CN111518865B - Method for screening skin repair active ingredients based on mesenchymal stem cells - Google Patents
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Abstract
The application provides a method for screening skin repair active ingredients based on mesenchymal stem cells, and belongs to the technical field of biology. The screening method comprises the following steps: performing conventional culture and induction culture on active mesenchymal stem cells, and filtering the obtained cell culture solution to obtain a mesenchymal stem cell conditioned medium suitable for skin repair; and (3) performing activity screening on the plant crude extract to be screened by utilizing a mesenchymal stem cell conditioned medium, gradually separating the plant crude extract by combining a natural plant separation technology, performing activity screening on components obtained after each separation, and locking a separation target until the monomer active components are obtained. The method can screen out plant crude extracts with certain activity from complex systems such as traditional Chinese medicines, and then the crude extracts are separated into monomer compounds by adopting a phytochemistry research means, and the monomer compounds can promote mesenchymal stem cells to generate more bioactive substances which are beneficial to repairing damaged skin in the growth process.
Description
Technical Field
The application relates to the technical field of biology, in particular to a method for screening skin repair active ingredients based on mesenchymal stem cells.
Background
Stem cells (stem cells) are undifferentiated cells that have self-replication ability and are capable of differentiating into specific cells under specific conditions. Human stem cells can be broadly divided into two categories depending on their stage of development: embryonic stem cells (embryonic stem cell) and adult stem cells (somatic stem cell). Embryonic stem cells are derived from the inner cell mass of the embryo sac, and adult stem cells are present in various organs of the adult. Mesenchymal stem cells (Mesenchymal stem cell, MSC) are multipotent adult stem cells derived from early stages of development, mesoderm and ectoderm, first found in bone marrow by Friedenstein and his colleagues in the last 70 th century. Subsequently, through intensive and extensive research, it was found that mesenchymal stem cells exist in many tissues of the human body during the occurrence and development processes, such as umbilical cord, subcutaneous adipose tissue, brain, liver, lung, dental pulp, and the like. The mesenchymal stem cells belong to non-terminally differentiated cells, can be differentiated into various tissue cells such as fat, cartilage, bone, muscle, nerve, liver, cardiac muscle, islet beta cells, endothelium and the like under specific in-vitro induction conditions, and still have multidirectional differentiation potential after continuous subculture and cryopreservation. In addition, mesenchymal stem cells are convenient to obtain, are not ethically restricted, and generally do not elicit an immune response in the host. Therefore, although embryonic stem cells have strong self-renewal and differentiation ability into different types of cells, the application range of the embryonic stem cells is limited due to ethical problems, and mesenchymal stem cells in adult stem cells are outstanding in terms of safety and effectiveness of treatment, and have wide clinical application prospects in various alternative treatments and the like.
Mesenchymal stem cells can secrete immune-modulating factors (HGF, LIF), chemokines (RANTES, SDF-1 alpha, fractalkine, MIP-1 alpha, MCP-1, MCP-2), nutritional factors (IL-6, FGF-2, PDGF-AA, PDGF-BB, EGF), angiogenic factors (VEGF 165, FGF-2, PDGF-AA, PDGF-BB, EGF), scar-inhibiting factors (HGF, FGF-2), anti-apoptotic factors (VEGF 165, FGF-2, HGF), wound-healing-related factors (IL-6, IL-8, TGF-beta 1, MCP-1, VEGF, GM-CSF, TIMP-1), various collagens (I, II, III, IV type collagens), and the like. The cytokines can effectively regulate and control the cell signal transmission of the organism and activate the stem cells of the human body, thereby physiologically repairing or replacing the cells of the injury, pathological changes and aging of the organism. For example, fibronectin (Fibronectin) has wound healing efficacy as a skin constituent; vascular Endothelial Growth Factor (VEGF) promotes skin regeneration, tissue recovery and wound treatment, and regulates nutrients and epidermal cell proliferation; fibroblast growth factor (bFGF) promotes skin regeneration, recovery of aged tissues, treats wounds, regulates nutrients and epidermal cell proliferation, moves, and regulates differentiation; epithelial cell growth factor (EGF) promotes proliferation and migration of endothelial cells, enhancing regeneration and function of skin tissue; hepatocyte Growth Factor (HGF) activates cells, treating wound effects; transforming growth factor (TGF- β) restores damaged tissue and regulates proliferation of immune cells.
The cosmetic at the present stage basically has no biological activity, can not radically improve the living environment and metabolism of epidermal cells, has no repairing effect on skin injury caused by external factors, and is not suitable for the defects of skin sensitivity or pregnant women. By adding the cytokines of the mesenchymal stem cells into the beauty cosmetics, the beauty cosmetics not only have the moisturizing and whitening effects of common cosmetics, but also can repair damaged skin, eliminate skin wrinkles, shrink pores, improve complexion and the like, and are beneficial to fundamentally repairing injured tissues, promoting skin regeneration and improving skin elasticity and smoothness.
In view of the above, the application provides a method for screening skin repair active ingredients based on mesenchymal stem cells, which screens natural plant ingredients to obtain a small molecular compound capable of pertinently regulating and controlling the mesenchymal stem cells and having repair effect on damaged skin.
Disclosure of Invention
The application aims to provide a screening method of skin repair active ingredients based on mesenchymal stem cells, by the method, plant crude extracts with certain activity can be screened out from a complex system of traditional Chinese medicinal materials or compound medicines, and then the crude extracts are separated into monomer compounds by adopting a phytochemistry research means, and the monomer compounds can promote the mesenchymal stem cells to generate more bioactive substances which are beneficial to repairing damaged skin in the growth process.
In order to achieve the above object of the present application, the following technical solutions are specifically adopted:
a method for screening skin repair active ingredients based on mesenchymal stem cells, comprising:
performing conventional culture and induction culture on active mesenchymal stem cells, and filtering the obtained cell culture solution to obtain a mesenchymal stem cell conditioned medium suitable for skin repair;
and (3) performing activity screening on the plant crude extract to be screened by utilizing the mesenchymal stem cell conditioned medium, gradually separating the plant crude extract by combining a natural plant separation technology, performing activity screening on the components obtained after each separation, and locking a separation target until the monomer active components are obtained.
Further, in a preferred embodiment of the present application, the method for activity screening comprises: mixing the mesenchymal stem cell conditioned medium with a drug to be tested, and performing CO at 35-38 DEG C 2 After the culture is continued for 30-36 hours under the concentration of 7-10%, the absorbance of the culture solution at 490nm is detected by adopting an MTT method.
Further, in a preferred embodiment of the present application, the mesenchymal stem cells are derived from umbilical cord, placenta, fat, bone marrow or dental pulp.
Further, in a preferred embodiment of the present application, theThe conventional culturing steps include: inoculating the mesenchymal stem cells into DMEM culture medium, and performing CO at 35-38deg.C 2 Culturing at a concentration of 7-10% until the growth density of mesenchymal stem cells is above 90%.
Further, in a preferred embodiment of the present application, the step of inducing culture includes: adding astragaloside IV into DMEM medium to obtain induction medium, mixing the mesenchymal stem cells with the induction medium, and CO-culturing at 35-38deg.C 2 Culturing at 7-10% concentration for 36-48 hr.
Further, in a preferred embodiment of the present application, the method for preparing the crude plant extract to be screened comprises: mixing the dried plant powder with 60-80% ethanol, reflux extracting at 70-80deg.C for 1-2 hr, and concentrating the extractive solution.
The application has the following effects:
the inventor researches and discovers that a plurality of traditional Chinese medicines and compound medicines have promotion effect on proliferation and differentiation of mesenchymal stem cells, but because traditional Chinese medicine components are complex, different kinds of compounds can have obvious different effects on the mesenchymal stem cells, in order to screen and separate monomer compounds with specific promotion effect on proliferation and differentiation of the mesenchymal stem cells, the application provides a screening method of skin repair active ingredients based on the mesenchymal stem cells.
The method adopts a screening scheme guided by biological activity, firstly screens out crude plant extracts with certain activity, and then separates the crude plant extracts into single compounds by adopting a phytochemistry research means, wherein the process is guided by the activity. The natural micromolecules extracted from the plants obtained by screening are used for regulating the biological characteristics, states and functions of the mesenchymal stem cells, even the fate of the mesenchymal stem cells in vitro through regulating the specific targets related to cell metabolism, transcription and epigenetic, so that more various bioactive substances such as nerve growth factors, matrix erythrocyte-derived growth factors, vascular endothelial growth factors, epidermal growth factors, interleukin-6, interleukin-7, megakaryocyte colony stimulating factors, tumor necrosis factors, interferons and the like with active action on injury repair are generated in the growth process.
Meanwhile, the natural micromolecules extracted from the plants obtained by screening through the method have the capacity of adjusting the biological characteristics of mesenchymal stem cells. Compared with the genetically modified mesenchymal stem cells, the plant extracted natural small molecules have the characteristic advantages in operation and use: the extraction of natural small molecule regulated mesenchymal stem cells from plants provides dual controllability in time and space. In the action time, the natural small molecules extracted from the plants take effect rapidly, and the reversible effect can be achieved by removing the natural small molecules; in the space of action, the action of the plant to extract natural small molecules may not be limited to a single cell and tissue type. Furthermore, the concentration, the action time, the action mode, the single use or the combination and the like of natural small molecules extracted from plants can be regulated to effectively regulate the action effect. In view of the fact that different plants extract natural small molecules in a mode and target points have specificity, the natural small molecules extracted from the plants acting on the specific target points and regulating the phenotype of the mesenchymal stem cells can be found through reasonable design and screening, so that the effect of repairing damaged skin is better exerted.
Drawings
Fig. 1 is a flowchart of screening five Chinese medicinal materials based on mesenchymal stem cells in an embodiment of the present application.
Detailed Description
Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The present embodiment provides a method for screening skin repair active ingredients based on mesenchymal stem cells, and the flow chart is shown in fig. 1, and specifically, examples 1 and 2.
Example 1
The primary screening of the proliferation activity of the mesenchymal stem cells is carried out, and the specific method is as follows:
1. preparation of mesenchymal Stem cell conditioned Medium:
selecting active umbilical cord mesenchymal stem cells at 1x10 6 Inoculating the cells into DMEM medium, and culturing at 37deg.C and CO 2 Culturing at concentration of 8% until the growth density of mesenchymal stem cells is above 90%, discarding culture medium supernatant, and washing with PBS buffer solution for 2-3 times.
Adding astragaloside IV into DMEM medium, wherein the concentration of astragaloside IV is 0.05 μmol/L, to obtain induction medium, mixing the cleaned mesenchymal stem cells with induction medium, and mixing at 37deg.C and CO 2 Culturing for 42h at concentration of 8%, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain the final product.
2. Preparing a plant crude extract for screening:
5 Chinese medicinal materials are selected, which are kudzuvine root, red sage root, chinese angelica, szechuan lovage rhizome and officinal magnolia bark respectively. Respectively collecting 10g of each medicinal material dry powder, mixing with 50ml of 70% ethanol, heating and reflux-extracting at 75deg.C for 1.5h, repeating for 2 times, filtering the obtained extractive solution, concentrating, and re-suspending in 10ml of DMSO to obtain crude plant extractive solutions of five medicinal materials.
3. And (3) active primary screening:
mixing the mesenchymal stem cell conditioned medium prepared in the step 1 with crude plant extracts of five medicinal materials of radix puerariae, radix salviae miltiorrhizae, chinese angelica, rhizoma ligustici wallichii and magnolia officinalis respectively, wherein the volume of the plant extracts is 2% of that of the mesenchymal stem cell conditioned medium. The mixed culture medium is placed at 37 ℃ and CO 2 After incubation at 8% concentration for 32h, 5mg/mL MTT 10. Mu.L was added and incubation was continued for 4h, followed by addition of lysate (10% SDS,0.1% NH) 4 Cl) 100 μl, incubated overnight, the supernatant separated, and the absorbance measured at 490 nm.
4. Screening results:
TABLE 1 screening results for proliferation promoting Activity of mesenchymal Stem cells
| Radix Puerariae | Radix Salviae Miltiorrhizae | Chinese angelica | Ligusticum wallichii | Cortex Magnolia officinalis | Blank control | |
| Absorbance value | 0.22 | 0.29 | 0.18 | 0.24 | 0.16 | 0.17 |
From table 1, it can be seen that, of the five medicinal materials, the absorbance values of the four medicinal materials except magnolia officinalis are higher than that of the blank group, so that the four medicinal materials of kudzuvine root, red sage root, chinese angelica and szechuan lovage rhizome all have certain proliferation promoting activity of mesenchymal stem cells. Wherein, the promoting activity of the salvia is optimal.
Example 2
The method for separating the compound with the effect of promoting the proliferation of the mesenchymal stem cells from the red sage root comprises the following steps:
1. and (3) heating reflux extraction:
taking 2kg of red sage root medicinal material, mixing with water after crushing, mixing the mixture with water according to a feed-liquid ratio of 1:7, adding 0.5% sodium bicarbonate to adjust the pH value to 9, heating and reflux extracting for 1.5h, and separating an extracting solution; adding water into the residue according to a feed-liquid ratio of 1:7, adjusting pH, reflux-extracting for 1h, separating the extracting solution, combining the two extracting solutions, and filtering. 1% hydrochloric acid was added to the filtrate, the pH of the solution was adjusted to 3, the supernatant was kept by filtration, and concentrated under reduced pressure. Continuously adding ethanol to make the alcohol concentration of the solution be 75%, standing overnight, filtering to remove precipitate, concentrating the supernatant under reduced pressure until no alcohol smell exists, and obtaining the Saviae Miltiorrhizae radix crude extract.
2. Extracting with an organic solvent:
sequentially extracting the obtained Saviae Miltiorrhizae radix crude extract with sequentially increased polarity organic solvents respectively including petroleum ether, ethyl acetate and n-butanol, and concentrating to obtain petroleum ether extract, ethyl acetate extract and n-butanol extract.
3. Screening different extraction sites by using a mesenchymal stem cell conditioned medium:
the mesenchymal stem cell conditioned medium (100 mL) prepared in step 1 of example 1 was mixed with the crude extract of red sage root (0.5 mg), petroleum ether extract fraction (0.5 mg), ethyl acetate extract fraction (0.5 mg), n-butanol extract fraction (0.5 mg), respectively. The obtained mixed culture medium was placed at 37℃and CO 2 After incubation at 8% concentration for 32h, 5mg/mL MTT 10. Mu.L was added and incubation was continued for 4h, followed by addition of lysate (10% SDS,0.1% NH) 4 Cl) 100 μl, incubated overnight, the supernatant separated, and the absorbance measured at 490 nm.
The screening results were as follows:
TABLE 2 Activity screening results for different isolated parts of Salvia Miltiorrhiza
As is clear from table 2, although the absorbance values of the several extraction sites of the petroleum ether extraction site, the ethyl acetate extraction site and the n-butanol extraction site were all improved as compared with the blank group, the absorbance values of the petroleum ether extraction site and the n-butanol extraction site were decreased as compared with the extract of salvia miltiorrhiza, and the absorbance value of the ethyl acetate extraction site was significantly increased, thereby indicating that the compounds having the promoting effect on the proliferation of mesenchymal stem cells in salvia miltiorrhiza were mainly concentrated on the ethyl acetate site.
4. Separating the ethyl acetate extraction part:
concentrating ethyl acetate extraction part of Saviae Miltiorrhizae radix, mixing with 200-300 mesh silica gel, loading into column by dry method, performing silica gel column chromatography, gradient eluting with ethyl acetate-ethanol (10:1-0:1), monitoring by TLC in real time, mixing the same fractions to obtain 5 polar segments with sequentially increased polarity, which are respectively first polar segment, second polar segment, third polar segment, fourth polar segment, and fifth polar segment.
The 5 polarity segments were screened using the activity screening method of step 3, and the results are shown in Table 3:
TABLE 3 Activity screening results for different polarity segments of Salvia Miltiorrhiza
As can be seen from table 3, among the five polar segments separated from the ethyl acetate extraction site of salvia miltiorrhiza, the absorbance values of the second polar segment and the third polar segment are increased compared with the ethyl acetate extraction site, wherein the absorbance of the second polar segment is the maximum, thereby suggesting that the compound having the promoting effect on the proliferation of mesenchymal stem cells in salvia miltiorrhiza is mainly concentrated in the second polar segment.
5. Separation of the second polarity segment:
four monomer compounds are obtained by Sephadex LH-20 and reversed phase semi-preparative high performance liquid chromatography separation, and are identified by MS and NMR to be ferulic acid, rosmarinic acid methyl ester and salvianic acid methyl ester respectively.
The four monomer compounds were screened using the activity screening method in step 3, and the results are shown in table 4:
TABLE 4 Activity screening results for isolated four monomer Compounds
As can be seen from table 4, the absorbance value of methyl salvianolic acid was maximum when screening the four monomeric compounds isolated from the second polar stage, indicating that it has the effect of promoting mesenchymal stem cell proliferation, methyl rosmarinate had a weaker effect, whereas ferulic acid and rosmarinic acid did not exhibit this activity. Therefore, the main drug effect substance of the salvianolic acid methyl ester has the activity of promoting the proliferation of mesenchymal stem cells and is beneficial to repairing damaged skin.
In conclusion, the active micromolecular methyl salvianolic acid is tracked by performing primary screening on odorless medicinal materials of radix puerariae, radix salviae miltiorrhizae, angelica sinensis, ligusticum chuanxiong and cortex magnoliae officinalis, locking the proliferation activity of mesenchymal stem cells of radix salviae miltiorrhizae, separating radix salviae miltiorrhizae, and performing activity detection by adopting a similar method in each step of separation. Therefore, the screening method of the skin repair active ingredient based on the mesenchymal stem cells can effectively guide the separation of natural products, is favorable for screening more small-molecule monomer compounds with promotion effect on the proliferation of the mesenchymal stem cells, and provides a material guarantee for further developing cosmetics capable of adjusting the biological characteristics of the mesenchymal stem cells and improving damaged skin.
The above examples are only illustrative of the preferred embodiments of the present application and are not intended to limit the scope of the present application, and various modifications and improvements made by those skilled in the art to the technical solution of the present application should fall within the scope of protection defined by the claims of the present application without departing from the spirit of the present application.
Claims (4)
1. A method for screening skin repair active ingredients based on mesenchymal stem cells, comprising:
performing conventional culture and induction culture on active mesenchymal stem cells, and filtering the obtained cell culture solution to obtain a mesenchymal stem cell conditioned medium suitable for skin repair;
selecting medicinal materials of radix puerariae, radix salviae miltiorrhizae, angelica sinensis, ligusticum wallichii and magnolia officinalis, and obtaining plant crude extract of five medicinal materials by an alcohol extraction method;
mixing the mesenchymal stem cell conditioned medium with the plant crude extract of five medicinal materials, respectively, and cooling at 35-38deg.C with CO 2 Culturing for 30-36h at a concentration of 7-10%, detecting absorbance of the culture solution at 490nm by adopting an MTT method, and performing activity screening;
gradually separating the crude plant extract by combining with a natural plant separation technology, and performing activity screening on the components obtained after each separation again, and locking a separation target until a monomer active component is obtained;
wherein the step of induction culture comprises the following steps: adding astragaloside IV into DMEM medium to obtain induction medium, mixing the mesenchymal stem cells with the induction medium, and CO-culturing at 35-38deg.C 2 Culturing at 7-10% concentration for 36-48 hr.
2. The method for screening a skin repair active ingredient based on mesenchymal stem cells according to claim 1, wherein the mesenchymal stem cells are derived from umbilical cord, placenta, fat, bone marrow or dental pulp.
3. The method for screening a mesenchymal stem cell-based skin repair active ingredient according to claim 1, wherein the step of conventional culturing comprises: inoculating the mesenchymal stem cells into DMEM culture medium, and performing CO at 35-38deg.C 2 Culturing at a concentration of 7-10% until the growth density of mesenchymal stem cells is above 90%.
4. The method for screening a mesenchymal stem cell-based skin repair active ingredient according to claim 1, wherein the method for preparing the crude plant extract to be screened comprises: mixing the dried plant powder with 60-80% ethanol, reflux extracting at 70-80deg.C for 1-2 hr, and concentrating the extractive solution.
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| 不同来源间充质干细胞条件培养基对内源性衰老细胞作用的比较;郑桂纯等;中国组织工程研究;第23卷(第21期);第3357-3363页 * |
| 体外培养人骨髓间充质干细胞向表皮细胞分化及表皮细胞角蛋白的表达;李凌云等;中国组织工程研究与临床康复;第15卷(第36期);第6661-6664页 * |
| 周健洪等.龟板组份及配伍对体外培养骨髓间充质干细胞增殖的影响.中药材.2006,第29卷(第5期),摘要，材料和方法，讨论. * |
| 赵瑞东等.黄芪甲苷对间充质干细胞生物活性的影响.世界最新医学信息文摘.2015,第15卷(第19期),第10-11页. * |
| 陈洪超等.中药单体诱导间充质干细胞定向分化研究进展.医学综述.2015,第21卷(第22期),第4075-4077页. * |
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