CN114451357B - 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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- 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
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- 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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Abstract
The invention is thatA method for constructing a model of heart failure in adult mice based on myocardial retinol metabolic disorder is provided, which comprises first making RDH10 fl/fl The mice were mated with MYH6-iCre mice to obtain mice bearing RDH10 at the same time fl/fl F2 generation mice of the gene and MHY6-iCre gene; and then, when the F2 generation mice are 5-6 weeks old, carrying out specific knockout on RDH10 genes in myocardial cells, and continuously feeding until the left ventricular ejection fraction is less than 60%, thus obtaining the heart failure model of the adult mice based on myocardial retinol metabolic disorder. The construction of the model increases operability and convenience for further researching the relationship between myocardial retinol metabolic disorder and heart failure, provides a replication and research tool for heart diseases based on myocardial retinol metabolic disorder, and has quite necessity for heart disease related research.
Description
Technical Field
The invention belongs to the technical field of medicines. More particularly, it relates to a method for constructing an adult mouse heart failure model based on myocardial retinol metabolic disorder.
Background
Heart failure is the final stage of various cardiovascular diseases, and patients with heart failure can have symptoms such as dyspnea, fatigue, poor exercise endurance, fluid retention and the like, and seriously affect the life quality of the patients. Furthermore, heart failure is hidden in onset and extremely high in mortality, and survival rate of patients after diagnosis is even lower than that of various malignant tumors within five years. Therefore, it is important to find early diagnosis indexes and treatment methods of heart failure and to develop basic researches on pathogenesis of heart failure.
The application of disease animal models to simulate human diseases is a main way for searching 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 blockage method, pressure load heart failure models mainly constructed by an aortic stenosis method and a salt load method, capacity pre-load heart failure models constructed by an arteriovenous fistula method and a heart valve insufficiency method, drug-induced heart failure models constructed by myocardial damage drugs and gene modification heart failure models constructed by specific gene modification according to different modeling modes and pathogenesis. Although the construction of these animal models has provided great help for research in the prevention and treatment of heart failure-related diseases, the increasing prevalence and mortality indicate that the current research on heart failure is still insufficient.
Recently, ni Yang et al pointed out that retinol metabolic disorders characterized by a significant decrease in retinoic acid content are prevalent in cardiac muscle of heart failure patients (Yang Ni, parker Lauren, yu Jianshi, jones Jace W, liu Ting, papanicolaou Kyriakos N, talbot C over, margulies Kenneth B, O' rouke Brian, kane Maureen, foster D Brian. Cardioac retinoic acid levels decline in heart failure [ J ]. JCI insight, 2021:), but the relationship between myocardial retinol metabolic disorders and heart failure is not clear, and therefore a need exists to construct an appropriate animal model for research.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a construction method of an adult mouse heart failure model based on myocardial retinol metabolic disorder, which increases operability and convenience for further researching the relationship between the myocardial retinol metabolic disorder and heart failure and provides a tool for copying and researching heart diseases based on the myocardial retinol metabolic disorder.
The primary object of the invention is to provide the application of RDH10 gene myocardial specificity knockout in constructing adult mice heart failure model based on myocardial retinol metabolic disorder.
The invention also aims to provide a construction method of an adult mouse heart failure model based on myocardial retinol metabolic disorder.
The above object of the present invention is achieved by the following technical scheme:
the invention is realized by carrying RDH10 towards 5 weeks of age fl/fl The mice with the gene and the MHY6-iCre gene are injected with tamoxifen in the abdominal cavity, the iCre protein activated by tamoxifen can perform specific induction knockout on the RDH10 gene in the cardiac muscle of the mice, so that the mice spontaneously generate the phenomena of myocardial retinol metabolic disorder and heart failure at 15 weeks after induction is finished, and an adult mice heart failure model based on the myocardial retinol metabolic disorder is successfully constructed, which shows that the RDH10 gene myocardial specific knockout plays a vital role in constructing the adult mice heart failure model based on the myocardial retinol metabolic disorder. Therefore, the application of RDH10 gene myocardial specificity knockout in constructing adult mice heart failure model based on myocardial retinol metabolic disorder should be within the scope of the invention。
The invention also provides a construction method of the adult mouse heart failure model based on myocardial retinol metabolic disorder, which firstly leads RDH10 to be fl/fl The mice were mated with MYH6-iCre mice to obtain mice bearing RDH10 at the same time fl/fl F2 generation mice of the gene and MHY6-iCre gene; and then, when the F2 generation mice are 5-6 weeks old, carrying out specific knockout on RDH10 genes in myocardial cells, and continuously feeding until the left ventricular ejection fraction is less than 60%, thus obtaining the heart failure model of the adult mice based on myocardial retinol metabolic disorder.
The invention is firstly carried out by carrying RDH10 towards the 5-week-old fl/fl The mice of the gene and MHY6-iCre gene are injected with tamoxifen in the abdominal cavity, the iCre protein activated by tamoxifen completes the specific induction knockout of RDH10 gene which is a key enzyme in the retinol metabolic process in the myocardium of the mice, after 15 weeks of induction, the retinol and retinoic acid content in the myocardium of the mice is measured by LC-MS/MS, the condition that the mice have myocardial retinol metabolic disorder is proved, and then the heart failure is found by the detection of heart function and pathology, which proves that the method can successfully construct an adult mice heart failure model based on the myocardial retinol metabolic disorder.
Preferably, the mice are C57BL/6 background mice.
Preferably, the specific knockout is a tamoxifen-induced knockout.
Further preferably, the preparation method of tamoxifen comprises the following steps: dissolving with absolute ethanol, and diluting with organic solvent. The organic solvent needs to be used without toxic or side effect.
More preferably, the dilution is to 10mg/mL.
More preferably, the organic solvent is corn oil.
Preferably, the tamoxifen is induced by intraperitoneal injection.
Further preferably, the intraperitoneal injection is: 48-52 mg/kg per day Weight of body Is injected once for 5 days. More preferably, the amount is 50mg/kg Weight of body . Most preferably, the injectionThe irradiation is performed at a fixed time of day, such as 9 o' clock per day.
Preferably, the knockout is performed at 5 weeks of age in F2 mice.
Preferably, the adult mice are fed with maintenance mouse feed, fed with free water, during the construction of the heart failure model. 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 presents the phenomena of myocardial retinol metabolic disorder and heart failure at 15 weeks after tamoxifen induction is completed, has the advantage of rapid modeling, is obtained by specifically knocking out RDH10 genes 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 myocardial retinol metabolic disorder. The model is constructed to further study the relationship between the myocardial retinol metabolic disorder and heart failure, increase operability and convenience, provide a replication and research tool for heart diseases based on the myocardial retinol metabolic disorder, and be favorable for developing diagnostic methods and therapeutic drugs for heart failure based on the myocardial retinol metabolic disorder, and have considerable necessity for heart disease related research.
Drawings
FIG. 1A is a Western blot of myocardial RDH10 and a statistical chart thereof, and FIG. 1B is a tissue immunofluorescent staining result of myocardial RDH10 protein. (data in statistical plots are presented as mean ± standard error, representing statistically analyzed RDH10 myocardial specific knockout group values versus RDH10 fl/fl Group number less than 0.001 compared to P value
Fig. 2A is a graph of myocardial retinol content and fig. 2B is a graph of myocardial retinoic acid content. (data in statistical plots are presented as mean ± standard error:. Means statistically analyzed, RDH10 myocardial specific knockout group values and RDH10 fl/fl Group number vs P value less than 0.05)
FIG. 3A is a schematic diagram of cardiac ultrasound for three groups of mice, FIG. 3B is a statistical plot of left ventricular ejection fraction for mice, and FIG. 3C is a statistical plot of left ventricular shortening fraction for miceAnd (5) counting. (data in statistical plots are presented as mean ± standard error, where # represents statistically analyzed RDH10 myocardial-specific knockout group values less than 0.001P compared to wild group values, and # represents statistically analyzed RDH10 myocardial-specific knockout group values and RDH10 fl/fl Group number less than 0.001 compared to P value
Fig. 4A is an observation result of a neutral resin patch in the detection of myocardial cell hypertrophy, and fig. 4B is an analysis result of myocardial cell area. (data in statistical plots are presented as mean ± standard error:, representing statistically analyzed RDH10 myocardial specific knockout group values less than 0.05P compared to wild group values; # represents statistically analyzed RDH10 myocardial specific knockout group values and RDH10 fl/fl Group number vs P value less than 0.05)
Fig. 5A is an observation result of a neutral resin patch in the detection of myocardial interstitial fibrosis, and fig. 5B is an analysis result of myocardial collagen area ratio. (data in statistical plots are presented as mean ± standard error, where # represents statistically analyzed RDH10 myocardial-specific knockout group values less than 0.001P compared to wild group values; # represents statistically analyzed RDH10 myocardial-specific knockout group values and RDH10 fl/fl Group number less than 0.01 compared to P value
FIG. 6A is a photograph of a gel-coated neutral gel sheet in the detection of the degree of apoptosis, and FIG. 6B is a result of analysis of the number of myocardial positive cells. (data in statistical plots are presented as mean ± standard error, # indicates that the P value is less than 0.01 for the RDH10 myocardial-specific knockout group value compared to the wild group value after statistical analysis, # # indicates that the RDH10 myocardial-specific knockout group value and RDH10 after statistical analysis fl/fl Group number less than 0.01 compared to P value
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 construction of adult mouse heart failure model based on myocardial retinol metabolism disorder
1. Experimental materials
1) RDH10 for gestational age fl/fl Mice (university of ruketma health center, usa) and MYH 6-cre mice (Jiangsu Ji zhikang); the mice were all C57BL/6 background mice.
2) Tamoxifen: dissolving tamoxifen powder with the purity of more than or equal to 99% by using absolute ethyl alcohol, and diluting the tamoxifen powder to 10mg/mL by using corn oil with the volume of 9 times.
2. Experimental grouping
RDH10 myocardial specific knockout group: let RDH10 first fl/fl The mice were mated with MYH6-iCre mice, and screened to obtain 15 mice with RDH10 at the same time fl/fl F2 generation mice of the gene and MHY6-iCre gene; further at 5 weeks of age in F2 mice, 50mg/kg was given at 9 morning daily Weight of body Tamoxifen is injected once in the abdominal cavity, and after continuous injection for 5 days, the feed is further fed for 15 weeks, so that an RDH10 myocardial specificity knockout group is obtained;
RDH10 fl/fl group: RDH10 of 15 weeks of age 5 fl/fl Mice, 9 a day in the morning at 50mg/kg Weight of body Tamoxifen is injected once by abdominal cavity, and after continuous injection for 5 days, the feed is continuously fed for 15 weeks, thus obtaining RDH10 fl/f A group;
wild group: 15 wild-type C57BL/6J mice, 5 weeks old, were not injected with tamoxifen and used as wild-type controls.
EXAMPLE 2 myocardial RDH10 knockout Condition
1. Verification method
(1) immunoWestern blotting
On week 4 after completion of tamoxifen induction, myocardial specific knockdown groups and RDH10 from RDH10 of example 1, respectively fl/fl 3 mice were randomly extracted from the group, and their myocardial tissue proteins were extracted, respectively, and subjected to immunowestern 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 on ice for 30min, centrifuging at 15000rpm and 4 ℃ for 15min, and transferring the supernatant into an ep tube to obtain the mouse myocardial tissue protein solution.
b. Detecting the protein concentration:
the concentration of the myocardial tissue protein fluid is measured by using a BCA kit, and the method specifically comprises the following steps: protein standard solutions were diluted to 1000 ng/. Mu.L, 500 ng/. Mu.L, 250 ng/. Mu.L, 125 ng/. Mu.L, 25 ng/. Mu.L and 0 ng/. Mu.L, respectively, and 25. Mu.L to 96 well plates were taken to 2.5. Mu.L of protein solution to be tested together with 22.5. Mu.L of ultrapure water into 96 well plates to 200: preparing a detection solution according to the volume ratio of 4, adding 200 mu L of the detection solution into a hole added with a standard protein solution and a protein solution to be detected, slightly oscillating and uniformly mixing, incubating for half an hour at 37 ℃, detecting an absorbance (OD) value at 562nm by using an enzyme-labeled instrument, and establishing a standard curve (R) 2 And the standard protein liquid OD value is not less than 0.99, the standard protein liquid OD value is on the abscissa, the concentration is on the ordinate), the OD value of the protein liquid to be detected is substituted, and the protein concentration is calculated.
c. Immunowestern blot:
after the corresponding tissue protein solution and the concentration thereof are obtained, 35 mug of protein solution is added into ultrapure water and 5 Xprotein loading buffer solution, the mixture is loaded into SDS-PAGE gel after boiling, the first electrophoresis (60V constant voltage) is carried out for 30 minutes, the second electrophoresis (120V constant voltage) is carried out for 90 minutes, the electrophoresis is carried out for 90 minutes again (250 mA constant current), the converted membrane is put into 5% BSA for sealing, and then the analysis is monitored and carried out according to the sequence: cutting a film, incubating for 16 hours at the temperature of 4 ℃ for the first antibody, rinsing with TBST, incubating for 60 minutes at the temperature of 25 ℃ for the second antibody, rinsing with TBST, developing with a developing solution, developing and photographing with a gel imager, and performing Image J gray scale scanning analysis.
(2) Tissue immunofluorescence staining method
On week 4 after completion of tamoxifen induction, myocardial specific knockdown groups from RDH10, respectively fl/f 3 mice were randomly drawn from the group and wild group, and the heart was fixed and embedded for post tissue immunofluorescence staining analysis. The specific method comprises the following steps:
a. preparation of cardiac paraffin sections:
after 15 weeks of tamoxifen induction, mice were fasted for 12 hours (without water) in advance. After the end of fasting, mice were anesthetized with pentobarbital, respectively, and then the thoracic cavity was cut off with ophthalmology, exposing and cutting off the right auricle of the mice. Subsequently, sterile PBS was perfused from the left ventricle of the mice with a syringe until no blood was present in the effluent, the hearts of the mice were then excised, rinsed with PBS, and fixed in 4% paraformaldehyde for 16h. The next day, PBS wash for 30 minutes, repeated 3 times; subsequently, the heart of the mice is immersed in 25 percent ethanol, 50 percent ethanol, 75 percent ethanol, 90 percent ethanol, absolute ethanol (I) and absolute ethanol (II) for 30 minutes respectively; immersing the mixture into xylene (I) and xylene (II) for 15min respectively; then immersing the mixture into paraffin (I) and paraffin (II) for 60min respectively; placing the heart in paraffin for embedding; finally, the embedded paraffin blocks were sliced (5 μm thick) using a Leica paraffin slicer.
b. Immunohistochemical fluorescent staining of sections:
taking the corresponding slice 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 (10 mM, PH7.4) for 10min in a microwave oven, PBS for 5min for three times, 3 milltriton-100 for 10min, running water for 20min, distilled water for 1-2s, 0.5% eosin for 2min, distilled water for 1-2s, PBS for three times, 5% BSA (PBS) for 30min at 37 ℃, corresponding primary antibody for four degrees of 16h, 25 ℃ for 30min for rewarming, PBS for 5 h for three times, corresponding secondary antibody for 2h at 37 ℃ for 5min for three times, hoechst for 33342 25 ℃ for 15min, PBS for three times, anti-quenching sealing tablet for three times, and fluorescent microscope photographing.
2. Verification result
The experimental results are shown in FIG. 1, wherein FIG. 1A is a myocardial RDH10 protein immunoblotting result and a statistical chart thereof, and FIG. 1B is a tissue immunofluorescence staining result of myocardial RDH10 protein.
As can be seen from FIG. 1A, the myocardial RDH10 protein expression in RDH10 myocardial-specific knockout mice is significantly lower than RDH10 fl/fl A group; as can be seen from FIG. 1B, the myocardial RDH10 protein expression in RDH10 myocardial-specific knockout mice was significantly lower than RDH10 fl/fl Group and wild group. The method of the invention is demonstrated to obtain the obvious effect on the RDH10 gene knockout in the RDH10 myocardial specific knockout group mouse myocardial cellsThe effect is remarkable, and the specific knockout is realized.
EXAMPLE 3 Metabolic Condition of myocardial retinol
1. Detection method
(1) Sample collection
After 15 weeks of tamoxifen induction, mice were fasted for 12 hours (without water), anesthetized, chest opened, pre-chilled PBS perfused and rinsed, and hearts were harvested. After heart harvest, excess tissue was washed and removed in a petri dish filled with pre-chilled PBS, 20-30mg of tissue was excised from the apex, weighed, snap frozen in liquid nitrogen and stored. Because ROL and atRA chemical property are unstable, the ROL and the atRA are extremely easy to decompose by visible light, and the sample collection should be carried out in a light-proof way.
(2) Sample pretreatment
The myocardial tissue of the mice after sub-packaging was taken out, 200. Mu.L of a pre-chilled 0.9% sodium chloride solution was added, homogenized for 5 seconds by using a homogenizer at 4 th gear, then 100. Mu. L D4-ROL internal standard was added, and the homogenization was continued for 5 seconds. After vortexing for 5min, 1mL of extractant (1% n-hexane in formic acid) was added, vortexing for 5min, and centrifugation at 13000rpm for 5min. 900. Mu.L of the supernatant was dried in the absence of light at 25℃with nitrogen, and the dried sample was reconstituted with 100. Mu.L of 70% methanol. All manipulations were performed protected from light and on ice. And (3) sucking 95 mu L of the 70% methanol compound solution to a 96-well sample plate, and carrying out LC-MS/MS sample injection analysis, wherein each sample is injected with 10 mu L.
(3) LC-MS/MS condition setting
a. Chromatographic conditions:
chromatographic column: phenomenex Kinetex C18 (50×2.1mm 2.6 μm);
mobile phase a: acetonitrile: methanol: water: formic acid=200: 150:150:0.5 (V/V);
mobile phase B: acetonitrile: methanol: water: formic acid=275: 150:75:0.5 (V/V);
column temperature: 25 ℃; flow rate: 0.2mL/min; gradient elution was performed for 6min.
Gradient elution procedure is shown in table 1:
TABLE 1 LC-MS/MS chromatography gradient elution program settings
b. Mass spectrometry conditions:
the Ion Source IS an electrospray ionization Source (Turbo Spray), a positive Ion detection mode IS adopted, a Gas Curtain Gas (CUR) IS 30psi, a Collision Gas (CAD) IS 10psi, a Spray Voltage (IS) IS 4000V, a drying Gas Temperature (TEM) IS 400 ℃, an atomization Gas (Ion Source Gas1, GS 1) IS 35psi, and an auxiliary Gas (Ion Source Gas2, GS 2) IS 40psi.
The conditions for ro and RA mass spectrometry detection optimization are shown in table 2:
TABLE 2 LC-MS/MS Mass Spectrometry optimization parameters
Retinol detection limit: 0.02ng/mL; all-trans retinoic acid detection limit: 0.02ng/mL
(4) Standard Curve establishment
Accurately weighing 1mg of ROL and atRA standards respectively to a brown sample injection small bottle, and adding 70% methanol to fix the volume to 1mg/mL of storage liquid. The ROL and the atRA stock solution are diluted to 100ng/mL, 50ng/mL, 20ng/mL, 5ng/mL, 1ng/mL and 0ng/mL in a gradient way, equivalent internal standards are mixed, the peak area is detected and recorded by an upper machine, the peak area ratio is calculated, and a standard curve is established.
2. Detection result
The LC-MS/MS analysis results are shown in FIG. 2, wherein FIG. 2A is a graph of myocardial retinol content and FIG. 2B is a graph of myocardial retinoic acid content. As can be seen from FIGS. 2A and 2B, with RDH10 fl/fl Compared with the group, the RDH10 myocardial specificity knockout group has no significant difference on the mouse myocardial retinol, but has the increasing trend, and the content of the myocardial retinoic acid is significantly reduced. Indicating that after myocardial RDH10 is specifically knocked out, mice have myocardial retinol metabolism disorder, and retinol to retinoic acid metabolism conversion is inhibited.
Example 4 detection of cardiac function in mice
1. Detection method
Cardiac function of mice for useThe left ventricular contractile function related data measured by the Vevo 3100 small animal ultrasonic instrument are taken as judgment standards. The specific operation method comprises the following steps: myocardial specific knockout of RDH10 group and RDH10 fl/fl The mice in the group and the wild group are respectively inhaled and anesthetized by isoflurane, the chest of the mice is depilated by depilatory cream under the anesthetic state, the mice are placed on a constant temperature heating plate at 37 ℃ in the supine position, isoflurane is continuously inhaled, the limbs of the mice are connected with an electrocardiogram electrode for detecting and recording heart rate, and a 30MHz probe is used for detecting the left sternum part of the mice. Short axis, measuring left ventricular ejection fraction and left ventricular shortening fraction with M-type ultrasonic,
2. detection result
The measurement results are shown in fig. 3, wherein fig. 3A is a cardiac ultrasound schematic diagram of three groups of mice, fig. 3B is a left ventricular ejection fraction statistical graph of mice, and fig. 3C is a left ventricular shortening fraction statistical graph of mice.
As can be seen from FIG. 3, with RDH10 fl/fl Compared with the wild group, the RDH10 myocardial specific knockout mice have significantly reduced left ventricular ejection fraction and shortening fraction at 15 weeks after tamoxifen induction is completed, 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 the moment, and heart failure occurs.
Example 5 detection of pathologies
1. Detection method
(1) Heart paraffin sections were prepared as in example 2 (2).
(2) Detection of cardiomyocyte hypertrophy:
after fluorescent staining of the cardiomyocyte marker protein cTNT in the sections according to the method in example 2 (2), the sections were stained in WGA staining solution with green fluorescence for 30min at 37 ℃, rinsed with PBS and sealed with anti-fluorescent quenching sealing tablets. The sealed sections were photographed and stored using a fluorescence microscope (FIG. 4A), and the cardiomyocyte areas of each group of mice were analyzed by Image J software (FIG. 4B).
(3) Detection of myocardial cell interstitial fibrosis:
the method comprises the main steps of carrying out Masson staining on the heart paraffin sections of the mice: 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, weibert's hematoxylin for 5min, running water for hematoxylin removal for 1-3s, 1% hydrochloric acid for 1-2s, running water for 20min, distilled water for 1-2s, ponceau acid fuchsin for 5-10min, distilled water for 2-5s, phosphomolybdic acid for 3-5min, aniline blue for counterstain for 2min, 1% glacial acetic acid for 1min, 95% ethanol (II) for 2-3s, absolute ethanol (III) for 3-5s, absolute ethanol (IV) for 5-10s, xylene (I) for 2min, xylene (II) for 2min, neutral resin sealing plate observation, image J software analysis of collagen deposition area (FIG. 5B).
(4) Detection of the degree of apoptosis in myocardium:
apoptotic cells in paraffin sections of mouse hearts were labeled using Tunel staining (DAB chromogenic method) as follows: baking at 60deg.C 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, proteinase K without DNase for 20min at room temperature, PBS for 3 times, 3%H 2 O 2 (PBS) 20min at room temperature, PBS 3 times, biotin labeling solution incubation at 37 ℃ in the dark for 60min, PBS 1 time, labeling reaction termination solution at room temperature for 10min, PBS 3 times, strepitavidin-HRP working solution at room temperature for 30min, PBS 3 times, DAB color development solution at room temperature for 30min, PBS 3 times, hematoxylin at room temperature for 30s, running water flushing, soaking in 80%, 95% and 100% ethanol in sequence, 2min each, xylene twice, neutral resin sealing, photographing and preserving by an optical microscope (figure 6A), and statistical analysis of the number of labeled positive cells (figure 6B).
2. Detection result
From FIGS. 4-6, it can be seen that the RDH10 myocardial-specific knockout group had significantly higher myocardial cell area, collagen area ratio, and Tunel staining positive cell number per field than RDH10 at 15 weeks after tamoxifen induction was completed fl/fl Group and wild group, demonstrating that the RDH10 myocardial specific knockout group at this time exhibited severe cardiomyocyte hypertrophy, cardiomyocyte interstitial fibrosis and apoptosis in the myocardium, with severe myocardial structural remodeling.
In summary, the invention is first achieved by providing RDH10 at the same time fl/fl The gene and the MHY6-iCre gene are injected into the abdominal cavity of a mouse, the ICRE protein activated by the tamoxifen can perform specific induction knockout on RDH10 gene serving as key enzyme in the retinol metabolic process in the myocardium of the mouse, after 15 weeks of induction, the retinol and retinoic acid content in the myocardium of the mouse is measured by LC-MS/MS, the condition that the myocardial retinol metabolic disorder occurs in the mouse is proved, and then the heart failure is detected by detecting the heart function and pathology, so that the mouse is also accompanied by the heart failure, and the method can successfully construct an adult mouse heart failure model based on the myocardial retinol metabolic disorder.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
- Application of RDH10 gene myocardial specificity knockout in constructing adult mice 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 adopted fl/fl The mice were mated with MYH6-iCre mice to obtain mice bearing RDH10 at the same time fl/fl F2 generation mice of the gene and MHY6-iCre gene; and then, when the F2 generation mice are 5-6 weeks old, carrying out specific knockout on RDH10 genes in myocardial cells, and continuously feeding until the left ventricular ejection fraction is less than 60%, thus obtaining the heart failure model of the adult mice based on myocardial retinol metabolic disorder.
- 3. The method of claim 2, wherein the mouse is a C57BL/6 background mouse.
- 4. The method of construction according to claim 2, wherein the specific knockout is a tamoxifen-induced knockout.
- 5. The construction method according to claim 4, wherein the tamoxifen is formulated by: dissolving with absolute ethanol, and diluting with organic solvent.
- 6. The method according to claim 5, wherein the organic solvent is corn oil.
- 7. The method according to claim 4, wherein the tamoxifen is induced by intraperitoneal injection.
- 8. The method of claim 7, wherein the intraperitoneal injection is: 48-52 mg/kg per day Weight of body Is injected once for 5 days.
- 9. The method of claim 8, wherein the amount is 50mg/kg Weight of body 。
- 10. The method of claim 2, wherein the knockout is performed in F2 mice 5 weeks of age.
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