CN114451357A - Construction method of adult mouse heart failure model based on myocardial retinol metabolic disorder - Google Patents
Construction method of adult mouse heart failure model based on myocardial retinol metabolic disorder Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/05—Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0375—Animal model for cardiovascular diseases
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention provides a construction method of an adult mouse heart failure model based on myocardial retinol metabolic disorder, which comprises the following steps of firstly enabling RDH10fl/flThe mice were mated with MYH6-iCre mice to obtain mice carrying RDH10fl/flF2 generation mice of gene and MHY6-iCre gene; and then specifically knocking out the RDH10 gene in the cardiac muscle cells of the F2 generation mice when the mice are 5-6 weeks old, and continuously feeding the mice until the left ventricular ejection fraction is less than 60 percent to obtain the adult mouse heart failure model based on the myocardial retinol metabolic disorder. The construction of the model increases the operability and convenience for further researching the relation between the myocardial retinol metabolic disorder and the heart failure, provides a replication and research tool for heart diseases based on the myocardial retinol metabolic disorder, and has considerable necessity for the heart disease related research.
Description
Technical Field
The invention belongs to the technical field of medicines. More particularly, relates to a construction method of an adult mouse heart failure model based on myocardial retinol metabolic disorder.
Background
Heart failure is the terminal stage of various cardiovascular diseases, and patients with heart failure can have various symptoms such as dyspnea, fatigue, poor exercise tolerance, fluid retention and the like, and the life quality of the patients is seriously affected. Moreover, the heart failure is hidden and has extremely high fatality rate, and the five-year survival rate of patients after confirmed diagnosis is even lower than that of various malignant tumors. Therefore, it is important to find early diagnosis indicators and treatment methods of heart failure and to develop basic research aiming at the pathogenesis of heart failure.
The application of animal models of diseases to simulate human diseases is a main way to find potential molecular mechanisms and therapeutic targets of human diseases. The existing heart failure animal models can be divided into ischemic cardiomyopathy heart failure models mainly constructed by a coronary artery ligation method and a plugging method, pressure load type heart failure models mainly constructed by an aortic stenosis method and a salt load method, pre-volume load type heart failure models constructed by an arteriovenous fistula method and a heart valve insufficiency method, drug-induced heart failure models constructed by using myocardial damage drugs and gene-modified heart failure models constructed by specific gene modification according to different model making modes and pathogenesis. Although the construction of these animal models has provided great help for the research on the prevention and treatment of heart failure-related diseases, the increasing morbidity and mortality rate indicates that the existing research on heart failure is still insufficient.
Recently, Ni Yang et al have pointed out that retinol metabolic disorder phenomenon (Yang Ni, Parker Lauren, Yu Jianshi, Jones Jace W, Liu Ting, Papanicolaou Kyrakos N, Talbot C Conover, Margulies Kenneth B, O' RourBrian, Kane Mauren, Foster D Brian. Cardiac reoic acid levels deckle in heart failure) mainly characterized by significant decrease of retinoic acid content generally exists in cardiac muscle of heart failure patients, but the relationship between myocardial retinol metabolic disorder and heart failure is not clear, so that a suitable animal model needs to be constructed for research.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for constructing an adult mouse heart failure model based on myocardial retinol metabolic disorder, which increases operability and convenience for further researching the relation between myocardial retinol metabolic disorder and heart failure and provides a tool for copying and researching heart diseases based on myocardial retinol metabolic disorder.
The invention mainly aims to provide application of RDH10 gene myocardial specific knockout in construction of an adult mouse heart failure model based on myocardial retinol metabolic disorder.
The invention also aims to provide a construction method of the heart failure model of the adult mouse based on the metabolic disorder of the myocardial retinol.
The above purpose of the invention is realized by the following technical scheme:
the invention is provided with the 5-week-old beltRDH10fl/flThe gene and MHY6-iCre gene are injected into the abdominal cavity of a mouse, tamoxifen is injected into the abdominal cavity of the mouse, iCre protein activated by tamoxifen can carry out specific induction knockout on RDH10 gene in the cardiac muscle of the mouse, so that the phenomenon of myocardial retinol metabolic disorder and heart failure can spontaneously occur in the 15 th week after induction of the mouse is completed, an adult mouse heart failure model based on the myocardial retinol metabolic disorder is successfully constructed, and the RDH10 gene myocardial specific knockout plays a vital role in constructing the adult mouse heart failure model based on the myocardial retinol metabolic disorder. Therefore, the application of the RDH10 gene myocardial specific knockout in the construction of an adult mouse heart failure model based on the metabolic disorder of myocardial retinol is within the protection scope of the invention.
The invention also provides a construction method of the heart failure model of the adult mouse based on the metabolic disorder of the myocardial retinol, which comprises the following steps of firstly enabling RDH10fl/flMating the mouse with MYH6-iCre mouse to obtain a mouse carrying RDH10fl/flF2 generation mice of gene and MHY6-iCre gene; and when the F2 generation mouse is 5-6 weeks old, specifically knocking out the RDH10 gene in the cardiac muscle cells of the F2 generation mouse, and continuously feeding the F2 generation mouse until the left ventricular ejection fraction is less than 60%, thus obtaining the heart failure model of the adult mouse based on the metabolic disorder of the cardiac retinol.
The invention firstly carries out the operation of carrying RDH10 to 5 weeks oldfl/flThe gene and MHY6-iCre gene are injected into the abdominal cavity of a mouse, the iCre protein activated by the tamoxifen completes the specific induction knockout of RDH10 gene which is used as key enzyme in the retinol metabolic process in the cardiac muscle of the mouse, after the induction is carried out for 15 weeks, the contents of the retinol and retinoic acid in the cardiac muscle of the mouse are determined through LC-MS/MS, the fact that the mouse has myocardial retinol metabolic disorder is proved, and then the detection of the cardiac function and pathology finds that the mouse is accompanied with heart failure, which indicates that the method can successfully construct an adult mouse heart failure model based on the myocardial retinol metabolic disorder.
Preferably, the mouse is a C57BL/6 background mouse.
Preferably, the specific knockout is induced by tamoxifen.
Further preferably, the preparation method of tamoxifen comprises the following steps: firstly, absolute ethyl alcohol is used for promoting dissolution, and then an organic solvent is used for diluting. The organic solvent does not need to be an organic solvent without toxic and side effects.
More preferably, the dilution is to 10 mg/mL.
More preferably, the organic solvent is corn oil.
Preferably, the induction mode of tamoxifen is intraperitoneal injection.
Further preferably, the intraperitoneal injection is: 48-52 mg/kg per dayBody weightThe dosage of the composition is injected once and continuously for 5 days. More preferably, the amount is 50mg/kgBody weight. Most preferably, the injection is performed at a fixed time per day, such as 9 am per day.
Preferably, the knockout is performed at 5 weeks of age in F2 generation mice.
Preferably, the adult mouse heart failure model is constructed during periods of time to maintain rat feed, free water intake. The maintenance mouse feed is a basic feed eaten by experimental mice in daily experiments.
The invention has the following beneficial effects:
the model mouse constructed by the invention spontaneously generates the phenomena of myocardial retinol metabolic disorder and heart failure in 15 weeks after tamoxifen induction is completed, has the advantage of fast modeling, is obtained by specifically knocking out the RDH10 gene in myocardial cells, namely the retinol metabolic disorder phenomenon of the model mouse only exists in cardiac muscle, and is an adult mouse heart failure model completely based on the myocardial retinol metabolic disorder. The construction of the model increases operability and convenience for further researching the relation between the myocardial retinol metabolic disorder and the heart failure, provides a tool for copying and researching the heart disease based on the myocardial retinol metabolic disorder, is favorable for developing a diagnosis method and a treatment medicine for the heart failure based on the myocardial retinol metabolic disorder, and has considerable necessity for the research related to the heart disease.
Drawings
FIG. 1A is the results of the myocardial RDH10 Western blot and its statistical chart, and FIG. 1B is the myocardial RDH10 protein tissue immunofluorescence staining results. (data in the statistical plots are presented as means ± sem, representing the values of the RDH10 myocardial-specific knockout group and RDH10, as analyzed statisticallyfl/flGroup number comparison P value less than 0.001)
Fig. 2A is a graph of myocardial retinol content, and fig. 2B is a graph of myocardial retinoic acid content. (data in the statistical plots are presented as means ± sem, representing the values of the RDH10 myocardial-specific knockout group and RDH10, analyzed statisticallyfl/flGroup number comparison P value less than 0.05)
Fig. 3A is a schematic representation of cardiac ultrasound of three groups of mice, fig. 3B is a statistical representation of left ventricular ejection fraction of mice, and fig. 3C is a statistical representation of left ventricular shortening fraction of mice. (data in the statistical plots are presented as means ± standard errors, which indicate that, upon statistical analysis, the P value for the RDH10 myocardial-specific knockout group compared to the wild group was less than 0.001; ###indicates that, upon statistical analysis, the RDH10 myocardial-specific knockout group compared to RDH10fl/flGroup number comparison P value less than 0.001)
Fig. 4A is an observation result of a neutral gum patch in the myocardial cell hypertrophy test, and fig. 4B is an analysis result of a myocardial cell area. (data in the statistical plots are presented as means. + -. standard error, which indicates that the P-value for RDH10 myocardial-specific knockout group compared to the wild-type group was less than 0.05 by statistical analysis; # indicates that RDH10 myocardial-specific knockout group compared to RDH10 by statistical analysisfl/flGroup number comparison P value less than 0.05)
Fig. 5A is an observation result of a neutral gum patch in a myocardial interstitial fibrosis test, and fig. 5B is an analysis result of an area ratio of myocardial collagen. (data in the statistical plots are presented as means. + -. standard errors, which indicate that, upon statistical analysis, the P value for the RDH10 myocardial-specific knockout group values compared to the wild-type group values was less than 0.001; # indicates that, upon statistical analysis, the RDH10 myocardial-specific knockout group values compared to RDH10fl/flGroup number comparison P value less than 0.01)
FIG. 6A is a photograph of a neutral gum patch in the apoptosis assay, and FIG. 6B is the result of analysis of the number of myocardial positive cells. (data in the statistical plots are presented as means ± semThe RDH10 myocardial-specific knockout group value is less than 0.01 compared with the wild group value; # indicates the values of the RDH10 myocardial-specific knockout group and RDH10 by statistical analysisfl/flGroup number comparison P value less than 0.01)
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1 construction of adult mouse heart failure model based on myocardial retinol metabolic disorder
First, experimental material
1) RDH10 for reproductive yearsfl/flMice (the university of Oklahoma health center, Oklahoma USA) and MYH6-iCre mice (Jiangsu Jiejiaokang); the mice were all C57BL/6 background mice.
2) Tamoxifen: firstly, dissolving the tamoxifen powder with the purity of more than or equal to 99% by using absolute ethyl alcohol, and then diluting the tamoxifen powder to 10mg/mL by using corn oil with 9 times of volume.
Second, grouping experiments
RDH10 myocardial-specific knockout group: first let RDH10fl/flMating the mice with MYH6-iCre mice, and screening to obtain 15 mice simultaneously carrying RDH10fl/flF2 generation mice of gene and MHY6-iCre gene; at 9 am, 50mg/kg of the F2 mouse 5 weeks oldBody weightThe dose of tamoxifen is intraperitoneally injected once, and after continuous injection is carried out for 5 days, the feeding is continued for 15 weeks, so that an RDH10 myocardial specificity knockout group is obtained;
RDH10fl/flgroup (2): 15 RDH10 aged 5 weeksfl/flMice, 9 o' clock in the morning at 50 mg/kg/dayBody weightThe dosage of the preparation is to inject tamoxifen into the abdominal cavity once, and the mixture is continuously fed for 15 weeks after being injected for 5 days, thus obtaining RDH10fl/fGroup (d);
wild group: 15 wild-type C57BL/6J mice, 5 weeks old, were not injected with tamoxifen and served as wild-type controls.
EXAMPLE 2 myocardial RDH10 knockout
Verification method
(1) Western blotting method
On week 4 after completion of tamoxifen induction, the myocardial-specific knockout group from RDH10 and RDH10 of example 1, respectivelyfl/fl3 mice are randomly extracted from the group, and then myocardial tissue proteins of the mice are respectively extracted for immune western blot analysis. The specific method comprises the following steps:
a. extracting myocardial tissue protein:
taking 20mg of corresponding myocardial tissue, adding 600 mu L of RIPA lysate containing PMSF, homogenizing on ice, standing the obtained homogenate liquid on ice for 30min, centrifuging at the rotating speed of 15000rpm and the temperature of 4 ℃ for 15min, and transferring the supernatant to an ep tube to obtain the mouse myocardial tissue protein liquid.
b. And (3) detecting the protein concentration:
the BCA kit is used for measuring the concentration of the myocardial tissue protein liquid, and specifically comprises the following steps: the protein standard solution is respectively diluted into 1000 ng/muL, 500 ng/muL, 250 ng/muL, 125 ng/muL, 25 ng/muL and 0 ng/muL in a gradient way, and 25 muL to 96-hole plates → 2.5 muL of protein solution to be detected and 22.5 muL of ultrapure water are respectively added into the holes of the 96-hole plates → 200: 4 preparing detection solution by volume ratio → adding 200 μ L detection solution to the well added with standard protein solution and protein solution to be detected → slightly shaking and mixing, incubating at 37 deg.C for half an hour → detecting absorbance (OD) value at 562nm with microplate reader → establishing standard curve (R)2Not less than 0.99, the abscissa is the OD value of the standard protein liquid, and the ordinate is the concentration), and substituting the OD value of the protein liquid to be detected to calculate the protein concentration.
c. And (3) performing western blotting:
after obtaining the corresponding tissue protein liquid and the concentration thereof, adding 35 mu g of protein liquid into ultrapure water and 5 Xprotein sample buffer solution, boiling, then loading the protein liquid on SDS-PAGE gel, carrying out electrophoresis for 30 minutes for the first time (60V constant voltage) and 90 minutes for the second time (120V constant voltage), then carrying out electrotransfer (250 mA constant current) for 90 minutes after the electrophoresis is finished, placing the transferred membrane into 5% BSA for sealing, and then monitoring and analyzing in sequence: cutting membrane → primary antibody incubation at 4 ℃ for 16 hours → TBST rinsing → secondary antibody incubation at 25 ℃ for 60 minutes → TBST rinsing → developer development → gel imager development photographing → Image J gray scale scanning analysis.
(2) Tissue immunofluorescence staining method
Myocardial-specific knockout from RDH10, RDH10, respectively, at week 4 after completion of tamoxifen inductionfl/f3 mice were randomly selected from the group and the wild group, and tissues were analyzed by immunofluorescence staining after the heart was fixed and embedded. The specific method comprises the following steps:
a. preparing a heart paraffin section:
after 15 weeks of completion of tamoxifen induction, mice were fasted for 12 hours in advance (without water deprivation). After fasting was complete, mice were anesthetized with pentobarbital, the thorax was cut open with ophthalmic surgery, and the right atrial appendage of the mice was exposed and cut open. Subsequently, the mice were perfused with sterile PBS from their left ventricle with a syringe until the effluent was bloodless, and the hearts of the mice were cut, rinsed with PBS, and fixed in 4% paraformaldehyde for 16 h. The next day, PBS wash for 30 minutes, repeat 3 times; then sequentially immersing the heart of the mouse in 25% ethanol, 50% ethanol, 75% ethanol, 90% ethanol, absolute ethanol (I) and absolute ethanol (II) for 30min respectively; then soaking in dimethylbenzene (I) and dimethylbenzene (II) for 15min respectively; then immersing into paraffin (I) and paraffin (II) for 60min respectively; then, the heart is horizontally placed in paraffin for embedding; finally, the embedded paraffin blocks are sliced with a laika paraffin slicer (slice thickness 5 μm).
b. Fluorescent staining of the sections with immunohistones:
taking the corresponding section obtained in the step a, and dyeing according to the following steps: baking at 60 ℃ for 30min → xylene (I) for 10min → xylene (II) for 10min → absolute ethanol (I) for 3min → absolute ethanol (II) for 3min → 95% ethanol (I) for 1min → 70% ethanol for 1min → distilled water for 2min for three times → citric acid buffer solution (10mM, pH7.4) microwave oven fire for 10min → PBS5min for three times → 3% triton-10010 min → flowing water washing for 20min → distilled water washing for 1-2s → 0.5% eosin for 2min → distilled water washing for 1-2s → PBS5min for three times → 5% BSA for three times → PBS (37 ℃) for 30min → corresponding one-fourth antibody for 16h → 25 ℃ for 30min rewarming → PBS5min for three times → PBS (37 ℃) for 2h → 5min → three times → hoechst 3334225 ℃ for 15min → PBS5min → three times → anti-fluorescence quenching tablet → fluorescent microscope.
Second, verification result
The experimental results are shown in fig. 1, wherein fig. 1A is the myocardial RDH10 protein immunoblotting results and the statistical chart thereof, and fig. 1B is the tissue immunofluorescence staining results of the myocardial RDH10 protein.
As can be seen from FIG. 1A, the myocardial RDH10 protein expression of RDH10 myocardial-specific knockout mice is significantly lower than that of RDH10fl/flGroup (d); as shown in FIG. 1B, the myocardial RDH10 protein expression of the RDH10 myocardial-specific knockout mice is significantly lower than that of RDH10fl/flGroup and wild group. The method provided by the invention has a remarkable effect on the knockout of the RDH10 gene in the myocardial cells of the RDH10 myocardial specific knockout group mice, and realizes the specific knockout.
Example 3 Metabolic Condition of cardiac retinol
Detection method
(1) Sample collection
After 15 weeks of completion of tamoxifen induction, mice were fasted for 12h (without water), anesthetized, opened to the chest, pre-cooled PBS perfusion rinsed, and hearts were harvested. After heart harvesting, the cells were washed in a petri dish filled with pre-cooled PBS and excess tissue was removed, 20-30mg of tissue was excised from the apex, weighed, snap frozen in liquid nitrogen and stored. Because the ROL and the atRA are unstable in chemical property and are easy to decompose by visible light, the sample collection should be carried out in a dark place in the whole process.
(2) Sample pretreatment
The divided mouse myocardium tissue was removed, 200 μ L of pre-cooled 0.9% NaCl solution was added, homogenized using a homogenizer 4 bar for 5 seconds, followed by addition of 100 μ L D4-ROL internal standard and continued homogenization for 5 seconds. After vortex mixing for 5min, 1mL of an extractant (1% formic acid in n-hexane) was added, vortex extracted for 5min, and centrifuged at 13000rpm for 5 min. mu.L of the supernatant was dried in the dark at 25 ℃ with nitrogen and the dried sample was redissolved with 100. mu.L of 70% methanol. All manipulations were protected from light and performed on ice. And sucking 95 mu L of the 70% methanol complex solution to a 96-well sample feeding plate, and performing LC-MS/MS sample injection analysis, wherein 10 mu L of sample is injected into each sample.
(3) LC-MS/MS Condition setting
a. Chromatographic conditions are as follows:
a chromatographic column: phenomenex kinex Kinetex C18(50 x 2.1mm 2.6 μm);
mobile phase A: acetonitrile: methanol: water: formic acid 200: 150: 150: 0.5 (V/V/V/V);
mobile phase B: acetonitrile: methanol: water: formic acid 275: 150: 75: 0.5 (V/V/V/V);
column temperature: 25 ℃; flow rate: 0.2 mL/min; gradient elution for 6 min.
The gradient elution procedure is shown in table 1:
TABLE 1 LC-MS/MS chromatographic gradient elution program set-up
b. Mass spectrum conditions:
the Ion Source IS an electrospray ionization Source (Turbo Spray) and adopts a positive Ion detection mode, air Curtain Gas (CUR) IS 30psi, Collision Gas (CAD) IS 10psi, Spray Voltage (Ionspray Voltage, IS) IS 4000V, Temperature of dry Gas (TEM) IS 400 ℃, atomization Gas (Ion Source Gas1, GS1) IS 35psi, and auxiliary Gas (Ion Source Gas2, GS2) IS 40 psi.
The optimal conditions for the Rol and RA mass spectrometric detection are shown in Table 2:
TABLE 2 LC-MS/MS Mass Spectrometry optimization parameters
Retinol detection limit: 0.02 ng/mL; detection limit of all-trans retinoic acid: 0.02ng/mL
(4) Standard curve establishment
Accurately weigh 1mg of ROL and atRA standards into brown injection vials, respectively, and add 70% methanol to a volume of 1mg/mL stock. ROL and atRA stock solutions are diluted into 100ng/mL, 50ng/mL, 20ng/mL, 5ng/mL, 1ng/mL and 0ng/mL in a gradient manner, an equivalent internal standard is mixed, a computer is used for detecting and recording peak wave areas, peak area ratios are calculated, and a standard curve is established.
Second, the detection result
The results of LC-MS/MS analysis are shown in FIG. 2, in which FIG. 2A is a graph showing the content of myocardial retinol, and FIG. 2B is a graph showing the content of myocardial retinoic acid. As can be seen from fig. 2A and 2B, RDH10fl/flCompared with the group, although the mouse myocardial retinol in the RDH10 myocardial specific knockout group has no obvious difference, the content of the myocardial retinoic acid tends to be increased, and the content of the myocardial retinoic acid is obviously reduced. The result shows that after the myocardial RDH10 is specifically knocked out, the mice have metabolic disorder of myocardial retinol, and the metabolic conversion of the retinol to retinoic acid is inhibited.
Example 4 measurement of cardiac function in mice
Detection method
The heart function of the mice was judged by using the data related to the contraction function of the left ventricle measured by a Vevo 3100 small animal ultrasound apparatus. The specific operation method comprises the following steps: RDH10 myocardial-specific knockout group, RDH10fl/flMice of the group and the wild group are respectively anesthetized by isoflurane inhalation, the breasts of the mice are depilated by depilatory cream under the anesthesia state, the mice are placed on a constant-temperature heating plate at 37 ℃ in a supine position, isoflurane is inhaled continuously, the limbs of the mice are connected with electrocardiogram electrodes for detecting and recording the heart rate, and a 30MHz probe is used for detecting the left sternum of the mice. Short axis, measuring left ventricular ejection fraction and left ventricular shortening fraction with M-type ultrasound,
second, the detection result
The measurement results are shown in fig. 3, in which fig. 3A is a schematic diagram of cardiac ultrasound of three groups of mice, fig. 3B is a statistical diagram of left ventricular ejection fraction of mice, and fig. 3C is a statistical diagram of left ventricular shortening fraction of mice.
As can be seen from fig. 3, with RDH10fl/flCompared with the wild group, in 15 weeks after tamoxifen induction is completed, the left ventricular ejection fraction and the shortening fraction of the RDH10 myocardial specific knockout mice are both obviously reduced, and the left ventricular ejection fraction is less than 60%, which indicates that the heart function of the RDH10 myocardial specific knockout mice is seriously damaged at this time, and the heart failure occurs.
Example 5 detection of pathologies
Detection method
(1) Paraffin sections of the heart were prepared as in example 2 (2).
(2) Detection of cardiomyocyte hypertrophy:
after the myocardial cell marker protein cTNT in the sections was fluorescently stained according to the method described in example 2(2), the sections were stained in WGA staining solution with green fluorescence at 37 ℃ for 30min, rinsed with PBS, and mounted with an anti-fluorescence quenching mounting agent. The sealed sections were stored by fluorescence microscopy (FIG. 4A), and then the cardiomyocyte area of each group of mice was analyzed by Image J software (FIG. 4B).
(3) Detection of myocardial interstitial fibrosis:
masson staining is carried out on paraffin sections of mouse hearts, and the main steps are as follows: baking at 60 ℃ for 30min → xylene (I) 10min → xylene (II) 10min → absolute ethanol (I) 3min → absolute ethanol (II) 3min → 95% ethanol (I) 1min → 70% ethanol 1min → distilled water 2min → Weigart's iron hematoxylin staining for 5min → flowing water washing hematoxylin 1-3s → 1% hydrochloric acid alcohol 1-2s → flowing water washing for 20min → distilled water washing for 1-2s → poncey acid fuchsin staining for 5-10min → distilled water quick rinsing for 2-5s → phosphomolybdic acid aqueous solution treatment for 3-5min → aniline blue solution counterstaining for 2min → 1% glacial acetic acid treatment for 1min → 95% ethanol (II) 2-3s → absolute ethanol (III) 3-5s → absolute ethanol (IV) 5-10s → xylene (I) 2 → xylene (II) 2min → neutral mounting observation (FIG. 5A) and collagen deposition area for 5J → collagen deposition area for 5B (FIG. 5A).
(4) Detection of the degree of apoptosis in the myocardium:
apoptotic cells in paraffin sections of mouse hearts were labeled by Tunel staining (DAB staining) as follows: baking at 60 ℃ for 30min → xylene (I) 10min → xylene (II) 10min → absolute ethanol (I) 3min → absolute ethanol (II) 3min → 95% ethanol (I) 1min → 70% ethanol 1min → distilled water 2min → protease K without DNase at room temperature for 20min → PBS washing for 3 times → 3% H2O2(PBS) Room temperature 20min → PBS Wash 3 times → Biotin labeling solution 37 deg.C lightproof incubation 60min → PBS Wash 1 time → labeling reaction stop solution Room temperature 10min → PBS Wash 3 times → Streptavidin-HRP working solution Room temperature 30min → PBS Wash 3 times → DAB color development solution Room temperature 30min → PBS Wash 3 times → hematoxylin Room temperature 30s → flowing Water Wash →Sequentially immersed in 80%, 95%, 100% ethanol, 2min each → xylene twice → neutral gum seal → preservation by optical microscopy for photographing (fig. 6A) → statistical analysis of the number of labeled positive cells (fig. 6B).
Second, the detection result
As can be seen from FIGS. 4-6, in the RDH10 myocardial specific knockout group, the myocardial cell area, the collagen area ratio and the Tunel staining positive cell number per visual field were all significantly higher than those of RDH10 at 15 weeks after tamoxifen induction was completedfl/flGroup and wild group, indicating that the RDH10 myocardium-specific knockout group at this time exhibited severe cardiomyocyte hypertrophy, cardiomyocyte interstitial fibrosis and apoptosis in myocardium, with severe remodeling of myocardial structure.
To sum up, the present invention first proceeds by bringing together RDH10fl/flTamoxifen is injected into the abdominal cavity of a mouse with genes and MHY6-iCre genes, iCre protein activated by tamoxifen can specifically induce and knock out RDH10 gene which is used as key enzyme in a retinol metabolic process in the cardiac muscle of the mouse, after induction is carried out for 15 weeks, the contents of retinol and retinoic acid in the cardiac muscle of the mouse are determined through LC-MS/MS, the fact that the mouse has myocardial retinol metabolic disorder is proved, and then the heart function and pathology detection shows that the mouse is accompanied with heart failure, which indicates that the method can successfully construct an adult mouse heart failure model based on the myocardial retinol metabolic disorder.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
- Application of RDH10 gene myocardial specific knockout in construction of an adult mouse heart failure model based on myocardial retinol metabolic disorder.
- 2. A method for constructing an adult mouse heart failure model based on myocardial retinol metabolic disorder is characterized in that RDH10 is firstly usedfl/flThe mice were mated with MYH6-iCre mice to obtain mice carrying RDH10fl/flF2 generation mice of gene and MHY6-iCre gene; and when the F2 generation mouse is 5-6 weeks old, specifically knocking out the RDH10 gene in the cardiac muscle cells of the F2 generation mouse, and continuously feeding the F2 generation mouse until the left ventricular ejection fraction is less than 60%, thus obtaining the heart failure model of the adult mouse based on the metabolic disorder of the cardiac retinol.
- 3. The method of claim 2, wherein the mouse is a C57BL/6 background mouse.
- 4. The method of construction of claim 2, wherein the specific knockout is induced by tamoxifen.
- 5. The construction method according to claim 4, wherein the tamoxifen is prepared by the following method: firstly, absolute ethyl alcohol is used for promoting dissolution, and then an organic solvent is used for diluting.
- 6. The method for constructing according to claim 5, wherein the organic solvent is corn oil.
- 7. The construction method according to claim 4, wherein the induction mode of tamoxifen is intraperitoneal injection.
- 8. The construction method according to claim 7, wherein the intraperitoneal injection is: 48-52 mg/kg per dayBody weightThe dosage of the composition is injected once and continuously for 5 days.
- 9. The method of claim 8, wherein the amount is 50mg/kgBody weight。
- 10. The construction method according to claim 2, wherein the knockout is performed in F2 generation mice at 5 weeks of age.
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