CN113439711B - Modified macrophage and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biology, in particular to a macrophage Adar gene knockout mouse model and a construction method and application thereof. According to the invention, Adar genes of a mouse are knocked out based on a gene knocking-out technology, and then the Adar genes are mated with a tool mouse, so that two mouse models with different genotypes are finally constructed, and an ideal disease model is provided for mechanism research of liver-related diseases and drug screening and application of liver diseases. On the other hand, the macrophage is modified by knocking out a mouse animal model by macrophage Adar gene. Compared with the method for knocking out Adar gene in tumor cells, the method has the advantages that severe autoimmune reaction of patients is caused, the feasibility of macrophage modification is stronger, the side effect is lower, and a new target is provided for clinical tumor treatment.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a macrophage Adar gene knockout mouse model and a macrophage Adar gene knockout cell model, as well as a construction method and application thereof.
Background
Immunotherapy is to stimulate the immune function of human body to overcome tumor, and macrophage is one of the main immune cells, and the immunotherapy designed aiming at the macrophage shows huge potential in tumor treatment. Unlike traditional chemoradiotherapy, immunotherapy and targeted therapy have gradually demonstrated their powerful advantages and prospects, making tumor treatment more individualized and targeted. Macrophages have plasticity and pluripotency, and show obvious functional differences under the influence of different microenvironments in vivo and in vitro. Many macrophage-directed anticancer applications against tumors have been reported.
Adar genomic DNA is located on mouse chromosome 3, has 15 exons, encodes 1178 amino acids, and has an expression product length of 136.066 KDa. Adar is mainly localized in the nucleus, is a well-known RNA editing enzyme, has the capability of editing adenosine (A) of RNA into creatinine (I), is up-regulated in various tumors, and plays a role in resisting tumor immunity in tumor cells. Corresponding to the mouse-derived Adar gene is the human-derived gene ADAR1, the research of the human-derived gene ADAR1 as an RNA editing enzyme in tumor cells is very deep, but the role of the human-derived gene ADAR1 in regulating and controlling the tumor cells in macrophages is worthy of further exploration, and the influence of the absence of the ADAR1 in the macrophages on the livers of the human-derived gene ADAR is not reported.
Because of ethical limitations, many studies cannot be conducted in humans, and experimental animal disease models are indispensable research tools for studying the causes and pathogenesis of human diseases, and developing prevention and treatment technologies and drugs. The most common animal models of disease and pharmacological experiments are rodent mice. In order to study the important pathophysiological function of ADAR1 in macrophages more deeply, it is imperative to simulate the human condition with the aid of corresponding animal models. At present, no report about liver disease correlation and tumor immunity and immunotherapy correlation exists in a macrophage Adar gene knockout mouse model.
Therefore, exploring the role of ADAR1 in regulating tumor and liver in macrophages, and finding its upstream and downstream regulatory mechanisms would help develop therapeutic regimens directed against macrophages.
Disclosure of Invention
In order to solve the problems, the invention provides a macrophage Adar gene knockout mouse model and a construction method and application thereof. In addition, the modified macrophage is obtained by knocking out a macrophage Adar gene from a mouse model. The modified macrophage inhibits the generation of tumor large blood vessels and the growth of tumors under the intervention of an immunomodulator.
In order to achieve the above object, the present invention provides the following technical solutions.
The invention provides a macrophage Adar gene knockout mouse model.
Further, the mouse model comprises a Flox/Flox, Lyz2-Cre gene knockout type.
Further, the mouse model includes Flox/Flox knockout.
The invention also provides a construction method of the macrophage Adar gene knockout mouse model, which is characterized in that the Adar gene of a mouse is knocked out based on a gene knockout technology, and then the Adar gene is mated with a tool mouse, and finally, two mouse models with different genotypes are constructed.
Furthermore, the knockout gene in the method is the 4 th-6 th exon of the Adar gene as a conditional knockout region, and the nucleotide sequence of the knockout gene is shown as SEQ NO: 1.
Further, the method comprises the following specific steps:
and 5, backcrossing the recombinant heterozygous young mouse and the Flox/Flox young mouse to obtain an Adar gene knockout Flox/Flox, Lyz2-Cre mouse.
The invention also provides application of the macrophage Adar gene knockout mouse model, which is characterized by comprising application in the research of pathogenesis of liver diseases and screening of liver disease drugs.
The invention also provides application of the macrophage Adar gene knockout mouse model, which is characterized by comprising research on a mechanism of drug resistance of an immunomodulator applied to tumor cells and relieving the drug resistance of the immunomodulator.
The invention also provides a modified macrophage, which is characterized in that the cell is an Adar gene knockout macrophage.
The invention also provides the application of the cell, which is characterized by comprising research on the aspects of inhibiting the generation of tumor macroangiogenesis and inhibiting the growth of tumors under the intervention of the immunomodulator.
Compared with the prior art, the invention has the following beneficial effects:
the invention constructs a mouse model for specifically knocking out Adar gene in macrophage, which can simulate human liver-related and tumor immunity-related diseases and an immunotherapy method. Mice with macrophage Adar gene knockout show larger liver appearance, and provide ideal disease models for mechanism research of liver-related diseases and drug screening and application of liver diseases. In addition, the macrophage is modified and the immunomodulator is combined to effectively inhibit the generation of tumor macroangiogenesis and obviously inhibit the growth of tumors, and no lethal adverse reaction is observed in mice subjected to Adar gene knockout before and after the administration period. Compared with the method for knocking out the ADAR1 gene in the tumor cell, the method can cause serious autoimmune reaction of a patient, provides a new way for modifying macrophage, and has the advantages of stronger feasibility, wider application range and lower side reaction. Provides a new strategy and means for clinical treatment of liver-related diseases and tumors, and has great clinical significance.
Drawings
FIG. 1 is a schematic diagram of a macrophage Adar gene knockout mouse model constructed in the manner of
FIG. 2Adar knock-out and control mouse genotype identification electropherograms
FIG. 3 comparison of liver size and weight of Adar knockout and control mice
FIG. 4 comparison of subcutaneous tumor size and growth trend following immunomodulator intervention in Adar knockout and control mice
FIG. 5 immunohistochemical graphs and statistical graphs of subcutaneous tumor vascular markers in Adar gene knockout and control mice
FIG. 6 is a figure of identification of human macrophages with reduced ADAR1 gene knockdown, and results and analysis of experiments affecting human vascular endothelial cell vascularization.
Detailed Description
The embodiments of the invention are not intended to limit the scope of the invention, which is defined by the claims and their equivalents.
Example 1 construction of Adar Gene knockout mice.
1. Inquiring gene information: the Adar gene (NCBI reference sequence: NM-001146296; Ensembl: ENSMUSG 00000027951) is located on mouse chromosome 3, 15 exons are identified in total, starting from the start codon ATG of exon 1 to the end of the stop codon TGA of exon 15 (Transcript: ENSMUST 00000107405).
2. Designing a knockout position: exons 4-6 were selected as conditional knock-out regions (region cKO). Deletion of this region should result in loss of function of the mouse Adar gene.
3. Selecting an implementation technology: to engineer the targeting vector, homology arms and the cKO region were generated by PCR primers, reaction system and reaction conditions in tables 1-3 using BAC clone RP23-146K23 as template. In targeting vectors, the Neo cassette will be flanked by auto-deleting anchor Sites (SDA) and DTA will be used for negative selection. And (3) electrically transferring the targeting vector into C57BL/6N embryonic stem cells, and obtaining Adar gene knockout after recombining with Cre genes.
TABLE 1 PCR amplification primers
TABLE 2 PCR reaction System
TABLE 3 PCR reaction conditions
Example 2
Genotyping of Adar Gene knock-out mice. Extracting mouse tail genome DNA, PCR amplifying (see tables 4-6 for primers, reaction system and reaction conditions), and electrophoretic identification (as shown in figure 3).
Step of extracting genomic DNA (One Step Mouse Genotyping Kit):
1) 2 mm Mouse tail tips were taken, and 1 Xnew Lysis Buffer (200. mu.L L. times. Mouse tissue Lysis Buffer plus 4. mu.L of protease K) was added to each Mouse tail for vortex oscillation.
2) Followed by incubation at 55 ℃ for 30 minutes with shaking
3) Heating at 95 ℃ for 5 minutes to inactivate Proteinase K
4) Centrifuging at 12000 rpm for 5 minutes, and taking the supernatant as a PCR template
TABLE 4 PCR amplification primers
TABLE 5 PCR reaction System
TABLE 6 PCR reaction conditions
Example 3 comparison of the liver of Adar gene knock-out mice and control mice.
After 5 mice of 10 weeks old Adar knock-out C57BL/6N and control C57BL/6N mice were anesthetized and sacrificed, livers were removed and photographed and weighed (see FIG. 4).
Example 4 comparison of subcutaneous tumor size and growth trends in Adar knock-out and control mice.
C57BL/6N mice and control mice were knocked out for 5 each of 12 weeks old Adar genes. Each mouse was injected subcutaneously with 150 ten thousand mouse lung cancer cells (LLC). The tumor length and the tumor length were measured every 3 days beginning on the 9 th day after cell injection, and murine interferon g (2.5 μ g/mouse) was injected. On day 18, the mice were euthanized and measured by photography to count the growth trend of the nodules.
Example 5 vascular comparison of subcutaneous tumors in Adar knockout and control mice.
Adar knock-out C57BL/6N mice at 12 weeks of age and 5 mice in the control group were used. Each of the mice was injected subcutaneously with 150 million lung cancer cells (LLC). The murine interferon g (2.5 mug/mouse) was injected every 3 days, starting on day 9 after cell injection. At day 18, the mice are killed by anesthesia, the nodules are picked and embedded and fixed, the mice are photographed after immunohistochemistry is carried out by using a blood vessel marker CD31 (Abcam: ab28364) antibody, blood vessels with the diameter smaller than 40 mu m in 5 pictures shot by each mouse nodule are defined as small blood vessels, blood vessels with the diameter of 40-100 mu m are defined as large blood vessels, the blood vessels with the diameter larger than 100 mu m are defined as special large blood vessels, counting is carried out on the blood vessels with the diameter larger than 100 mu m respectively, the sum of the 5 pictures is counted for each mouse, and then single-factor variance analysis is carried out on the number of the blood vessels with different sizes of the two groups of mice respectively by using the self-carried statistical function of Graphpad software.
Example 6 effect of human macrophage ADAR1 knockdown on blood vessels.
The ADAR 1-knocked low plasmid and a control vector plasmid thereof are introduced into human mononuclear macrophage (THP1) cells in a lentivirus infection mode, and protein is collected for western identification after PMA (100ng/ml) is added for inducing for 48 hours after the puromycin drug screening is successful. Inducing successfully constructed low ADAR1 cells and control group cells PMA (100ng/ml) for 48 hours, inducing with human interferon gamma (100ng/ml) for 48 hours after liquid changing, treating with 1640 complete culture medium containing 10% serum for 48 hours, collecting corresponding condition culture mediums, adding 150 muL of the condition culture mediums into 96 pore plates pre-paved with matrix glue, and placing in a 96 pore plate containing 5% CO2After 5 hours of culture in an incubator at 37 ℃, 2 mu L of calcein (Thermo Fisher S) with the concentration of 10 mu g/mu L is added into each holeScientific 65-0853-39), incubated at 37 ℃ for 20 minutes, photographed using a fluorescence microscope and analyzed with Angio Tool software.
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cctctttgac taatttctcc ttagtctgga ggtgcgaaca cagactcact tgatgaatct 960
atggctccca acaagatcag gaggattggt gagctcgtca ggtacctgaa caccaacccc 1020
gtaggcggct tgttggagta cgcccgatct catggctttg ctgctgagtt caagctcatt 1080
gaccagtctg gacctcctca cgaacccaa 1109
<210> 2
<211> 23
<212> DNA
<213> Artificial Synthesis
<400> 2
tccttatatc agcgaaacag cca 23
<210> 3
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 3
agcaagctct cccagacagt c 21
<210> 4
<211> 24
<212> DNA
<213> Artificial Synthesis
<400> 4
catctaatga gctgagaggc tgaa 24
<210> 5
<211> 27
<212> DNA
<213> Artificial Synthesis
<400> 5
gtgacttctt actaatgttc tctgagc 27
<210> 6
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 6
<210> 7
<211> 21
<212> DNA
<213> Artificial Synthesis
<400> 7
cttgggctgc cagaatttct c 21
Claims (5)
1. A construction method of a macrophage Adar gene knockout mouse model is characterized in that Adar genes of a C57BL/6N mouse are knocked out based on a gene knockout technology, and then the Adar genes mate with a Lyz2-Cre tool mouse, and finally two mouse models with different genotypes are constructed; the mouse model comprises a Flox/Flox, Lyz2-Cre gene knockout type and a Flox/Flox gene knockout type; in the method, the knockout gene is the 4 th-6 th exon of Adar gene as a conditional knockout region, and the nucleotide sequence of the knockout gene is shown as SEQ NO: 1; the method comprises the following specific steps:
step 1, for engineering targeting vectors, homology arms and conditional knock-out regions were generated by PCR using BAC clone RP23-146K23 as template with PCR primer sequences:
F1：5’-TCCTTATATCAGCGAAACAGCCA-3’，R1：5’-AGCAAGCTCTCCCAGACAGTC-3’；
step 2, in the targeting vector, two sides of the Neo box are automatic deletion anchor sites, and DTA is used for negative screening;
step 3, the targeting vector is electrically transferred into embryonic stem cells of a C57BL/6N mouse and recombined with Cre gene to obtain an Adar gene knockout Flox/Flox mouse;
step 4, mating the Flox/Flox mouse with Lyz2-Cre tool mice to obtain a recombinant heterozygous newborn mouse;
and 5, backcrossing the recombinant heterozygous young mouse and the Flox/Flox young mouse to obtain an Adar gene knockout Flox/Flox, Lyz2-Cre mouse.
2. Use of the macrophage Adar gene knockout mouse model according to claim 1, comprising use in the study of pathogenesis of liver disease and drug screening of liver disease.
3. Use of the macrophage Adar knock-out mouse model according to claim 1, wherein said use comprises studying the mechanism of resistance of tumor cells to chemotherapeutic drugs and relieving chemotherapeutic drug resistance.
4. A macrophage engineered according to the macrophage Adar knock-out mouse model of claim 1, wherein said cell is an Adar knock-out macrophage.
5. A macrophage cell according to claim 4, wherein said cell is for use in research applications in inhibiting tumor macroangiogenesis and inhibiting tumor growth.
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