CN115058385A - Isolated culture method and application of myocardial fibroblasts of hypertensive mice - Google Patents

Abstract

The invention provides a method for separating and culturing myocardial fibroblasts of hypertensive mice, which comprises the following steps: a. selecting a homozygous male mouse with a GEFT gene knocked out, and constructing a hypertension model by using an angiotensin II micropump; b. cutting ventricle of successfully modeled mouse, shearing, transferring to digestive juice containing trypsin and collagenase II, and digesting; c. digesting the tissue blocks in digestive juice, standing, and removing redundant digestive juice; d. and (3) uniformly paving the digested tissue blocks at the bottom of a culture dish, air-drying, slowly adding a fibroblast culture medium, and incubating in a cell incubator. After culturing in fibroblast culture medium for 4-5 days, its purity can be up to above 97% by immunofluorescence identification, and the separated myocardial fibroblast is subcultured with fibroblast culture medium, and its cell proliferation is quick and purity is high.

Description

Isolated culture method and application of myocardial fibroblasts of hypertensive mice

This application is a divisional application of patent application having application number 2021109134092 and application date 2021, 8/10.

Technical Field

The invention belongs to the technical field of cell separation culture, and particularly relates to a method for efficiently separating and culturing cardiac fibroblast cells of a hypertensive mouse.

Background

Hypertension is the first risk factor causing heart failure, myocardial infarction, arrhythmia and other events of urban and rural residents in China, and is an independent risk factor increasing the morbidity and mortality of cardiovascular diseases of urban and rural residents. In China, the number of hypertension patients is as high as 2.7 hundred million, and one hypertension patient exists in every three adults. Patients with hypertension may develop different degrees of cardiac dysfunction as the course of the disease is prolonged. Long-term hypertension causes decompensated ventricular dilatation, cardiac interstitial fibrosis and increased apoptotic necrosis of the cardiomyocytes, which in turn causes cardiac remodeling. In addition, the main pathological feature of hypertensive cardiac remodeling is myocardial fibrosis, which directly reflects the severity of cardiac pathology and cardiac dysfunction.

Therefore, how to improve the prevention and treatment level of the hypertensive myocardial remodeling injury in China becomes a significant medical problem and a public health problem. The solution to the above-mentioned key pathophysiology basis-myocardial fibrosis reconstruction injury mechanism is very urgent. At present, the pathogenesis of hypertensive cardiac remodeling has not been fully elucidated. Numerous studies have shown that cardiac fibroblasts play an important role in the process of myocardial remodeling injury through proliferation, differentiation, migration, autocrine or paracrine means. In hypertensive states, prolonged pressure load increases lead to the secretion of a variety of bioactive substances by the heart locally or other organs, which play an important role in the process of cardiac hypertrophy and fibrosis formation.

Guanine nucleotide conversion factor (GEFT) has been shown to belong to the Rho family member, and its physiopathological role plays a close relationship with the small G proteins of the Rho family. GEFT is widely expressed in tissues such as heart and skeletal muscle, and specifically activates Rho-GTP enzyme. In recent years, studies prove that the myocardial-specific GEFT knockout mice are easy to suffer from myocardial hypertrophy under pathological stimulation, and the mechanism of the mice is mainly related to the increase of myocardial hypertrophy-related genes p21, SRF and ANF. In addition, recent studies found that the expression of the small G protein Cdc42 in myocardium was significantly down-regulated after isoproterenol stimulation of GEFT knockout mice. The small G protein Cdc42 has the effect of inhibiting myocardial hypertrophy, and RhoA and Racl have the effect of promoting myocardial hypertrophy in hypertensive mice. Studies suggest that GEFT may function by regulating signal pathways such as proliferation and differentiation of cardiac fibroblasts, but the specific molecular mechanism is not clear. The GEFT gene plays far more than these roles in heart pathological injury, however, what role and the specific mechanism of the role is not clear. At present, related researches of GEFT genes in the heart are few, and the further exploration is worth to be conducted on the basis of the pathophysiological mechanism of the GEFT gene knockout in myocardial structural and functional damage in a hypertensive state. The myocardial fibroblasts play an important role in pathological damage and cardiac remodeling. In order to research the pathophysiological mechanism of the GEFT gene playing a role in the remodeling of the hypertension heart fibrosis, the hypertension mouse model with the GEFT gene knockout is constructed by using an angiotensin II (ang II) micropump technology. The isolated culture is based on that the GEFT gene knocks out myocardial fibroblasts of a hypertensive mouse induced by an Ang II micropump, and is favorable for deeply researching the hypertensive cardiac hypertrophy and fibrosis reconstruction damage mechanism and the important function of the GEFT gene in the pathophysiological process at the cellular molecular level.

Most of the methods for extracting cardiac fibroblasts reported in the literature at present are differential centrifugation methods after stepwise enzymatic digestion. The suckling mouse heart was removed, placed in a petri dish containing D-Hanks solution, minced, and transferred to a 15mL centrifuge tube. Adding 10 times of trypsin, gently blowing, mixing, placing in incubator, digesting for 4min, and collecting supernatant. The above procedure was repeated 4-5 times, and the cells were collected after about 5min of each digestion, and the pancreatin digestion was terminated with an equal amount of complete medium containing 10% fetal calf serum and 1% streptomycin. Centrifuging the collected cell suspension at 1000r/min for 5min, discarding the supernatant, resuspending the cells with complete culture medium, inoculating into a culture flask, placing into an incubator for 1h, replacing with new complete culture medium, and placing into the incubator for continuous culture. The extraction of the cardiac fibroblasts reported in the past is based on common suckling mice which are not subjected to gene knockout and drug intervention modeling, the operation steps are complicated, the consumed time is long, the number of the required suckling mice is large, the trypsin consumption is high, the cost is high, and the number of the extracted cardiac fibroblasts is small.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a method for separating and culturing the cardiac fibroblast of the hypertensive mouse, which has the advantages of convenient material acquisition, simple operation, less time consumption, cost saving, quick cell proliferation and high purity.

The invention also provides a cell cultured according to the method and application of the cell in the research of hypertensive myocardial remodeling injury.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention also provides a method for separating and culturing the myocardial fibroblasts of the hypertensive mouse, which comprises the following steps:

a. selecting a homozygous male mouse with a GEFT gene knocked out, and constructing a hypertension model by using an angiotensin II micropump;

b. cutting ventricle of mouse, shearing, transferring to digestive juice composed of trypsin and type II collagenase, and digesting;

c. digesting the tissue blocks in digestive juice, standing, and removing redundant digestive juice;

d. and (3) uniformly paving the digested tissue blocks at the bottom of a culture dish, air-drying, slowly adding a fibroblast culture medium, and incubating in a cell incubator.

In one embodiment of the present invention, the concentration of trypsin and collagenase type II is in the range of 0.1% to 0.2% (w/v) trypsin and 0.08% to 0.15% (w/v) collagenase type II, respectively.

In another embodiment of the present invention, wherein the volume ratio of the digestive juice to the tissue mass is 3: 1.

in still another embodiment of the present invention, wherein the tissue mass is digested in the digestive fluid for 25 min.

In another embodiment of the invention, the invention also provides the cells cultured according to the method and the application of the cells in the research of the hypertensive myocardial remodeling injury.

In an embodiment of the invention, the cells are prepared by the isolated culture method of the hypertensive mouse myocardial fibroblasts.

In one embodiment of the present invention, the cell is used in the research of diseases such as hypertension.

Compared with the prior art, the invention has the following advantages:

(1) the invention relates to primary extraction of myocardial fibroblasts of a hypertensive mouse based on GEFT gene knockout, wherein the primary extracted myocardial fibroblasts are closer to the pathological state of hypertension, and the GEFT gene plays an important role in the myocardial hypertrophy process after pathological stimulation according to existing research, but the specific mechanism of the GEFT gene playing a role is not clear yet. GEFT genes are now known to be widely expressed in heart and skeletal muscle, and the existing studies have focused mainly on the role of GEFT genes in skeletal muscle-related diseases. The GEFT gene plays a role in hypertension-related heart injury, and the research on specific action mechanisms is few, so that the GEFT gene is worthy of deep exploration. Constructing a primary myocardial fibroblast of a special GEFT gene knockout hypertensive mouse, and facilitating the in-depth research on a specific mechanism of the GEFT gene in the development process of hypertension-related heart disease;

(2) according to the method, the cardiac tissue of the hypertension mouse with the GEFT gene knockout is directly taken to separate the cardiac fibroblasts, the material taking is easy, and compared with the conventional differential centrifugation method for extracting the primary cardiac fibroblasts, the method is simple to operate, the experimental conditions are easy to control, the separation time is shortened compared with that of the existing method, the number of the needed mice is small, the digestive juice is less, and the separation cost is reduced compared with that of the existing method;

(3) compared with the method using single trypsin for digestion, the method adopts the combined digestion of the trypsin and the collagenase type II, determines the proper digestion time and the proper enzyme action concentration, has milder digestion effect, and obtains cells with less damage, and has higher activity and more quantity than the cells obtained by the prior method;

(4) after the method is adopted for culturing for 4-5 days, a large amount of myocardial fibroblasts can be obtained, the purity is over 97 percent through immunofluorescence identification, the cell purity is high, and the method is convenient for industrially separating the myocardial fibroblasts.

The method for separating and subculturing cardiac fibroblast cells of the mouse with the GEFT gene knockout hypertension, provided by the invention, has the advantages of convenient material acquisition, simple operation, less time consumption, cost saving, rapid cell proliferation and high purity, is an ideal and reliable method for separating and subculturing cardiac fibroblast cells of the mouse with the hypertension, provides high-quality cell resources for experiments, and is suitable for industrially separating and centrifuging the cardiac fibroblast cells.

Drawings

FIG. 1 is a microscope image (40X) of a myocardial fibroblast cultured for 3d according to the isolation culture method of the present invention;

FIG. 2 is a microscopic picture (40X) of P2 generation cardiac fibroblasts obtained by the isolated culture method of the present invention; FIG. 3 is a chart (100X) of immunofluorescence identification of cardiac fibroblast Vimentin and alpha-SMA obtained by the isolated culture method of the present invention.

Detailed Description

The invention is further illustrated with reference to the following specific examples, which are not to be construed in any way as imposing limitations upon the invention.

Example 1

Experiment implementation procedure

(1) Experimental Material

Tissue forceps 2, ophthalmic scissors 2, tissue scissors 2, culture bottles 1, 10cm petri dishes, pasteur pipettes, pipette tips, 15ml centrifuge tubes, sealing membranes, water baths (Shanghai Boxun, Inc.), implantable osmotic pump tubes (ALZETDURECT Corporation, USA), non-invasive caudal artery manometry, desktop thermostats (Shanghai Jinghong experiment, Inc.), fibroblast cell culture media (Science Inc., USA), 1XPBS (Sigma, UK.), streptomycin (Science, USA), 0.25% trypsin (Gibco, Canada), collagenase type II (collagenase, USA), isoflurane (Hebei, Jiu Pai, Inc.), angiotensin II (Sigma, Japan), DAPI (gold bridge, North), anti-protein-antibody (Proteinch, USA), rat-antibody (Kyoto Japan), and mouse, Rabbit anti-alpha-SMA primary antibody (CST corporation, USA), Alexa Fluor 647 labeled goat anti-rabbit IgG (H + L) (Shanghai Biyun Co., Ltd.), Alexa Fluor 488 labeled goat anti-mouse IgG (H + L) (Shanghai Biyun Co., Ltd.), 1% Triton-X100 solution (Shenzhen Lidong science and technology Co., Ltd., China), 4% paraformaldehyde fixing solution (Shanghai Biyun Co., Ltd.), 5%

BSA blocking solution (Solarbio, China) and 1XPBST (Solarbio, China) (all experimental products except for the dyeing reagent were autoclaved)

(2) Experimental procedure

First, 10 male homozygous GEFT gene knockout mice of 6 weeks of age were taken and anesthetized with isoflurane. The method comprises the following steps of anesthetizing a mouse, depilating the neck, cutting the skin of the neck, carrying out blunt separation on the skin and subcutaneous muscle tissues of the mouse by using forceps, implanting an implanted osmotic pressure pump tube (hereinafter referred to as a micro pump) filled with angiotensin II into the subcutaneous tissue along an incision, and adjusting the position of the micro pump on the back of the mouse to reduce the influence of the micro pump on the daily behaviors of the mouse. After the micropump is implanted, the incision is sutured and disinfected. The dose of angiotensin II was 1.5mg/kg/d for four weeks.

Measuring the blood pressure of the mice on days 1, 3, 7, 14, 21 and 28 after the pump is buried, finding that the blood pressure starts to rise on day 3, reaching 170mmHg by day 7, and then entering a plateau stage, wherein the blood pressure value is basically unchanged.

And thirdly, selecting 5 mice successfully modeled 28 days after the pump is buried, anesthetizing with isoflurane, disinfecting with 75% alcohol, cutting skin and sternum along the midline of the sternum by using tissue scissors, replacing ophthalmic scissors to obtain the heart, and placing the heart into a culture dish which contains precooled phosphate buffer solution (PBS for short) containing 1% of penicillin/streptomycin.

Transferring the culture dish into a cell super clean bench, cleaning peripheral redundant tissues and left and right atria by using an ophthalmic scissors, flushing the heart by using precooled PBS containing 1% of cyan/streptomycin on the residual ventricular muscle, and cleaning blood cells in the heart. (the invention firstly uses the ophthalmology scissors to cut off the atria, is more beneficial to cleaning the rest ventricular musculature and cleaning the blood cells, and avoids the interference of the blood cells to the experiment in the subsequent experiment)

Fifthly, the tissue scissors are replaced to cut the ventricular muscle tissue to 1mm ³ About the size of the tissue piece, 1ml of a digestion solution (ready to use) containing 0.125% trypsin and 0.1% collagenase type II was added to the tissue piece, and the tissue piece was transferred to a culture flask using a Pasteur pipette. (the invention researches the optimal action time and concentration of trypsin and collagenase type II, insufficient digestion can be caused by too low enzyme concentration and too short digestion time, the damage to cells can be increased by too high enzyme concentration and too long digestion time, the cell activity is influenced, and the digestive juice is prepared at present.)

Sixthly, adding digestive juice of tissues with 3 times volume, gently blowing and beating the digestive juice in a culture bottle to uniformly mix the digestive juice, and placing the mixture in a desktop constant temperature oscillator at 37.0 ℃ and 100r/min for digestion for 25 min. (firstly, the addition of 3 times of digestive juice in volume is more beneficial to subsequent experiments, the insufficient digestion can be caused by less digestive juice, the subsequent natural air-drying adherence process can be not facilitated by excessive digestive juice; secondly, the temperature of a desktop constant temperature oscillator at 37 ℃ is the optimal temperature for enzyme action and cell culture, multiple experiments show that the rotating speed of 100r/min can be more beneficial to the contact and digestion of the digestive juice and tissue blocks, and the subsequent cell climbing out of the tissue blocks can be more facilitated by 25min for digestion.)

Seventhly, after digestion, placing the culture bottle into a cell super clean bench, standing for 3min, and removing the supernatant. (standing for 3min can make the tissue block better precipitate, and discarding the supernatant to avoid the influence of long time on cell activity caused by subsequent air drying)

Eighthly, sucking out the precipitated tissue blocks, uniformly spreading the tissue blocks at the bottom of a culture dish, naturally drying the tissue blocks in a cell super clean bench for 15min (the surfaces of the tissue blocks are wet and cannot be dried), slowly adding a fibroblast culture medium, and putting the tissue blocks into a culture dish at 37.0 ℃ and 5% CO ₂ And (5) changing the liquid in the incubator after 48 hours. Then 1 liquid change is carried out every 48 h. (firstly, the operation and the wind speed of a fan on a cell super clean bench are combined, the air drying is carried out for 15min, so that the cells can be attached to the wall better, the surface of a tissue block is still kept moist, the activity of the tissue block and the subsequent cell climbing are not influenced.) No culture dish is moved for the first 48h, the tissue block attached to the wall caused by the flow of liquid is prevented from shaking down from the bottom of the dish, the liquid is changed after the first 48h, the PBS is not needed to be used for washing, and the fibroblast culture medium is used for primary culture, so that the environment which is more beneficial to the growth of the cardiac fibroblasts is provided, the cell growth characteristics are provided according to the advantage that the cardiac fibroblasts can proliferate fast, and the purity of the cardiac fibroblasts is further improved)

Ninthly, when a large number of fibroblasts grow around the tissue blocks under the observation of a microscope, the fibroblasts between the tissue blocks are partially fused, 0.25 percent of trypsin is used for digestion and passage (1: 2), and the cells of the 2 nd generation to the 3 rd generation are used for experiment and identification.

Identification of the cells in the red: taking the cells growing to 90% of confluence, adding 0.25% trypsin, observing the cells under a microscope, adding 2ml of fibroblast culture medium to stop digestion after the cells shrink and become round, obtaining cell suspension, and performing digestion by using 2X10 ⁵ Inoculation in/ml

And climbing a sheet, and performing Vimentin and alpha-SMA immunofluorescence staining identification, wherein the result shows that the Vimentin positive rate is 97%, namely the cell purity is 97%.

(3) Results of the experiment

First, it can be seen from fig. 1 that, on day 3 of primary culture, it is seen that cardiac fibroblasts climb out around the adherent tissue mass, the cells near the tissue mass are dense, and the cells between the tissue mass and the tissue mass tend to gradually converge.

② from figure 2, the passage of the extracted primary myocardial fibroblasts can show that the adherent cells form fusiform and polygon, the pseudopodium extends out from the periphery of the cells, the cell body is larger, the cytoplasm is transparent, the nucleus is clear, the spontaneous pulsation does not exist, and the cells grow rapidly.

And thirdly, as can be seen from the figure 3, the identification of the primary cells is carried out on the cell plates of the generations P2 and P3 through immunofluorescence, and the fact that Vimentin in the extracted primary cells is high in expression, alpha-SMA is low in expression and accords with the molecular biological characteristics of myocardial fibrosis is found, and the purity of the extracted primary myocardial fibroblasts is proved to be high. Purity (%) of cardiac fibroblasts is 100% of the number of Vimentin-positive stained cells/total number of nuclei.

It should be understood that the above-described embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. Not all embodiments are exhaustive. All obvious changes and modifications which are obvious to the technical scheme of the invention are covered by the protection scope of the invention.

Claims (8)

1. A method for separating and culturing myocardial fibroblasts of hypertensive mice comprises the following steps:

a. selecting a homozygous male mouse with a GEFT gene knocked out, and constructing a hypertension model by using an angiotensin II micropump;

b. cutting ventricle of successfully modeled mouse, shearing, transferring to digestive juice composed of trypsin and type II collagenase, and digesting;

c. digesting the tissue blocks in digestive juice, standing, and removing redundant digestive juice;

d. and (3) uniformly paving the digested tissue blocks at the bottom of a culture dish, air-drying, slowly adding a fibroblast culture medium, and incubating in a cell incubator.

2. The isolated culture method of the myocardial fibroblasts of the hypertensive mouse as set forth in claim 1, wherein the purity of the isolated culture method can reach more than 97% by immunofluorescence assay after culturing in a fibroblast culture medium for 4-5 days.

3. The isolated culture method of the hypertensive mouse cardiac fibroblast according to claim 1 or 2, wherein the isolated cardiac fibroblast is subcultured with a fibroblast culture medium.

4. The isolated culture method of myocardial fibroblasts in hypertensive mice as set forth in claim 1, wherein the concentrations of trypsin and collagenase type II are in the range of 0.1% to 0.2% (w/v) trypsin and 0.08% to 0.15% (w/v) collagenase type II, respectively.

5. The isolated culture method of myocardial fibroblasts of hypertensive mice as set forth in claim 1, wherein the volume ratio of the digestive juice to the tissue mass is 3: 1.

6. the isolated culture method of myocardial fibroblasts in hypertensive mice according to claim 1, wherein the tissue mass is digested in the digestive fluid for 25 min.

7. The isolated culture method of the hypertensive mouse cardiac fibroblast cells according to any of claims 1-6.

8. Use of the cell according to claim 7 in the study of hypertensive disorders.

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