Application of Cthrc1 gene in preparation of medicine for treating cardiac hypertrophy

Abstract

The invention provides an application of a Cthrc1 gene in preparation of a medicine for treating cardiac hypertrophy. The Cthrc1 expression of a cardiac hypertrophy model mouse is remarkably up-regulated, the cross sectional area and fibrosis of cardiac muscle cells of a Cthrc1 gene knockout mouse are remarkably reduced, and the cardiac contraction function is remarkably improved. Based on the discovery, the Cthrc1 gene can be used as an action target of a medicine for treating the cardiac hypertrophy and the heart failure, and a reagent for knocking out the Cthrc1 gene or inhibiting the expression of the Cthrc1 gene can be used for preparing the medicine for preventing, relieving and / or treating the cardiac hypertrophy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to application of Cthrc1 gene in preparation of medicine for treating myocardial hypertrophy. BACKGROUND

[0002] At present, the morbidity of cardiovascular disease is continuously increasing, and the cardiovascular disease accounts for the first place of total death causes of urban and rural residents. Myocardial hypertrophy is a pre-stage lesion of heart failure, and is an adaptive response to physiological and pathological overload. In order to cope with overload, initially, the myocardial cells are enlarged and the ventricle is grown towards the heart, and then develops into myocardial cell elongation, ventricular wall thinning, ventricular cavity expansion and heart contraction function decline. The pathological myocardial hypertrophy is in the compensatory stage at the initial stage, and the clinical manifestation is not obvious and is easy to be ignored, but with the slow development of the disease, the pathological myocardial hypertrophy is accompanied by myocardial fibrosis, and then leads to adverse ventricular remodeling, causing the decrease of heart function and heart failure.

[0003] In the fibrosis process, the fibroblasts are transformed into collagen-producing fibroblasts and myofibroblasts, which are characterized by high expression of α-smooth muscle actin (α-SMA) and increased contraction activity. The activation, proliferation and sustained existence of these cells are driven by various cytokines, including interleukin-1 (IL-1), tumor necrosis factor (TNF) and transforming growth factor-β (TGF-β), and connective tissue growth factor (CTGF, also known as CCN2), platelet-derived growth factor (PDGFs) and various matrix factors, such as hyaluronic acid and matrix metalloproteinases (MMPs). Cthrc1 (collagen triple helix repeat containing 1) is a secreted protein, and previous studies have found that activated fibroblasts after myocardial infarction in mice express high levels of Cthrc1 and are located in the scar, indicating the potential role of Cthrc1 in regulating collagen matrix deposition and cell migration. However, the role of Cthrc1 in myocardial cell hypertrophy has not been reported. SUMMARY

[0004] In order to solve the problems in the background art, the present application provides application of Cthrc1 gene in preparation of medicine for treating myocardial hypertrophy, aiming at solving the problem that the prior art lacks effective means for preventing and intervening myocardial hypertrophy.

[0005] The technical scheme for solving the above technical problem of the present application is as follows: In a first aspect, the present application provides application of Cthrc1 gene in preparation of medicine for preventing, alleviating and / or treating myocardial hypertrophy, wherein the sequence of the Cthrc1 gene is shown as SEQ ID NO: 1.

[0006] In a second aspect, the present application provides a use of a reagent for knocking out a Cthrc1 gene or inhibiting expression of a Cthrc1 gene in the preparation of a drug for preventing, alleviating and / or treating cardiac hypertrophy.

[0007] According to the above scheme, the reagent knocks out the Cthrc1 gene, reduces or inhibits transcription of the Cthrc1 gene, and / or reduces or inhibits translation of the mRNA product of the Cthrc1.

[0008] According to the above scheme, the reagent is a gene editing system or a Cthrc1 inhibitor.

[0009] According to the above scheme, the Cthrc1 inhibitor is an shRNA lentivirus expression vector for inhibiting expression of the Cthrc1 gene.

[0010] According to the above scheme, the cardiac hypertrophy is pathological cardiac hypertrophy.

[0011] In a third aspect, the present application provides a drug for preventing, alleviating and / or treating cardiac hypertrophy, the drug comprising a reagent for knocking out a Cthrc1 gene or inhibiting expression of a Cthrc1 gene.

[0012] In a fourth aspect, the present application provides a method for screening a drug for preventing, alleviating and / or treating cardiac hypertrophy, the method comprising screening a Cthrc1 inhibitor.

[0013] According to the above scheme, the Cthrc1 inhibitor can inhibit or reduce expression of the Cthrc1 gene or inhibit or block activity of the Cthrc1 protein.

[0014] According to the above scheme, the step of screening the Cthrc1 inhibitor comprises: detecting whether a test reagent can inhibit expression of the Cthrc1 gene or activity of the Cthrc1 protein; selecting a test reagent capable of inhibiting expression of the Cthrc1 gene or activity of the Cthrc1 protein as a candidate drug.

[0015] The present application has the following beneficial effects: the present application identifies the function and role of Cthrc1 in cardiac hypertrophy through experiments, and further proposes a use of a Cthrc1 gene, a reagent for knocking out a Cthrc1 gene or inhibiting expression of a Cthrc1 gene in the preparation of a drug for preventing, alleviating and / or treating cardiac hypertrophy, so as to provide a new means for effectively protecting heart function and suppressing or alleviating cardiac hypertrophy. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Figure 1 shows protein expression of Cthrc1 in serum of normal people and clinical cardiac hypertrophy patients (heart failure patients) in Example 1 of the present application (p<0.0001 vs normal people group); Figure 2 Figure 2 is a schematic diagram of wild-type C57BL / 6 mice (WT mice) and Cthrc1 KO mice in sham operation and aortic arch constriction operation (TAC) modeling in Example 2 of the present application, and a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling, wherein A is a modeling schematic diagram, and B is a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling; Figure 3 Figure 2 is a schematic diagram of wild-type C57BL / 6 mice (WT mice) and Cthrc1 KO mice in sham operation and aortic arch constriction operation (TAC) modeling in Example 2 of the present application, and a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling, wherein A is a modeling schematic diagram, and B is a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling; Figure 4 Figure 2 is a schematic diagram of wild-type C57BL / 6 mice (WT mice) and Cthrc1 KO mice in sham operation and aortic arch constriction operation (TAC) modeling in Example 2 of the present application, and a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling, wherein A is a modeling schematic diagram, and B is a Western blot diagram of Cthrc1 protein expression in the heart 4 weeks after modeling; Figure 5Results of M-mode echocardiography detection of Cthrc1 KO and littermate wild-type (WT) mice in Example 2 of the present application after 4 weeks of sham operation (Sham) or aortic arch constriction (TAC), wherein A shows the left ventricular short-axis section ultrasound images of different groups of mice, and the yellow arrow indicates the end-diastolic and end-systolic internal diameter measurement position; B is a quantitative statistical diagram of ejection fraction (EF), left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) (*: p<0.05 vs WT+TAC group, ***: p<0.001 vs WT+TAC group, ****: p<0.0001 vs WT+Sham group); Figure 6 Structure schematic diagram of lentiviral vector GV493 in Example 3 of the present application; Figure 7 Results of immunofluorescence staining of primary cardiac fibroblasts of suckling mice infected with adenovirus Ad-shRNA (Sh-Ctrl) and Ad-shCthrc1 (Sh-Cthrc1) in Example 3 of the present application after 48 hours of PBS or angiotensin II (AngII, 1 μM) stimulation, wherein A is the result of immunofluorescence staining, and B is the quantitative analysis result of fluorescence staining (*: p<0.05 vs Sh-Ctrl+AngII group, ****: p<0.0001 vs Sh-Ctrl+PBS group); Figure 8 Results of immunofluorescence staining of primary cardiac fibroblasts of suckling mice infected with adenovirus Ad-shRNA (Sh-Ctrl) and Ad-shCthrc1 (Sh-Cthrc1) in Example 3 of the present application after 48 hours of PBS or angiotensin II (AngII, 1 μM) stimulation, wherein A is the result of immunofluorescence staining, and B is the quantitative analysis result of fluorescence staining (*: p<0.05 vs Sh-Ctrl+AngII group, ****: p<0.0001 vs Sh-Ctrl+PBS group). DETAILED DESCRIPTION

[0017] The principles and characteristics of the present application are described below in combination with the drawings and specific examples, and the examples are only used to explain the present application and are not used to limit the scope of the present application.

[0018] Cthrc1 (collagen triple helix repeat containing 1) is a secreted protein, the present application takes Cthrc1 knockout mice (Cthrc1 KO) and its littermate wild-type control mice (WT) as the research object, and adopts aortic arch constriction (TAC) to construct a pathological myocardial hypertrophy model. The research results show that the expression of Cthrc1 in the heart tissue of mice after TAC is significantly up-regulated; compared with the WT model group, the Cthrc1 gene knockout can significantly reduce the heart / body weight ratio, lung / body weight ratio, myocardial cell cross-sectional area and degree of cardiac fibrosis, and significantly improve the cardiac systolic function.

[0019] The sequence of the gene Cthrc1 in mice is shown in SEQ ID NO: 1: The above results show that Cthrc1 gene has a promoting effect in myocardial remodeling process, and its deletion can delay the pathological process of myocardial hypertrophy and fibrosis. Therefore, Cthrc1 can be used as a potential drug target, and an in vitro cell model or animal model of Cthrc1 knockout can be constructed for screening drugs or biological reagents with the effects of preventing, alleviating or treating pathological myocardial hypertrophy and fibrosis, and achieving the purpose of preventing, alleviating and / or treating pathological myocardial hypertrophy through genetic engineering means. In addition, Cthrc1 inhibitors can be designed targeting Cthrc1 to inhibit or reduce the expression of Cthrc1 gene or inhibit or block the activity of Cthrc1 protein, thereby providing new therapeutic molecules for the treatment of myocardial hypertrophy.

[0020] The following is a specific embodiment.

[0021] Experimental animals and feeding: Experimental animals: 8-10 week-old, 25.5±2.0 g weight, Cthrc1 KO and littermate wild WT male mice were selected as experimental objects.

[0022] Feeding environment: All experimental mice were fed in the Specific Pathogen Free (SPF) level experimental animal center of the Institute of Cardiovascular Disease, Wuhan University.

[0023] Feeding conditions: The room temperature was between 22-24℃, the humidity was between 50-70%, the light and dark alternating lighting time was 12 hours, and the water and food were free.

[0024] Example 1 Difference in expression amount of Cthrc1 in normal people and myocardial hypertrophy patients The serum of normal people and myocardial hypertrophy patients (heart failure patients) was selected for Cthrc1 Elisa detection. The protein expression of Cthrc1 in the serum of normal people (Non-HF) and myocardial hypertrophy patients (heart failure patients, HF) was as shown in Figure 1 The expression of Cthrc1 in the myocardial hypertrophy patients was up-regulated.

[0025] Example 2 Construction of myocardial hypertrophy model of Cthrc1 knockout mice and wild mice and pathological detection (1) Construction of myocardial hypertrophy model of Cthrc1 knockout mice and wild mice 8-10 week-old Cthrc1 heterozygous mice (purchased from Sanyei (Suzhou) Biotechnology Co., Ltd.) were self-crossed to obtain Cthrc1 KO mice, and littermate wild WT mice were used as controls.

[0026] Cthrc1 KO and littermate wild type (WT) male mice were used as experimental subjects, and aortic arch constriction operation (TAC) was used to construct a mouse model of myocardial hypertrophy. The operation process is as follows: 1) Preoperative preparation: ① Anesthesia: First, weigh the mouse, calculate the amount of anesthetic (3% sodium pentobarbital) needed according to 90 mg / kg body weight, and record the injection time point. The success criteria for anesthesia are that the mouse has no obvious reaction after clamping the tail and toes, and the mouse is in good condition (usually 10 minutes after injection, the mouse has no obvious reaction, and the mouse has a reaction after 50 minutes of anesthesia, and the optimal operation time is about 30 minutes after anesthesia); ② Operation area preparation: Shave the skin of the left chest, left side of the chest and left forelimb under the axilla of the mouse. After shaving, wipe the operation area with a wet gauze to remove the mouse hair without affecting the surgical field; ③ Tracheal intubation: Use a rubber band to fix the mouse's upper incisors on the inclined surface of the V-shaped plate, and quickly insert the tracheal tube through the glottis into the trachea. Then, place the mouse in a right lateral position on a heating pad (the heating pad needs to be preheated), then connect the tracheal tube to the ventilator, and fix the mouse. If the mouse's chest rises and falls in sync with the ventilator's frequency, it means the tracheal tube is successfully inserted.

[0027] 2) TAC operation: Model group (TAC): Take the right lateral position, place the mouse's left forelimb above the right forelimb, and fix the two forelimbs with medical tape. Pad a cotton swab under the right chest, elevate the chest, and disinfect the skin of the operation area with iodine and alcohol with a volume fraction of 75%. Hold the left chest skin with an ophthalmic forceps in the left hand, hold an ophthalmic scissors in the right hand, and cut the skin about 1 cm. Separate the muscles and soft tissues in turn, open the chest at the 2-3 rib level, use a cotton swab to slightly push the left lung, and free the descending branch of the aortic arch. Pass a 7-0 surgical suture through the blood vessel, and place a 26G (25.0-27.5g mouse) or 27G (23.5-25.0g) syringe needle parallel above the blood vessel. Ligate the blood vessel and needle together, then pull out the needle to achieve the corresponding degree of blood vessel constriction. After ligation, suture in turn, close the chest, and insert a syringe into the chest from the suture opening and extract 1cc gas to restore the negative pressure in the chest. After pulling out the syringe, quickly suture the skin incision. Sham group: After freeing the descending branch of the aorta, only thread without ligation, and the rest of the steps are the same as the myocardial hypertrophy model group.

[0028] 3) Postoperative care: After TAC operation, when the mouse has spontaneous respiration and a strong reaction to toe clamping, remove the tracheal tube, and place the mouse in a cage with high-pressure sterilized bedding, feed and drinking water, and continue to feed and observe in the feeding room. Cthrc1 KO mice and WT mice were detected for various indicators 4 weeks after surgery.

[0029] (2) Detection of Cthrc1 knockout mouse and wild type mouse myocardial hypertrophy model Specific process includes: 1) preliminary work: prepared in advance with 20 mL volume fraction 10% formaldehyde urine cup, and paste label (mouse number, group, type of operation and sampling date). Pour full mass fraction 10% KCl solution of culture dish in sampling place. Open the analytical balance, adjust zero for standby. Again weigh the mice; 2) sampling: ophthalmic curved forceps clamps the blood vessel pedicle below the auricle, cut off the heart, quickly put into the mass fraction 10% KCl solution. After the heart stops beating in diastole, place on sterile gauze, gently squeeze the liquid in the heart cavity, after wiping the surface liquid, weigh and record, and detect as follows.

[0030] 1) The heart and lung of the mouse were taken out, trimmed, and dried with filter paper. The tibia length of the mouse hind leg was measured and recorded. The ratio of heart weight to body weight (HW / BW) and the ratio of heart weight to tibia length (HW / TL) were calculated. The results are shown in Figure 4 B and 4C, the differences between HW / BW and HW / TL of Cthrc1 KO mice and WT mice in the sham operation group were not statistically significant; the HW / BW and HW / TL of Cthrc1 KO mice 4 weeks after TAC operation were higher than those in the sham operation group; 4 weeks after TAC operation, the HW / BW and HW / TL of Cthrc1 KO mice were lower than those of WT mice.

[0031] 2) The expression of Cthrc1 in the heart of the sham operation group and the model group was measured respectively Part (1) obtained the sham operation group (Sham) and the model group (TAC) of wild type C57BL / 6 mice (WT mice) and Cthrc1 knockout mice (Cthrc1 KO mice). Four weeks after operation, the heart of the mouse was taken out, the protein extracted from the heart was subjected to SDS-PAGE- Western blot, and the expression of the protein was detected by combining the antibodies specific for Cthrc1 protein and the markers COL-1 and α-SMA of cardiac fibroblast fibrosis. The expression of the markers ANP and BNP of myocardial hypertrophy and the marker α-SMA of fibroblast fibrosis was detected by qPCR, and GAPDH was used as an internal reference. The results are shown in Figure 2 and Figure 3 After 4 weeks of TAC operation, the expression of the markers BNP, ANP, COL-1 and α-SMA related to myocardial hypertrophy and fibrosis in the myocardial tissue of WT mice was significantly up-regulated, while that in Cthrc1 KO mice was significantly down-regulated. At the same time, the expression of Cthrc1 protein was significantly up-regulated in WT mice after TAC operation, and no expression was found in Cthrc1 KO mice, suggesting that Cthrc1 has a promoting effect in the process of myocardial remodeling.

[0032] 3) Pathological detection Paraffin sections were prepared and then subjected to HE staining, wheat germ agglutinin (WGA) staining and Sirius red (PSR) staining. HE staining was used to observe the overall structure of the heart tissue, WGA staining was used to evaluate the degree of hypertrophy of cardiomyocytes, and PSR staining was used to detect collagen deposition in the heart and evaluate the degree of fibrosis. These staining methods collectively help analyze the pathological changes of myocardial hypertrophy and fibrosis. The specific experimental steps are as follows: ①Preparation of paraffin sections: trim the heart → handle the embedding frame → rinse with running water → dehydrate → transparentize → immerse in wax → embed → section → spread the slice → dry or bake for standby; ②Wheat germ agglutinin (WGA) staining: Place the paraffin section baked at 60°C for 30 min in xylene for 5 min x 3 times → 100% ethanol for 5 min x 2 times → 95% ethanol for 5 min → 70% ethanol for 5 min → distilled water rinse for 5 min x 2 times → PBS rinse for 5 min → PBS rinse for 10 min → discard PBS, add trypsin working solution (DIG-3008, Fuzhou Mayxin) 37°C for 20 min in the dark → PBS rinse for 5 min x 3 times → remove the section and wipe off the liquid around the tissue with filter paper (do not dry the tissue), place it flat in a wet box with a histological pen → add WGA-AlexaFlour488 working solution (10 μg / mL) 37°C for 2 h in the dark → discard the staining solution, PBS rinse for 5 min x 3 times → SlowFadeGoldantifadereagentwithDAPI mounting → observe under a fluorescence microscope and take photos with a microscope.

[0033] ③Sirius red (PSR) staining: bake at 55°C for 30 min → xylene for 2 min, 3 times → 100% alcohol for 1 min → 95% alcohol for 1 min → 70% alcohol for 1 min → rinse with running water for 10 min → distilled water for 1 min → 0.2% phosphomolybdic acid for 5 min → 0.1% picric acid Sirius red dye for 1.5 h → remove residual liquid → 0.01N hydrochloric acid for 4 times, each for about 1 s → 70% alcohol once → 90% alcohol once → 100% alcohol for 30 s, 3 times → cover the slide with a cover glass immediately while the xylene is still wet, take photos with a microscope.

[0034] ④HE staining: 55℃ baking for 30 min → xylene immersion for 5 min x 2 times → removing paraffin → 100% ethanol immersion for 5 min → 95% ethanol immersion for 5 min → 70% ethanol immersion for 5 min → distilled water rinsing for 5 min x 2 times → staining with hematoxylin solution for 10-15 min → rinsing with running water → 0.1% hydrochloric acid alcohol solution rinsing for 1-2 times → rinsing with running water for 5 min x 2 times → staining with eosin solution for 3-5 min → rinsing with running water → 70% ethanol dehydration → 95% ethanol dehydration → 100% ethanol dehydration → xylene transparency for 5 min x 2 times → sealing → observing under a microscope and taking photos.

[0035] The staining results after TAC operation modeling of Cthrc1 KO and littermate WT mice are shown in Figure 4 A, the gross image of the heart is shown in Figure 4 D, the left ventricular collagen volume fraction (LV collagen volume fraction) and the cross-sectional area of myocardial cells of each group were quantitatively counted, and the results are shown in Figure 4 E and 4F.

[0036] From the gross image of the heart, it can be seen that in the sham operation group (Sham), there is no difference in the heart between the Cthrc1 knockout mice and the littermate WT mice. The HE staining results show that the heart of the model group is larger than that of the sham operation group, and the heart of the Cthrc1 KO mice is significantly smaller than that of the WT mice. WGA staining slices can be observed: the cell hypertrophy of the Cthrc1 KO group is significantly reduced compared with the WT group, and the difference is statistically significant. After PSR staining, it is found that the collagen content in the myocardial interstitium of the model group is increased compared with the sham operation group, and the collagen around the arterial blood vessels is increased more obviously, the collagen is thickened, and the arrangement is disordered into a network. The collagen content in the myocardial interstitium and the collagen content around the blood vessels of the Cthrc1 KO mice after TAC operation are reduced compared with those of the WT mice after TAC operation. The WT mice in the TAC model group show typical ventricular remodeling phenotypes such as cardiac hypertrophy, pulmonary congestion, interstitial collagen deposition, and myocardial cell hypertrophy and myocardial fibrosis, while the degree of cardiac hypertrophy, pulmonary congestion, interstitial collagen deposition, and myocardial cell hypertrophy and myocardial fibrosis of the Cthrc1 KO mice is significantly lower than that of the WT mice, indicating that Cthrc1 gene knockout can significantly alleviate the above pathological changes, suggesting that Cthrc1 gene plays a key promoting role in the process of myocardial hypertrophy and myocardial fibrosis.

[0037] 4) Ultrasonic cardiogram detection of mouse heart function Preparation: Anesthesia machine preparation: first connect the oxygen cylinder and the gas inlet interface on the anesthesia machine, then unscrew the seal cover on the anesthesia machine, quickly add isoflurane to the safe scale, and then tighten the seal cover. Unscrew the total valve on the oxygen cylinder, adjust the knob of the flow control valve, and maintain the outlet gas pressure at 0.2-0.3 mPa. Preparation of the test mouse: after the test mouse is anesthetized with isoflurane, the left chest area is shaved, and the head of the treated mouse is inserted into the anesthesia conduit sleeve. Maintain the mouse in a stable anesthetic state with 1.5-2.0% isoflurane.

[0038] Heart function detection: The mouse is placed in a left lateral position or supine position, and the shaved area is evenly coated with an ultrasonic coupling agent. A high-frequency ultrasonic diagnostic instrument is used, with a frequency of 15 MHz. The standard left ventricular papillary muscle short-axis section is selected to measure the left ventricular mass (LVMass) and ejection fraction (EF) and fractional shortening (FS).

[0039] The results are shown in Figure 5 Compared with the sham operation group WT mice, the WT mice showed decreased heart function and myocardial hypertrophy 4 weeks after TAC operation, mainly manifested as increased left ventricular end-diastolic diameter and left ventricular end-systolic diameter, and decreased ejection fraction and fractional shortening. Four weeks after TAC operation, the heart dysfunction of Cthrc1 KO mice was relieved compared with WT mice. The results showed that the EF and FS of the TAC model group WT mice were significantly reduced, and the LVMass was increased, indicating impaired heart function. The Cthrc1 KO mice showed improvement in these indicators, indicating that Cthrc1 deletion helps maintain heart function and structural stability.

[0040] Example 3 Effect of Cthrc1 on cardiac fibroblast fibrosis (1) Expression of Cthrc1 in myocardial fibroblasts after stimulation by control group (PBS) or angiotensin II (AngII) Newborn Sprague-Dawley (SD) mice (1-3 days) were cultured, and the primary myocardial fibroblasts were cultured for 48 h. After the cells were synchronized by adding serum-free DMEM / F12 to the starved myocardial fibroblasts for 12 h, PBS and angiotensin II (AngII, 1 μM) were added for 48 h. Western blot was performed on the myocardial cells to detect the expression of Cthrc1 protein, and GAPDH was used as an internal control.

[0041] The culture method of primary neonatal SD rat myocardial cells is as follows: 1) Take 10 neonatal 1-3 day Sprague-Dawley rats, disinfect the neck below 75% alcohol, and take out the heart with ophthalmic scissors and microforceps and put it into a glass dish containing 10 mL of DMEM / F12 liquid. Repeat the above process with another one; 2) Wash the heart with DMEM / F12 medium, and cut the heart into 1-2 mm3 fragments. Transfer to a serum bottle with a rotor, remove the DMEM / F12, and add trypsin digestion solution. The rotation speed is 120 r / min, and the digestion time is 15 min. Rest for a few seconds, and discard the supernatant; 3) Add trypsin digestion solution, rotate at 120 r / min for 15 min. Rest for a few seconds, aspirate the supernatant, terminate the digestion with 20% calf serum DMEM / F12 medium, and store in a 4°C refrigerator. Repeat the step for several cycles. When collecting the supernatant, try to take as much as possible. When the tissue pieces become white and significantly smaller, stop the digestion; 4) Centrifuge the collected myocardial cell suspension at 1500 rpm for 8 min, discard the supernatant. Add an appropriate amount of medium to the centrifuge tube, gently blow the cells to resuspend them, concentrate them into one 50 mL centrifuge tube, and filter the cell suspension with a 40 μm cell filter; 5) Seed the cells in a 100 mm culture dish, and let them adhere to the wall for 90 min. Filter the unadhered cell suspension. According to the total amount of cell suspension, add Brdu (final concentration 0.1 mM), mix well, and then add it to the 0.1% gelatin coated dish; 6) Disperse the cells by gentle shaking, not vortex shaking. Incubate at 37°C, 5% CO2 for 48 hours, wash once with PBS, and replace the medium.

[0042] The culture method of primary neonatal SD rat myocardial fibroblast cells is as follows: 1) After the first time the mixed cell suspension is seeded into a normal 100 mm culture dish, incubate at 37°C, 5% CO2 in an incubator for 60-90 minutes; 2) During this period, fibroblasts adhere to the bottom of the dish due to their strong adhesion ability, while most myocardial cells are still in suspension; 3) After incubation, aspirate the supernatant (for myocardial cell culture) and retain the adhered cells, which are mainly fibroblasts; 4) For the adhered cells, gently wash once with PBS, add fresh DMEM or DMEM / F12 medium (containing 10-20% FBS), and continue to culture at 37°C, 5% CO2; 5) When the cells reach 80-90% confluence, they can be passaged to obtain a stable primary cardiac fibroblast cell line. To obtain primary cardiac fibroblasts synchronously, separate and culture the early adhered cells during the adhesion step.

[0043] The detection results show that the expression of Cthrc1 in cardiac fibroblasts after stimulation by angiotensin II (AngII) is significantly up-regulated.

[0044] (2) Effect of Cthrc1 interference (Ad-shCthrc1) on AngII-stimulated fibrosis of primary cardiac fibroblasts and cardiomyocytes After infecting the in-vitro cultured primary cardiac fibroblasts with adenovirus Ad-shRNA (Sh-Ctrl) and Ad-shCthrc1 (Sh-Cthrc1), a control group and a Cthrc1 interference group were constructed by stimulating with angiotensin II (AngII), and the effect of Cthrc1 interference (Ad-shCthrc1) on AngII-stimulated fibrosis of primary cardiac fibroblasts was detected. The specific experimental method is as follows: 1) Cthrc1 expression interference (Ad-shCthrc1) The GV lentivirus vector (such as GV493) carrying the shRNA sequence of interest, the packaging plasmid pHelper1.0 (containing gag / pol / rev genes) and the envelope plasmid pHelper2.0 (containing VSV-G genes) were co-transfected into HEK293T cells by a three-plasmid system. The cell supernatant containing virus particles was collected 48 hours after transfection, concentrated and purified by ultracentrifugation (25,000 rpm, 2h, 4°C), and high-titer lentivirus LV-Cthrc1-shRNA-EGFP-Puro, referred to as Ad-shCthrc1, was obtained. Finally, the proportion of EGFP positive cells was observed under a fluorescence microscope to calculate the virus titer (TU / mL), and the titer and the expression level of the target gene Cthrc1 were detected for standby use.

[0045] Figure 6 The structure of the lentivirus vector GV493 is shown in the schematic diagram (10.9 kb), which is used to construct the interference lentivirus carrying shRNA-Cthrc1. The vector contains multiple functional elements: the 5' LTR region driven by the PCMV promoter; the MCS region downstream of the PhU6 promoter for connecting the target shRNA sequence; the gCGFP and Puro expression cassette driven by the PCBh promoter downstream, connected by the IRES element, which can be used for fluorescence detection of virus infection efficiency and positive screening. The plasmid also contains a pBRori replication origin and an Amp^r resistance selection marker, suitable for plasmid amplification. This vector is suitable for the three-plasmid system to package and prepare high-titer lentivirus in HEK293T cells.

[0046] 2) Effect of Cthrc1 expression interference on AngII-induced cardiomyocyte hypertrophy model Primary cardiomyocyte fibroblasts cultured for 3 days were infected with 10 MOIs of Ad-shRNA (Sh-Ctrl) and Ad-shCthrc1 (Sh-Cthrc1) (lentiviruses containing shRNA-Cthrc1). After 12 hours, the cells were stimulated with 1 μM angiotensin II (AngII) (purchased from ENZO, ALX-151-039-M025) or PBS for 48 hours. Immunofluorescence assays were then performed, and cells were stained with anti-α-SMA antibody (red) and DAPI (blue) to observe myofibroblast formation. Results are as follows: Figure 7 As shown, the surface area of ​​myocardial fibroblasts infected with Ad-shCthrc1 was significantly smaller than that of the control group, suggesting that Cthrc1 interference can inhibit the process of myocardial fibrosis. AngII stimulation significantly enhanced α-SMA expression and promoted the transformation of fibroblasts into myofibroblasts, while Cthrc1 interference significantly weakened this change, suggesting that Cthrc1 plays a promoting role in the process of cardiac fibroblast fibrosis.

[0047] 3) The effect of Cthrc1 paracrine secretion in fibroblasts on cardiomyocyte hypertrophy To further clarify the regulatory role of Cthrc1 paracrine secretion in cardiomyocyte hypertrophy, primary cardiomyocytes were immunofluorescence stained with α-actinin as a cytoskeletal marker, combined with DAPI staining, to define the cardiomyocyte boundaries and nuclei. Cross-sectional areas were measured under a fluorescence microscope using image analysis software to assess changes in cross-sectional area under different conditioned media treatments. Specifically, cardiomyocytes infected with Ad-shRNA (control) or Ad-shCthrc1 were collected. The supernatant obtained after 24 hours of stimulation with AngII (1 μM) or PBS was used as conditioned media for the synchronized primary cardiomyocytes. After 48 hours of treatment, the cardiomyocytes were fixed and stained with both α-actinin and DAPI. Microscopic images were taken, and the cross-sectional area of ​​the cardiomyocytes was quantitatively analyzed using image analysis software.

[0048] The results are as follows Figure 8 As shown, cardiomyocytes treated with the supernatant of AngII-stimulated Ad-shRNA fibroblasts (with normal Cthrc1 expression) exhibited significant hypertrophy and a markedly increased cross-sectional area; while cardiomyocytes treated with the supernatant of Ad-shCthrc1 fibroblasts showed a significantly reduced α-actinin-labeled cell outline and a significantly decreased cross-sectional area compared to the control group. These results further indicate that Cthrc1 mediates cardiomyocyte hypertrophy through paracrine factors secreted by fibroblasts, and interfering with its expression can effectively alleviate pathological structural remodeling of cardiomyocytes, further establishing the application value of Cthrc1 as a potential drug target for anti-cardiomyocyte hypertrophy.

[0049] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. The use of the Cthrc1 gene in the preparation of drugs for the prevention, relief and / or treatment of myocardial hypertrophy, wherein the sequence of the Cthrc1 gene is shown in SEQ ID NO:

1.

2. Application of reagents that knock out the Cthrc1 gene or inhibit Cthrc1 gene expression in the preparation of drugs for the prevention, relief and / or treatment of myocardial hypertrophy.

3. The application according to claim 2, characterized in that, The reagents knock out the Cthrc1 gene, reduce or inhibit the transcription of the Cthrc1 gene, and / or reduce or inhibit the translation of the Cthrc1 mRNA product.

4. The application according to claim 2, characterized in that, The reagents are gene editing systems or Cthrc1 inhibitors.

5. The application according to claim 4, characterized in that, The Cthrc1 inhibitor is a shRNA lentiviral expression vector that inhibits Cthrc1 gene expression.

6. The application according to any one of claims 2-5, characterized in that, The myocardial hypertrophy mentioned is pathological myocardial hypertrophy.

7. A drug for preventing, alleviating, and / or treating myocardial hypertrophy, characterized in that, The drug includes reagents that knock out or inhibit the expression of the Cthrc1 gene.

8. A method for screening drugs for the prevention, relief, and / or treatment of myocardial hypertrophy, characterized in that, The method includes screening for Cthrc1 inhibitors.

9. The method for screening drugs for the prevention, relief, and / or treatment of myocardial hypertrophy according to claim 8, characterized in that, The Cthrc1 inhibitor can inhibit or reduce the expression of the Cthrc1 gene, or inhibit or block the activity of the Cthrc1 protein.

10. The method for screening drugs for the prevention, relief, and / or treatment of myocardial hypertrophy according to claim 8, characterized in that, The steps for screening Cthrc1 inhibitors include: The test reagent was used to detect whether it could inhibit the expression of the Cthrc1 gene or the activity of the Cthrc1 protein. Select test agents that can inhibit Cthrc1 gene expression or Cthrc1 protein activity as candidate drugs.

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