A non-human mammal model simulating heart-kidney metabolic syndrome and heart-kidney co-injury and a construction method and application thereof
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NANJING GENERAL HOSPITAL NANJING MILLITARY COMMAND P L A
- Filing Date
- 2026-06-08
- Publication Date
- 2026-08-07
AI Technical Summary
目前尚未有研究将CYP7B1基因敲除与瘦素受体缺陷（db/db）结合用于构建CKM模型
[0014]1、本发明的Cyp7b1-/-db/db双基因突变模型，并非简单的病理叠加，而是通过脂质/胆汁酸代谢异常（Cyp7b1敲除）与全身性糖脂代谢紊乱（db/db）的强烈协同作用，产生了意想不到的技术效果。相比于单基因 db/db 小鼠，本发明的模型在 UACR、SCr 等肾脏核心排泄滤过指标，以及 EF%、FS% 等心脏核心收缩指标上呈现出极显著的恶化，自发形成了重度的心肌间质纤维化和肾小球硬化。
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Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedicine and laboratory animal technology, specifically to a non-human mammalian model simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys, its construction method, and its application. Background Technology
[0002] Cardio-kidney and metabolic syndrome (CKM) is a complex pathological condition involving the heart, kidneys, and metabolic system, commonly seen in patients with advanced diabetes. As metabolic disorders worsen, patients often develop severe myocardial fibrosis and glomerulosclerosis, resulting in an extremely high mortality rate.
[0003] Currently, in preclinical studies, animal models commonly used to simulate metabolic syndrome (such as simple db / db mice) have characteristics such as obesity and hyperglycemia, but they often fail to simulate severe human CKM, especially "progressive organic damage to the heart and kidneys driven by severe lipid metabolism abnormalities," with long modeling cycles and milder phenotypes.
[0004] CYP7B1 (cytochrome P450 7B1) plays a central role in the alternative pathway of bile acid synthesis, and its dysfunction leads to the accumulation of toxic metabolites and severe lipid metabolism disorders. Currently, no studies have combined CYP7B1 gene knockout with leptin receptor deficiency (db / db) to construct a CKM model. Therefore, there is an urgent need for a novel animal model that can accurately simulate the cardiorenal co-injury caused by the abnormal metabolic microenvironment of CKM, in order to overcome the bottleneck of existing preclinical research. Summary of the Invention
[0005] The purpose of this invention is to provide a non-human mammalian model that simulates cardiorenal metabolic syndrome and co-injury of the heart and kidneys; the second purpose of this invention is to provide a method for constructing a non-human mammalian model that simulates cardiorenal metabolic syndrome and co-injury of the heart and kidneys; the third purpose of this invention is to provide an application of a non-human mammalian model that simulates cardiorenal metabolic syndrome and co-injury of the heart and kidneys.
[0006] To achieve the above objectives, the technical solution provided by the present invention is as follows:
[0007] A non-human mammalian model simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys, wherein the model is a non-human mammalian genome containing both functional loss of the Cyp7b1 gene and functional loss of the leptin receptor gene.
[0008] A method for constructing a non-human mammalian model of simulated cardiorenal metabolic syndrome and co-injury of the heart and kidneys, as described above, wherein the method involves mating a non-human mammal with a Cyp7b1 gene knockout with a non-human mammal carrying a db mutation, and screening by PCR genotyping to obtain offspring non-human mammals that are homozygous for Cyp7b1 gene knockout and are db / db homozygous.
[0009] Preferably, the PCR genotype identification is performed using a specific primer combination; the primer combination includes an upstream primer as shown in SEQ ID No. 1 and a downstream primer as shown in SEQ ID No. 2 for amplifying the Cyp7b1 gene knockout fragment, and an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4 for amplifying the Cyp7b1 gene wild-type fragment.
[0010] Preferably, when using the upstream and downstream primers for amplifying the Cyp7b1 gene knockout fragment, the amplification product length for wild-type non-human mammals is 1747 bp, and the amplification product length for Cyp7b1 gene knockout homozygous non-human mammals is 254 bp; when using the upstream and downstream primers for amplifying the Cyp7b1 gene wild-type fragment, the amplification product length for wild-type non-human mammals is 556 bp, and the amplification product length for Cyp7b1 gene knockout homozygous non-human mammals is 0 bp.
[0011] Preferably, the non-human mammal is a rat.
[0012] The use of a non-human mammalian model, as described above, simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys, in screening or evaluating drugs for the prevention or treatment of cardiorenal metabolic syndrome, wherein the use is for non-therapeutic purposes.
[0013] Compared with the prior art, the present invention has the following beneficial effects:
[0014] 1. The Cyp7b1 of the present invention - / - The db / db dual-gene mutation model is not a simple superposition of pathologies, but rather produces unexpected technical effects through the strong synergistic effect of lipid / bile acid metabolism abnormalities (Cyp7b1 knockout) and systemic glucose and lipid metabolism disorders (db / db). Compared with single-gene db / db mice, the model of this invention shows extremely significant deterioration in renal core excretory filtration indicators such as UACR and SCr, and cardiac core contractile indicators such as EF% and FS%, spontaneously leading to severe myocardial interstitial fibrosis and glomerulosclerosis.
[0015] 2. This model successfully overcomes the technical bias of traditional metabolic animal models that "have mild phenotypes and are difficult to spontaneously develop severe fibrosis". It perfectly reproduces the dual organic failure characteristics of clinically advanced CKM refractory at the tissue and organ level, providing an in vivo assessment tool for preclinical mechanism research.
[0016] 3. The model of this invention significantly aggravates the pathological phenotype of co-injury of the heart and kidneys, effectively shortens the modeling window period and improves the sensitivity of evaluation indicators, which helps to accelerate the research and development and high-throughput screening of drugs for anti-CKM complications (especially innovative drugs targeting multi-organ fibrosis and organ remodeling). Attached Figure Description
[0017] Figure 1 A schematic diagram of the Cyp7b1 gene knockout strategy and homozygous knockout (KO) alleles;
[0018] Figure 2 Agarose gel electrophoresis image of PCR products for mouse genotyping;
[0019] Figure 3 For the double-gene mutant mouse (Cyp7b1) - / - Schematic diagram of db / db) breeding strategy;
[0020] Figure 4 A statistical chart comparing the renal function (UACR, SCr) of three groups of mice;
[0021] Figure 5 A quantitative comparison of echocardiograms, histopathological findings, and core cardiac function indicators of three groups of mice.
[0022] Figure 6 The images show the pathological assessment and quantitative statistical data of fibrosis in the heart and kidney tissues of three groups of mice. Detailed Implementation
[0023] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.
[0024] This invention provides a Cyp7b1 - / - A db / db dual-gene mutant mouse model was developed. This model was obtained by crossbreeding mice with systemic Cyp7b1 gene knockout (genetic background: C57BL / 6J) with db / db mice. This invention utilizes the dual-gene mutation to induce severe systemic metabolic disorders (dual abnormalities in glucose, lipid, and bile acid metabolism), successfully inducing a significant co-damage phenotype in target organs (heart and kidneys). This invention also provides the application of this model in the preparation, screening, and evaluation of drugs for the treatment of CKM and related cardiorenal complications.
[0025] Example 1
[0026] Model construction and genotyping
[0027] like Figure 1 As shown, the Cyp7b1 gene is located on chromosome 3 of mice and contains 7 exons. The basic Cyp7b1 knockout mouse of this invention was constructed using CRISPR / Cas9 gene editing technology. The specific strategy is as follows: Specific upstream and downstream sgRNAs were designed to target the Cyp7b1 gene. These sgRNAs include an upstream sgRNA (target sequence as shown in SEQ ID No. 5: 5'-CTAAGCCCTAATCAGAGGGT-3', PAM sequence TGG) and a downstream sgRNA (target sequence as shown in SEQ ID No. 6: 5'-GAAGGTTGCTGCTGATCCAC-3', PAM sequence AGG). The Cas9 nuclease, guided by these sgRNAs, precisely knocks out a 1493 bp region, including exon 2 (coding region 137 bp). The deletion of this region disrupts normal splicing or causes frameshift mutations in translation, thereby completely eliminating the normal function of the CYP7B1 protein and achieving a systemic gene knockout effect. Subsequently, the knockout mouse was crossbred with db / db mice to construct a dual-gene mutation model. The offspring were then identified by PCR using genomic DNA extracted from tail tissue.
[0028] Knockout fragment amplification primers:
[0029] Upstream primer SEQ ID No. 1: CAAACCCAACTTTGATGAGAAGGAGT;
[0030] Downstream primer SEQ ID No.2: AAACTAATGCTGCCTGTTCAAGGG.
[0031] The product length of wild-type (WT) mice was 1747 bp; the product length of knockout (KO) mice was 254 bp.
[0032] Primers for wild-type fragment amplification:
[0033] Upstream primer SEQ ID No. 3: GAGCTATATTTGTGGGAAGATGTG;
[0034] Downstream primer SEQ ID No. 4: TTGGCAAGTGGGATGGCTGATC.
[0035] like Figure 2 As shown, PCR amplification of mouse genomic DNA was performed using two pairs of primers for verification.
[0036] Knockout allele testing, such as Figure 2 As shown in the Primer A panel, when using knockout fragment amplification primers, wild-type (WT) primers fail to amplify or have extremely low amplification efficiency within a specific extension time due to the excessively large fragment size (1747 bp), while knockout (KO) primers can stably amplify a specific band of 254 bp.
[0037] Wild-type allele testing, such as Figure 2 As shown in the Primer B panel, using primers for wild-type fragment amplification, the wild-type (WT) was able to amplify a specific band of 556 bp, while the knockout type could not be amplified (0 bp) due to the absence of the binding site.
[0038] Example of identification result determination:
[0039] 1. Wild type (WT): such as Figure 2 As shown in lanes 65 and B6 (C57BL / 6 wild-type control), only Primer B amplified a 556 bp band, while Primer A showed no band.
[0040] 2. Heterozygote (Het): such as Figure 2 As shown in the middle lanes (e.g., lanes 66, 67, and 68), Primer A amplified a 254 bp band, while Primer B simultaneously amplified a 556 bp band. This identification result provides technical support for screening offspring carrying the knockout gene in subsequent breeding steps of this invention.
[0041] 3. Homozygous knockout (KO): such as Figure 2 As shown in lane 69, only Primer A amplified a 254 bp band, while Primer B showed no band. The basis for establishing a dual-gene mutation model subsequently used in this invention for breeding db / db mice is the homozygous knockout type (Cyp7b1) rigorously screened using this method. - / - )individual.
[0042] Example 2
[0043] To obtain stably inherited double-mutant mice with a severe cardiorenal metabolic syndrome phenotype, this invention establishes a scientific hybridization breeding route. For example... Figure 3 As shown, the specific breeding steps are as follows:
[0044] 1. Obtaining homozygous knockout first-generation mice: First, mice with the genotype Cyp7b1... + / - Heterozygous mice were interbred (Cyp7b1) + / - × Cyp7b1 + / - Cyp7b1 in the offspring was screened using the aforementioned PCR identification method. - / -Homozygous knockout mice.
[0045] 2. Introduce the db mutant gene: The Cyp7b1 gene selected above... - / - Homozygous mice were mated with classic leptin receptor-deficient heterozygous mice (db / m) (Cyp7b1) - / - ×db / m) to incorporate the pathogenic gene background of metabolic syndrome into the model system.
[0046] 3. Breeding double heterozygous / carrier offspring: From the offspring of step 2, select transitional mice that simultaneously carry the homozygous knockout of Cyp7b1 and the recessive db mutation, with the genotype Cyp7b1. - / - db / m.
[0047] 4. Obtain the final double-mutation model: The Cyp7b1 obtained in step 3... - / - db / m mice were subjected to male-female sibling mating (Cyp7b1) - / - db / m × Cyp7b1 - / - (db / m). In its offspring, rigorous genotyping was performed according to Mendelian inheritance laws, ultimately selecting the Cyp7b1 genotype. - / - The db / db bigenic homozygous mutant mouse is the core animal model used in this invention to simulate severe cardiac and renal damage.
[0048] Example 3
[0049] Experimental group setup and validation of co-injury phenotype of heart and kidney
[0050] I. Grouping of Experimental Animals
[0051] Control group (C57BL / 6J normal mice, 20 weeks old);
[0052] db / db group (single gene mutation model group, 20 weeks old);
[0053] Cyp7b1 - / - db / db group (double gene mutation model group, 20 weeks old).
[0054] II. Phenotypic Verification of Cardiac and Renal Co-injury
[0055] (1) Significantly aggravated damage to kidney structure and function.
[0056] like Figure 4 As shown, this invention quantitatively detected the core excretion and filtration function indicators of the kidneys in three groups of mice.
[0057] Urinary albumin excretion rate (UACR): UACR is a sensitive indicator reflecting early damage to the glomerular filtration barrier. For example... Figure 4The UACR results showed that although the UACR of the db / db group mice with a single gene mutation was higher than that of the normal control group, Cyp7b1 - / - The UACR level of mice in the db / db model group showed an explosive increase, which was significantly higher than that of the db / db group alone (p < 0.01).
[0058] Serum creatinine (SCr): SCr is the gold standard reflecting overall renal excretory function. For example... Figure 4 The Serum creatinine results show that Cyp7b1 - / - The serum creatinine concentration in the db / db model group mice reached its highest value, showing a statistically significant difference in deterioration compared to the db / db group. The quantitative comparison of these biochemical indicators conclusively demonstrates that the superimposed Cyp7b1 knockout against the background of db / db metabolic disorder is not a simple pathological superposition, but rather produces a strong synergistic destructive effect, leading to accelerated failure of end-stage renal filtration function.
[0059] (2) Significant exacerbation of cardiac structural and functional damage, such as Figure 5 As shown, this invention systematically evaluated the cardiac function and morphology of mice in each group using small animal ultrasound and HE tissue staining, confirming that the double mutation caused severe heart failure.
[0060] In vivo echocardiographic assessment of cardiac function, such as Figure 5 As shown in the M-mode echocardiography, the ventricular wall motion amplitude was normal in the normal control group mice; the ventricular wall motion was slightly weakened in the db / db group mice with the single gene mutation; while the Cyp7b1 of this invention... - / - In the db / db model group mice, the amplitude of the free wall motion of the ventricle became extremely flat, showing extremely low cardiac contractile activity.
[0061] Quantitative indicators of cardiac function (EF% and FS%): Left ventricular ejection fraction (EF%) and fractional shortening (FS%) are core quantitative indicators for evaluating cardiac pumping function. For example... Figure 5 The statistical results of left ventricular ejection fraction (EF%) and fractional shortening (FS%) in Cyp7b1 showed that... - / - The EF% and FS% of the db / db model group mice were not only significantly lower than those of the normal control group, but also showed a trend of extremely significant decrease compared to the single-mutant db / db group. This indicates that the superimposed Cyp7b1 gene knockout led to catastrophic cardiac systolic dysfunction.
[0062] Cardiac histopathological changes: such as Figure 5 The HE-stained sections showed that the control group's cardiomyocytes were neatly arranged and clearly structured; the db / db group showed slight compensatory cell thickening; while the Cyp7b1 group... - / - The myocardial tissue of the db / db model group mice exhibited typical severe myocardial pathological changes, including significant hypertrophy of cardiomyocytes, extremely disordered arrangement, and obvious tissue structural damage. These results strongly demonstrate that the Cyp7b1 of this invention... - / - The db / db mouse successfully broke the phenotypic limitations of single-gene metabolic models, exhibiting severe organic failure in both the heart and kidneys, making it an excellent animal model for simulating late-stage severe cardiorenal metabolic syndrome.
[0063] (3) Pathological confirmation of severe fibrosis in target organs of the heart and kidneys
[0064] To reveal the organic basis of severe cardiac and renal failure caused by dual gene mutations at the microscopic pathological level, this invention further performed Masson trichrome staining (for specifically labeling collagen fiber deposition) on the heart and kidney tissues of mice in each group, such as... Figure 6 As shown.
[0065] Severe renal fibrosis: such as Figure 6 Masson staining results of the kidneys showed that the glomeruli and renal interstitial structures were clear in the normal control group, with very little blue collagen fiber distribution; the db / db group mice showed a certain degree of fibrosis deposition; while the Cyp7b1 of this invention... - / - In the db / db model group mice, large areas of deep blue collagen fiber deposition appeared in the kidney tissue, with severe glomerular sclerosis and diffuse interstitial fibrosis. Figure 6 The quantitative statistical plot of the area of kidney fibrosis (%) further confirms that Cyp7b1 - / - The proportion of renal fibrosis in the db / db group was significantly higher than that in the db / db group alone (p < 0.001, marked with *** in the figure).
[0066] Severe cardiac fibrosis: Similarly, as Figure 6 Masson staining of the heart showed that in the normal control group, myocardial fibers were tightly packed with no obvious blue collagen in the interstitium; slight collagen deposition was observed in the myocardial interstitium of the db / db group; while Cyp7b1... - / - In the db / db model group mice, thick, dense networks of blue collagen fibers appeared in the myocardial interstitium and perivascular space, indicating severe damage to the myocardial tissue structure. Figure 6The quantitative statistical plot of the area of heart fibrosis (%) further confirmed that the area of myocardial fibrosis in the double mutation group was significantly higher than that in the single mutation db / db group (p < 0.01, marked with ** in the figure).
[0067] Pathological conclusion: The above results conclusively demonstrate that the deletion of the Cyp7b1 gene in the context of db / db systemic glucose and lipid metabolism disorder not only leads to the deterioration of physiological indicators but also induces an extremely strong and irreversible pro-fibrotic storm at the tissue level, ultimately resulting in severe cardiorenal interstitial fibrosis and glomerulosclerosis. This perfectly reproduces the refractory pathological characteristics of clinically advanced cardiorenal metabolic syndrome (CKM), endowing this animal model with extremely high value for drug screening and translational medicine.
Claims
1. A non-human mammalian model simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys, characterized in that, The model is a non-human mammalian genome that simultaneously contains the functional loss of the Cyp7b1 gene and the functional loss of the leptin receptor gene.
2. A method for constructing a non-human mammalian model simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys as described in claim 1, characterized in that, The construction method involves mating a Cyp7b1 gene knockout non-human mammal with a non-human mammal carrying the db mutation, and then screening by PCR genotype identification to obtain offspring non-human mammals that are homozygous for Cyp7b1 gene knockout and are db / db homozygous.
3. The construction method according to claim 2, characterized in that, The PCR genotype identification is performed using a specific primer combination; the primer combination includes an upstream primer as shown in SEQ ID No. 1 and a downstream primer as shown in SEQ ID No. 2 for amplifying the Cyp7b1 gene knockout fragment, and an upstream primer as shown in SEQ ID No. 3 and a downstream primer as shown in SEQ ID No. 4 for amplifying the Cyp7b1 gene wild-type fragment.
4. The construction method according to claim 3, characterized in that, When amplifying the Cyp7b1 gene knockout fragment using the aforementioned upstream and downstream primers, the amplification product length for wild-type non-human mammals is 1747 bp, and the amplification product length for Cyp7b1 gene knockout homozygous non-human mammals is 254 bp; when amplifying the Cyp7b1 gene wild-type fragment using the aforementioned upstream and downstream primers, the amplification product length for wild-type non-human mammals is 556 bp, and the amplification product length for Cyp7b1 gene knockout homozygous non-human mammals is 0 bp.
5. The construction method according to claim 2, characterized in that, The non-human mammal in question is a rat.
6. The application of a non-human mammalian model simulating cardiorenal metabolic syndrome and co-injury of the heart and kidneys as described in claim 1 in screening or evaluating drugs for the prevention or treatment of cardiorenal metabolic syndrome, characterized in that, The application is for non-therapeutic purposes.