable model is provided for the mechanism research of the occurrence and the development of lung cancer.

Claims (10)

1. An animal model, characterized in that the model is the preparation of alveolar type II epithelial cell specific deletion by using a mouse with a C57BL/6 genetic backgroundNrf1Gene mice [ ]Nrf1(ATII)-KO）。
2. The animal model of claim 1, wherein the mouse isNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- Triple transgenic mice.
3. The animal model of claim 2, wherein the animal model comprisesTeto-Cre +/- The gene should be inherited by female mice.
4. Use of an animal model according to any one of claims 1-3 for the preparation of a mouse lung adenocarcinoma model, wherein the mouse model is combined with urethane.
5. A method of constructing an animal model according to any one of claims 1 to 3, comprising the steps of:
(1) Preparation of F1 mice: mice with a C57BL/6 genetic background,Nrf1 f/f (female) andSPC-rtTA +/- (Male) mating to obtain F1 filial generation, and after genotyping, retaining genotype of F1 generation asNrf1 f/- /SPC-rtTA +/- Is a mouse of (2);Nrf1 f/f (Male) andTeto-Cre +/- (female) mating to obtain F1 filial generation, and after genotyping, retaining genotype of F1 generation asNrf1 f/- /Teto-Cre +/- Is a mouse of (2);
(2) F2 generation mice preparation: f1-generation mice prepared in (1)Nrf1 f/- /SPC-rtTA +/- (Male) and then toNrf1 f/f Mating (female) to obtain F2 filial generation, and after genotyping, retaining genotype of F2 generation asNrf1 f/f /SPC-rtTA +/- Is a mouse of (2); f1-generation mice prepared in (1)Nrf1 f/- /Teto-Cre +/- (female) andNrf1 f/f (Male) mating to obtain F2 filial generation, and after genotyping, retaining genotype of F2 generation asNrf1 f/f /Teto-Cre +/- Is a mouse of (2);
(3) Preparation of F3 mice: (2) F2-substituted mice obtained in the aboveNrf1 f/f /SPC-rtTA +/- (Male) andNrf1 f/f /Teto-Cre +/- (female) mating to obtain F3 filial generation, and after genotyping, retaining genotype of F3 generationNrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- is a mouse of (2);
(4) Alveolar type II epithelial cell specific deletionNrf1Preparation of mice:f3-substituted mice obtained in (3)Nrf1 f/f 、Nrf1 f/f /SPC-rtTA +/- 、Nrf1 f/f /Teto-Cre +/- AndNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- mice of several genotypes were given drinking water containing doxycycline 2mg/mL and sucrose 5% at 6 weeks of age for 2 weeks to give genotypeNrf1 f/f /SPC-rtTA +/- /Teto-Cre +/- A kind of electronic deviceNrf1(ATII) -KO mice.
6. The method according to claim 5, wherein the feeding conditions of all mice are: SPF-level environment feeding is carried out, the feeding temperature is controlled to be 23+/-1 ℃, the humidity is controlled to be 55-70%, and the circadian rhythms are alternated.
7. A lung adenocarcinoma mouse model, characterized in that the animal model of claim 1-3 is combined with urethane to prepare the lung adenocarcinoma mouse model.
8. The mouse model of claim 7, comprising the specific steps of:
selecting a weight within the range of 18-22 gNrf1(ATII) -KO and control mice were initially subjected to lung adenocarcinoma modeling: the urethane was injected intraperitoneally at a dose of 1g/kg, 1 time per week for 10 weeks continuously, and the pulmonary adenocarcinoma mouse model was obtained by further observation for 17 weeks after stopping the injection.
9. Use of the lung adenocarcinoma mouse model of claim 7, wherein the use comprises use in research of pathogenesis of lung adenocarcinoma disease.
10. Use of the lung adenocarcinoma mouse model of claim 7, wherein the use comprises use in screening for lung adenocarcinoma disease drugs.
CN202310968710.2A 2023-08-03 2023-08-03 Construction method of mouse lung adenocarcinoma model with Nrf1 alveolus II type epithelial cells specifically knocked out Pending CN116949096A (en)

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