Aortic aneurysm model and construction method thereof

Abstract

The present invention provides an aortic aneurysm model and a method for constructing the same. The present application constructs an aortic aneurysm model by administering angiotensin II (AngII) to vascular smooth muscle cell-specific Echs1 gene knockout mice. The model has typical abdominal aortic segment vasodilation and matrix metalloproteinase MMP expression, providing a new research tool for the study of the etiology and pathological mechanism of aortic aneurysms and the screening of drugs for the prevention and treatment of aortic aneurysms, and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to an aortic aneurysm model and a construction method thereof. Background Art

[0002] Aortic aneurysm (AA) is an aortic dilatation disease that seriously threatens human life and health. It is mainly manifested as progressive, permanent, and irreversible dilatation of the aorta (exceeding 50% of the normal diameter). Epidemiological data show that the mortality rate of aortic aneurysm rupture is as high as 70%, and 21% of patients with acute aortic events die before arriving at the hospital; if emergency surgical intervention is not performed for those who are admitted to the hospital in time, more than 50% of patients will still die within the next 48 hours, showing an extremely high risk of death and disability. Short-chain enoyl-CoA hydratase Echs1 is an important protein involved in fatty acid oxidation metabolism and plays an important role in growth and development and many pathological and physiological processes. Disorders in fatty acid metabolism can cause vascular inflammatory responses and imbalances in the phenotypic conversion of vascular smooth muscle cells.

[0003] Laboratory animals are indispensable tools in biomedical research. They play a very important role in exploring the basic functions of genes, exploring the pathogenesis of diseases, and preclinical screening of applied drugs. Mice have great similarities with humans in development and metabolism, and their genes are also highly similar to human genes (99% of mouse genes can be found in the human genome). They have a short life cycle and are considered to be ideal model organisms among mammals. Therefore, it is very necessary to provide a method for constructing an aortic aneurysm model for basic and clinical research on aortic aneurysms. Summary of the invention

[0004] To overcome the deficiencies of the prior art, the present invention provides an aortic aneurysm model and a construction method thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides a method for constructing an aortic aneurysm model, comprising administering AngII to an animal with vascular smooth muscle cell-specific Echs1 gene knockout to obtain an aortic aneurysm model.

[0007] Furthermore, the method of administering AngII is selected from a subcutaneous implanted pump.

[0008] Further, the administration dose of AngII is 1.1-1.5 mg / kg / d.

[0009] Further, the administration dose of AngII was 1.44 mg / kg / d.

[0010] Further, the animal is CreER T2 Genetic animals.

[0011] Furthermore, the CreER T2 Genetic animal is Itga8-CreER T2 Genetic animals.

[0012] Furthermore, Echs1 knockout animals were generated by Itga8-CreER T2 Genetic Animals and Echs1 flox / flox Obtained from Animal Crossing.

[0013] Furthermore, the specific method of producing an Echs1 knockout animal includes:

[0014] (1) Itga8-CreER T2+ / - Animals and Echs1 flox / flox Animal hybridization to obtain Itga8-CreER T2+ / - ;Echs1 flox / - animal;

[0015] (2) Itga8-CreER T2+ / - ;Echs1 flox / - Animals and Echs1 flox / flox Animal hybridization to obtain Itga8-CreER T2+ / - ;Echs1 flox / flox Animals, i.e., Echs1 gene knockout animals.

[0016] Furthermore, the method further comprises administering a gene knockout activator to activate CreER T2 system.

[0017] Furthermore, the method of administering the gene knockout activator is selected from intraperitoneal injection.

[0018] Furthermore, the gene knockout activator is selected from tamoxifen.

[0019] Furthermore, the concentration of tamoxifen is 5 mg / mL.

[0020] Furthermore, the administration dosage of tamoxifen is 0.01 mL / g.

[0021] Furthermore, the animal is selected from non-human mammals.

[0022] Furthermore, the non-human mammal is selected from rodents.

[0023] Furthermore, the rodent is selected from mice.

[0024] Furthermore, the Echs1 flox / flox Animals are Echs1flox / flox Mouse.

[0025] Furthermore, the Echs1 flox / flox The mice were of C57BL / J6 background.

[0026] Furthermore, the aortic aneurysm is selected from abdominal aortic aneurysm.

[0027] The second aspect of the present invention provides a method for screening candidate drugs for preventing / treating aortic aneurysm, the method comprising administering the drug to be tested to the aortic aneurysm model prepared by the method described in the first aspect of the present invention, and selecting the drug to be tested that can improve the symptoms of aortic aneurysm.

[0028] The third aspect of the present invention provides any of the following applications:

[0029] (1) Application of the aortic aneurysm model prepared by the method described in the first aspect of the present invention in the study of the pathogenesis or pathogenic factors of aortic aneurysm;

[0030] (2) Use of the aortic aneurysm model prepared by the method described in the first aspect of the present invention in screening drugs for preventing / treating aortic aneurysms or developing new drugs for aortic aneurysms.

[0031] Advantages and beneficial effects of the present invention:

[0032] The present application constructs an aortic aneurysm model by administering angiotensin II (AngII) to vascular smooth muscle cell-specific Echs1 gene knockout mice. The model has typical abdominal aortic segment vasodilation and matrix metalloproteinase (MMP) expression, providing a new research tool for studying the etiology and pathological mechanism of aortic aneurysm and screening drugs for preventing and treating aortic aneurysm, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is a flow chart of the model building method in Example 1;

[0034] Figure 2 is a statistical graph of the total number of mice of each generation in Example 1;

[0035] Figure 3 is a flow chart for constructing the Echs1 homozygous knockout mouse in Example 2;

[0036] Figure 4 Itga8-CreER T2 Gene identification PCR result diagram;

[0037] Figure 5 It is Echs1 loxp Gene identification PCR result diagram;

[0038] Figure 6 is a flowchart of aortic aneurysm model construction;

[0039] Figure 7 is the ultrasound image of the abdominal aorta segment blood vessels of mice in each group;

[0040] Figure 8 8A is a statistical diagram of the maximum inner diameter of the abdominal aorta segment of the mice, wherein 8B is a statistical diagram of the maximum inner diameter of the abdominal aorta segment of the male mice, and 8C is a statistical diagram of the maximum inner diameter of the abdominal aorta segment of the female mice;

[0041] Fig. 9 This is a statistical diagram of the maximum vascular diameter of the mouse thoracic aorta segment;

[0042] Fig.10 is a general diagram of the vascular anatomy of each group of mice;

[0043] Fig.11 11A is a graph of the blood vessel weight ratio of total mice, 11B is a graph of the blood vessel weight ratio of male mice, and 11C is a graph of the blood vessel weight ratio of female mice;

[0044] Fig.12 This is a graph showing the mRNA expression of MMP2 in mouse vascular tissue. DETAILED DESCRIPTION

[0045] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0046] The present invention provides a method for constructing an aortic aneurysm model. The method comprises administering AngII to an Echs1 gene knockout animal to obtain an aortic aneurysm model.

[0047] In some embodiments, Echs1 includes wild type, mutant or fragments thereof. The term encompasses full-length, unprocessed Echs1, as well as any form of Echs1 derived from processing in cells, and naturally occurring variants of Echs1 (e.g., splice variants or allelic variants). The term encompasses, for example, human Echs1 as well as Echs1 from any other vertebrate source, including mammals, such as primates and rodents (e.g., mice and rats), Gene ID: 93747.

[0048] In some embodiments, the methods of administering AngII include, but are not limited to, subcutaneous implantation of a pump, injection (including intraperitoneal injection, intravenous injection, etc.), oral administration, etc.

[0049] In a specific embodiment, the method of administering AngII is selected from subcutaneous implantation of a pump, that is, implanting a sustained-release pump under the skin of an animal.

[0050] In some embodiments, the Echs1 gene knockout animal is a vascular smooth muscle cell-specific Echs1 gene knockout animal.

[0051] In a preferred embodiment, the animal is a CreER T2 Genetic animals.

[0052] In some embodiments, CreER T2 Genetic animals are transgenic animals constructed by fusing Cre recombinase with the hormone binding domain (HBD) of the estrogen receptor. T2 Genetic animals need to use the Cre-loxP system, which is a conditional gene editing technology. The Cre recombinase gene is combined with a specific promoter (such as Itga8 promoter) to achieve the knockout or modification of specific genes in VSMCs.

[0053] In a specific embodiment, the CreER T2 Genetic animal is Itga8-CreER T2 Genetic animals.

[0054] In some embodiments, Itga8-CreER T2 Genetically modified animals were created by inserting CreER after the promoter of the Itga8 gene using gene editing technology. T2 Genetically constructed animal models.

[0055] In some embodiments, the Echs1 gene knockout animal can be constructed by the method disclosed in Example 2 of the present application, or by any other method disclosed in the prior art.

[0056] The method further comprises administering a gene knockout activator to activate CreER T2 system.

[0057] In some embodiments, the method of administering the gene knockout activator includes, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, or intramuscular injection.

[0058] In a specific embodiment, the mode of administering the knockout activator is selected from intraperitoneal injection.

[0059] In some embodiments, the gene knockout activator comprises tamoxifen or 4-hydroxytamoxifen.

[0060] In a specific embodiment, the gene knockout activator is selected from tamoxifen.

[0061] The animal is selected from non-human mammals.

[0062] In some embodiments, the non-human mammals include primates (such as rhesus monkeys, bear monkeys, red-faced macaques, crab-eating macaques, etc.), rodents (such as mice, rats, squirrels, beavers, marmots, gophers, voles, woodchucks, hamsters, guinea pigs, agouti, etc.), lagomorphs (such as rabbits), and ungulates (such as cattle, sheep, horses, etc.).

[0063] In a preferred embodiment, the non-human mammal is selected from rodents.

[0064] In a specific embodiment, the rodent is selected from a mouse.

[0065] The present invention provides a method for screening candidate drugs for preventing / treating aortic aneurysms, the method comprising applying the drug to be tested to the aortic aneurysm model prepared by the above method, and selecting the drug to be tested that can improve the symptoms of aortic aneurysm.

[0066] In some embodiments, the specific method includes: (1) administering the drug to be tested to the aortic aneurysm model prepared by the above method; (2) detecting whether the aortic aneurysm model has changes in abnormal symptoms related to aortic aneurysm after the administration of the drug to be tested; (3) when the drug to be tested improves the symptoms of aortic aneurysm / has a therapeutic effect, the drug to be tested is identified as a candidate drug capable of treating aortic aneurysm.

[0067] In some embodiments, the drug to be tested may be a low molecular weight compound, a protein (e.g., an antibody), DNA, RNA, a low molecular weight interfering RNA, or an antisense oligonucleotide. The drug to be tested may be, for example, a drug for treating a disease other than aortic aneurysm or cancer. The test drug may be, for example, a mixture of one or more than two. The test drug is preferably a substance.

[0068] In some embodiments, treatment refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity and / or incidence of, one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be applied to subjects who do not show signs of the disease and / or only show early signs of the disease, with the goal of reducing the risk of developing a condition associated with the disease.

[0069] The present invention provides application of the aortic aneurysm model prepared by the above method in the study of the pathogenesis or pathogenic factors of aortic aneurysm.

[0070] In some embodiments, the study of the pathogenesis or pathogenic factors of aortic aneurysm includes but is not limited to the effects of Echs1 on vascular metabolism, vascular smooth muscle phenotype conversion, inflammation, aging, and their molecular mechanisms in aortic aneurysm models.

[0071] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0072] Example 1

[0073] Echs1 flox / flox Mice were purchased from Saiye Biotechnology, SM22α-Cre + / + Mice were purchased from Jackson Laboratory.

[0074] 1. SM22α-Cre + / - Echs1 flox / flox Method for constructing homozygous mice

[0075] Utilizing SM22α-Cre + / + Mice (129 background) were backcrossed with wild-type C57BL / J6 mice for more than 10 generations to obtain SM22α-Cre in C57BL / J6 background. + / - Mouse. flox / flox (C57BL / J6 background) and ♂SM22α-Cre + / - The vascular smooth muscle-specific Echs1 heterozygous knockout mouse SM22α-Cre was obtained by hybridization with C57BL / J6 background. + / - ;Echs1 + / - To obtain vascular smooth muscle-specific Echs1 homozygous knockout mice, SM22α-Cre + / - ;Echs1 flox / - With ♀Echs1 flox / flox (C57BL / J6 background) for hybridization, the method flow is as follows Figure 1 shown.

[0076] 2. SM22α-Cre gene identification

[0077] The primer sequences used for SM22α-Cre gene identification are shown in Table 1 , and the PCR program is shown in Table 2 .

[0078] Table 1 Primer sequences

[0079]

[0080] Table 2 PCR program

[0081]

[0082] PCR product characteristics:

[0083] Mutant=758bp; Heterozygote=480bp and 758bp; Wide type=480bp.

[0084] 3. Echs1 loxp Gene identification

[0085] Echs1 loxp The primer sequences used for gene identification are shown in Table 3 , and the PCR program is shown in Table 4 .

[0086] Table 3 Primer sequences

[0087]

[0088] Wildtype: 322 bp Targeted: 372 bp.

[0089] Table 4 PCR program

[0090]

[0091] 4. Experimental results

[0092] Five cages of mice ♂SM22α-Cre + / - 、Echs1 flox / - With ♀Echs1 flox / flox (C57BL / J6 background) In the past year, there were 141 offspring in total. The statistical results are as follows Figure 2 As shown, vascular smooth muscle-specific homozygous Echs1 knockout mice (SM22α-Cre + / - Echs1 flox / flox ) 3 died within two weeks after birth (* represents death within two weeks after birth); vascular smooth muscle-specific Echs1 heterozygous knockout mice (SM22α-Cre + / - Echs1 flox / - ) 44 in total, Echs1 flox / flox 35 mice, Echs1 flox / - There were 59 mice, and all of them were in good condition.

[0093] The method used in Example 1 resulted in the death of mice after homozygous knockout of ECHS1, ie, the method of Example 1 was lethal, so the applicant explored other methods to construct a model.

[0094] Example 2

[0095] Echs1flox / flox Mice were purchased from Saiye Biotechnology, Itga8-CreER T2 The genetic mice came from Professor Lu Qing's laboratory.

[0096] 1. Construction of Echs1-V-KO mice

[0097] Itga8-CreER T2 Genetic mice. Using ♂ Itga8-CreER T2 Genetic mice with ♀ Echs1 flox / flox The vascular smooth muscle-specific Echs1 heterozygous knockout mouse Itga8-CreER was obtained by hybridization of mice with C57BL / J6 background. T2+ / - ;Echs1 flox / - . ♂Itga8-CreER T2+ / - ;Echs1 flox / - With ♀Echs1 flox / flox (C57BL / J6 background) were hybridized to further obtain vascular specific Echs1 Homozygous knockout mice (Echs1-V-KO) and littermate controls Echs1 flox / flox (WT) mice, the method flow is as follows Figure 3 shown.

[0098] 2. Itga8-CreER T2 Gene identification

[0099] Itga8-CreER T2 The primer sequences used for gene identification are shown in Table 5 , and the PCR program is shown in Table 6 .

[0100] Table 5 Primer sequences

[0101]

[0102] Table 6 PCR program

[0103]

[0104] 3. Echs1 loxp Gene identification

[0105] Echs1 loxp The primer sequences used for gene identification are shown in Table 7 , and the PCR program is shown in Table 8 .

[0106] Table 7 Primer sequences

[0107]

[0108] Table 8 PCR program

[0109]

[0110] 4. Construction of mouse aortic aneurysm model

[0111] ♂Itga8-CreER T2+ / - ;Echs1 flox / - With ♀Echs1 flox / flox (C57BL / J6 background) were further crossed to obtain vascular-specific Echs1 homozygous knockout mice (Echs1-V-KO) and littermate control Echs1 flox / flox (WT) mice. The final concentration of tamoxifen solution was 5 mg / mL. When the mice reached a body weight of (18-23 g), they were given 0.01 mL / g of the prepared tamoxifen solution by intraperitoneal injection for 5 consecutive days. Two weeks later, AngII (1.44 mg / kg / d) was administered by subcutaneous implantation of a sustained-release pump for 28 days as the experimental group. The control group mice were given normal saline ( Figure 6 ).

[0112] 5. Ultrasound vascular images of mice in each group (TA)

[0113] After the experiment started, the survival of the mice was observed daily. The maximum diameter of the thoracic and abdominal aorta segments of the mice was detected by ultrasound on the day before the 28th day after the pump treatment. The data were statistically analyzed to obtain the expansion degree of the vascular diameter during the formation of AA in the mice. After dissection, the blood vessels were stripped and the weight of the mouse blood vessels was weighed to obtain the blood vessel weight to body weight ratio.

[0114] 6. Experimental results

[0115] The identification results of experimental mice are as follows Figure 4 As shown, 1, 2, 4, 5, 6, 9, 11, 13, 14, 15, 16 are Itga8-CreER T2+ / - , and the rest are Itga8-CreER T2- / - . Echs1 flox / flox The identification results are as follows Figure 5 shown.

[0116] Figure 7 Mice homozygous for vascular-specific Echs1 knockout (Echs1-V-KO) and littermate controls Echs1 flox / flox Ultrasound image of the abdominal aorta segment of (WT) mice after 28 days of AngII stimulation (scale: 2 mm). The blue dotted line marks the abdominal aorta segment. The results showed that Echs1-V-KO had significant vascular dilation after AngII administration, while there was no significant change in the other groups.

[0117] After 28 days of Ang II stimulation, the maximum vascular diameter of mice was detected by ultrasound. It can be seen that the maximum diameter of the abdominal main segment of Echs1-V-KO mice was significantly expanded compared with that of the Saline-treated group and the Ang II-treated WT mice ( Figure 8 , P values ​​were obtained using one-way ANOVA and Bonferroni correction, and data are expressed as mean ± standard error. ***P < 0.001, ****P < 0.0001, ns, no statistical difference, A: all mice; B: male mice; C: female mice), there was no significant difference in the maximum inner diameter of the thoracic main segment among the groups ( Fig. 9 , P values ​​were obtained using one-way ANOVA with Bonferroni correction, and data are expressed as mean ± standard error).

[0118] Fig.10 This is a gross anatomy of the blood vessels in each group of mice (scale: 250 mm). The mice were dissected, the extra thoracic and abdominal organs were removed, and the aorta was exposed. If the thoracic aorta segment was bleeding, it was considered to be ruptured; if the abdominal aorta segment was bleeding, it was considered to be ruptured; if there was no bleeding, the vascular structure was considered intact. After Echs1-V-KO was given AngII, the rupture of the abdominal aorta segment was more obvious than that of the Saline-treated group and the Ang II-treated WT mice.

[0119] Fig.11 is the vascular weight to body weight ratio of each group of mice. The aorta was weighed and recorded, and the vascular weight to body weight ratio (aorta weight / body weight) was calculated. It was found that the vascular weight and body weight of the Echs1-V-KO group treated with AngII were significantly increased compared with the Saline group and the WT mice treated with Ang II (P values ​​were obtained using one-way ANOVA with Bonferroni correction, and the data are expressed as mean ± standard error. *P<0.05, **P<0.01, ns, no statistical difference, A: all mice; B: male mice; C: female mice).

[0120] In order to detect the changes in matrix metalloproteinases (MMPs) in mice of each treatment group, RNA was extracted from the dissected aorta after removing the adventitia, and the expression of MMP2 was detected. Fig.12 As shown, after AngII stimulation, MMP2 was highly expressed in the Echs1-V-KO group mice compared with the Saline-fed mice (P values ​​were obtained using one-way ANOVA with Bonferroni correction, and the data are expressed as mean ± standard error. *P < 0.05).

[0121] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing an aortic aneurysm model, characterized in that: The method comprises administering AngII to an animal with vascular smooth muscle cell-specific Echs1 gene knockout to obtain an aortic aneurysm model. Echs1 knockout animals were generated by Itga8-CreER T2 Genetic mice with Echs1 flox / flox Vascular smooth muscle cell-specific Echs1 gene homozygous knockout mice obtained by crossbreeding mice.

2. The method according to claim 1, characterized in that The method further comprises administering a gene knockout activator to activate CreER T2 system.

3. The method according to claim 2, characterized in that The gene knockout activator is selected from tamoxifen.

4. A method for screening candidate drugs for preventing / treating aortic aneurysm, characterized in that: The method comprises administering a drug to be tested to an aortic aneurysm model prepared by the method according to any one of claims 1 to 3, and selecting a drug to be tested that can improve the symptoms of aortic aneurysm.

5. Any of the following applications: (1) Application of the aortic aneurysm model prepared by the method according to any one of claims 1 to 3 in the study of the pathogenesis or pathogenic factors of aortic aneurysm; (2) Use of the aortic aneurysm model prepared by the method according to any one of claims 1 to 3 in screening drugs for preventing / treating aortic aneurysms or developing new drugs for aortic aneurysms.

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