Traditional Chinese medicine composition for promoting differentiation of embryonic stem cells to myocardial cells and application of traditional Chinese medicine composition

Abstract

The invention provides a traditional Chinese medicine composition for promoting differentiation of embryonic stem cells to myocardial cells and application of the traditional Chinese medicine composition, and relates to the technical field of biological medicines. The traditional Chinese medicine composition provided by the invention comprises a radix salviae miltiorrhizae extract and a lucid ganoderma extract, and a culture medium containing the traditional Chinese medicine composition is used for culturing embryonic stem cells, so that autologous stem cells can be promoted to be induced and differentiated into myocardial cells, and the differentiation rate of converting the stem cells into the myocardial cells is increased.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a traditional Chinese medicine composition for promoting the differentiation of embryonic stem cells into cardiomyocytes and an application thereof. Background Art

[0002] Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and the more recently emerging induced pluripotent stem cells (iPSCs), can replicate and expand indefinitely in vitro and, in theory, can be directed to differentiate into all tissue cells of the human body under specific conditions, providing an inexhaustible source of cells that do not require donors for human regenerative medicine and cell replacement therapy. Currently, human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells, have successfully achieved in vitro differentiation into cardiomyocytes (CMs). Cardiomyocytes differentiated from stem cells have broad application prospects and are often used in regenerative treatments related to heart disease, exploration of the mechanisms of hereditary heart disease, discovery of new drugs, and evaluation of drug cardiac toxicity safety. Therefore, establishing an efficient method for directed differentiation of cardiomyocytes is the key to obtaining cardiomyocytes.

[0003] Cardiac diseases such as heart failure caused by cardiomyocyte dysfunction are often treated with cardiomyocyte transplantation. This approach can help patients restore myocardial function and the pumping capacity of regenerated myocardial tissue. Studies have also shown that injected cardiomyocytes can form new myocardial tissue. However, a key factor in cell transplantation therapy is identifying the appropriate donor cell type. Therefore, inducing stem cells to differentiate into cardiomyocytes and improving the rate and speed of stem cell differentiation into cardiomyocytes are urgent technical challenges for medical treatment.

[0004] Fang Haiqin et al. found that resveratrol can promote the differentiation of mouse ES-D3 cells into cardiomyocytes by stimulating peroxisome proliferator-activated receptor (PPARγ) and upregulating mitochondrial biogenesis mediated by PGC1α. However, the differentiation rate was slow when induced by resveratrol. There was no significant increase in the expression of myocardial-related markers on the 7th day of culture, and there was no significant difference from the control group until the 14th day of culture. The differentiation efficiency was low and the time was too long. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a traditional Chinese medicine composition for promoting the differentiation of embryonic stem cells into cardiomyocytes and its application.

[0006] In one aspect, the present invention provides a traditional Chinese medicine composition for promoting the differentiation of embryonic stem cells into cardiomyocytes, the composition comprising a Ganoderma lucidum extract and a Salvia miltiorrhiza extract.

[0007] Specifically, the mass ratio of the Ganoderma lucidum extract to the Salvia miltiorrhiza extract is: 0.01-0.1:0.05-0.5.

[0008] More specifically, the preparation method of the Ganoderma lucidum extract comprises the following steps: crushing the Ganoderma lucidum fruiting bodies, performing ultrasonic-assisted extraction, concentrating, and then freeze-drying.

[0009] Preferably, the ultrasonic-assisted extraction conditions are: 35-45 kHz, 55-65 °C.

[0010] More specifically, the preparation method of the salvia miltiorrhiza extract comprises the following steps: taking dried rhizomes of salvia miltiorrhiza, adding water at a material-liquid ratio of 1 g:15-20 mL, boiling, filtering through a membrane, and then freeze-drying.

[0011] In another aspect, the present invention provides the use of the above-mentioned traditional Chinese medicine composition in the preparation of a product for promoting the differentiation of embryonic stem cells into cardiomyocytes.

[0012] In another aspect, the present invention provides a culture medium for promoting the differentiation of embryonic stem cells into cardiomyocytes, wherein the culture medium comprises any one of the above-mentioned traditional Chinese medicine compositions.

[0013] Specifically, the culture medium further comprises: at least one of a basal culture medium, antibiotics, serum, polysaccharide and growth factors.

[0014] In another aspect, the present invention provides a method for inducing and cultivating cardiomyocytes from stem cells in vitro, characterized in that the stem cells are cultured in a culture medium containing the traditional Chinese medicine composition provided by the present invention.

[0015] In another aspect, the present invention provides a method for inducing and cultivating cardiomyocytes from stem cells in vitro, wherein the stem cells are cultured in any of the above-mentioned culture media.

[0016] Specifically, the culturing comprises the following steps: (1) Stem cells are made into single cell suspension; (2) After counting the single cell suspension, dilute it with differentiation medium to a cell concentration of 10 3 -10 5 cells / mL stem cell suspension, inoculated and cultured; (3) Collect the embryoid bodies formed in the culture, transfer them to a container containing differentiation medium, and culture them in an incubator to promote further proliferation of the embryoid bodies; (4) The further proliferated embryoid bodies are transferred and culture medium that promotes the differentiation of embryonic stem cells into cardiomyocytes is added for intervention differentiation culture.

[0017] In another aspect, the present invention provides a cell line for detecting differentiation of embryonic stem cells into cardiomyocytes.

[0018] Specifically, the cell line is constructed by knocking in a fluorescent protein gene upstream of the start codon of the Tnnt2 gene, so that Tnnt2 and the fluorescent protein form a fusion protein through an H2B connecting peptide segment for fusion expression.

[0019] Compared with the prior art, the present invention has the following advantages: 1) Inducing autologous stem cells to differentiate into cardiomyocytes and using the obtained cardiomyocytes as cell donors for cardiomyocyte transplantation therapy can reduce the rejection reaction of the original cells to the transplanted cardiomyocytes and improve the success rate of cardiomyocyte transplantation; using the Ganoderma lucidum extract, Salvia miltiorrhiza extract or their combination of the present invention to induce stem cells to transform into cardiomyocytes can improve the differentiation rate of stem cells into cardiomyocytes.

[0020] 2) Autologous stem cells are induced to differentiate in culture medium with Ganoderma lucidum extract, Salvia miltiorrhiza extract or a combination thereof to obtain sufficient cardiomyocytes that can effectively improve the success rate of cell transplantation therapy.

[0021] 3) In the present invention, the mechanism by which Ganoderma lucidum and Salvia miltiorrhiza extracts promote the differentiation of embryonic stem cells into cardiomyocytes provides a basis for further research on the mechanism by which Ganoderma lucidum and Salvia miltiorrhiza extracts promote the differentiation of embryonic stem cells into cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the principle of knocking in Tnnt2 H2B-mCherry using CRISPR-Cas9 technology in the present invention, A is a schematic diagram of the cell line construction; B is the PCR identification result; C is the expression of Oct4 determined by immunofluorescence; D is the expression of Sox2 determined by immunofluorescence; E is the expression of SSEA1 determined by immunofluorescence; F is the expression of Pou5f1, Brachyury, lsl1 and Tnnt2 determined by RT-qPCR; G is the expression of Tnnt2 determined by immunofluorescence.

[0023] Figure 2 Validation and pluripotency analysis of the Tnnt2 H2B-mCherry reporter cell line. A is a schematic diagram of the cTnT H2B-mCherry plasmid; B is the PCR identification result; C is the Sanger sequencing result; D is the expression of Pou5f1, Sox2, and Nanog in the cell line pluripotency.

[0024] Figure 3 The CCK-8 method was used to determine the effects of 48-hour culture with different concentrations of extracts on the activity of mESCs. A is the result of Ganoderma lucidum extract administration; B is the result of Salvia miltiorrhiza extract administration; and C is the result of combination administration.

[0025] Figure 4Figure 2 shows the effects of Ganoderma lucidum, Salvia miltiorrhiza extracts and their combination on the in vitro differentiation of mouse embryonic stem cells. A is a schematic diagram of the induction differentiation method of hanging drop-suspension-adherent culture; B is the percentage of beating EBs in each group; C is the proportion of fluorescent cells in each group; is the expression level of the cardiomyocyte-specific gene Tnnt2; E is the expression level of the cardiomyocyte-specific gene Myh6; F is the expression level of the cardiomyocyte-specific gene Myh7. * P <0.05, *** P <0.001, **** P <0.0001.

[0026] Figure 5 The effects of Ganoderma lucidum, Salvia miltiorrhiza extracts and their combination on cardiomyocyte differentiation, A is the expression level of Isll gene; B is the expression level of Mef2c gene; C is the expression level of Gata4 gene, * P <0.05, *** P <0.001, **** P <0.0001.

[0027] Figure 6 This is a differential analysis of the effects of Ganoderma lucidum, Salvia miltiorrhiza extracts and their combination on promoting myocardial differentiation. A is the expression levels of cardiac development-related genes Tnnt2, Tbx20 and Gata4; B is the expression levels of digestion-related genes Gata5, Hoxd13 and Lefty2. *** P <0.001, **** P <0.0001. DETAILED DESCRIPTION

[0028] The present invention will be further described in detail below with reference to specific examples. The following examples are not intended to limit the present invention but are merely intended to illustrate the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified.

[0029] Example 1 Construction of a vector for the Tnnt2 H2B-mCherry reporter cell line Using CRISPR-Cas9 genome editing technology, a fluorescent protein gene was knocked into the upstream of the start codon of the Tnnt2 gene by site-specific integration in mouse embryonic stem cells R1. Tnnt2 and fluorescent protein formed a fusion protein through the H2B linker peptide. The presence of H2B ensured that mCherry could be specifically expressed in the cell nucleus, enabling fusion expression. The Tnnt2 H2B-mCherry reporter cell line was screened and obtained. The specific steps are as follows: (1) Before transfection, the R1 cell confluency in a 35 mm culture dish reached 80%. 0.25% trypsin solution was added to digest the cells into single cells. 125 μL opti-MEM was mixed with 2.5 μg sgRNA expression vector and recombinant vector (plasmid mass ratio 1:2) in a ratio of 4 μL lipo8000 transfection reagent. The mixture was gradually added dropwise to the digested mouse embryonic stem cell R1. The cells were gently mixed and cultured in a 37°C 5% CO2 constant temperature incubator.

[0030] (2) 12 hours after transfection, replace the culture medium with fresh medium. After 36 hours of culture, digest the cells with 0.25% trypsin and select the cells with culture medium containing 500 μg / mL neomycin.

[0031] (3) After 10 days, single clones were selected under a microscope, passaged into 24-well plates, and the genomes of the single clones were amplified using PCR forward and reverse primers P1 and P2, and P3 and P4, respectively; P1 (SEQ ID NO.1): 5'-CCTTAATTAACAGTCCCTGTTCAGAGGTAAGACA-3'; P2 (SEQ ID NO.2): 5'-CAGTCACTATGGTCGACCGTAAGATACATTGATGAGTTTGG-3'; P3 (SEQ ID NO.3): 5'-CCGCTTCCTCGTGCTTTACG-3'; P4 (SEQ ID NO. 4): 5'-TTGGCGCGCCGTGACAGGACATCAAGACTCACTG-3'.

[0032] The PCR thermal cycle program was as follows: 98°C for 10 s, 60°C for 30 s, 72°C for 1 min, repeated 35 times, and 72°C for 5 min. The products were detected by 1% agarose gel electrophoresis. Based on the above PCR and identification results, a cell line with a single copy of the H2B-mCherry-polyA-mclNeo-polyA gene was obtained.

[0033] The construction model is as follows Figure 1As shown in A, a cell model with stable expression of fluorescent protein was formed. To ensure the site-specific insertion and accuracy of the protein, we used PCR ( Figure 1 B, Figure 2 B) and Sanger sequencing comparison ( Figure 2 C) The expression level of the Tnnt2 gene is directly indicated by fluorescence intensity, which allows real-time tracking and visualization of cardiomyocyte expression. ESCs are pluripotent cells derived from the inner cell mass of the pre-implantation blastocyst of a mouse or human host. ESCs show the ability to differentiate into cells of all three germ layers, so maintaining cell pluripotency is a prerequisite for inducing differentiation. To this end, we detected the expression of Pou5f1, Sox2, and SSEA1 in the Tnnt2 H2B-mCherry reporter cell line by immunofluorescence and RT-qPCR; the results showed that there was no significant difference in the expression of pluripotency genes in this cell line compared with the control ( P >0.05) ( Figure 1 CE, Figure 2 D). The differentiation of embryonic stem cells into cardiomyocytes in vitro goes through a continuous but staged process of gradually developing into mesoderm cells, cardiac progenitor cells, and functional cardiac myocytes. We used the hanging drop differentiation method to induce stem cells to differentiate into cardiac myocytes, such as Figure 1 As shown in F, the expression of the pluripotency transcription factor Pou5f1 continued to downregulate during the differentiation process, while the expression of the Brachyury gene showed a trend of first increasing and then decreasing, especially increasing significantly on days D4-D6 of differentiation ( P <0.01). Isl1 is a LIM-homeodomain transcription factor that is transiently expressed in SHF progenitor cells. Isl1 variants and deletions are associated with congenital heart disease because Isl1 is involved in the proliferation, survival, migration, and differentiation of SHF CPCs, which are important for the formation of the right ventricle, outflow tract, and most of the atrium. During differentiation, we found that Isl1 is gradually upregulated in the late mesoderm and is stably expressed from Day 6 to Day 10, which is consistent with previous studies ( Figure 1 F). As differentiation progresses, the cardiomyocyte marker Tnn2 gene gradually becomes expressed, accompanied by regular cell beating. Immunofluorescence results show that mCherry fluorescent protein colocalizes with the cell nucleus and that the green fluorescent protein Tnnt2 is expressed in the cytoplasm. These results demonstrate the successful construction of a Tnnt2 H2B-mCherry reporter cell line capable of indicating cardiomyocyte expression, enabling real-time observation of cardiomyocyte generation ( Figure 1 G).

[0034] Example 2 CCK-8 method to determine the effect of Danshen and Ganoderma lucidum water extracts on cell growth Preparation method of Danshen extract: Take the dried rhizome of Danshen, add water at a material-liquid ratio of 1 g:20 mL, boil for 1 hour, filter through a 0.22 μm filter membrane, and freeze-dry. The final purity is ≥80%, and the Danshen extract is obtained.

[0035] Preparation method of Ganoderma lucidum extract: After the Ganoderma lucidum fruiting body was crushed to 100 mesh, 100 g of Ganoderma lucidum fruiting body was added to 1 L of water and ultrasonic-assisted extraction was performed (40 kHz, 60°C, 30 min). The residue was added to 0.8 L of water and the extraction was repeated (40 kHz, 60°C, 30 min). The extracts were combined.

[0036] After the extraction is completed, the extract is concentrated and freeze-dried to obtain Ganoderma lucidum extract.

[0037] The preparation method of the traditional Chinese medicine composition is as follows: the salvia miltiorrhiza extract and the ganoderma lucidum extract are mixed in a mass ratio of 1:1 to obtain the composition.

[0038] To examine the effects of different traditional Chinese medicine extracts on mouse embryonic stem cells (mESCs), we first screened cell viability using the CCK-8 assay. 100 μL of serially diluted Salvia miltiorrhiza extract, Ganoderma lucidum extract, and the traditional Chinese medicine combination (at concentrations of 0, 0.1, 0.3, 0.5, 0.8, 1, and 1.5 mg / mL) was added to a 96-well plate and incubated for 48 hours. After 48 hours, CCK-8 reagent was added at a ratio of 1:10 per well to the volume of cell suspension. After incubation for 2 hours at 37°C, the OD values ​​were measured at 450 nm using a microplate reader. Cell morphology and cell number changes were recorded using photographs.

[0039] The results are as follows Figure 3 As shown, Salvia miltiorrhiza ( Figure 3 A), Ganoderma lucidum water extract ( Figure 3 B) and its combination ( Figure 3 C) caused a dose-dependent reduction in cell proliferation. At a Ganoderma lucidum aqueous extract concentration of 1.0 mg / mL, almost no cells survived, and at a Ganoderma lucidum aqueous extract concentration of 0.1 mg / mL, cell viability was unaffected. When the Salvia miltiorrhiza aqueous extract concentration was below 0.5 mg / mL, cell viability was unaffected. When the Salvia miltiorrhiza aqueous extract concentration exceeded 0.5 mg / mL, cell viability began to decrease with increasing concentration. At a concentration of 0.3 mg / mL, cell viability was unaffected, but when the concentration of the composition exceeded 0.3 mg / mL, cell viability began to decrease with increasing concentration.

[0040] Example 3 Effects of Ganoderma Lucidum and Salvia Miltiorrhiza Water Extracts on Cardiomyocyte Differentiation In this embodiment, the concentration of the Ganoderma lucidum water extract is 0.3 mg / mL; the concentration of the Salvia miltiorrhiza water extract is 0.1 mg / mL; the concentration of the composition is 0.3 mg / mL, and the composition is obtained by mixing the Salvia miltiorrhiza extract and the Ganoderma lucidum extract in a mass ratio of 1:1.

[0041] like Figure 4 Figure A shows the induction differentiation method using a hanging drop-suspension-adherent culture approach. After 2 days of differentiation culture, ES cells aggregated to form spherical or nearly spherical embryonic bodies (EBs). After 5 days of suspension culture, the EBs gradually increased in size, with stratification of the suspended bodies and a dense concentration of cells in the middle. As the EBs continued to adhere to the wall, one or more contracted cell clusters appeared in the EBs. The proportion of beating EBs was observed and counted under an inverted fluorescence microscope. To further validate the promoting effects of Danshen and Ganoderma on cardiomyocyte differentiation, we examined the expression of cardiomyocyte-specific genes by RT-PCR and real-time PCR.

[0042] The results are as follows Figure 4 The percentage of beating EBs in the group with Danshen, Ganoderma lucidum extract and their combination shown in B was significantly higher than that in the control group. The proportion of fluorescent cells also showed a higher proportion ( Figure 4 C), and the effect of the mixed administration group was the most significant ( P <0.01). In addition, the aqueous extracts of Danshen and Ganoderma lucidum significantly increased the expression of myocardial-specific genes Tnnt2, Myh6, and Myh7 ( Figure 4 DF). In conclusion, both Danshen and Ganoderma lucidum aqueous extracts can promote the differentiation of ESCs into cardiomyocytes, and the effect of Danshen is relatively more significant.

[0043] according to Figure 4 C The results showed that after 15 days of differentiation culture, the differentiation ratio of cardiomyocytes in the control group was 18%, the differentiation ratio of cardiomyocytes in the Salvia miltiorrhiza extract group reached 36%, the differentiation ratio of cardiomyocytes in the Ganoderma lucidum water extract group increased to 43%, and the differentiation ratio of cardiomyocytes in the mixed administration group was the highest, reaching 51%.

[0044] Example 4: Aqueous extracts of Salvia miltiorrhiza and Ganoderma lucidum promote the expression of CPC myocardial-related genes To understand how Ganoderma lucidum aqueous extract and Salvia miltiorrhiza extract affect the induction of ESC-derived cardiomyocytes, we performed RNA-seq analysis on cell samples at day 15 of differentiation. The results showed that both Ganoderma lucidum aqueous extract and Salvia miltiorrhiza extract modulated the Wnt signaling pathway compared to the control group. It is well known that the Wnt / β-catenin signaling pathway is an evolutionarily conserved pathway that plays a crucial role in cardiac development, with its activation crucial for inducing cardiomyocyte differentiation. Several studies investigating the in vitro differentiation of embryonic stem cell lines into cardiomyocytes have demonstrated the role of canonical Wnt molecules in promoting cardiomyocyte differentiation. Activating the Wnt / β-catenin signaling pathway during the early stages of differentiation promotes the differentiation of mouse embryonic stem cells into mesoderm and cardiomyocytes. Furthermore, the genes induced by Ganoderma lucidum aqueous extract and Salvia miltiorrhiza extract were enriched in cardiac-related signaling pathways, including ventricular development, cardiac contraction, and myocardial development.

[0045] To further reveal the stage of action of the extracts, we detected the expression of Isll, Mef2c, and Gata4, genes related to the development of myocardial progenitor cells (CPCs). Interestingly, these genes were upregulated after the addition of Ganoderma lucidum water extract and Salvia miltiorrhiza extract ( Figure 5 AB). The review results show that Danshen and Ganoderma lucidum water extracts can promote the formation of CPC, thereby producing more cardiomyocytes.

[0046] Example 5: Differential Analysis of the Effects of Salvia miltiorrhiza and Ganoderma lucidum Water Extracts on Promoting Cardiac Differentiation Previous results showed that both Ganoderma lucidum water extract and Salvia miltiorrhiza extract can induce more cardiomyocytes. The two may have similar induction mechanisms, but the induction effect of Salvia miltiorrhiza extract seems to be stronger than that of Ganoderma lucidum extract. For this reason, we further explored the different mechanisms of the two in cardiomyocyte induction. Heat map analysis showed that the genes induced by Ganoderma lucidum water extract and Salvia miltiorrhiza extract can be divided into three categories. Compared with the control, Salvia miltiorrhiza extract had the highest number of activated genes, followed by Ganoderma lucidum extract. GO analysis showed that the genes activated by Ganoderma lucidum extract were related to anterior-posterior axis distance, digestive tract / system development, and carbon dioxide transport. However, the genes induced by Salvia miltiorrhiza extract were enriched in DNA replication, chromosome organization, and cell cycle regulation, which will contribute to gene expression activation and cell proliferation. Consistent with this finding, genes related to heart development, such as Tnnt 2, Tbx20, and Gata4, were expressed at higher levels after treatment with Salvia miltiorrhiza extract than those with Ganoderma lucidum extract, and the expression level in the drug combination group was the highest ( Figure 6 A). The expression of genes related to anterior-posterior pattern regionalization and digestion in the Ganoderma lucidum extract group was significantly higher than that in the Salvia miltiorrhiza extract group and the control group, and the expression level in the drug combination group was the highest ( Figure 6B). The above results suggest that Ganoderma lucidum extract has an effect on cardiac development, while Salvia miltiorrhiza extract can activate gene expression and promote cardiac development by regulating DNA replication, cell cycle and chromosome organization.

[0047] The present invention discovered for the first time that Ganoderma lucidum, Salvia miltiorrhiza and their combination can promote the differentiation of mouse embryonic stem cells into cardiomyocytes. The present invention uses Ganoderma lucidum, Salvia miltiorrhiza and their combination to efficiently and selectively induce the differentiation of undifferentiated mouse embryonic stem cells into cardiomyocytes. By adding Ganoderma lucidum, Salvia miltiorrhiza and their combination, mouse embryonic stem cells can be differentiated into beating cardiomyocytes in 5-7 days. After 15 days of culture, the differentiation rate of cardiomyocytes in the control group was 18%, the differentiation rate of cardiomyocytes in the Salvia miltiorrhiza extract group reached 36%, the differentiation rate of cardiomyocytes in the Ganoderma lucidum water extract group increased to 43%, and the mixed administration group had the highest differentiation rate of cardiomyocytes, reaching 51%. The above experimental data show that the Ganoderma lucidum, Salvia miltiorrhiza and their combination provided by the present invention can significantly improve the efficiency of inducing mouse embryonic stem cells to differentiate into cardiomyocytes, which provides a good cell resource for heart-related medical research such as human heart development, disease research, drug development, toxicity analysis and cell replacement therapy, and also provides new insights into the development of culture medium strategies for cardiomyocyte differentiation.

[0048] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A Chinese medicine composition for promoting the differentiation of embryonic stem cells into cardiomyocytes, characterized in that: The composition comprises Ganoderma lucidum extract and Salvia miltiorrhiza extract.

2. The Chinese medicine composition according to claim 1, characterized in that The mass ratio of the Ganoderma lucidum extract to the Salvia miltiorrhiza extract is 0.01-0.1:0.05-0.

5.

3. The Chinese medicine composition according to any one of claims 1 to 2, characterized in that The preparation method of the ganoderma lucidum extract comprises the following steps: crushing the ganoderma lucidum fruiting bodies, performing ultrasonic-assisted extraction, concentrating, and then freeze-drying.

4. The Chinese medicine composition according to claim 3, characterized in that The ultrasonic-assisted extraction conditions are: 35-45 kHz, 55-65°C.

5. The Chinese medicine composition according to any one of claims 1 to 2, characterized in that The preparation method of the salvia miltiorrhiza extract comprises the following steps: taking dried salvia miltiorrhiza rhizomes, adding water according to a material-liquid ratio of 1 g:15-20 mL, boiling, filtering through a membrane, and then freeze-drying.

6. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a product for promoting the differentiation of embryonic stem cells into cardiomyocytes.

7. A culture medium for promoting differentiation of embryonic stem cells into cardiomyocytes, characterized in that: The culture medium comprises the traditional Chinese medicine composition according to any one of claims 1 to 6.

8. A method for inducing and cultivating cardiomyocytes from stem cells in vitro, characterized in that: The stem cells are cultured in the culture medium according to claim 7.

9. The method according to claim 8, characterized in that The following steps are involved: (1) Stem cells are made into single cell suspension; (2) After counting the single cell suspension, dilute it with differentiation medium to a cell concentration of 10 3 -10 5 cells / mL stem cell suspension, inoculated and cultured; (3) Collect the embryoid bodies formed in the culture, transfer them to a container containing differentiation medium, and culture them in an incubator to promote further proliferation of the embryoid bodies; (4) The further proliferated embryoid bodies are transferred and culture medium that promotes the differentiation of embryonic stem cells into cardiomyocytes is added for intervention differentiation culture.

10. A cell line for detecting differentiation of embryonic stem cells into cardiomyocytes in the method according to any one of claims 8 to 9, characterized in that: The cell line is constructed by knocking a fluorescent protein gene into the upstream of the start codon of the Tnnt2 gene, forming a fusion protein with the fluorescent protein via an H2B connecting peptide segment, and expressing the fusion protein in the cell.

Images