阅读说明：本技术 Myog基因作为靶点在制备治疗心肌细胞凋亡相关的心血管疾病的药物中的应用 (Application of MYOG gene as target in preparation of medicine for treating cardiovascular diseases related to myocardial apoptosis ) 是由 曾嵘 林彬 于 2020-12-21 设计创作，主要内容包括：本发明属于生物医药技术领域,特别是指MYOG基因作为靶点在制备治疗心肌细胞凋亡相关的心血管疾病的药物中的应用。本发明通过体外构建血管紧张素II诱导的人诱导多能干细胞分化的心肌细胞凋亡模型,首次揭示了转录因子MYOG在抑制心肌细胞凋亡方面的作用,为心肌细胞凋亡相关的心血管疾病的药物研发提供了理论基础和科学依据,是心血管疾病药物研发的新靶点。(The invention belongs to the technical field of biological medicine, and particularly relates to an application of MYOG gene as a target spot in preparation of a medicine for treating cardiovascular diseases related to myocardial apoptosis. The invention discloses the effect of transcription factor MYOG in inhibiting the myocardial cell apoptosis for the first time by constructing an angiotensin II induced myocardial cell apoptosis model of human induced pluripotent stem cell differentiation in vitro, provides theoretical basis and scientific basis for the research and development of cardiovascular diseases related to the myocardial cell apoptosis, and is a new target for the research and development of cardiovascular disease medicines.)

Application of MYOG gene as target in preparation of medicine for treating cardiovascular diseases related to myocardial apoptosis.

2. The use of claim 1, wherein the inhibition of cardiomyocyte apoptosis is achieved by drug-regulated expression of the MYOG gene.

3. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is essential hypertension.

4. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is ischemic heart disease, or reperfusion injury.

5. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is myocarditis.

6. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is cardiomyopathy.

7. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is arrhythmia.

8. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is heart failure.

9. The use of claim 1 or 2, wherein the cardiovascular disease associated with cardiomyocyte apoptosis is congenital heart disease.

Technical Field

The invention belongs to the technical field of biological medicine, and particularly relates to an application of MYOG gene as a target spot in preparation of a medicine for treating cardiovascular diseases related to myocardial apoptosis.

Background

Cardiovascular disease (CVD) is currently the first killer of human health, and the number of people dying from cardiovascular disease worldwide every year is greater than any other cause. According to the World Health Organization (WHO), over 1700 million people die globally annually from CVD, accounting for 31% of the total number of deaths worldwide. Along with the improvement of living standard of people and the change of dietary structure, the morbidity and mortality of cardiovascular diseases are in a remarkably rising trend.

Apoptosis of myocardial cells is involved in the pathophysiological processes of various cardiovascular diseases, including essential hypertension, ischemic heart disease and reperfusion injury, myocarditis, cardiomyopathy, arrhythmia, heart failure, congenital heart disease, and the like. Apoptotic dysfunction of cardiomyocytes is an important mechanism for the development of a variety of severe cardiovascular diseases. Through intervening the apoptosis program, the method inhibits the occurrence of myocardial apoptosis and saves the heart and cardiac function, and has become one of the important directions for the research of cardiovascular disease drugs. At present, the drugs for clinically inhibiting the myocardial cell apoptosis comprise statins, beta-receptor blockers, angiotensin converting enzyme inhibitors and the like, but have side effects of different degrees.

Due to the difficulty in obtaining human cardiomyocyte samples, the short survival time of primary cells in vitro culture, ethical problems in the research process and the like, the research on the aspect of directly using the cardiomyocytes of patients to carry out cardiovascular diseases is almost impossible to realize. In recent years, with the development of iPS (induced pluripotent stem cell) technology and the establishment of a method for differentiating and purifying cardiac muscle cells, I make the in vitro preparation and culture of human cardiac muscle cells possible. The method for inducing the myocardial cells in vitro by using the iPS can prepare a sufficient number of human myocardial cells in vitro to perform various functional experiments, simulate the occurrence and development processes of cardiovascular diseases, and greatly promote the research progress of the mechanism of the cardiovascular diseases.

Currently, the model of myocardial apoptosis in vitro is generally via hydrogen peroxide (H)₂O₂) Or angiotensin ii (angiotensin ii). Angiotensin II (angiotensin II) causes apoptosis of cardiac muscle cells by activating angiotensin II receptor, and H₂O₂Compared with the normal physiological process, the induction is closer to the normal physiological process and is a common inducer for establishing a myocardial apoptosis model at present.

The transcription factor gene MYOG encodes Myogenin (Myogenin) protein, and is one of the members of the Myogenic Regulatory Factor (MRFs) gene family. The MRFs family of transcription factors (including Myod, Myf5, Mrf4 and MYOG) play a key role in every stage of skeletal myogenesis. All members of this family share a conserved helix-loop-helix (bHLH) motif that can bind to the E-box of downstream genes, thereby activating expression of downstream muscle-specific genes. Studies have shown that MYOG plays a key role in the process of muscle differentiation by controlling, initiating myoblast fusion and myofiber formation. Studies in mice have shown that deletion of the MYOG gene leads to severe muscle differentiation defects, resulting in perinatal death. MYOG is therefore an essential regulatory factor in skeletal muscle development and is not replaceable. At present, researches prove that MYOG genes are expressed in hearts of animals such as mice, ducks, grass carps, Jinghai yellow chickens and the like and are possibly related to the growth and development of cardiac muscles. However, the expression of the MYOG gene in human heart tissues and the role in cardiac development have not been reported.

Disclosure of Invention

The invention discloses the effect of a transcription factor MYOG in inhibiting the myocardial cell apoptosis for the first time by constructing a hipSC-CM (human induced pluripotent stem cell differentiated myocardial cell) apoptosis model induced by angiotensin II (angiotensin II) in vitro, provides theoretical basis and scientific basis for the research and development of medicaments for treating cardiovascular diseases related to the myocardial cell apoptosis, and is a new target for the research and development of medicaments for treating the cardiovascular diseases.

The invention inhibits the myocardial apoptosis by regulating the expression of MYOG genes. Therefore, the medicine can be a traditional medicine, and can also be a gene regulation medicine, such as packaging lentivirus capable of regulating MYOG gene expression and the like.

Drawings

FIG. 1 is a technical flow chart of the present invention;

FIG. 2 is a graph of the determination of the expression level of MYOG in example 2;

FIG. 3 is a graph showing the effect of angiotensin II treatment on cardiomyocyte activity in example 4;

FIG. 4 is a flow chart showing the results of detecting the myocardial apoptosis by Annexin V method after treatment with angiotensin II at different concentrations;

FIG. 5 is a graph showing the statistical results of the Annexin V method for detecting the myocardial apoptosis after the treatment of angiotensin II with different concentrations;

FIG. 6 is a graph showing the immunofluorescence staining results of TUNEL to detect myocardial apoptosis after treatment with angiotensin II at different concentrations;

FIG. 7 is a graph showing the statistical results of TUNEL method for detecting myocardial apoptosis after treatment with angiotensin II at different concentrations;

FIG. 8 is a graph showing the results of detecting the activity of cardiomyocytes with the Prestoblue cell activity detection reagent;

FIG. 9 is a graph of immunofluorescence staining results for MYOG inhibition of angiotensin II-induced apoptosis;

FIG. 10 is a graph of statistics of MYOG inhibition of angiotensin II-induced apoptosis.

Detailed Description

In order to make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following detailed description is given with reference to specific embodiments.

As shown in fig. 1, the technical process of the present invention is as follows:

1. constructing pCW-MYOG vector.

2. And (4) packaging the lentivirus.

3. The hiPSC cells were transfected.

4. The hiPSC and pCW-MYOG were differentiated into cardiomyocytes and purified.

5. Doxycycline hydrochloride (Dox) was used to induce hiPSC-MYOG cells to express the MYOG gene at a Dox concentration of 2 μ g/mL.

6. And inducing apoptosis of hipSC-CM and pCW-MYOG-CM by using angiotensin II, wherein the concentration of the angiotensin II is 1 nM-1 mM.

7. Cell viability was determined.

8. Determining apoptosis.

Example 1: obtaining hIPSC-MYOG cell strain

1.1 lentivirus expression vector construction: MYOG cDNA and a puromycin resistance gene are subcloned into a pCW-Cas9-Blast vector (Addgene, 83481) by using a conventional molecular cloning method to replace Cas9 and Blast genes in an original vector, so that pCW-MYOG is obtained.

1.2 Lentiviral packaging

1.2.1 HEK293T cells were seeded into 6-well plates and cultured in D10 medium (DMEM medium + 10% fetal bovine serum) ready for transfection when the cell confluence reached 70% -80%.

1.2.2 Prior to transfection, 1h, the original medium was discarded and 2 mL/well of pre-warmed serum-free OptiMEM medium was added.

1.2.3 transfection was performed with Lipofectamine 2000 reagent as per the instructions. pCW-MYOG (20. mu.g), pVSVg (10. mu.g) (Addgene), psPAX2 (15. mu.g) (Addgene) were co-transfected into HEK293T cells.

1.2.46 h later, the culture was changed to D10 medium (DMEM medium + 10% fetal bovine serum + 1% BSA).

1.2.5 after further culturing for about 60h, the culture broth was centrifuged at 3000rpm at 4 ℃ for 10min to remove cell debris.

1.2.6 the supernatant was filtered through a 0.45 μm low protein binding filter (Millipore Steriflip HV/PVDF) to remove cellular debris.

1.2.7 the virus-containing culture broth was centrifuged at 10000g and 4 ℃ for 4h in a volume ratio of 4:1 with 10% sucrose buffer (50mM Tris-HCl, pH 7.4, 100mM NaCl, 0.5mM EDTA). Carefully discard the supernatant, drain the tube on absorbent paper for 3min, add 1 × PBS to resuspend, and store at-80 ℃.

1.3 transfection of hiPSC cells

1.3.1 hiPSC culture: human induced pluripotent stem cells (hiPSC) DYR0100(ATCC) were seeded on a Matrigel matrix (corning, 354277) -coated plate, followed by culture with stemup (nissan Chemical corporation). The STEMUP medium was changed every two days. ipscs were passaged every 3 days, or when cell cultures reached 80-90% confluence. During passaging, it was rinsed 1 time with 1 XDPBS (Gibco, 14040133) and then treated with 0.5mM EDTA (Invitrogen, 15575020) diluted with 1 XDPBS (Gibco, 14190144) for 10min at room temperature. The generation ratio is 1:3-1: 6.

1.3.2 transfection: transfection was performed when the hiPSC cells reached 70% -80% confluence. Multiplicity of infection (MOI) is about 0.3-0.5. 24h after transfection, the culture medium was replaced with fresh STEMUP (containing a final concentration of 2. mu.g/mL of tetracycline hydrochloride (Dox)). After 2 days, the culture broth was replaced with STEMUP (containing Dox 2. mu.g/mL + puromycin (puromycin) (InvivoGen)) for selection. After 2-3 days of screening, about 30% conversion efficiency can be obtained. Selecting single clone and inoculating the single clone into different dishes for culture to obtain the hiPSC-MYOG cell strain.

Example 2: dox induced MYOG expression

2.1 inducing: MYOG expression was induced by addition of doxycycline hydrochloride (Dox) (Sigma, D9891) to STEMUP at a final concentration of 2. mu.g/mL, and DMSO as a control. Control group, without DOX only DMSO, labeled C1, added to STEMUP; the test group contained DOX, which was first added to DMSO, labeled C2, and then to STEMUP; DOX was at a final concentration of 2. mu.g/mL in STEMUP; c1 and C2 are added in the same amount;

2.2 Total RNA extraction: total RNA from cells was extracted using UNlQ-10 column Trizol Total RNA extraction kit (Sangon Biotech, B511321-0100). (samples were treated with DNase I (DNase I, Sangon Biotech, B618252) for 30min in advance);

2.3 reverse transcription: RNA was Reverse transcribed using the Reverse Transcription kit iScript Reverse Transcription Supermix (Bio-Rad, 1708841).

2.4 qPCR assay MYOG mRNA expression levels: according to SsoAdvanced^TM UniversalGreen Supermix (Bio-Rad,1725271) instructions, using the Pikoreal Real-Time PCR System (Thermo Fisher) System, with NAPDH as an internal reference, primers were designed to detect MYOG expression levels in hipSC and hipSC-MYOG (Dox-induced group, DMSO control group). The primer sequences are as follows:

MYOG-RT-F:GCCCAAGGTGGAGATCCT；

MYOG-RT-R:GGTCAGCCGTGAGCAGAT；

GAPDH-RT-F:TGGGTGTGAACCATGAGAAG；

GAPDH-RT-R:GTGTCGCTGTTGAAGTCAGA.

and (4) analyzing results: as shown in fig. 2, expression levels of MYOG in Dox-induced group were 100-fold higher than DMSO control group. The result shows that the hipSC-MYOG cell strain capable of expressing MYOG genes at high level is successfully constructed.

Example 3: acquisition of hipSC-CM/hipSC-MYOG-CM

3.1 differentiation of hipscs: the hipscs were treated for 24h with the addition of small molecule CHIR99021(Tocris, 4423, final concentration 10mM) in RPMI-BSA medium [ RPMI1640 medium (HyClone, SH30027.01) +213 μ g/mL AA2P (l-ascorbic acid 2-magnesium phosphate) (Sigma, a8960) and 0.1% bovine serum albumin (Sigma, a1470) ], followed by 48h incubation with RPMI-BSA medium. On day 4 of differentiation, cells were treated with small molecule IWP2(Tocris, 3533, final concentration 5. mu.M) in RPMI-BSA medium. After 48h, the medium was replaced with RPMI-BSA. At this stage, the hipsc is differentiated into hipsc-cm. In subsequent experiments, cardiomyocytes were cultured in RPMI1640 medium plus 3% serum replacement (Gibco, 10828-028).

3.2 purification of hiPSC-MYOG-CM: the hiPSC-CM was purified using a metabolic selection method. The metabolic selection medium was DMEM medium (sugar-free) supplemented with 0.1% bovine serum albumin (Sigma, A1470) and 1 Xlinoleic acid-oleic acid-albumin (Sigma, L9655) (Gibco, 11966-. Cells were treated with metabolic selection medium for 3-6 days. The culture medium was changed every 2 days. The purity of the cardiomyocytes purified by the method can reach 99%.

Example 4: establishment of angiotensin II-induced hiPSC-CM apoptosis model

2.1 angiotensin II induces hiPSC-CM apoptosis: the purified hiPSC-CM cells were treated with angiotensin ii by adding different amounts (1nM, 10nM, 100nM, 1 μ M, 10 μ M, 100 μ M, 1mM) of angiotensin ii in cardiomyocyte medium (MedChemExpress, HY-13948), and the angiotensin ii medium was changed every 2 days.

2.2 hiPSC-CM apoptosis validation:

2.2.1 myocardial cell activity was assayed at 24h, 48h, 6d, and 10d using PrestoBlue cell activity assay reagent (Invitrogen, A13261), respectively, and the results are shown in FIG. 3.

2.2.2 application of apoptosis detection kit Annexin V, Alexa Fluor^TM488conjugate (Invitrogen, V13201) assay for angiotensin II treatment(100. mu.M, 1mM) and untreated 10d cardiomyocyte apoptosis. Labeling cardiomyocytes with the apoptosis marker Annexin V, followed by FACSAria^TMII flow cytometry (BD) was used for the analysis. The results are shown in FIGS. 4 and 5.

2.2.3 TUNEL experiments: the apoptotic status of the cardiomyocytes treated with angiotensin II (1nM, 1. mu.M, 100. mu.M, 1mM) and untreated for 10D was examined using the TdT In Situ Apoptosis Detection Kit (TdT In Situ Apoptosis Detection Kit (R & D Systems, 4812-30-K)). Cardiomyocytes were digested and re-plated on glass slides, stained according to kit instructions, and nuclei were labeled with DAPI.

And (4) analyzing results: PrestoBlue cell viability assay results (fig. 3) show that cell viability is significantly reduced after hiPSC-CM treated with high concentrations of angiotensin ii (100 μ M, 1mM) for long periods of time (6d, 10 d); the Annexin V, TUNEL experimental results show (fig. 4-7) that the proportion of apoptotic cells was significantly increased after prolonged (10d) treatment of hiPSC-CM with high concentrations of angiotensin ii (100 μ M, 1 mM). The results show that the hiPSC-CM apoptosis can be induced by using high-concentration angiotensin II (100 mu M and 1mM) for a long time of treatment, and the myocardial cell apoptosis model is successfully constructed.

Example 5: MYOG inhibits angiotensin II-induced hipSC-CM apoptosis

5.1 angiotensin II induces apoptosis of hipSC-MYOG-CM cells: purified hiPSC-MYOG-CM cells were treated in 4 groups (Ang II (+) & Dox (+), Ang II (+) & Dox (-), Ang II (-) & Dox (+), and Ang II (-) & Dox (-), wherein the Dox concentration was 2 μ g/mL angiotensin II concentration at 1mM and the treatment time was 6 d.

5.2 cell viability assay: the myocardial cell activity was detected using Prestoblue cell activity detection reagent (Invitrogen, A13261), and the results are shown in FIG. 8.

5.3 detection of apoptosis: TUNEL experiment: apoptosis of 4 groups (Ang II (+) & Dox (+), Ang II (+) & Dox (-), Ang II (-) & Dox (+), and Ang II (-) & Dox (-)) of cardiomyocytes was detected using TdT In Situ Apoptosis Detection Kit (R & D Systems, 4812-30-K). Cardiomyocytes were digested and re-plated on glass slides, stained according to kit instructions, and nuclei were labeled with DAPI.

And (4) analyzing results: the Ang II (+) & Dox (+) group showed significantly increased cell activity compared to Ang II (+) & Dox (-) group (fig. 8) and significantly decreased apoptotic cell proportion (1.49-fold and 2.01-fold of the control group, respectively) (fig. 9-10), indicating that MYOG can inhibit angiotensin II induced hiPSC-CM apoptosis. Meanwhile, there was no significant difference in the apoptosis ratio between the Ang II (-) & Dox (+) group and the Ang II (-) & Dox (-) group, indicating that MYOG had no effect on apoptosis in cells in the absence of angiotensin II.

Thus, it can be seen that:

(1) the invention successfully constructs the iPSC-MYOG cell strain, and through verification, Dox can induce the iPSC-MYOG cell to highly express the MYOG gene.

(2) The invention establishes an angiotensin II-induced myocardial cell apoptosis model.

(3) The high expression MYOG gene can inhibit cardiac muscle cell apoptosis induced by angiotensin II.

The invention discovers the effect of MYOG on the aspect of inhibiting myocardial cell apoptosis for the first time, and provides a new idea for the research and development and clinical treatment of cardiovascular disease medicaments related to myocardial cell apoptosis.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.