Traditional Chinese medicine extract for improving homing ability of mesenchymal stem cells and application of traditional Chinese medicine extract

Abstract

The invention belongs to the technical field of stem cell treatment, and discloses a traditional Chinese medicine extract for improving the homing ability of mesenchymal stem cells and application thereof.After a beautiful millettia root extract obtained through simple water extraction and umbilical cord mesenchymal stem cells are subjected to mixed culture, the migration and / or homing ability of the obtained umbilical cord mesenchymal stem cells is remarkably improved, and the homing ability of the umbilical cord mesenchymal stem cells is improved. Therefore, the beautiful millettia root extract can be used for preparing a preparation for improving migration and / or homing of the umbilical cord mesenchymal stem cells. Through cell tests, it is studied that the beautiful millettia root extract does not have obvious cytotoxicity to umbilical cord mesenchymal stem cells and does not change functions and phenotypes of the umbilical cord mesenchymal stem cells, and therefore the beautiful millettia root extract can be used as a natural extract for improving the treatment capacity of the umbilical cord mesenchymal stem cells. The invention further researches the application effect of the umbilical cord mesenchymal stem cells subjected to mixed culture pretreatment of the beautiful millettia root extract, and when the umbilical cord mesenchymal stem cells are applied to scalded wounds of mice, the wound treatment and healing capabilities of the umbilical cord mesenchymal stem cells can be remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cell therapy, and more specifically, relates to a traditional Chinese medicine extract for improving the homing ability of mesenchymal stem cells and an application thereof. Background Art

[0002] Although it is generally believed that mesenchymal stem cells (MSCs) have certain therapeutic effects in the treatment of various diseases, there are still urgent problems to be solved before they can be promoted and applied clinically, such as the low migration rate of MSCs to damaged tissues. The low migration rate and colonization rate of MSCs in damaged areas may have a direct impact on the efficacy of their therapeutic effects. Researchers used animal in vivo tracing technology to observe the distribution of MSCs in the body. The results showed that MSCs can be scattered in multiple organs such as the liver, lungs, and spleen in the body, and only some cells can migrate to the damaged area. It has been confirmed in multiple liver injury treatment models that the reduced homing rate of MSCs has greatly affected its therapeutic effect. Therefore, how to improve the ability of MSCs to migrate to damaged areas in the body and reduce their distribution in other "ineffective" organs is an urgent problem to be solved to improve the efficacy of MSCs.

[0003] One of the primary functions of the SDF-1 / CXCR4 axis is regulating the migration, chemotaxis, and homing of progenitor cells to sites of injury. This evolutionarily conserved signal mediates stem cell migration and distribution, playing a crucial role in the recruitment and directional migration of mesenchymal stem cells. In the study of mesenchymal stem cell homing, SDF-1 exhibits a stronger chemotactic ability than several other chemokines. Therefore, the study of the effects of SDF-1 and its receptor CXCR4 on mesenchymal stem cell chemotaxis is currently a hot topic. Several chemical substances have been shown to enhance the homing of mesenchymal stem cells by increasing the responsiveness of the CXCR4 receptor on the stem cell surface to SDF-1. However, these chemicals still face significant safety hurdles, necessitating the development of safer pretreatment methods that can enhance mesenchymal stem cell homing.

[0004] Medicinal plants are widely distributed worldwide, but many remain largely unexplored in stem cell research. These plants hold a wealth of new active ingredients that could potentially benefit stem cell therapy. Therefore, identifying new medicinal plants with beneficial effects plays a crucial role in stem cell therapy. Niu Dali (Gibberish-like Herb), a popular medicinal and edible plant in the Lingnan region of my country, has numerous aliases, such as "Dali Niu," "Jinzhonggen," "Xueteng," and "Inverted Golden Hammer." These all refer to the beautiful Millettia vine, belonging to the genus Millettia in the Leguminosae family. Its dried roots are known as Niu Dali (Gibberish-like Herb). Niu Dali is primarily produced in Guangxi, Hainan, and Guangdong. It has a sweet taste and a neutral nature, and boasts tonifying, moistening the lungs, and strengthening tendons and activating blood circulation. Modern research has demonstrated that Niu Dali possesses varying degrees of protective and therapeutic effects on the immune, respiratory, digestive, and endocrine systems, including modulating immune function, combating fatigue, depression, relieving asthma, protecting the liver, and lowering blood sugar.

[0005] Currently, there are no research reports in domestic and foreign literature on the use of Radix Codonopsis pilosulae to improve the homing ability of mesenchymal stem cells. Summary of the Invention

[0006] In order to overcome the deficiencies of the prior art, the present invention aims to provide a use of a Herba Lycopodii extract in improving the homing ability of mesenchymal stem cells.

[0007] The second object of the present invention is to provide mesenchymal stem cells pretreated with the extract of Bovis spp.

[0008] The third object of the present invention is to provide the use of the mesenchymal stem cells pretreated with the Herba Lycopodii extract in the preparation of a wound healing drug.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] Use of a Herba Lycopodii extract in preparing a preparation for improving the migration and / or homing of umbilical cord mesenchymal stem cells.

[0011] The present invention has found through research that after the Botrytis cinerea extract obtained by simple water extraction is mixed and cultured with umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells obtained significantly improve their migration and / or homing ability. Therefore, the Botrytis cinerea extract can be used to prepare a preparation that improves the migration and / or homing of umbilical cord mesenchymal stem cells.

[0012] The present invention studies, through cell experiments, that the extract of Botrytis cinerea has no obvious cytotoxicity to umbilical cord mesenchymal stem cells and does not change the function and phenotype of umbilical cord mesenchymal stem cells. Therefore, it can be used as a natural extract to improve the therapeutic ability of umbilical cord mesenchymal stem cells.

[0013] Preferably, Herba Cynanchii extract of the present invention is that Herba Cynanchii root is obtained through preliminary water extraction. More preferably, the extracting method of Herba Cynanchii extract of the present invention is: put into pot after getting Herba Cynanchii pulverizing, add 8-10 times of volume of distilled water, soak 24h, boil to boiling and then turn to simmer 2-3h, collect first time extracting solution. Add 8-10 times of volume of distilled water in medicinal residues, boil to boiling and then turn to simmer 2-3h, collect second time extracting solution, repeat 2 times, collect all extracting solutions, concentrate extracting solution by rotary evaporator, concentrate 40-45 times (to 200mL), 3500r / min centrifugal 5min, take supernatant, use absolute ethanol that supernatant is adjusted to containing alcohol amount 70-80%, 4 ℃ of standing 4-6h, centrifugal precipitation, 45-55 ℃ of oven dry, obtain crude extract.

[0014] The present invention also studies the efficacy of umbilical cord mesenchymal stem cells pretreated with a co-cultured extract of Botrytis cinerea. Application to burn wounds in mice significantly enhanced the ability of umbilical cord mesenchymal stem cells to treat wounds and heal. Therefore, the present invention also provides the use of Botrytis cinerea extract in the preparation of a synergist for enhancing wound healing with umbilical cord mesenchymal stem cells.

[0015] Preferably, in the above application, the Herba Lycopersicon esculentum extract and umbilical cord mesenchymal stem cells are mixed and cultured for pretreatment, and the Herba Lycopersicon esculentum extract has the following effects on the umbilical cord mesenchymal stem cells after the mixed culture:

[0016] (1) Improve the migration and / or homing ability of umbilical cord mesenchymal stem cells;

[0017] (2) Improve the ability of umbilical cord mesenchymal stem cells to treat wound healing.

[0018] On the premise of discovering the therapeutic ability of Botrytis cinerea extract on umbilical cord mesenchymal stem cells, it can be used to prepare umbilical cord mesenchymal stem cell culture medium or biological preparations to broaden the range of umbilical cord mesenchymal stem cell related biological culture products.

[0019] Therefore, the present invention also provides a culture medium for improving the migration and / or homing ability of umbilical cord mesenchymal stem cells, wherein the culture medium contains a Herba Strengthis Extract.

[0020] Preferably, in the culture medium, the cell density of the umbilical cord mesenchymal stem cells is 1×10 6 / ml, and the added amount of the Herba Lycopodii extract is 1.5-4 mg.

[0021] The present invention also provides a method for improving the migration and / or homing ability of umbilical cord mesenchymal stem cells, comprising the following steps:

[0022] S1. Inoculate umbilical cord mesenchymal stem cells into a culture vessel. After the cells are fully attached, replace the culture medium with complete culture medium containing Botrytis cinerea extract.

[0023] S2. After culturing in a cell culture incubator for a period of time, umbilical cord mesenchymal stem cells with enhanced migration and / or ability are obtained.

[0024] The present invention also provides the use of the umbilical cord mesenchymal stem cells obtained by the above-mentioned culture method in preparing a preparation for treating wound healing.

[0025] Preferably, the wound is a wound caused by burns.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention has discovered that when a simple aqueous extract of Botrytis cinerea extract is co-cultured with umbilical cord mesenchymal stem cells, the resulting umbilical cord mesenchymal stem cells significantly enhance their migration and / or homing abilities. Therefore, the Botrytis cinerea extract can be used to prepare a formulation that enhances the migration and / or homing abilities of umbilical cord mesenchymal stem cells. Cell-based experiments have also demonstrated that the Botrytis cinerea extract has no significant cytotoxicity to umbilical cord mesenchymal stem cells and does not alter their function or phenotype. Therefore, it can be used as a natural extract to enhance the therapeutic potential of umbilical cord mesenchymal stem cells.

[0028] The present invention also studies the application effect of umbilical cord mesenchymal stem cells pretreated by mixed culture with Botrytis cinerea extract. Applying it to the burn wound of mice can significantly improve the ability of umbilical cord mesenchymal stem cells to treat wounds and heal. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The effect of the extract of Botrytis cinerea on the survival rate of umbilical cord mesenchymal stem cells;

[0030] Figure 2 Effect of Bovis serrata extract on the migration of umbilical cord mesenchymal stem cells (microscope);

[0031] Figure 3 The effect of the extract of Botrytis cinerea on the expression of SDF-1a and CXCR4 in umbilical cord mesenchymal stem cells;

[0032] Figure 4 This is the effect of umbilical cord mesenchymal stem cells pretreated with Botrytis cinerea extract on wound healing (animal experiment). DETAILED DESCRIPTION

[0033] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and examples. In the examples, the experimental methods used are conventional methods unless otherwise specified, and the materials and reagents used are all commercially available unless otherwise specified.

[0034] Example 1 Preparation of Herba Lycopodii Extract and Its Effect on the Physiological Function of Mesenchymal Stem Cells

[0035] 1. Preparation of Herba Cynanchifoliae extract: 200g of Herba Cynanchifoliae was crushed and placed in a pot. 8-10 times the volume of distilled water was added and soaked for 24 hours. After boiling, the mixture was simmered for 2-3 hours. The first extract was collected. 8-10 times the volume of distilled water was added to the medicinal residues. After boiling, the mixture was simmered for 2-3 hours. The second extract was collected. This was repeated 2 times. All extracts were collected and concentrated by rotary evaporation. The extract was concentrated 40-45 times (to 200mL). The extract was centrifuged at 3500r / min for 5 minutes. The supernatant was adjusted to 70-80% alcohol content using anhydrous ethanol. The extract was allowed to stand at 4°C for 4-6 hours. The precipitate was centrifuged and dried at 45-55°C to obtain a crude extract. When used, a 10g / L solution was prepared (pure water was used as the solvent).

[0036] 2. Culture of umbilical cord mesenchymal stem cells

[0037] The umbilical cord of a clinically healthy full-term newborn was sterilized twice with 75% alcohol in a clean bench, placed in a culture dish, and the Warburg jelly was removed with tweezers and cut into 1 mm pieces with scissors. 3 Tissue blocks of different sizes were attached to the bottom wall of a 50ml culture flask in a dotted pattern with a spacing of 1 cm. Ultra CULTURE medium containing serum replacement was added and cultured in a 5% CO2, 37°C incubator. The medium was changed every 3 days. When the cells reached 70%-80% confluence, they were passaged and expanded, and the cell morphology was observed under an inverted microscope. When the cells reached P3, culture medium was added and cultured for 48 hours. The culture supernatant was collected and centrifuged at 3000g / 10min to obtain the supernatant. The supernatant was filtered through a 220-mesh filter to remove cell debris. The filtrate obtained was the mesenchymal stem cell culture supernatant. The P3 cells at the bottom of the flask were digested with 0.25% trypsin and the cell density was adjusted to 1×10 6Cells were plated at 400 nmol / ml and placed in flow cytometry tubes. Anti-mouse and anti-human monoclonal antibodies (CD29, CD44, CD90, CD105, IgG1-FITC, and HLADR) were added, respectively, and incubated at 4°C in the dark for 30 minutes. The cells were washed twice with PBS and analyzed by flow cytometry at 1000 rpm for 3 minutes. Cells were identified as umbilical cord mesenchymal stem cells when the expression rates of CD73, CD44, CD90, and CD105 were above 98% and the expression rates of IgG1-FITC and HLADR were less than 1%.

[0038] 3. Mixed culture of bovine radix extract and umbilical cord mesenchymal stem cells

[0039] (1) P3 umbilical cord mesenchymal stem cells were digested with trypsin to prepare a single cell suspension at a cell density of 1×10 6 / ml, inoculated in a 96-well plate, and randomly divided into a control group (the amount of Herba Lycopodii extract added was 0 μL) and experimental groups 1-9 (100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, 500 μL), with 8 replicate wells in each group.

[0040] (2) After 24 h of culture, the control group was replaced with complete culture medium, and the experimental groups 1-9 were replaced with complete culture medium supplemented with 100 μL, 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, 400 μL, 450 μL, and 500 μL of Herba Lysimachiae extract, respectively, according to the settings.

[0041] (3) After culturing for 24 h, the cell viability was determined using the MTT assay, and the cell phenotype was analyzed using flow cytometry.

[0042] 3.1 Cytotoxicity assay

[0043] like Figure 1 It can be seen that when the amount of Botrytis cinerea extract added is less than 400 μL, it has no significant effect on the activity of umbilical cord mesenchymal stem cells. When the amount of Botrytis cinerea extract added is greater than 400 μL, the effect on the activity of umbilical cord mesenchymal stem cells is also small, indicating that Botrytis cinerea extract has no obvious toxicity to umbilical cord mesenchymal stem cells within the range of addition used.

[0044] 3.2 Effects on Mesenchymal Stem Cell Migration

[0045] (1) P4 umbilical cord mesenchymal stem cells were digested with trypsin to prepare a single-cell suspension and seeded into a 6-well plate;

[0046] (2) When the cell density reaches above 90%, remove the culture medium and use a 200 μL yellow pipette tip to slide across the cell layer in the vertical direction of the culture well;

[0047] (3) PBS was used to rinse off the detached cells. Complete culture medium was added to the control group, and complete culture medium containing 200 μL, 250 μL, 300 μL, and 350 μL of Herba Lycopodii extract was added to experimental groups 1-9, respectively. The scratch marks were recorded under a microscope.

[0048] (4) After culturing in a cell culture incubator for 24 h, the scratch marks were recorded again under a microscope.

[0049] from Figure 2 It can be seen that after treatment with the extract of Botrytis cinerea, the migration distance of umbilical cord mesenchymal stem cells was significantly increased, indicating that the extract of Botrytis cinerea can significantly promote the migration of umbilical cord mesenchymal stem cells.

[0050] 3.3 Effects on homing of umbilical cord mesenchymal stem cells

[0051] (1) P3 umbilical cord mesenchymal stem cells were digested with trypsin to prepare a single-cell suspension and seeded into a 6-well plate;

[0052] (2) After the cells were completely attached, a portion of the cells were pretreated with complete medium containing different amounts of Botrytis cinerea extract for 24 h, and the other portion was added with complete medium for a further 24 h, followed by trypsin digestion, and then a cell suspension was prepared using serum-free medium (suspension 1: cultured in complete medium, suspension 2: cultured in complete medium containing 100 μL of Botrytis cinerea extract, suspension 3: cultured in complete medium containing 150 μL of Botrytis cinerea extract, suspension 4: cultured in complete medium containing 200 μL of Botrytis cinerea extract, suspension 5: cultured in complete medium containing 250 μL of Botrytis cinerea extract, suspension 6: cultured in complete medium containing 300 μL of Botrytis cinerea extract, suspension 7: cultured in complete medium containing 350 μL of Botrytis cinerea extract, suspension 8: cultured in complete medium containing 400 μL of Botrytis cinerea extract, suspension 9: cultured in complete medium containing 450 μL of Botrytis cinerea extract, suspension 10: cultured in complete medium containing 500 μL of Botrytis cinerea extract);

[0053] (3) Process according to the following groups:

[0054] Control group: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 1 was added to the upper chamber of the chamber, containing 1×10 cells. 5 indivual;

[0055] Experimental group 1: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 2 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0056] Experimental group 2: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 3 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0057] Experimental group 3: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 4 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0058] Experimental group 4: Complete culture medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 5, containing 1×10 cells, was added to the upper chamber of the chamber. 5 indivual;

[0059] Experimental group 5: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 6 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0060] Experimental group 6: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 7 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0061] Experimental group 7: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 8 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0062] Experimental group 8: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension 9 containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual;

[0063] Experimental group 9: Complete medium was added to the wells of the 24-well plate, and a Transwell chamber was placed. 200 μl of cell suspension containing 1×10 cells was added to the upper chamber of the chamber. 5 indivual.

[0064] (4) Place the 24-well transwell chamber in a cell culture incubator and incubate for 12 hours. Cut the polycarbonate membrane on the bottom of the transwell upper chamber to remove the stem cells that failed to migrate to the upper side of the membrane. Wash with PBS and repeat three times. (5) Add paraformaldehyde to fix for 15 minutes, add crystal violet to stain, and gently rinse the polycarbonate membrane with PBS after 15 minutes to wash away the excess dye. Count the number of cells that migrated to the lower side of the chamber membrane under a microscope (×200). Randomly select 5 fields of view to calculate the average number of cells that migrated and the migration promotion rate compared with the control group.

[0065] Table 1

[0066] Group Average number of cell migration Migration promotion rate (%) control group 105 Experimental Group 1 111 5.7 Experimental Group 2 123 17.1 Experimental Group 3 149 41.9 Experimental Group 4 178 78.8 Experimental Group 5 214 103.6 Experimental Group 6 199 89.5 Experimental Group 7 177 68.6 Experimental Group 8 156 48.6 Experimental Group 9 108 2.85

[0067] As can be seen from Table 2, compared with the control group, the addition of the Herba Lycopersicum Extract in the experimental group significantly promoted the homing of mesenchymal stem cells. Specifically, when the amount of Herba Lycopersicum Extract added was less than 350 μL, the migration rate of mesenchymal stem cells increased with the increase in the amount of Herba Lycopersicum Extract added. When the amount of Herba Lycopersicum Extract added was 300 μL, the migration rate of mesenchymal stem cells was greater than 100%, indicating that the Herba Lycopersicum Extract can significantly improve the homing ability of mesenchymal stem cells. However, when the amount of Herba Lycopersicum Extract added was greater than 350 μL, the migration rate of mesenchymal stem cells showed a significant downward trend, which may be related to the slight inhibitory effect of Herba Lycopersicum Extract on the activity of mesenchymal stem cells at high concentrations.

[0068] 3.4 Effects of Pretreatment with Radix Glehniae Extract on SDF-1α and CXCR4 Protein Expression in Mesenchymal Stem Cells

[0069] (1) P3 umbilical cord mesenchymal stem cells were digested with trypsin to prepare a single-cell suspension and seeded into a 6-well plate. When the cell density reached above 80%, the control group was replaced with new complete medium, and the experimental group was replaced with complete medium containing 350 μL of Botrytis cinerea extract. Three replicate wells were set up for each group.

[0070] (2) After culturing for 24 h, the culture medium was removed, the cells were washed twice with cold PBS, and pre-chilled lysis buffer (containing protease inhibitors) was added. The cells were lysed on ice for 20 min and centrifuged at 12,000 g for 15 min.

[0071] (3) Take a small amount of supernatant and use the BCA kit to detect the protein concentration. Add 4× loading buffer to the supernatant and place it in a water bath at 100°C for 5 minutes.

[0072] (4) WB experiment: After installing the electrophoresis tank, prepare 10 ml of 12% lower layer gel, fill the gel and seal it with anhydrous ethanol. After solidification, discard the upper layer liquid and gently rinse with deionized water, and dry it with absorbent paper; (5) Prepare 6 ml of 5% upper layer gel, fill the gel and insert the comb. After solidification, pull out the comb; (6) Fix the gel on the electrophoresis rack, add electrophoresis buffer, add protein sample and protein marker, and add 30 μg to each well; (7) Adjust the power supply to a constant voltage of 80V until the protein marker separates, then adjust the power supply to a constant voltage of 120V. Stop electrophoresis when bromophenol blue reaches the bottom of the electrophoresis gel; (8) Place the PVDF membrane in anhydrous methanol for 15 seconds, then place it in electrotransfer buffer together with the filter paper, and place them in the order of sponge, filter paper, gel, PVDF membrane, filter paper, and sponge. Then lock the electrotransfer clamp, put it in the ice box, and adjust the power supply to a constant current of 300 mA for 1.5 hours; (9) After the electrotransfer is completed, The PVDF membrane was removed and rinsed with TBST solution for 3 times, 10 min each time, and then placed in blocking solution for 1 h; (10) The protein bands of SDF-1a, CXCR4 and GAPDH were cut and placed in the corresponding primary antibody diluent, and incubated at 4°C overnight; (11) The membrane was removed and rinsed with TBST solution for 3 times, 10 min each time, and the secondary antibody diluent was prepared. The bands were placed in the secondary antibody diluent and incubated at room temperature with gentle shaking for 1 h; (12) The membrane was removed and rinsed with TBST solution for 3 times, 10 min each time, and the secondary antibody diluent was prepared. The bands were placed in the secondary antibody diluent and incubated at room temperature with gentle shaking for 1 h; (13) The membrane was removed and rinsed with TBST solution for 3 times, 10 min each time, and the PVDF membrane was spread flat on a plate with the front side facing up. An appropriate amount of freshly prepared chemiluminescent substrate was added. After incubation at room temperature in the dark for 5 min, the membrane was photographed and recorded using a BIO-RAD gel imaging system.

[0073] The results are shown in Figure 3 It can be seen that after treatment with the Herba Lycopersicon esculentum extract, the protein expression levels of SDF-1a and CXCR4 in the cells were upregulated, indicating that treatment with the Herba Lycopersicon esculentum extract can effectively promote the expression of SDF-1a and CXCR4 in umbilical cord mesenchymal stem cells. Therefore, the present invention speculates that the Herba Lycopersicon esculentum extract can promote the migration and homing of umbilical cord mesenchymal stem cells by promoting the expression of SDF-1a and CXCR4.

[0074] Example 2: Promoting the healing of skin wounds by umbilical cord mesenchymal stem cells

[0075] 1. Mesenchymal stem cell biologics for skin wound healing

[0076] (1) P3 umbilical cord mesenchymal stem cells were digested with trypsin to prepare a single cell suspension at a cell density of 1×10 6 / ml, inoculated into 96-well plates, cultured for 24 h, the control group was replaced with complete culture medium, and experimental groups 1-6 were set as per the settings, with 150 μL, 200 μL, 250 μL, 300 μL, 350 μL, and 400 μL of Herba Lysimachiae extract added to the complete culture medium, respectively.

[0077] (3) After culturing for 24 h, the culture medium was removed, the cells were digested with trypsin, and DMEM was added to terminate the digestion. The cells were collected into a centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant was removed, and the cells were resuspended in PBS and centrifuged at 1000 rpm for 5 min. The supernatant was removed, and the cells were resuspended in physiological saline and the cell concentration was adjusted to 5 × 10 6 / ml, and evenly disperse carbomer powder into physiological saline containing cells to obtain mesenchymal stem cell biogels 1-6 for treating skin wound healing. The control group biogel was obtained by the same culture method except that no Herba Lycopodii extract was added.

[0078] 1. Construction of mouse scald model

[0079] Nude mice aged 16 weeks and weighing approximately 200 ml were selected and fasted for 12 hours before surgery. They were also deprived of water. After anesthesia, the mice were fixed on a foam board. Their backs were depilated and routinely disinfected. Boiled water was applied and a cylindrical hollow tube (2 cm diameter) was placed vertically on the skin on the right side of the mouse's spine. 10 ml of boiling water was added and held for 20 seconds to create a 2 cm circular burn wound. The wound was then disinfected with iodine.

[0080] 2. Observation of Wound Healing in Mice

[0081] The scalded mice were randomly divided into 6 experimental groups, namely experimental groups 1-6 (using the mesenchymal stem cell biogels 1-6 prepared as described above), a blank group (blank group gel was obtained by evenly dispersing carbomer powder in physiological saline), and a control group (using the control group biogel); each group had 5 mice.

[0082] Animals in the experimental group: Biogel (0.2 mL) was applied to the burn wound surface once in the morning, noon and evening every day. The blank group and control group were applied with blank group gel and control group gel on the wound surface in the same way.

[0083] The day of gel application was designated as day 0 (0d). The local wound area of ​​mice in each group was measured at different time points (3d, 5d, 8d, and 12d) after injury, and the wound healing rate was calculated according to the following formula.

[0084] Healing rate / %=(original wound area-unhealed wound area) / original wound area×100%.

[0085] Effective evaluation criteria: healing rate ≥75% is considered cured, ≥50% is considered significantly effective, ≥25% is considered effective, and ≤25% is considered ineffective.

[0086] Total effective rate / %=(number of markedly effective cases + number of effective cases) / total number of mice in each group×100%.

[0087] Table 2 Comparison of wound healing rates in mice

[0088]

[0089]

[0090] Table 2 and Figure 4 As can be seen, experimental group 4 achieved a 96% wound healing rate on day 12, while the blank and control groups achieved wound healing rates of 56% and 75%, respectively, on day 12. This indicates that umbilical cord mesenchymal stem cells pretreated with Botrytis cinerea extract can significantly promote wound healing, increase wound healing rates, and shorten wound healing cycles. This also demonstrates that Botrytis cinerea extract can significantly promote the homing of umbilical cord mesenchymal stem cells to wounds, thereby increasing the number of umbilical cord mesenchymal stem cells recruited for wound healing.

[0091] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of a Herba Lycopodii extract in the preparation of a preparation for enhancing the migration and / or homing of umbilical cord mesenchymal stem cells.

2. Application of Herba Lycopodii Extract in the preparation of a synergist for improving wound healing by umbilical cord mesenchymal stem cells.

3. The use according to claim 1 or 2, characterized in that The extract of Botrytis cinerea was mixed and cultured with umbilical cord mesenchymal stem cells for pretreatment. The extract of Botrytis cinerea had the following effects on the umbilical cord mesenchymal stem cells after mixed culture: (1) Improve the migration and / or homing ability of umbilical cord mesenchymal stem cells; (2) Improve the ability of umbilical cord mesenchymal stem cells to treat wound healing.

4. A culture medium for improving the migration and / or homing ability of umbilical cord mesenchymal stem cells, characterized in that: The culture medium contains a Herba Strengthis Extract.

5. The culture medium according to claim 4, characterized in that The cell density of the umbilical cord mesenchymal stem cells is 1×10 6 / ml, and the added amount of the Herba Lycopodii extract is 1.5-4 mg.

6. A method for improving the migration and / or homing ability of umbilical cord mesenchymal stem cells, characterized in that: The following steps are involved: S1. Inoculate umbilical cord mesenchymal stem cells into a culture vessel. After the cells are fully attached, replace the culture medium with complete culture medium containing Botrytis cinerea extract. S2. After culturing in a cell culture incubator for a period of time, umbilical cord mesenchymal stem cells with enhanced migration and / or ability are obtained.

7. Use of the umbilical cord mesenchymal stem cells obtained by the method of claim 6 in preparing a preparation for treating wound healing.

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