CN108271740B - Method for establishing neutrophilic granulocyte-deficient atherosclerosis model mouse - Google Patents
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Abstract
The invention relates to a method for establishing a neutrophile granulocyte-deleted atherosclerosis model mouse, belonging to the technical field of genetic engineering and genetic modification. According to the invention, Gfi1 point mutation is introduced into ApoE knockout mouse genome for the first time, and the first neutrophilic granulocyte deficiency atherosclerosis mouse genetic model is constructed. Experiments prove that in the model mouse, atherosclerotic plaques are obviously reduced, and the atherosclerotic symptoms are obviously relieved, which shows that the neutrophile granulocyte-deficient atherosclerosis model mouse is successfully constructed, and the neutrophile granulocyte deficiency can relieve the pathological change degree of atherosclerosis. The model mouse with neutrophilic granulocyte deficiency and atherosclerosis obtained by the invention has very important potential application value for uncovering the role of neutrophilic granulocyte in formation and repair of atherosclerosis and diagnosis and treatment of atherosclerosis.
Description
Technical Field
The invention relates to a method for establishing a neutrophile granulocyte-deleted atherosclerosis model mouse, belonging to the technical field of genetic engineering and genetic modification.
Background
Cardiovascular and cerebrovascular diseases are difficult to cure and high in death rate, and become the disease causing the most death worldwide, atherosclerosis is one of the main causes of cardiovascular and cerebrovascular diseases, the pathogenesis of the atherosclerosis is complex, the degree of harm to health is serious, and the mechanism of the atherosclerosis needs to be deeply researched. Atherosclerosis (AS) is a major cause of coronary heart disease, cerebral infarction, and peripheral vascular disease, but there is no suitable animal model for studying its pathogenesis and clinical treatment.
Neutrophils (neutrophiles) are one of the blood cell components in the blood, and have chemotactic, phagocytic and bactericidal effects. Atherosclerosis is a chronic inflammatory disease that severely threatens human health, while neutrophils are the main cell population for the inflammatory response. The relation between the carotid atherosclerosis of the essential hypertension patients and the ratio of the neutrophils to the lymphocytes and the total bilirubin level of the serum, the higher the index bleed atherosclerosis in southern medical university, Liudongfeng, etc., the higher the patient's neutrophil/lymphocyte ratio NLR, and the result is also confirmed by multi-factor analysis, wherein the NLR is an independent risk factor of the carotid atherosclerosis of the essential hypertension patients, and the suggestion that the inflammation mediated by peripheral blood cells plays an important role in the reaction of intimal thickening of arteries.
However, the role of neutrophils in atherogenesis has been overlooked due to the lack of a suitable animal model that can be used to study the association between atherosclerosis and neutrophils. Research on atherosclerosis requires long-term follow-up of animal models, whereas currently existing neutrophilic granulocytopenia mice survive only for weeks and require antibiotic feeding in an SPF environment. At present, no suitable animal model can be used when researching the problems of whether neutrophils are needed in the process of atherosclerosis formation, whether the neutrophils participate in the repair of atherosclerosis and the like.
Disclosure of Invention
The invention aims to provide a method for establishing a neutrophil-deficient atherosclerosis model mouse, which is simple and rapid, and the obtained model mouse can be conveniently used for researching the effect of neutrophil on atherosclerosis.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for establishing a neutrophil-deficient atherosclerosis model mouse comprises the following steps:
1) hybridizing an Gfi1 point mutation mouse with an ApoE gene knockout mouse to obtain an F1 generation mouse with heterozygote of double genes;
2) selfing an F1 generation mouse with heterozygote double genes to obtain an F2 generation mouse, and carrying out genotype identification on the F2 generation mouse; selecting male mice of which both Gfi1 point mutation and ApoE gene knockout are homozygote, female mice of which Gfi1 point mutation is heterozygote and ApoE gene knockout is homozygote from F2 generation mice;
3) and hybridizing the male mouse and the female mouse, wherein Gfi1 point mutation and ApoE gene knockout in offspring are homozygote mice, namely the model mouse of neutrophilic granulocyte deletion atherosclerosis.
The Gfi1 point mutation mouse selected in the invention is a neutrophil-deleted mouse model which can survive for a long time, and in order to determine whether neutrophil is needed in the process of atherosclerosis formation and whether neutrophil participates in the repair of atherosclerosis, Gfi1 point mutation is introduced into ApoE knockout mouse genome for the first time, and the first neutrophil-deleted atherosclerosis mouse genetic model is constructed. According to the breeding method, a female mouse with Gfi1 point mutation as a heterozygote and ApoE gene knockout as a homozygote is selected for breeding, the immune system of the female mouse is consistent with the wild type, the female mouse is completely normal in performance and can be normally bred and suckled, and therefore it is guaranteed that a sufficient number of model mice with both Gfi1 point mutation and ApoE gene knockout as homozygotes can be obtained in subsequent experiments.
The Gfi1 genotype of the F2 mouse is identified by the following primers in the step 2):
gfi 1-f: GGAGCTACGCTTTTGTCCTG, Gfi 1-r: CCTGTGTGGATGAAGGTGTG are provided. The nucleotide sequence of the normal Gfi1 gene fragment amplified by the pair of primers is shown as SEQ ID NO.3, and the 288 th site of the nucleotide sequence of the point mutation Gfi1 gene fragment is mutated from G to A. Specifically, the point mutation of the Gfi1 gene can be detected by Sanger sequencing, and the Gfi1 genotype of the mouse can be conveniently and quickly detected by using the method. If the 288 th nucleotide is only G, the gene is a normal gene; if the 288 th nucleotide is only A, the mutant is a point mutation homozygote; and the 288 th nucleotide is G/A, and is heterozygote for point mutation.
Identifying the ApoE genotype of the F2 mouse by adopting the following primers in the step 2):
ApoE-1：GCCTAGCCGAGGGAGAGCCG，
ApoE-2：TGTGACTTGGGAGCTCTGCAGC，
ApoE-3: GCCGCCCCGACTGCATCT, which are supplied by the Jackson laboratory when purchasing ApoE mice.
Wherein, ApoE-1 is a common primer, wherein the amplified fragments of ApoE-1 and ApoE-2 are shown in SEQ ID NO.7, and the total length is 155 bp; the fragments amplified by ApoE-1 and ApoE-3 are shown in SEQ ID NO.8 as 245bp in total length. Detection of ApoE genes can be performed using gel electrophoresis. If the amplification product is only a band of 155bp, the gene is a normal gene; if the amplification product is only one band of 245bp, the gene is knocked out and homozygote exists; if the amplification product has two bands, one is 155bp, and the other is 245bp, the gene is a knockout heterozygote.
Specifically, in step 2), after genotyping F2 generation mice, immunophenotype analysis was performed on F2 generation mice using flow cytometry. The antibodies are CD4, CD5, CD8, CD11B, CD11C, CD19, CD25, CD44, CD45, LY6G, and NK 1.1. More specifically, firstly preparing an antibody mixture, then sucking 40 mul of fresh blood, adding 10 mul of the antibody mixture, fully shaking and uniformly mixing, placing in a refrigerator at 4 ℃ for dark incubation for 30min, after the incubation is finished, adding 400 mul of erythrocyte lysate, fully shaking and uniformly mixing, placing at room temperature for dark incubation for 10min, after the reaction is finished, and then detecting by adopting a flow cytometer. By adopting the method, whether the neutrophils in the F2 generation mouse are deleted can be further determined, and a mouse individual with completely deleted neutrophils can be further screened.
In the breeding process of the model mouse, a male mouse with Gfi1 point mutation and ApoE gene knockout both homozygote, a female mouse with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected to breed offspring. The immune system of the female mouse is completely normal, and breeding is not influenced, so that enough offspring mice can be obtained, and up to 50% of the offspring mice are homozygotes, thereby ensuring the smooth development of subsequent experiments.
The present invention will result in mouse neutrophilic granulocyte deficiency but normal survival mouse mutation site Gfi1 for the first timeC318YMouse model of atherosclerosis with genetic background introduced by hybridization of ApoE gene deletion (ApoE)-/-) Through hybridization and screening, the homozygote mouse ApoE with double gene deletion is obtained-/-Gfi1C318Y/C318YIt was experimentally confirmed that in ApoE-/-Gfi1C318Y/C318YIn mice, the atherosclerosis symptom is obviously relieved, which shows that the neutrophilic granulocyte-deficient atherosclerosis model mouse is successfully constructed.
The neutrophil-loss atherosclerosis model mouse obtained by the establishing method is applied to research of interaction between neutrophil loss and atherosclerosis. In particular, the application of the neutrophile granulocyte deficiency atherosclerosis model mouse in researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis. Or the application of the neutrophile granulocyte deficiency atherosclerosis model mouse in researching a diagnosis and treatment method of atherosclerosis.
The obtained model mouse with neutrophilic granulocyte deficiency and atherosclerosis is further subjected to experimental study, and the result shows that the neutrophilic granulocyte deficiency can obviously reduce the formation of atherosclerotic plaques. The model mouse with neutrophilic granulocyte deficiency and atherosclerosis obtained by the invention has very important potential application value for uncovering the role of neutrophilic granulocyte in formation and repair of atherosclerosis and diagnosis and treatment of atherosclerosis.
The invention has the following beneficial effects:
1. obtaining an atherosclerosis model mouse which is deficient in neutrophils and can normally survive for the first time;
2. it is proposed for the first time to use as male parent (ApoE genotype) male mice which are immunodeficient but can normally breed in breeding mice with severe immunodeficiency-/-Gfi1C318Y/C318Y) Heterozygote mice with normal immunophenotype are used as female parents (ApoE genotype)-/-Gfi1+/C318Y) And hybridizing to obtain enough homozygous immune-deficient mice (ApoE) while ensuring smooth breeding-/-Gfi1C318Y/C318Y) The method is used for subsequent experiments, and provides a new idea for breeding mice with similar immunodeficiency;
3. the relation between the neutrophils and the atherosclerosis is determined for the first time, namely the neutrophilic granulocyte deletion can obviously relieve the symptoms of the atherosclerotic disease, which opens a new direction for deeply developing the research of the atherosclerosis, provides more choices for the clinical treatment of the atherosclerosis and has very important theoretical and practical significance.
Drawings
FIG. 1 is a graph showing the sequencing verification result of Gfi1 point mutation site of F2 mouse;
FIG. 2 is a diagram showing the result of ApoE genotype determination in mouse generation F2;
FIG. 3 is a graph of immunophenotypic results for a sex granulocyte deficient atherosclerotic model mouse;
FIG. 4 is a graph showing the results of serum biochemical indicator measurements after the model and control mice were fed high-fat diet for 12 weeks;
FIG. 5 is a graph showing the results of oil red O and H & E staining of heart cryosections after the model and control mice were fed with high-fat diet for 12 weeks;
FIG. 6 is a graph showing the results of aortic oil red O staining after the model and control mice were fed with high-fat diet for 12 weeks.
Detailed Description
The present invention will be described in further detail with reference to specific examples. Gfi1 point mutation mouse (genotype Gfi 1) in the following examples and experimental examplesC318Y/C318Y) Supplied by Bernard Malissen laboratory, France; ApoE knockout mice (genotype is ApoE)-/-) Purchased by Jackson Laboratory, usa. The formula of the high-fat feed is referred to the Research Diets company No. D12108C. Preparation of oil red O stock solution: 100% isopropanol 100mL +0.5g oil red O, storing in a 4 degree refrigerator, preparing oil red O working solution (stock solution and distilled water are diluted according to a ratio of 3: 2) when in use, diluting and filtering before use, wherein the oil red O working solution should be wine red and have no precipitate). Other experimental articles are not described and are all commercial products.
Example 1
The method for establishing the neutrophil-deficient atherosclerosis model mouse comprises the following steps:
1) gfi1 point mutation mouse (genotype Gfi 1)C318Y/C318Y) With ApoE knockout mice (genotype is ApoE)-/-) Hybridizing to obtain F1 generation mouse, wherein the double gene sites of the F1 generation mouse are heterozygote (the genotype is ApoE)+/-Gfi1+/C318Y)。
2) Mice with heterozygote double gene loci of F1 generation are selfed to obtain mice of F2 generation.
A. By using sanger sequencing, mice with Gfi1 point mutation sites as heterozygotes and homozygotes can be selected, and the detection primer sequence is as follows: gfi 1-f: GGAGCTACGCTTTTGTCCTG (shown as SEQ ID NO. 1), Gfi 1-r: CCTGTGTGGATGAAGGTGTG (shown as SEQ ID NO. 2), and the size of the amplified product is 429 bp.
The PCR system is as follows: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each, Gfi1-F (5 μ M) and Gfi1-R (5 μ M), 0.5 μ L; genomic DNA: 10 ng; supplemental ddH2O to the total volume of 20 mu L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min; the number of PCR cycles was 35.
The detection result is shown in SEQ ID NO.3, wherein the 288 th nucleotide is G only, and the gene is a normal gene (WT); the 288 th nucleotide is only A, and is a point mutation homozygote (Gfi 1)C318Y/C318Y) (ii) a The 288 th nucleotide is G/A, and is a point mutation heterozygote (Gfi 1)+/C318Y)。
B. By using 1.5% agarose gel electrophoresis, a mouse with an ApoE gene locus as a homozygote can be selected, and the detection primer sequence is as follows: ApoE-1: GCC TAG CCG AGG GAG AGC CG (shown in SEQ ID NO. 4), ApoE-2: TGTGAC TTG GGA GCT CTG CAG C (shown in SEQ ID NO. 5), ApoE-3: GCC GCC CCG ACT GCA TCT (shown in SEQ ID NO. 6).
The PCR system is as follows: (Vazyme, P111)2 Taq Master PCR MIX: 10 μ L of each, Gfi1-F (5 μ M) and Gfi1-R (5 μ M), 0.5 μ L; genomic DNA: 10 ng; supplemental ddH2O to the total volume of 20 mu L; the procedure is as follows: 95 ℃ for 5 min; 95 ℃, 30s, 60 ℃, 30s, 72 ℃, 40 s; 72 ℃ for 10 min; the number of PCR cycles was 35.
The detection result is shown in FIG. 2, wherein the amplification product is only a band of 155bp, which is a normal gene (WT); the amplification product is only one band of 245bp, and is a knockout gene homozygote (ApoE)-/-) (ii) a The amplification product has two bands, one is 155bp, the other is 245bp, and the amplification product is a knockout gene heterozygote (ApoE)+/-)。
C. Using flow cytometry, homozygous mice with neutrophilic granulocyte depletion can be further tested and selected using a loss cytometer model number CANTO II (BD, USA) with antibody combinations as shown in Table 1.
The method comprises the following steps: firstly preparing an antibody mixture, then sucking 40 mu l of fresh blood, adding 10 mu l of the antibody mixture, fully shaking and uniformly mixing, placing in a refrigerator at 4 ℃ for dark incubation for 30min, adding 400 mu l of erythrocyte lysate after incubation is finished, fully shaking and uniformly mixing, placing at room temperature for dark incubation for 10min, finishing reaction, and then detecting by adopting a flow cytometer.
Table 1 name and brand of antibody used for flow cytometry
The result of the detection is shown in FIG. 3, and it can be seen that CD11B+LY6G+The positive cells (i.e., neutrophils, marked in the boxes in the figure) had disappeared, indicating that the mature neutrophils in the model mice obtained from the construction had been completely deleted.
3) Because the Gfi1 point mutation site is homozygousMouse (genotype Gfi 1)C318Y/C318Y) Since neutrophilic granulocytes are deleted, serious immunodeficiency is caused, the fecundity is low, and if the neutrophilic granulocytes are used as parents for breeding, enough bigene homozygote mice are difficult to obtain for experiments, so that the invention selects male mice (the genotype is ApoE) with both Gfi1 point mutation and ApoE gene homozygote from F2 generation mice-/-Gfi1C318Y/C318YImmunodeficient but normal breeding), Gfi1 point mutation to heterozygous and ApoE gene homozygote female mouse (ApoE genotype)-/-Gfi1+/C318YNormal neutrophilic granulocyte, no immunodeficiency and good health state) to obtain Gfi1 point mutation and ApoE gene homozygote mouse (ApoE gene type is ApoE)-/-Gfi1C318Y/C318Y) The mouse is a new neutrophilic granulocyte-deficient atherosclerosis mouse model when used for subsequent experiments.
The F2 mice were successfully selected for double gene deletion (genotype is ApoE) by genotyping (FIGS. 1 and 2) and immunophenotyping (FIG. 3)-/-Gfi1C318Y/C318Y) But also neutrophil-depleted progeny mice. In the subsequent breeding process of model mice, male mice with Gfi1 point mutation and ApoE gene knockout homozygote, female mice with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected for breeding offspring.
Test example 1
The mouse model of neutrophile-deficient atherosclerosis obtained in example 1 (genotype ApoE)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) And (5) performing high fat feeding.
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) High fat diet feeding was performed, during which the weight change was monitored weekly, taking care to weigh at the same time point every week. As a result, the weight of the neutrophilic granulocyte-deficient atherosclerosis model mouse is not significantly different from that of the control mouse.
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After feeding with high-fat diet for 12 weeks, serum was obtained, biochemical indicators such as serum Total Cholesterol (TC), Triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) (ADVIA 2400, SIEMENS were used as a measurement instrument), and statistical analysis was performed on the data.
And (5) counting results: there was no significant difference in serum biochemical indicators in the neutrophil-deficient atherosclerosis model mice compared to the control mice (as shown in fig. 4).
Test example 2
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After 12 weeks of high-fat diet feeding, heart tissue was harvested, frozen sections (10um) after fixation (4% paraformaldehyde, overnight fixation) and embedding (embedding using OCT), followed by oil red O staining and H&E, staining, counting the area of the plaque, and carrying out statistical analysis on the data.
Oil red O staining and H&The staining results are shown in FIG. 5-A, in which ApoE in control group is shown in FIG. 5-A-/-Mouse and experimental group ApoE-/-Gfi1C318Y/C318YSchematic representation of a sample of a heart portion. Therefore, the area of the heart plaque of the mice in the experimental group is obviously reduced; statistical results of plaque area As shown in FIG. 5-B, atherosclerosis was induced by feeding high-fat diet, and neutrophile-deficient atherosclerosis model mice (ApoE) were found-/-Gfi1C318Y/C318Y) And atherosclerosis model mice (ApoE)-/-) In contrast, there was a significant reduction in plaque area, and the results indicated that neutrophilic granulocytopenia reduced the extent of atherosclerotic lesions.
The neutrophile granulocyte-deleted atherosclerosis model mouse obtained by the establishing method can be used for researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis. The neutrophilic granulocyte-deleted atherosclerosis model mouse obtained by the establishing method can also be used for researching a diagnosis and treatment method of atherosclerosis.
Test example 3
Model mouse of neutrophilic granulocyte-deficient atherosclerosis (ApoE genotype)-/-Gfi1C318Y/C318Y) And control mice (genotype ApoE)-/-) After feeding with high-fat feed for 12 weeks, aortic tissues were obtained, and after fixation with 4% paraformaldehyde for development, oil red O staining was performed, plaque area was counted, and statistical analysis was performed on the data.
The oil red O staining results are shown in FIG. 6-A, which is ApoE of control group-/-Mouse and experimental group ApoE-/-Gfi1C318Y/C318YA schematic diagram of a mouse aorta sample, from which it can be seen that the aortic plaque area in the experimental group is significantly reduced; statistical results of plaque area As shown in FIG. 6-B, atherosclerosis was induced by feeding high-fat diet, and as such, neutrophile-deficient atherosclerosis model mice (ApoE) were found-/-Gfi1C318Y/C318Y) The plaque area of (A) is significantly smaller than that of an atherosclerosis model mouse (ApoE)-/-)。
From the above experimental results, it can be seen that, compared with the wild type control, in the mouse of Gfi1 and ApoE double gene homozygote, the atherosclerotic plaque is significantly reduced, and the atherosclerotic symptom is significantly reduced, which indicates that the construction of the neutrophil-deficient atherosclerosis model mouse is successful, and the neutrophil deficiency can reduce the atherosclerotic lesion degree.
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Claims (10)
1. A method for establishing a neutrophil-deficient atherosclerosis model mouse is characterized by comprising the following steps: the method comprises the following steps:
1) hybridizing an Gfi1 point mutation mouse with an ApoE gene knockout mouse to obtain an F1 generation mouse with heterozygote of double genes;
2) selfing an F1 generation mouse with heterozygote double genes to obtain an F2 generation mouse, and carrying out genotype identification on the F2 generation mouse; selecting male mice of which both Gfi1 point mutation and ApoE gene knockout are homozygote, female mice of which Gfi1 point mutation is heterozygote and ApoE gene knockout is homozygote from F2 generation mice;
3) and hybridizing the male mouse and the female mouse, wherein Gfi1 point mutation and ApoE gene knockout in offspring are homozygote mice, namely the neutrophilic granulocyte-deleted atherosclerosis model mouse.
2. The method of establishing according to claim 1, wherein: the Gfi1 genotype of the F2 mouse is identified by the following primers in the step 2):
Gfi1-f：GGAGCTACGCTTTTGTCCTG，Gfi1-r：CCTGTGTGGATGAAGGTGTG。
3. the method of establishing according to claim 1, wherein: identifying the ApoE genotype of the F2 mouse by adopting the following primers in the step 2):
ApoE-1：GCCTAGCCGAGGGAGAGCCG，
ApoE-2：TGTGACTTGGGAGCTCTGCAGC，
ApoE-3：GCCGCCCCGACTGCATCT。
4. the method of establishing according to claim 2, wherein: the Gfi1 gene point mutation was detected in step 2) using Sanger sequencing.
5. The method of claim 3, wherein: and 2) detecting the ApoE gene by using gel electrophoresis.
6. The method of establishing according to claim 1, wherein: after genotyping F2 generation mice in step 2), immunophenotype of F2 generation mice was analyzed by flow cytometry.
7. The method of establishing according to claim 6, wherein: mouse neutrophils were labeled in said flow cytometry using CD4, CD5, CD8, CD11B, CD11C, CD19, CD25, CD44, CD45, LY6G, and/or NK1.1 antibodies.
8. The method of establishing according to claim 1, wherein: in the breeding process of the model mouse, a male mouse with Gfi1 point mutation and ApoE gene knockout both homozygote, a female mouse with Gfi1 point mutation as heterozygote and ApoE gene knockout as homozygote are selected to breed offspring.
9. The use of the neutrophilic granulocyte deficient atherosclerosis model mouse obtained by the establishment method according to claim 1 for studying interaction between neutrophilic granulocyte deficiency and atherosclerosis.
10. Use according to claim 9, characterized in that: the application of the neutrophile granulocyte deficient atherosclerosis model mouse in researching the effect of neutrophile granulocytes on the formation and repair of atherosclerosis.
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