Application of Psmg4 gene in preparation of medicine for treating heart diseases and related diagnostic products

Abstract

The invention belongs to the field of gene medicines, and particularly relates to application of a Psmg4 gene in preparation of a medicine or a composition thereof for treating heart diseases and a kit for detecting the heart diseases. The inventor finds for the first time that the Psmg4 gene is cut in myocardial cells and heart tissues with hypertrophy, and overexpression of the Psmg4 gene has an inhibition effect on pathological hypertrophy of the myocardial cells. The invention provides a medicine or a composition thereof for treating related heart diseases and a kit for detecting the heart diseases. The medicine for preventing and treating the heart diseases or the composition of the medicine contains a Psmg4 coding sequence or an accelerant of the Psmg4 coding sequence or a pharmaceutical preparation of the Psmg4 coding sequence or the accelerant of the Psmg4 coding sequence and the composition of the Psmg4 coding sequence and the composition of the Psmg4 coding sequence. The medicine can well play a role in preventing and treating various heart diseases. The kit for detecting the heart diseases contains a primer pair for detecting the shearing condition of the Psmg4 gene.

Description

Technical Field

[0001] The present invention belongs to the field of gene medicine, and specifically relates to the application of Psmg4 gene in the preparation of heart disease therapeutic medicine and related diagnostic products. Background Art

[0002] Cardiovascular disease has become a health problem of universal concern worldwide. The pathogenesis of cardiovascular disease is extremely complex, affected by genetic and / or environmental factors, and involves various types of cells, such as cardiomyocytes, endothelial cells, fibroblasts, and immune cells. After myocardial injury, cardiomyocytes are widely affected, causing adverse remodeling of the heart. Pathological myocardial hypertrophy is caused by hypertension, myocardial infarction, structural heart disease, etc. The expression level of hypertrophy-related indicators (such as BNP) in hypertrophic cardiomyocytes will be significantly increased, and the cell surface area will increase. However, continuous pathological overload can cause maladaptation and cardiac remodeling, leading to heart failure. At present, the research on heart diseases related to pathological myocardial hypertrophy is not in-depth enough, and new technical methods are still urgently needed to be developed for the diagnosis and treatment of related heart diseases. In view of the current situation, the present invention is specially proposed. Summary of the invention

[0003] The inventors first discovered that the Psmg4 (proteasome assembly chaperone 4) gene is sheared in hypertrophic cardiomyocytes and cardiac tissues, and that overexpression of the Psmg4 gene has an inhibitory effect on pathological hypertrophy of cardiomyocytes. Based on this, the present invention provides the use of the Psmg4 gene in the preparation of drugs for treating heart diseases, the use of the Psmg4 gene as a biomarker for diagnosing heart-related diseases, and the use of the Psmg4 gene in the preparation of a detection kit.

[0004] In a first aspect, the present invention provides the use of the Psmg4 gene for preparing a drug for preventing or treating heart disease, for example, overexpressing the Psmg4 gene by means of a vector, gene editing, mRNA translation, etc.

[0005] Based on this application, the second aspect of the present invention provides a drug for preventing or treating heart disease, wherein the drug comprises a pharmaceutical preparation for enhancing the expression of the Psmg4 gene.

[0006] In some embodiments, the pharmaceutical agent is a viral vector that overexpresses the Psmg4 gene.

[0007] In some embodiments, the viral vector is selected from one or more of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, or a retroviral vector, preferably an adenoviral vector.

[0008] In some embodiments, the medicament further comprises a drug delivery formulation, and / or an excipient.

[0009] In some embodiments, the drug delivery formulation is selected from one or more of liposomes, chitosan, cholesterol, LNP or nanoparticles, preferably liposomes.

[0010] In some embodiments, the auxiliary material is selected from one or more of phosphate buffer, mannitol or physiological saline, preferably phosphate buffer; the pH of the phosphate buffer is preferably 6.5-8.0.

[0011] The third aspect of the present invention provides the use of the Psmg4 gene as a biomarker in the preparation of a product for diagnosing heart disease. The fourth aspect of the present invention also provides the use of a reagent for detecting a biomarker in the preparation of a product for diagnosing heart disease, wherein the biomarker is the Psmg4 gene.

[0012] The fifth aspect of the present invention also provides a product for diagnosing heart disease, comprising a reagent for detecting a biomarker, wherein the biomarker is the Psmg4 gene.

[0013] In some embodiments, the heart disease is selected from hypertrophic cardiomyopathy, dilated cardiomyopathy, myocardial fibrosis, heart failure, coronary heart disease, myocardial damage caused by ischemia, myocardial infarction caused by ischemia, etc., preferably hypertrophic cardiomyopathy.

[0014] In some embodiments, the product uses cells, tissues or blood as test samples and diagnoses diseases by detecting the splicing status of the Psmg4 gene.

[0015] In some embodiments, the reagent for detecting biomarkers includes a primer pair for amplifying the Psmg4 gene. In some embodiments, the nucleotide sequences of the upstream and downstream primers in the primer pair are shown in SEQ ID NOs: 1 to 2, respectively.

[0016] In some embodiments, the reagents for detecting biomarkers include reagents for detecting the splicing status of the Psmg4 gene, such as a buffer, a marker for detecting the gene, and the like.

[0017] In some embodiments, the reagents for detecting biomarkers include reagents for detecting the ratios of different splicing forms of the Psmg4 gene by RT-qPCR technology.

[0018] In some embodiments, in the present invention, the nucleotide sequence of the Psmg4 gene is as shown in SEQ ID NO:7.

[0019] Compared with the prior art, the beneficial effects of the present invention include at least:

[0020] The inventors first discovered that the Psmg4 gene is sheared in hypertrophic cardiomyocytes and cardiac tissues, and that overexpression of the Psmg4 gene has an inhibitory effect on pathological hypertrophy of cardiomyocytes. The present invention provides the use of the Psmg4 gene in the preparation of a drug for treating heart disease, which can effectively prevent and treat a variety of heart diseases; the therapeutic effect is obvious and the application range is wide. The use of the Psmg4 gene as a biomarker in the preparation of a product for diagnosing heart disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The experimental results related to the angiotensin II (Ang II)-induced myocardial hypertrophy in mice provided in the embodiments of the present invention;

[0022] Figure 2 The experimental results of cardiomyocytes treated with AngⅡ provided in the embodiments of the present invention;

[0023] Figure 3 Schematic diagram of the original plasmid vector used in Examples 1 and 2 of the present invention. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0025] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0026] The terms involved in the following embodiments and drawings are explained as follows:

[0027] Saline: saline-treated group, intraperitoneal injection of saline group, and control of AngⅡ group;

[0028] AngⅡ (angiotensin Ⅱ): AngⅡ-treated group refers to mice injected intraperitoneally with AngⅡ;

[0029] AngⅡ+CTRL: AngⅡ-treated group after tail vein injection of control adenovirus;

[0030] AngⅡ+Psmg4-FL: AngⅡ-treated group after tail vein injection of adenovirus targeting amplification of Psmg4 gene;

[0031] Heart / Body weight (mg / g): ratio of mouse heart to body weight;

[0032] Cross-sectional area(μm 2 ): myocardial cell cross-sectional area;

[0033] BNP mRNA levels: relative expression levels of BNP mRNA;

[0034] LV collagen volume fraction (%): left ventricular collagen volume fraction;

[0035] Ejection Fraction (%): left ventricular ejection fraction;

[0036] Control: normal control group, which is the control of AngⅡ group;

[0037] AngⅡ: AngⅡ-treated group, referring to AngⅡ-induced treatment of mouse cardiomyocytes;

[0038] AngⅡ+CTRL: group in which mouse cardiomyocytes were treated with AngⅡ after transfection with control adenovirus;

[0039] AngⅡ+Psmg4-FL: group in which mouse cardiomyocytes were treated with AngⅡ after being transfected with adenovirus that targeted and amplified the Psmg4 gene;

[0040] Percent spliced ​​in (%): percentage of splicing of Psmg4 gene;

[0041] %Conversion of protein (Psmg4-FL / total Psmg4): the percentage of Psmg4-FL protein in the total Psmg4 protein;

[0042] Cell area (fold of control): cardiomyocyte surface area relative to the control group.

[0043] Example 1

[0044] This example provides subsequent experimental animals and constructs an Ang II-induced myocardial hypertrophy model, specifically:

[0045] 1.1 Experimental animals: C57BL / 6 male mice were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd., and mice aged 8-10 weeks were selected for the experiment. All mice were housed in the temperature-controlled Animal Center of Qingdao University under normal light / dark cycles and had free access to food and water throughout the experiment. All animal studies were conducted in accordance with protocols approved by the Institute's Animal Care Committee and protocols approved by government authorities.

[0046] 1.2AngⅡ induced myocardial hypertrophy: Mice were intraperitoneally injected with AngⅡ for 4 consecutive weeks to induce myocardial hypertrophy. The control group of mice was intraperitoneally injected with normal saline for 4 consecutive weeks as a control. For the purpose of overexpressing the Psmg4 gene in vivo, adenovirus was used to construct a Psmg4 gene overexpression vector, which was usually injected into the tail vein 5 days before the injection of AngⅡ in mice, and the adenovirus vector was injected again 2 weeks after the injection of AngⅡ. The virus infection titer was 1×10 10～ 14 PFU, preferably 1×10 12 PFU.

[0047] The original plasmid vector used in constructing the Psmg4 gene overexpression vector using adenovirus is ADV6. The plasmid map is as follows: Figure 3 As shown, the Psmg4 CDS region sequence (SEQ ID NO: 7) was inserted between the EcoRI and BamHI elements of the original plasmid vector to construct a recombinant viral vector that overexpresses the target gene Psmg4. The inserted nucleotide sequence of the recombinant viral vector is shown below:

[0048]

[0049] Example 2

[0050] In this example, cardiomyocytes were prepared for experiment and angiotensin II (Ang II) was applied to the cardiomyocytes, specifically:

[0051] 2.1 Cardiomyocytes: The cardiomyocytes used in this experiment are primary mouse cardiomyocytes. The hearts of 1-2 day old newborn mice were taken and rinsed 3-4 times with pre-cooled PBS. Then the ventricular area was separated and minced in HEPES buffered saline. The tissue was dispersed in a digestion solution containing pancreatin and collagenase II at 37°C, and a series of repeated digestion treatments were performed to collect the first type of cardiomyocytes. On the basis of the first type of cardiomyocytes, adenovirus was used to construct a Psmg4 gene overexpression vector in the cells to achieve the purpose of overexpressing the Psmg4 gene. Adenovirus was usually added to cells in good growth condition, and the infection titer was 1×10 10 ～12 PFU, preferably 1×10 10 PFU, and obtain the second type of cardiomyocytes.

[0052] 2.2 AngⅡ treatment: The second type of cardiomyocytes were added to DMEM / F12 medium containing 5% serum of AngⅡ, and the control group was added to DMEM / F12 medium containing 5% serum of the first type of cardiomyocytes. They were placed in an incubator at 37℃, 5% CO2, and 95% O2 for reoxygenation for 24 hours, and the cells were collected for subsequent experiments.

[0053] Example 3

[0054] In this example, RNA extraction, RT-qPCR, PCR amplification and agarose gel electrophoresis were performed on the myocardial tissue of the animal model of Example 1 and the myocardial cells treated in Example 2, specifically:

[0055] 3.1 RNA extraction: Total RNA was isolated from cardiomyocytes and tissue samples using Trizol reagent.

[0056] Specifically, after mixing the cells or tissues with Trizol, add chloroform in a volume ratio of 1:0.2, shake vigorously, let it stand for 5 minutes, use a 4°C centrifuge, centrifuge at 12000 rpm / min for 15 minutes to obtain the supernatant; then add isopropanol in a volume ratio of 1:1, let it stand for 30-60 minutes, centrifuge at 12000 rpm / min for 15 minutes to separate and precipitate RNA; add 75% ethanol prepared with pre-cooled DEPC water, wash and dry to obtain total RNA.

[0057] 3.2RT-qPCR: Use the reverse transcription kit according to the instructions to first remove genomic DNA, then reverse transcribe cDNA; then use quantitative reagents to perform qRT-PCR in a fluorescent quantitative qPCR instrument; use GAPDH to standardize the results. The primer sequences involved in this experiment are shown in the table below.

[0058]

[0059] 3.3 PCR amplification: The cDNA obtained in 3.2 was amplified by PCR using PCR Mix in a PCR instrument. The primer sequences involved in this experiment are shown in the table below.

[0060]

[0061]

[0062] 3.4 Agarose gel electrophoresis: The PCR product obtained in 3.3 was electrophoresed in 1-2% agarose gel, and the band distribution was observed in a developer, and quantitative analysis was performed using imageJ image analysis software.

[0063] Example 4

[0064] In this example, the heart treated in Example 1 was subjected to frozen sectioning, WGA staining, and quantitative analysis of the cross-sectional area of ​​myocardial cells, specifically:

[0065] 4.1 Heart frozen sections: The mouse heart was removed and washed with PBS, embedded with OTC embedding agent, and then sliced ​​using a freezing microtome; the slices were 5-7 μm thick and stored in a refrigerator.

[0066] 4.2 WGA staining: After the frozen sections were washed with PBS at room temperature, they were fixed with 4% paraformaldehyde at room temperature for 15-25 minutes; after washing with PBS three times, they were stained with WGA dye for 30 minutes at 37°C in the dark; after washing with PBS three times, they were stained with DAPI solution and sealed.

[0067] 4.3 Quantitative analysis: The WGA-stained slides were observed and photographed under a confocal microscope; the length of the cardiomyocytes was measured along their long axis, and the width of the cells was measured along the midpoint cross section.

[0068] Example 5

[0069] In this example, the mice and their hearts treated in Example 1 were weighed and quantitatively analyzed, specifically:

[0070] First weigh the mouse, then take out the mouse heart and weigh the heart mass; calculate the heart-to-weight ratio based on the mouse heart mass / body weight.

[0071] Example 6

[0072] In this example, the heart treated in Example 1 was subjected to paraffin sectioning, Masson staining, and quantitative analysis of myocardial fibrosis area, specifically:

[0073] 6.1 Heart paraffin sectioning: The mouse heart was removed and washed with PBS, fixed with 4% paraformaldehyde at room temperature, and then embedded in paraffin; the heart was sliced ​​using a microtome with a thickness of 5-7 μm.

[0074] 6.2Masson staining: After dewaxing and rehydration of the paraffin sections, stain them using the Masson kit according to the instructions; then seal the sections with glycerol gelatin and air dry.

[0075] 6.3 Quantitative analysis of fibrosis: The Masson-stained slides were observed and photographed under a microscope; the imageJ image analysis software was used to calculate the area of ​​myocardial tissue fibrosis.

[0076] Example 7

[0077] In this example, cardiac ultrasound detection was performed on the mouse model constructed in Example 1, specifically:

[0078] After AngⅡ or Saline injection, the mice were subjected to M-mode echocardiography using Vevo2100 (Visual Sonic) imaging system to evaluate cardiac function, and the system was used to calculate cardiac ejection fraction (EF%).

[0079] Example 8

[0080] In this example, protein extraction, protein immunoblotting (WB) and quantitative analysis were performed on the myocardial tissue of the animal model of Example 1 and the myocardial cells after treatment in Example 2, specifically:

[0081] 8.1 Protein extraction: Total protein was isolated from cardiomyocytes or myocardial tissue samples using RIPA lysis buffer supplemented with protease inhibitors under low temperature conditions.

[0082] Specifically, add RIPA lysis buffer containing protease inhibitors to the disrupted tissue homogenate or cardiomyocytes, and lyse on ice for 30-60 minutes; then use a 4°C centrifuge, centrifuge at 12000rpm / min for 15 minutes, and transfer the supernatant; add 5× protein loading buffer to the supernatant at a volume ratio of 4:1, use a metal bath, and boil at 99°C for 10 minutes.

[0083] 8.2WB: The collected protein samples were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane. Blocked with 5% skim milk powder in PBS at room temperature for 1 hour; then incubated with PSMG4 and GAPDH primary antibodies at room temperature for 2-3 hours, and secondary antibodies at room temperature for 1 hour. GAPDH was used as an internal reference. The ECL chemiluminescence detection system was used to take photos to detect the signal.

[0084] 8.3 Quantitative analysis: The images obtained in 8.2 were used to calculate the protein signal intensity using imageJ image analysis software.

[0085] Example 9

[0086] In this example, the cardiomyocytes treated in Example 2 were stained with phalloidin and quantitatively analyzed, specifically:

[0087] 9.1 Phalloidin staining: The cardiomyocytes treated in Example 2 were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 15-25 min; washed with PBS three times, stained with phalloidin probe dye for 30 min at room temperature in the dark; washed with PBS three times, and then stained with DAPI solution to seal the slides.

[0088] 9.2 Quantitative analysis: Use a confocal fluorescence microscope to observe the staining results of the slide; the phalloidin dye will mark the cytoskeleton in red, and DAPI can mark all cell nuclei in blue; use imageJ image analysis software to measure the surface area of ​​cardiomyocytes.

[0089] The experimental data obtained from the above examples were statistically analyzed: all data were expressed as mean ± standard deviation (mean ± SD); Graphpad Prism was used for statistical analysis; One-way ANOVA followed by Tukey's multiple comparisons was used for comparison among multiple groups; t-test was used for comparison between two independent samples; the experiment was repeated at least 3 times independently to obtain similar results. p < 0.05 was considered statistically significant.

[0090] The experimental results and analysis of the above embodiments are as follows:

[0091] The experimental results related to the mouse model are as follows Figure 1 As shown in Figure 2, the results of cardiomyocyte-related experiments are shown in Figure 2. Figure 2 As shown in the figure, it can be seen that:

[0092] In vivo, after injection of AngⅡ, the mouse heart became larger and the heart-to-body weight ratio increased ( Figure 1 A), the cross-sectional area of ​​myocardial cells increases ( Figure 1 B), this situation was alleviated to a certain extent after overexpression of Psmg4; overexpression of Psmg4 gene alleviated the increase of BNP mRNA, a hypertrophy marker, in myocardial tissue induced by AngⅡ ( Figure 1 C), reduced cardiac fibrosis area ( Figure 1 D) and improved ventricular function ( Figure 1 E).

[0093] In vitro, the Psmg4 gene in cardiomyocytes treated with AngⅡ was sheared, resulting in long and short fragments, with the long fragment reduced ( Figure 2 A in the figure); protein level detection of PSMG4 expression in cardiomyocytes treated with AngⅡ, the short fragment protein increased ( Figure 2 Overexpression of Psmg4 blocked the increase in cardiomyocyte surface area induced by AngⅡ ( Figure 2 C) and an increase in the hypertrophy marker BNP mRNA ( Figure 2 D).

[0094] It can be seen that in damaged myocardial cells and hearts, the Psmg4 gene is sheared, the short fragments increase, and the long fragments decrease; overexpression of the Psmg4 gene can effectively reduce myocardial hypertrophy and fibrosis area and improve cardiac function. Therefore, the Psmg4 gene can be used as a biomarker in the preparation of related cardiac disease diagnostic products; and the preparation of therapeutic drugs for related cardiac diseases using the Psmg4 gene as a target has broad application prospects.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Psmg4 gene in the preparation of drugs for preventing or treating heart diseases.

2. A drug for preventing or treating heart disease, characterized in that: The medicine includes a pharmaceutical preparation for enhancing the expression of Psmg4 gene.

3. The drug according to claim 2, characterized in that The pharmaceutical preparation is a viral vector that overexpresses the Psmg4 gene; the viral vector is selected from one or more of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector or a retroviral vector, preferably an adenoviral vector.

4. The drug according to claim 2 or 3, characterized in that The drug also includes a drug delivery preparation, and / or an excipient; the drug delivery preparation is selected from one or more of liposomes, chitosan, cholesterol, LNP or nanoparticles, preferably liposomes; the excipient is selected from one or more of phosphate buffer, mannitol or physiological saline, preferably phosphate buffer.

5. Application of Psmg4 gene as a biomarker in the preparation of products for diagnosing heart disease.

6. Use of a reagent for detecting biomarkers in the preparation of a product for diagnosing heart disease, characterized in that: The biomarker is the Psmg4 gene.

7. A product for diagnosing heart disease, characterized in that: Contains reagents for detecting a biomarker, wherein the biomarker is the Psmg4 gene.

8. The product according to claim 7, characterized in that The product uses cells, tissues or blood as test samples and diagnoses diseases by detecting the splicing status of the Psmg4 gene.

9. The product according to claim 7, characterized in that The reagent for detecting biomarkers includes a primer pair for amplifying the Psmg4 gene; preferably, in the primer pair, the nucleotide sequences of the upstream and downstream primers are respectively as shown in SEQ ID NOs: 1 to 2.

10. The use according to claim 1, the medicine according to any one of claims 2 to 4, the product according to claim 5 or 6, or any one of claims 7 to 9, characterized in that: The heart disease is selected from hypertrophic cardiomyopathy, dilated cardiomyopathy, myocardial fibrosis, heart failure, coronary heart disease, myocardial damage caused by ischemia, and myocardial infarction caused by ischemia.

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