Composition for promoting stem cell proliferation and application

Abstract

The invention discloses a composition for promoting stem cell proliferation and application, and belongs to the technical field of stem cells. The preparation method comprises the following steps: firstly, preparing a mesona chinensis benth extract, and then mixing reduced glutathione and the mesona chinensis benth extract in proportion to obtain the proliferation compound. In EdU cell proliferation detection and cell cycle detection experiments, a compound group using the proliferation compound is superior to other experimental groups using a single additive, the proliferation compound is added to generate a synergistic effect, proliferation and growth of human mesenchymal stem cells are promoted, the division and proliferation speed of the human mesenchymal stem cells is increased, and the proliferation effect of the human mesenchymal stem cells is improved. And the method has very important value for cell culture.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a composition for promoting stem cell proliferation and its application. Background Art

[0002] Stem cells are a type of multipotent cells with the ability to self-renew. Under certain conditions, they can differentiate into a variety of functional cells or tissues and organs. The medical community calls them "universal cells." Mesenchymal stem cells (MSCs) are an important member of the stem cell family. They originate from the mesoderm and ectoderm in the early stages of development and can differentiate into a variety of interstitial tissues, such as bone, cartilage, fat, and bone marrow hematopoietic tissue. Although stem cells have multidirectional differentiation capabilities, stem cells from different sources have different functional properties and cannot be directly replaced. Stem cells are generally frozen at a deep cryogenic temperature of -196°C so that stem cells can be revived and used to treat diseases when clinically needed. How to resuscitate stem cells in a short period of time and obtain a large number of stem cells with the ability to divide rapidly, and to shorten the time it takes to proliferate to the required number, is an urgent problem to be solved.

[0003] Glutathione is a tripeptide composed of glutamic acid, cysteine ​​and glycine. Glutathione exists in the body in two forms: reduced glutathione (GSH) and oxidized glutathione (GSSG). Its active ingredient is reduced glutathione, which can participate in the redox process in the body. Glutathione can activate a variety of enzymes, such as sulfhydryl (-SH) enzymes and coenzymes in the body, thereby promoting the metabolism of carbohydrates, fats and proteins. It can also affect the metabolic process of cells and is an important regulatory metabolic substance in cells.

[0004] Mesona chinensis Benth. (or Platostoma palustre (Blume) AJ Paton), also known as Mesona chinensis Benth., is an annual herbaceous perennial plant of the genus Mesona in the family Lamiaceae. Mesona chinensis Benth is up to 1 meter tall, with branches and stems covered with soft hairs and fine bristles, which fall off later; leaves are narrow ovate or nearly round, 2-5 cm long; cymes form terminal racemes; small nuts are black and oblong. It is produced in southern my country, growing by ditches and in grass on dry sandy land. Mesona chinensis is both a medicine and a food, with the effects of relieving heat and clearing away heat, cooling blood and detoxifying. It is mainly used to treat heatstroke, diabetes, jaundice, toothache caused by wind and fire, burns, etc.

[0005] Although people in various circles have a certain understanding of the effects of glutathione and grass jelly, for a long time, there has been no report on the combined use of the above two substances, and the impact of adding them on bone marrow mesenchymal stem cells is also unknown. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a composition for promoting stem cell proliferation and its application.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] A first aspect of the present invention provides a composition for promoting stem cell proliferation, wherein the composition is prepared by mixing grass jelly extract and glutathione in a mass ratio of (0.5-5) g:(0.5-5) g.

[0009] Furthermore, the stem cells are human bone marrow mesenchymal stem cells.

[0010] Furthermore, the glutathione is reduced glutathione.

[0011] Furthermore, the mass ratio of the herb jelly extract to the reduced glutathione is 1g:1g.

[0012] Furthermore, the herb extract is prepared by the following method:

[0013] Take the whole plant of fresh grass jelly, remove the roots, wash it, cut it into long sections, dry it in an oven at 60°C, crush it to 400 mesh and sieve it to obtain grass jelly powder; extract the grass jelly powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:5ml, each extraction time is 8h, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 60°C, and then dried in an oven at 75°C to obtain grass jelly extract.

[0014] The second aspect of the present invention provides use of the composition in preparing a product for promoting stem cell proliferation.

[0015] Furthermore, according to the volume of the culture medium, the composition is added to the stem cell culture medium at a ratio of 10-100 ng / ml.

[0016] Beneficial effects of the present invention:

[0017] (1) The present invention takes fresh herb jelly, dries and crushes it, extracts it at room temperature, and concentrates it under reduced pressure, and dries it again to obtain herb jelly extract. Then, reduced glutathione and herb jelly extract are mixed in proportion to obtain a proliferation complex.

[0018] (2) The present invention uses the proliferation complex for experiments. In the verification of cell proliferation ability, the number of cells in each experimental group increased compared with the control group, indicating that the addition of the proliferation complex to the culture medium has a promoting effect on cell proliferation. Furthermore, in the EdU cell proliferation detection, the complex group using the proliferation complex was superior to other experimental groups using a single additive, and produced a 29.3% increase compared with the control group; in the cell cycle detection experiment, the complex group using the proliferation complex was superior to other experimental groups using a single additive, and the proportion of cells in the G2 / M phase and S phase increased by 34.8% compared with the control group. This shows that compared with the glutathione group and the fairy grass group, the addition of the proliferation complex produced a synergistic effect, promoted the proliferation and growth of stem cells, and accelerated their division and proliferation rate. DETAILED DESCRIPTION

[0019] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0020] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0021] Bone marrow mesenchymal stem cells are the most widely studied mesenchymal stem cells. However, the proliferation ability of bone marrow mesenchymal stem cells is not superior to that of other tissue sources. Bone marrow mesenchymal stem cells showed chromosomal abnormalities and telomerase shortening when cultured to the 18th generation. In addition, with the increase of age, the number and proliferation ability of bone marrow mesenchymal stem cells decreased significantly. Therefore, how to improve the ability of bone marrow mesenchymal stem cells by improving the culture medium environment is very important for the application prospects of bone marrow mesenchymal stem cells, thus forming the present invention.

[0022] The experimental materials used in the embodiments of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. The human bone marrow mesenchymal stem cells (article number CP-H166) and their complete culture medium (article number CM-H166) used in this application were purchased from Wuhan Pronosai Life Science Technology Co., Ltd.; the CAS number of the reduced glutathione (GSH) used is 70-18-8.

[0023] Example 1

[0024] Preparation of Herba Jelly Extract

[0025] Take the whole plant of fresh grass jelly, remove the roots, wash it, cut it into long sections, dry it in an oven at 60°C, crush it to 400 mesh and sieve it to obtain grass jelly powder; extract the grass jelly powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:5ml, each extraction time is 8h, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 60°C, and then dried in an oven at 75°C to obtain grass jelly extract.

[0026] Example 2

[0027] Preparation of proliferation complex

[0028] The Herba Immortalis extract prepared in Example 1 and reduced glutathione were mixed in a mass ratio of 1 g:1 g to obtain a proliferation complex.

[0029] Example 3

[0030] Preparation of proliferation complex

[0031] The Herba Immortalis extract prepared in Example 1 and reduced glutathione were mixed in a mass ratio of 2 g:1 g to obtain a proliferation complex.

[0032] Example 4

[0033] Preparation of proliferation complex

[0034] The Herba Immortalis extract prepared in Example 1 and reduced glutathione were mixed in a mass ratio of 1 g:2 g to obtain a proliferation complex.

[0035] Test Example 1: Verification of cell proliferation ability

[0036] In order to verify the cell proliferation ability, the proliferation complexes prepared in Examples 2 to 4 were used for proliferation experiments.

[0037] In the proliferation experiment, the cells selected were human bone marrow mesenchymal stem cells, the culture medium used in the control group was human bone marrow mesenchymal stem cell complete culture medium (containing 5% FBS and 1% penicillin-streptomycin double antibody), and each experimental group was respectively added with the proliferation complex prepared in Example 2-Example 4 at a ratio of 40ng / ml on the basis of human bone marrow mesenchymal stem cell complete culture medium (containing 5% FBS and 1% penicillin-streptomycin double antibody), and was named Example 2 Group-Example 4 Group. 5000 cells / well were inoculated into different culture media in 96-well plates, and cultured continuously under the same conditions. When the cells filled the well plate, they were all digested and transferred to larger well plates (48-well plate, 24-well plate, 12-well plate, 6-well plate) in turn. Cell counts were performed on the third day, the sixth day and the ninth day, respectively, to examine the number of cells in each group.

[0038] According to the results, on the third, sixth and ninth days, the number of cells in each experimental group increased compared with the control group, and the difference was significant (P < 0.05), indicating that the addition of the proliferation complex prepared in Examples 2 to 4 to the culture medium promoted cell proliferation, among which the Example 2 group had the best effect, and subsequent experiments were carried out according to this experimental ratio.

[0039] Experimental Example 2: Cell proliferation and cell cycle detection

[0040] (1) Cell proliferation assay

[0041] EdU is a thymine nuclear analog, and Cy3 azide has a biologically unique group. After the two are connected, they can fluorescently label the DNA of proliferating cells, and then quantitatively detect them by flow cytometry. The present invention uses Cy3azide and EdU to fluorescently label the DNA of proliferating cells after connection, and detects them by flow cytometry (the maximum excitation wavelength of Cy3 azide is 555nm, and the maximum emission wavelength is 570nm). The specific operation method is as follows:

[0042] An equal amount of human bone marrow mesenchymal stem cells were seeded in a 6-well plate. After the cells were cultured overnight and recovered to a normal growth state, the culture medium was replaced with a culture medium: each experimental group was respectively added with glutathione, the Herba Immortas extract prepared in Example 1, and the proliferation complex prepared in Example 2 at a ratio of 40 ng / ml based on the volume of the culture medium on the basis of a complete culture medium for human bone marrow mesenchymal stem cells (containing 5% FBS), and were named the glutathione group, the Herba Immortas extract group, and the complex group; the control group was added with PBS at a ratio of 40 ng / ml based on the volume of the culture medium on the basis of a complete culture medium for human bone marrow mesenchymal stem cells (containing 5% FBS).

[0043] After culturing to an appropriate cell density, the EdU Flow Cytometry Assay Kits (Cy3) from APE BIO was used for detection. The results are shown in Table 1:

[0044] Table 1 Proportion of EdU+ cells

[0045]

[0046] According to the results in Table 1, analysis of the cell proliferation detection results of the control group and the experimental group shows that, compared with the control group, the proportion of EdU+ cells in each experimental group is increased, especially in the complex group, which has an increase of 29.3% compared with the control group, indicating that the addition of the proliferation complex prepared in Example 2 promotes the proliferation and growth of stem cells and accelerates their division and proliferation rate.

[0047] (2) Cell cycle detection

[0048] An equal amount of human bone marrow mesenchymal stem cells were seeded in a 6-well plate. After the cells were cultured overnight and recovered to a normal growth state, the culture medium was replaced with a culture medium: each experimental group was respectively added with glutathione, the Herba Immortas extract prepared in Example 1, and the proliferation complex prepared in Example 2 at a ratio of 40 ng / ml based on the volume of the culture medium on the basis of a complete culture medium for human bone marrow mesenchymal stem cells (containing 5% FBS), and were named the glutathione group, the Herba Immortas extract group, and the complex group; the control group was added with PBS at a ratio of 40 ng / ml based on the volume of the culture medium on the basis of a complete culture medium for human bone marrow mesenchymal stem cells (containing 5% FBS).

[0049] After culturing to an appropriate cell density, the cells were tested using the Bio-Tech Cell Cycle and Apoptosis Analysis Kit, and the proportion of cells in the G2 / M phase and S phase was analyzed using flow cytometry. The results are shown in Table 2:

[0050] Table 2 Proportion of cells in G2 / M phase and S phase

[0051]

[0052] According to the results in Table 2, analysis of the cell cycle detection results of the control group and the experimental group shows that, compared with the control group, the proportion of cells in the G2 / M phase and the S phase in each experimental group is increased, especially in the complex group, which has an increase of 34.8% compared with the control group, indicating that the addition of the proliferation complex prepared in Example 2 promotes the proliferation and growth of stem cells and accelerates their division and proliferation rate.

[0053] Therefore, the results of EdU cell proliferation assay and cell cycle assay both showed that adding proliferation complex to the culture medium had a good promoting effect on the proliferation of human bone marrow mesenchymal stem cells.

[0054] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. 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. A composition for promoting stem cell proliferation, characterized in that: In the composition, the herb jelly grass extract and glutathione are mixed in a mass ratio of (0.5-5) g: (0.5-5) g.

2. The composition for promoting stem cell proliferation according to claim 1, characterized in that: The stem cells are human bone marrow mesenchymal stem cells.

3. The composition for promoting stem cell proliferation according to claim 1, characterized in that: The glutathione is reduced glutathione.

4. The composition for promoting stem cell proliferation according to claim 3, characterized in that: The mass ratio of the herb jelly extract to reduced glutathione is 1g:1g.

5. The composition for promoting stem cell proliferation according to claim 1, characterized in that: The herb jelly extract is prepared by the following method: Take the whole plant of fresh grass jelly, remove the roots, wash it, cut it into long sections, dry it in an oven at 60°C, crush it to 400 mesh and sieve it to obtain grass jelly powder; extract the grass jelly powder in 75% v / v ethanol at room temperature with a mass volume ratio of 1g:5ml, each extraction time is 8h, and the extraction is performed 3 times in total; the extracts obtained each time are combined, concentrated under reduced pressure at 60°C, and then dried in an oven at 75°C to obtain grass jelly extract.

6. Use of the composition according to any one of claims 1 to 5 in the preparation of a product for promoting stem cell proliferation.

7. The use according to claim 6, characterized in that: According to the volume of the culture medium, the composition of claim 1 is added to the stem cell culture medium at a ratio of 10-100 ng / ml.

Images