阅读说明：本技术 一种干细胞增殖促进剂及其应用 (Stem cell proliferation promoter and application thereof ) 是由 杨绪奎 赵姣 岳建辉 于 2021-04-26 设计创作，主要内容包括：本发明公开了一种干细胞增殖促进剂及其应用,属于干细胞培养技术领域。该干细胞增殖促进剂是使用微波反应釜从大豆中提取获得,具体制备方法包括以下步骤：(1)物料前处理：将大豆筛选并去掉杂质；(2)将准备好的物料放入微波反应釜中处理2-3min,微波频率为2045MHz,冷凝回收气化出的大豆精华,即获得干细胞增殖促进剂,其可以添加到完全培养基中混合后使用,其中完全培养基：干细胞增殖促进剂的体积比为3-5:1。(The invention discloses a stem cell proliferation promoter and application thereof, and belongs to the technical field of stem cell culture. The stem cell proliferation promoter is extracted from soybeans by using a microwave reaction kettle, and the specific preparation method comprises the following steps: (1) pretreatment of materials: screening soybean and removing impurities; (2) placing the prepared materials into a microwave reaction kettle for treatment for 2-3min, wherein the microwave frequency is 2045MHz, condensing and recovering the gasified soybean essence to obtain the stem cell proliferation promoter, which can be added into a complete culture medium for mixing and then using, wherein the complete culture medium: the volume ratio of the stem cell proliferation promoter is 3-5: 1.)

1. A stem cell proliferation promoter, which is extracted from soybean by using a microwave cleavage reaction kettle.

2. The stem cell proliferation promoter according to claim 1, wherein the specific production method of the stem cell proliferation promoter comprises the steps of:

(1) pretreatment of materials: screening soybean and removing impurities;

(2) and (3) placing the prepared materials into a microwave cracking reaction kettle for treatment for 2-3min, and condensing and recycling the gasified soybean essence to obtain the stem cell proliferation promoter.

3. The stem cell proliferation promoter according to claim 2, wherein the treatment frequency of the microwave reaction vessel in the step (2) is 2045 MHz.

4. The stem cell proliferation promoter according to any one of claims 1 to 3, wherein the stem cell is a mesenchymal stem cell.

5. Use of the stem cell proliferation promoter according to any one of claims 1 to 3 in stem cell culture.

6. A culture medium composition for stem cells, which comprises the complete medium DMEM F12+ 10% FBS and the stem cell proliferation promoter according to any one of claims 1 to 3.

7. The media combination of claim 6, wherein the total media: the volume ratio of the stem cell proliferation promoter is 3-5: 1.

8. A method for promoting the proliferation of stem cells, which comprises culturing stem cells in a medium containing the stem cell proliferation promoter according to any one of claims 1 to 3.

Technical Field

The invention belongs to the technical field of stem cell culture, and particularly relates to a stem cell proliferation promoter and application thereof.

Background

Stem cells are a type of pluripotent cells with self-replicating capacity (self-rejuvenating). Under certain conditions, it can differentiate into a variety of functional cells. The stem cells are classified into embryonic stem cells (ES cells) and adult stem cells (stromal cells) according to the developmental stage in which the stem cells are located. The stem cells are classified into three categories according to their developmental potential: totipotent Stem Cells (TSC), pluripotent stem cells (pluripotent stem cells) and unipotent stem cells (multipotent stem cells). Stem cells (Stem cells) are insufficiently differentiated and immature cells, have the potential function of regenerating various tissues, organs and human bodies, and are called "universal cells" in the medical field.

Mesenchymal Stem Cells (MSCs) are important members of embryonic stem cells, are derived from early-developing mesoderm, belong to pluripotent stem cells, and are originally found in bone marrow, so that MSCs are increasingly concerned by the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self-replication and the like. Under the specific induction condition in vivo or in vitro, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelium and the like, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes. Especially, umbilical cord Mesenchymal Stem Cells (MSCs) are multifunctional Stem Cells existing in umbilical cord tissues of newborn, can be differentiated into a plurality of tissue Cells, and have wide clinical application prospects.

In the prior art, multi-component promoters are usually adopted for stem cell culture, and the preparation and the use are relatively complicated.

Disclosure of Invention

The invention aims to provide a stem cell proliferation promoter, which is extracted from soybeans by using a microwave reaction kettle.

Further, the specific preparation method of the stem cell proliferation promoter comprises the following steps:

(1) pretreatment of materials: screening soybean and removing impurities;

(2) and (3) placing the prepared materials into a microwave reaction kettle for treatment for 2-3min, and condensing and recovering the gasified soybean essence to obtain the stem cell proliferation promoter.

Furthermore, the treatment frequency of the microwave reaction kettle in the step (2) is 2045 MHz.

Further, the stem cell is a mesenchymal stem cell.

The invention also aims to provide the application of the stem cell proliferation promoter in stem cell culture.

It is a further object of the present invention to provide a combination of a culture medium for stem cells, which comprises a complete medium and a stem cell growth promoter.

Further, the combination of media is complete media: the volume ratio of the stem cell proliferation promoter is 3-5: 1.

It is a fourth object of the present invention to provide a method for promoting the proliferation of stem cells, which comprises culturing stem cells in a medium containing a stem cell proliferation promoter.

The soybean extraction essence provided by the invention has a water molecular cluster structure, the size of the water molecular cluster is in direct relation with the physical properties of the water molecular cluster, water with small molecular clusters has the characteristics of strong permeability, high solubility, good cell affinity and the like, and the change of the size of the water molecular clusters can change the action of the water in organisms (Zhang Jianping, Zhao Lin, Tanxin, the change of the structure of the water molecular clusters and the biological effect thereof [ J]Chemical notice, 2004, (4): 278-283).¹⁷The O-nmr method is currently the main method used to characterize the structure of water clusters,¹⁷the half-width of O-NMR can be used to characterize the size of water clusters (Li R, Jiang Z, Shi S et al Raman spectra and¹⁷O-NMR study effects ofCaCl₂ andMgCl₂on water structu re[J]JMol Struct, 2003, 645 (1): 69-75 of ion-water-binding type for Liruihua, Jiangxingg, Yang Wei, etc¹⁷O-NMR chemical shift and water structure).

According to the existing latest and extensive plant targeted metabolic component detection technology, the soybean essence is analyzed to contain 656 small molecular compounds which are all small molecular compounds below 1000Da, and is rich in various secondary metabolite small molecular compounds of soybeans (90 phenolic acids, 90 amino acids and derivatives thereof, 90 alkaloids and terpenes, 82 organic acids, 80 flavonoids, 70 lipids, sugar and alcohols and other 67 types, 51 nucleotides and derivatives thereof, 23 lignin types and 13 vitamins).

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a method for culturing mesenchymal stem cells, which can improve the proliferation efficiency of stem cells by 3-4 times after conventional culture for 72 hours without adding biochemical activator components for culturing stem cells such as cytokines such as human epidermal growth factor, human basic fibroblast growth factor and the like, and simultaneously has the potential of reducing the adding proportion of animal serum.

Drawings

Fig. 1 is a report of the measurement of the size of water clusters of the soybean essence extracted in example 1.

Fig. 2 is a report of a broad targeted metabolome assay of the soy extract extracted in example 1.

FIG. 3 is a histogram of the statistical results of different concentrations of soy serum cultured cells in example 4.

Detailed Description

Example 1

The preparation method of the soybean essence extract comprises the following steps:

(1) pretreatment of materials: screening soybeans to remove impurities such as dust and the like, cleaning, draining and finishing until the sizes of the soybeans are consistent;

(2) placing the prepared materials into a microwave cracking reaction kettle, treating for 2-3min at microwave frequency of 2045MHz, and condensing to recover gasified soybean essence.

Distilling or gasifying soybean material under high frequency microwave, condensing and recovering to obtain soybean essence solution, collecting 500g of soybean essence solution from 5kg of soybean, adjusting pH of the soybean essence solution to about 8, and making the soybean essence solution be alkalescent¹⁷The O-NMR detection essence has a small molecular group structure size of 38.81HZ, and a specific detection report is shown in figure 1. The broad targeting metabolome assay report for soy serum is shown in figure 2.

Example 2

The safety performance test of the soybean essence comprises the following steps:

15 male mice and 15 female mice each having a body weight ranging from 20g to 23g were randomly selected and divided into 3 groups by a direct drinking method, one group was administered with no soybean essence (control group), one group was administered with 5mL of the soybean essence prepared in example 1 every 6 hours (test group 1), and the other group was administered with 1mL of the soybean essence prepared in example 1 every 6 hours (test group 2), and the feeding was continued for 6 months. The results show that in the test group 1 and the test group 2 within 1 week, animals do not die, and mice have normal activity and no difference from a control group; when the test group 1 and the test group 2 were tested at 6 months, the animals did not die and the mice were active normally. Through forced swimming experiments, the swimming time of the mice of the test group 1 and the test group 2 is obviously prolonged compared with that of the control group, and the mice show a little strength compared with the control group.

Example 3

The culture experiment of the soybean essence extract on the culture substitute serum of hirudo nipponia includes the following steps:

in the artificial culture of the hirudo nipponica, animal serum is needed to be used in the breeding period of the young seedlings to quickly grow so as to meet the requirement of hirudin production. Through comparison experiments, animal serum and soybean extracting solution are not added in 3 culture ponds respectively, the growth speed of the leech added with the soybean extracting solution is the fastest after one week of production, and the growth speed of the leech added with the animal serum is the second highest and exceeds the effect of promoting the leech growth by the serum. The soybean extract can replace animal serum to a certain extent to be used as a culture reagent of plant origin.

Example 4

The influence test of the soybean essence extract on cell proliferation comprises the following steps:

(1) taking skin mesenchymal stem cells (product number: BFN60808614) with good growth state, performing enzyme digestion by adopting a general passage method, and preparing the mesenchymal stem cells into a mesenchymal stem cell suspension by using a complete culture medium culture solution (DMEM F12+ 10% FBS).

(2) Counting with a cell counter, and adjusting cell density to 5 × 10⁴Per ml, 100. mu.L/well of cell suspension was added adherently to a 96-well plate (12 columns by 8 rows) to achieve a cell count of 5000 cells/well, consistent with the total cell count per well.

(3) After 24 hours, the medium in the 96-well plate was removed, and 100 μ L of medium containing samples of soybean essence (prepared in example 1) at different concentrations (soybean essence samples: complete medium volume ratios were 1:3, 1:5, 1:7, 1:9, 1:11, 1:13, 1:15, 1:17, 1:19, 1:21, respectively) was added to each 1 line, and 5 replicates of each volume ratio were set, while setting the complete medium as a blank control.

The complete medium is available under the trademark PM150312B, available from Wuhan Punuo Seiki Life technologies, Inc., and can be prepared by itself.

(4) The 96-well cell culture plate was placed at 37 ℃ in 5% CO₂And (5) incubating the incubator.

(5) After three days of cell culture, 10. mu.l of CCK-8 solution was added to each well of the 96-well plate, the plate was incubated in the incubator for 1-4 hours, absorbance at 450nm was measured with a microplate reader, and the mean value was calculated.

(6) After the detection of all the detection time points was completed, statistical analysis software was used, and a histogram was prepared with samples of different concentrations as the horizontal axis and the cell OD number as the vertical axis (logarithm), and the effect of the samples on the proliferation activity of mesenchymal stem cells was analyzed, and as a result, as shown in table 1 and fig. 3, as can be seen from table 1 and fig. 3, the OD value representing the stem cell concentration in the normal control was 0.429, the OD value representing the stem cell concentration in the combination of soybean essence samples at a ratio of 1:3 was 1.77, the efficiency was increased by 4.12 times, and the OD value representing the stem cell concentration in the combination of extract at a ratio of 1:5 was 1.386, and the efficiency was increased by 3.23 times. According to multiple experiments, the value-added efficiency of the extracting solution is best between 16.7% and 25% of DMEM F12+ 10% FBS.

In the above experiment, the addition ratio of the calf serum in FBS was 7.5%, which was lower than 10% in the normal case, and it was found that the soybean extract can replace FBS (calf serum) to some extent, and is an important culture reagent in stem cell culture.

TABLE 1

The invention provides a method for culturing mesenchymal stem cells, which can improve the proliferation efficiency of the stem cells by 3-4 times by conventional culture for 72 hours under the conditions of not adding biochemical activator components of stem cell culture such as cell factors and the like and reducing the addition of animal serum.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solution of the present invention is defined by the claims.