CN116920069B - Traditional Chinese medicine extract and application thereof in promoting umbilical cord stem cells to secrete VEGF - Google Patents
Traditional Chinese medicine extract and application thereof in promoting umbilical cord stem cells to secrete VEGF Download PDFInfo
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Abstract
The invention provides a traditional Chinese medicine extract and application thereof in promoting umbilical cord stem cells to secrete VEGF, and belongs to the technical field of medicines. According to the invention, the Chinese medicinal extract is prepared from the Chinese angelica, the polygonum multiflorum, the ligusticum wallichii, the biota orientalis and the dried ginger, so that the Chinese medicinal prescription is convenient to obtain materials and has low cost; the traditional Chinese medicine extract is used for promoting the umbilical cord stem cells to secrete VEGF, the traditional Chinese medicine technology is combined with the modern stem cell culture technology, umbilical cord stem cells capable of secreting more vascular endothelial growth factors are cultured, and the umbilical cord stem cells and the supernatant containing the vascular endothelial growth factors and the traditional Chinese medicine extract play a role in auxiliary repair of hair follicle injury and alopecia, so that the trouble of hair rarefaction and alopecia is solved.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a traditional Chinese medicine extract and application thereof in promoting umbilical cord stem cells to secrete VEGF.
Background
The umbilical cord stem cells generally refer to umbilical cord mesenchymal stem cells, are derived from umbilical cord tissues of newborns, are separated and extracted from Wharton's jelly, are stem cells with self-renewal and multiple differentiation potential, have low immunogenicity and strong proliferation capability, and can be used for repairing damaged or diseased tissue organs, strengthening the immunoregulatory capability of organisms, promoting hematopoietic function repair and the like. In recent years, the medicine is widely applied and researched in a plurality of medical treatment fields. Numerous studies have demonstrated that umbilical cord mesenchymal stem cells are capable of secreting different types of cytokines, such as vascular endothelial growth factor (Vascular Endothelial Growth Factor, VEGF), fibroblast growth factor (Fibroblast Growth Factor, FGF), etc., which can optimize the microenvironment surrounding damaged skin tissue, promote proliferation of damaged tissue cells, accelerate healing of the injured site, etc.
VEGF is widely distributed in organs of heart, brain, kidney, bones, skin and other tissues of human and animals, can enhance the permeability of venules and venules, promote the formation of new blood vessels and the growth of vascular endothelial cells, and is the factor with highest specificity and strongest activity for promoting angiogenesis at present. Research shows that VEGF plays a vital role in preventing and treating cardiovascular and cerebrovascular diseases and chronic kidney diseases, growing and developing bone tissues, generating and developing tumors, regenerating scalp hair and repairing wound surfaces.
For example, patent CN110327458a discloses that vascular endothelial growth factor B (VEGF-B) is a metabolic regulator of selective secretion of different subtypes of activated T cells, activating T cells by antibodies or peptides, and/or inducing activated T cells to secrete VEGF-B by ROS/AP-1 signaling pathway using agonists capable of promoting mitochondrial biosynthesis or agents that regulate mitochondrial related ROS/AP-1 signaling, thereby promoting fatty acid uptake, anti-apoptosis, anti-tumor activity, etc. of T cells.
In the history of the development of Chinese medicine with a great popularity, the application of Chinese herbal medicines is more and more extensive, and a plurality of Chinese herbal medicines are verified to have the pharmacological actions of anti-inflammatory, anti-infection, antimicrobial, antiviral, anti-tumor, nerve protection, immunoregulation, blood vessel protection and the like under the support of modern scientific experiment technology, so that the research background of modern pharmacy and pharmacology is also laid for treating various diseases by applying Chinese medicinal formulas. Research shows that the Chinese medicine can promote the secretion of vascular endothelial growth factor, and as patent CN111249293A discloses the new application of astragaloside in promoting EPCs to secrete vascular growth factor (VEGFa, VEGFb, VEGFc, FGF, ang-1) and exosome. However, due to the instability of the active ingredients of the herbal extracts at the laboratory level and the requirement for aseptic processing, traditional Chinese medicine techniques are less applicable in relevant cell culture experiments. And at present, no related technical report exists that in the umbilical cord stem cell culture process, the vascular endothelial growth factor is promoted to be secreted by the umbilical cord stem cell through the induction of traditional Chinese medicines, and the cell culture supernatant containing traditional Chinese medicine components and the vascular endothelial growth factor and the induced cells are applied to hair follicle repair and hair growth. Therefore, the application of the traditional Chinese medicine to promote the secretion of VEGF by umbilical cord mesenchymal stem cells has important significance.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides a traditional Chinese medicine extract and application thereof in promoting umbilical cord stem cells to secrete VEGF, combines a traditional Chinese medicine technology with a cell culture process, is used for promoting mesenchymal stem cells to secrete VEGF level, solves the problem of stability of traditional Chinese medicine components in experiments, improves the level of secreted vascular endothelial growth factor, and is combined with added traditional Chinese medicine components to promote hair follicle repair and hair regeneration, thereby helping people to solve the problem of alopecia.
In order to achieve the above purpose, the present invention provides the following technical solutions:
In one aspect, the invention provides a traditional Chinese medicine prescription, which comprises 5-18 parts of Chinese angelica, 12-16 parts of polygonum multiflorum, 3-6 parts of ligusticum wallichii, 8-12 parts of biota orientalis and 8-13 parts of dried ginger.
Preferably, the Chinese medicinal prescription comprises 10 parts of Chinese angelica, 15 parts of polygonum multiflorum, 5 parts of ligusticum wallichii, 10 parts of biota orientalis and 10 parts of dried ginger.
The extraction method of the traditional Chinese medicine prescription comprises the following steps:
mixing the above materials, dissolving with water, extracting, and filtering to obtain Chinese medicinal extractive solution.
Preferably, the filtering step comprises: multi-layer gauze filtration, quantitative filter paper filtration and 0.2-0.25um sterile filter membrane filtration.
The application of the traditional Chinese medicine prescription or the traditional Chinese medicine extract in repairing hair follicle injury and treating alopecia comprises the application in promoting the secretion of VEGF by umbilical cord stem cells.
In yet another aspect, the present invention provides a method for promoting the secretion of VEGF by umbilical cord stem cells, comprising the steps of:
A. Separating and preparing mesenchymal stem cells from umbilical cord tissues, and then carrying out passage amplification culture and freezing;
B. Adding the traditional Chinese medicine extract prepared by the extraction method into a mesenchymal stem cell culture medium for culture, and determining the addition concentration of the traditional Chinese medicine extract through experiments;
C. And B, using the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B for culturing the mesenchymal stem cells.
In some embodiments, in step a, the umbilical cord is a neonatal umbilical cord, stored in normal saline or Phosphate Buffered Saline (PBS) after collection, transported at low temperature (zero), and prepared for no longer than 24 hours from collection.
In other embodiments, the step a includes:
A1, separating Wharton's jelly tissue from a neonatal umbilical cord for later use;
A2, inoculating the Whatman gel tissue blocks into a culture dish, and placing the culture dish in an incubator for preparation;
A3, adding a preheated mesenchymal stem cell culture medium into the culture dish in the step A2, and performing primary culture;
a4, sucking tissue blocks in the culture dish, and collecting cells for subculturing;
A5, transferring the cells to 1 generation, digesting and collecting the cells after the cells grow fully, inoculating the cells into a culture dish, continuously subculturing the cells, and freezing the rest cells;
A6, transferring the cells to 3 generations, digesting and collecting the cells after the cells grow up, and freezing the cells.
Preferably, the step A1 includes: the umbilical cord tissue of the neonate is disinfected and cleaned by alcohol and normal saline, after no microbial contamination of bacteria, fungi, mycoplasma, viruses and the like is detected, the umbilical cord is longitudinally split, three blood vessels (two arteries and one vein) in the umbilical cord are removed by forceps, and then the Wharton's jelly is torn and sheared into tissue blocks with the size of about 1mm 3; wherein 0.5-2% of diabody (penicillin-streptomycin mixed solution) is added into physiological saline.
Preferably, in the step A2, the plating density of the Whatman tissue block is 60-75% of the bottom area of the culture dish.
Preferably, in step A2, the incubator environment is: 3-8% CO 2, 36.5-37.2 ℃, saturated humidity, more preferably 5% CO 2, 37 ℃, saturated humidity; the holding time is 20-50min, more preferably 30min.
Preferably, in the step A3, the mesenchymal stem cell culture medium is added in an amount of 20-35 mL/dish, and more preferably 30 mL/dish.
Preferably, in step A3, the mesenchymal stem cell medium is alpha-MEM, and 4-5.5% of serum replacement is added, and more preferably 5% of serum replacement is added.
Preferably, in the step A3, the culture time is 12-15 days, and the culture medium is half-changed every 3-5 days during the culture.
Preferably, in step A4, the cells grow to cover 45-50% of the area of the bottom of the dish when the tissue mass is aspirated.
Preferably, the step A5 is: cells were transferred to 1 passage, collected by digestion after confluence, plated 1.4X10 6 on 1 150mm dishes for further passage, and the remaining cells were frozen in liquid nitrogen at a density of 5X 10 6/mL as seed cells.
Preferably, in step A6, the cells are frozen at a density of 3-5X 10 6/mL.
In other embodiments, the step B includes:
b1, adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium;
And B2, resuscitating the frozen 3-generation mesenchymal stem cells, transferring to 5-generation mesenchymal stem cells, re-suspending and inoculating to a mesenchymal stem cell culture medium for culture, discarding the mesenchymal stem cell culture medium, and adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B1 for continuous culture.
Preferably, in the step B1, the concentration of the Chinese medicine extract added to the culture medium is 1-8mL/L.
Further preferably, in step B1, when the concentration of the extract of the traditional Chinese medicine added to the culture medium is 4mL/L, the secretion function of the mesenchymal stem cells is optimal, and the secretion amount of the vascular endothelial growth factor is maximized.
Preferably, the step B2 includes: resuscitate the frozen mesenchymal stem cells for 5 generations, re-suspend the mesenchymal stem cells in 96 well plate, discard the mesenchymal stem cells after 24 hr, replace the prepared mesenchymal stem cells culture medium containing Chinese medicine extract to continue culturing, detect the proliferation rate of the cells in each well, and thus determine the concentration of Chinese medicine extract.
In other embodiments, step C comprises:
Resuscitate the frozen mesenchymal stem cells for 3 generations, transfer to 5 generations, continue culturing, discard supernatant, add the mesenchymal stem cell culture medium containing Chinese medicinal extract, continue culturing.
Preferably, the step C includes:
c1, recovering the frozen 3-generation mesenchymal stem cells, and transferring to 5-generation mesenchymal stem cells;
c2, culturing for 24 hours, discarding supernatant, and adding a mesenchymal stem cell culture medium containing a traditional Chinese medicine extract for culturing;
C3, continuously culturing for 72 hours, collecting supernatant containing vascular endothelial growth factor and the traditional Chinese medicine extract, flushing residual cells with PBS, and adding PBS for continuously culturing;
C4, culturing for 96h, and collecting PBS.
In still another aspect, the invention also provides a composition, which is the supernatant containing the vascular endothelial growth factor and the Chinese medicine extract obtained in the step C3.
The composition can be used for repairing hair follicle injury and treating alopecia.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention discloses an application of a traditional Chinese medicine extract in promoting umbilical cord stem cells to secrete VEGF, which has strong operability and stable process, and can be suitable for general laboratory academic research and large-scale cell production;
(2) The traditional Chinese medicine material selects traditional Chinese medicine formula particles according to equivalent standard, so that the medicine extraction time is greatly shortened, meanwhile, the stability of different batches of medicinal materials is ensured, the effective components in the traditional Chinese medicine are extracted through a low-temperature sterile process, and the loss of the medicinal components is reduced; the addition amount of the effective component is evaluated on the molecular level, so that the stability of the action effect of the added traditional Chinese medicine component is ensured;
(3) The traditional Chinese medicine technology is combined with the modern stem cell culture technology, mesenchymal stem cells capable of secreting more vascular endothelial growth factors are cultured, and the mesenchymal stem cells and supernatant containing the vascular endothelial growth factors and the Chinese medicine extract play a role in auxiliary repair of hair follicle injury and alopecia, so that the trouble of hair rarefaction and alopecia is solved.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing the growth state of umbilical cord mesenchymal stem cells according to the present invention; in the figure, (a) represents primary cells before passage and (b) represents cells transferred to 3 passages;
FIG. 2 is a graph showing the effect of different concentrations of the Chinese medicinal extract on proliferation rate of umbilical mesenchymal stem cells;
FIG. 3 is a flow chart showing the use of the mesenchymal stem cell medium containing the extract of the Chinese medicine for culturing umbilical cord mesenchymal stem cells.
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way. The following is merely exemplary of the scope of the invention as claimed and many variations and modifications of the invention will be apparent to those skilled in the art in light of the disclosure, which are intended to be within the scope of the invention as claimed.
In the invention, the Phosphate Buffer Solution (PBS) is prepared by dissolving 8g of NaCl, 0.2gKCl g of Na 2HPO4 and 0.24g of gKH 2PO4 in 800mL of distilled water, regulating the pH value of the solution to 7.4 by using HCl, adding distilled water to a volume of 1L, and sterilizing.
The invention is further illustrated by means of the following specific examples. The various chemical reagents used in the examples of the present invention were obtained by conventional commercial means unless otherwise specified.
Example 1
The traditional Chinese medicine prescription comprises the following components: 10g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 10g of biota orientalis and 10g of dried ginger.
Mixing the components in the traditional Chinese medicine prescription, fully mixing with warm water of about 100mL and 50 ℃ for dissolving, filtering residues with multi-layer gauze, quantitatively filtering with filter paper to remove small particulate matters, and filtering with a 0.22um sterile filter membrane for sterilization to obtain the traditional Chinese medicine extract.
The method for promoting the umbilical cord stem cells to secrete VEGF by using the traditional Chinese medicine extract comprises the following steps:
1. Isolated culture of umbilical cord mesenchymal stem cells
(1.1) The neonatal umbilical cord is stored in normal saline after being collected and transported at low temperature, and the time from collection to preparation is not longer than 24 hours;
(1.2) sterilizing and cleaning umbilical cord tissue by using alcohol and normal saline (1% double antibody is added: penicillin-streptomycin mixed solution), longitudinally cutting the umbilical cord after no microbial contamination of bacteria, fungi, mycoplasma, viruses and the like is detected, removing three blood vessels in the umbilical cord, and then tearing Wharton's jelly and shearing into tissue blocks with the size of about 1mm 3;
(1.3) placing the Whatman gum tissue blocks into a 150mm culture dish, spreading uniformly, covering about 70% of the bottom area of the culture dish, and placing the culture dish in a saturated humidity incubator with 5% CO 2 at 37 ℃ for 30min; the dish was removed, and 30mL of a mesenchymal stem cell medium (α -MEM manufactured by Gibco Co., ltd., 5% Helios HPCFDCGL% serum replacement) preheated at 37℃was added; placing into an incubator with 5% CO 2 and 37 ℃ and saturated humidity for culturing;
(1.4) half-dose medium exchange every 5 days, and tissue mass was aspirated when cultured cells grew to cover 50% of the bottom area of the dish, while cells were collected for subculture. Observing the morphology of primary cells before passage by using a light microscope, and as shown in the result of FIG. 1 (a), the cells grow in a spindle-shaped adherent manner and spread along the group edge to the periphery;
(1.5) transferring the cells to 1 generation, digesting and collecting the cells after the cells grow fully, inoculating 1.4X10 6 to 1 culture dish with 150mm, continuing to passage, and freezing the rest cells in liquid nitrogen according to the density of 5X 10 6/mL to obtain seed cells;
(1.6) cells were transferred to 3 passages, and after confluence, the collected cells were digested, frozen in liquid nitrogen at a density of 5X 10 6/mL to give working cells, and the morphology of the 3 passages of cells was observed by a light microscope, and the results are shown in FIG. 1 (b).
2. Determination of additive concentration of Chinese medicine extract
Adding the Chinese medicine extract into a mesenchymal stem cell culture medium according to a plurality of concentrations; resuscitate the frozen mesenchymal stem cells for 5 generations, re-suspend and inoculate the mesenchymal stem cells in 96-well plates (3000 cells/well) for culture, discard the mesenchymal stem cells culture medium after 24 hours, replace the prepared mesenchymal stem cells culture medium containing traditional Chinese medicine extract, and continue to culture for 48 hours in 5 multiple wells per concentration.
Detecting the cell viability of each hole by using an MTT method, and determining the concentration of the traditional Chinese medicine extracting solution when the cell proliferation rate is maximum, wherein the concentration of the traditional Chinese medicine extracting solution added into a culture medium is divided into I groups: 1mL/L, II group: 2mL/L, III group: 4mL/L, IV group: 8mL/L, V group: 16mL/L, VI group: 32mL/L, the group without the Chinese medicine extract was used as a blank control group, the absorbance OD value of each group was tested by MTT kit, and the cell proliferation rate was calculated from the absorbance, and the results are shown in Table 1 and FIG. 2.
MTT assay detection of Medium cell absorbance (n=5, X.+ -. S)
| Group of | Concentration of Chinese medicine extract (mL/L) | Absorbance (OD/490 nm) |
| Blank control group | 0 | 0.3842±0.0122 |
| Ⅰ | 1 | 0.4024±0.0099 |
| Ⅱ | 2 | 0.4809±0.0134* |
| Ⅲ | 4 | 0.5031±0.0406* |
| Ⅳ | 8 | 0.4698±0.0322* |
| Ⅴ | 16 | 0.3804±0.0122 |
| Ⅵ | 32 | 0.2873±0.0144* |
Note that: * For comparison with the control group without added traditional Chinese medicine, P <0.05.
The results of Table 1 were observed: the cell viability is represented by the absorbance OD value of each group, the higher the absorbance, the stronger the cell viability and the maximum cell proliferation rate, as shown in Table 1, when the concentration of the traditional Chinese medicine extract is 1-4mL/L compared with that of the mesenchymal stem cell culture medium without the traditional Chinese medicine extract, the absorbance is increased along with the increase of the adding concentration, the cell viability is enhanced, and the absorbance is reduced and is lower than that of the mesenchymal stem cell culture medium without the traditional Chinese medicine extract along with the further increase of the adding concentration; observe fig. 2: the horizontal axis shows the concentration of the added Chinese medicine extract, the vertical axis shows the cell proliferation rate, and the result shows that compared with the traditional Chinese medicine extract without the addition of the Chinese medicine extract, the cell activity is obviously changed, the cell activity is obviously improved along with the increase of the addition concentration of the Chinese medicine extract within a certain range, and the cytoplasmic proliferation rate reaches the maximum when the addition concentration reaches 4mL/L, but the inhibiting effect can be shown along with the increase of the addition concentration of the Chinese medicine extract. The absorbance corresponds to the result of the cell proliferation rate test, so that the concentration of the traditional Chinese medicine extract added in the mesenchymal stem cell culture medium used in the method is 4 mL/L.
3. Mesenchymal stem cell culture medium containing Chinese medicinal components for culturing mesenchymal stem cells (flow chart shown in figure 3)
(1) Cells were resuscitated and passaged to 5 passages: resuscitate the cryopreserved 3 rd generation mesenchymal stem cells, transfer to 5 th generation;
(2) Cell culture: culturing the cells for 24 hours, discarding the supernatant, and dividing the cells into two groups;
(3) Adding a mesenchymal stem cell culture medium containing a traditional Chinese medicine extract for culture: adding a group of mesenchymal stem cell culture medium containing 4mL/L of traditional Chinese medicine extract, and continuously culturing for 72h;
(4) Adding a mesenchymal stem cell culture medium without a traditional Chinese medicine extract for culture: adding the other group of mesenchymal stem cell culture medium without adding the traditional Chinese medicine extract as a control group, and continuously culturing for 72 hours under the same condition as the step (3);
(5) Collecting supernatant containing vascular endothelial growth factor and Chinese medicinal extractive solution: collecting the supernatant containing the vascular endothelial growth factor and the traditional Chinese medicine extract in the steps (3) and (4), flushing the rest cells twice by using PBS, adding the PBS for continuous culture for 96 hours, collecting the PBS, and detecting the concentration of the vascular endothelial growth factor secreted by the two groups of mesenchymal stem cells by using an ELISA method.
Example 2
Example 2 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 14g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 8g of biota orientalis and 8g of dried ginger; the remainder was the same as in example 1.
Example 3
Example 3 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 5g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 12g of biota orientalis and 13g of dried ginger; the remainder was the same as in example 1.
Comparative example 1
Comparative example 1 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 15g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 10g of biota orientalis and 5g of dried ginger; the remainder was the same as in example 1.
Comparative example 2
Comparative example 2 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 6g of angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 10g of biota orientalis and 14g of dried ginger; the remainder was the same as in example 1.
Comparative example 3
Comparative example 3 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 14g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 6g of biota orientalis and 10g of dried ginger; the remainder was the same as in example 1.
Comparative example 4
Comparative example 4 differs from example 1 in that the traditional Chinese medicine formulation in this example comprises the following components: 7g of angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii, 13g of biota orientalis and 10g of dried ginger; the remainder was the same as in example 1.
Comparative example 5
Comparative example 5 differs from example 1 in that the traditional Chinese medicine formulation in this comparative example comprises the following components: 20g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii and 10g of dried ginger; the remainder was the same as in example 1.
Comparative example 6
Comparative example 6 differs from example 1 in that the traditional Chinese medicine formulation in this comparative example comprises the following components: 20g of Chinese angelica, 15g of polygonum multiflorum, 5g of ligusticum wallichii and 10g of biota orientalis; the remainder was the same as in example 1.
Comparative example 7
Comparative example 7 differs from example 1 in that the traditional Chinese medicine formulation in this comparative example comprises the following components: 30g of angelica, 15g of polygonum multiflorum and 5g of ligusticum wallichii; the remainder was the same as in example 1.
The results of the final measurement of the concentration of the secreted vascular endothelial growth factor by the method for promoting the secretion of VEGF by umbilical cord stem cells using the extract of traditional Chinese medicine described in the above examples and comparative examples are shown in Table 2 below.
ELISA method for measuring cell VEGF concentration in culture Medium (n=5, X.+ -. S)
Note that: * For comparison with the control group without added traditional Chinese medicine, P <0.05.
As shown in table 2, when the traditional Chinese medicine extract (control group) is not added into the mesenchymal stem cell culture medium, the concentration of the obtained Vascular Endothelial Growth Factor (VEGF) is 8.9882pg/mL, and after the traditional Chinese medicine extract is added, the concentration of the obtained vascular endothelial growth factor is obviously improved, so that the traditional Chinese medicine extract can promote umbilical mesenchymal stem cells to promote the secretion of vascular endothelial growth factor.
In summary, the invention provides a traditional Chinese medicine extract and an application thereof in promoting the secretion of VEGF by umbilical cord stem cells, a certain amount of traditional Chinese medicine components have a promoting effect on the proliferation of umbilical cord mesenchymal stem cells, but with the increase of the addition amount, the traditional Chinese medicine extract has a inhibiting effect, and has a certain instability in laboratory application due to the influence of various factors such as the production place, the collection time method, the storage method, the processing method and the like. The invention utilizes a non-high temperature and sterile process to extract the traditional Chinese medicine extract, and reserves the effective components of the traditional Chinese medicine to the maximum extent. The method combines the traditional Chinese medicine technology of China with the modern cell culture, and the related products can be further applied to the fields of repairing hair follicle injury, alopecia and the like.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Claims (3)
1. The application of the traditional Chinese medicine extract in promoting the secretion of VEGF by umbilical cord stem cells is characterized in that the traditional Chinese medicine extract is prepared according to the following method:
Mixing the components of the traditional Chinese medicine prescription, fully mixing with water, dissolving, extracting, and filtering to obtain traditional Chinese medicine extract;
The filtering step comprises the following steps: multi-layer gauze filtration, quantitative filter paper filtration and 0.2-0.25um aseptic filter membrane filtration,
The traditional Chinese medicine prescription consists of the following components: 10 parts of Chinese angelica, 15 parts of tuber fleeceflower root, 5 parts of szechuan lovage rhizome, 10 parts of biota orientalis leaf and 10 parts of dried ginger,
The application comprises the steps of:
A. Separating and preparing mesenchymal stem cells from umbilical cord tissues, and then carrying out passage amplification culture and freezing;
B. Adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium for culture, and determining the adding concentration of the traditional Chinese medicine extract through experiments;
C. the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract liquid obtained in the step B is used for culturing the mesenchymal stem cells,
Wherein:
The step A comprises the following steps:
A1, separating Wharton's jelly tissue from a neonatal umbilical cord for later use;
A2, inoculating the Whatman gel tissue blocks into a culture dish, and placing the culture dish in an incubator for preparation;
A3, adding a preheated mesenchymal stem cell culture medium into the culture dish in the step A2, and performing primary culture;
a4, sucking tissue blocks in the culture dish, and collecting cells for subculturing;
A5, transferring the cells to 1 generation, digesting and collecting the cells after the cells grow fully, inoculating the cells into a culture dish, continuously subculturing the cells, and freezing the rest cells;
a6, transferring the cells to 3 generations, digesting and collecting the cells after the cells grow up, freezing,
In the step A3, the mesenchymal stem cell culture medium is alpha-MEM, and 4-5.5% of serum substitute is added;
The step B comprises the following steps:
b1, adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium;
B2, resuscitating the frozen 3-generation mesenchymal stem cells, transferring to 5-generation mesenchymal stem cells, re-suspending and inoculating to a mesenchymal stem cell culture medium for culture, discarding the mesenchymal stem cell culture medium, adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B1 for continuous culture,
In the step B1, the concentration of the traditional Chinese medicine extracting solution added into the culture medium is 4mL/L;
The step C comprises the following steps:
resuscitate the frozen mesenchymal stem cells for 3 generations, transfer to 5 generations, continue to culture, discard supernatant, add the mesenchymal stem cell culture medium containing the Chinese medicine extract to continue to culture,
The step of adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract for continuous culture comprises the following steps:
c1, recovering the frozen 3-generation mesenchymal stem cells, and transferring to 5-generation mesenchymal stem cells;
c2, culturing for 24 hours, discarding supernatant, and adding a mesenchymal stem cell culture medium containing a traditional Chinese medicine extract for culturing;
C3, continuously culturing for 72 hours, collecting supernatant containing vascular endothelial growth factor and the traditional Chinese medicine extract, flushing residual cells with PBS, and adding PBS for continuously culturing;
C4, culturing for 96h, and collecting PBS.
2. A method for promoting secretion of VEGF by umbilical cord stem cells, comprising the steps of:
A. Separating and preparing mesenchymal stem cells from umbilical cord tissues, and then carrying out passage amplification culture and freezing;
B. Adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium for culture, and determining the adding concentration of the traditional Chinese medicine extract through experiments; the traditional Chinese medicine extract is prepared according to the following method:
Mixing the components of the traditional Chinese medicine prescription, fully mixing with water, dissolving, extracting, and filtering to obtain traditional Chinese medicine extract;
The filtering step comprises the following steps: multi-layer gauze filtration, quantitative filter paper filtration and 0.2-0.25um aseptic filter membrane filtration,
The traditional Chinese medicine prescription consists of the following components: 10 parts of Chinese angelica, 15 parts of polygonum multiflorum, 5 parts of ligusticum wallichii, 10 parts of biota orientalis leaves and 10 parts of dried ginger;
C. the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract liquid obtained in the step B is used for culturing the mesenchymal stem cells,
Wherein:
The step A comprises the following steps:
A1, separating Wharton's jelly tissue from a neonatal umbilical cord for later use;
A2, inoculating the Whatman gel tissue blocks into a culture dish, and placing the culture dish in an incubator for preparation;
A3, adding a preheated mesenchymal stem cell culture medium into the culture dish in the step A2, and performing primary culture;
a4, sucking tissue blocks in the culture dish, and collecting cells for subculturing;
A5, transferring the cells to 1 generation, digesting and collecting the cells after the cells grow fully, inoculating the cells into a culture dish, continuously subculturing the cells, and freezing the rest cells;
a6, transferring the cells to 3 generations, digesting and collecting the cells after the cells grow up, freezing,
In the step A3, the mesenchymal stem cell culture medium is alpha-MEM, and 4-5.5% of serum substitute is added;
The step B comprises the following steps:
b1, adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium;
B2, resuscitating the frozen 3-generation mesenchymal stem cells, transferring to 5-generation mesenchymal stem cells, re-suspending and inoculating to a mesenchymal stem cell culture medium for culture, discarding the mesenchymal stem cell culture medium, adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B1 for continuous culture,
In the step B1, the concentration of the traditional Chinese medicine extracting solution added into the culture medium is 4mL/L;
The step C comprises the following steps:
resuscitate the frozen mesenchymal stem cells for 3 generations, transfer to 5 generations, continue to culture, discard supernatant, add the mesenchymal stem cell culture medium containing the Chinese medicine extract to continue to culture,
The step of adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract for continuous culture comprises the following steps:
c1, recovering the frozen 3-generation mesenchymal stem cells, and transferring to 5-generation mesenchymal stem cells;
c2, culturing for 24 hours, discarding supernatant, and adding a mesenchymal stem cell culture medium containing a traditional Chinese medicine extract for culturing;
C3, continuously culturing for 72 hours, collecting supernatant containing vascular endothelial growth factor and the traditional Chinese medicine extract, flushing residual cells with PBS, and adding PBS for continuously culturing;
C4, culturing for 96h, and collecting PBS.
3. A composition, characterized in that it is prepared according to the following method:
A. Separating and preparing mesenchymal stem cells from umbilical cord tissues, and then carrying out passage amplification culture and freezing;
B. Adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium for culture, and determining the adding concentration of the traditional Chinese medicine extract through experiments; the traditional Chinese medicine extract is prepared according to the following method:
Mixing the components of the traditional Chinese medicine prescription, fully mixing with water, dissolving, extracting, and filtering to obtain traditional Chinese medicine extract;
The filtering step comprises the following steps: multi-layer gauze filtration, quantitative filter paper filtration and 0.2-0.25um aseptic filter membrane filtration,
The traditional Chinese medicine prescription consists of the following components: 10 parts of Chinese angelica, 15 parts of polygonum multiflorum, 5 parts of ligusticum wallichii, 10 parts of biota orientalis leaves and 10 parts of dried ginger;
C. B, using the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B for culturing mesenchymal stem cells, and collecting supernatant containing vascular endothelial growth factors and the traditional Chinese medicine extract to obtain the composition;
Wherein:
The step A comprises the following steps:
A1, separating Wharton's jelly tissue from a neonatal umbilical cord for later use;
A2, inoculating the Whatman gel tissue blocks into a culture dish, and placing the culture dish in an incubator for preparation;
A3, adding a preheated mesenchymal stem cell culture medium into the culture dish in the step A2, and performing primary culture;
a4, sucking tissue blocks in the culture dish, and collecting cells for subculturing;
A5, transferring the cells to 1 generation, digesting and collecting the cells after the cells grow fully, inoculating the cells into a culture dish, continuously subculturing the cells, and freezing the rest cells;
a6, transferring the cells to 3 generations, digesting and collecting the cells after the cells grow up, freezing,
In the step A3, the mesenchymal stem cell culture medium is alpha-MEM, and 4-5.5% of serum substitute is added;
The step B comprises the following steps:
b1, adding the traditional Chinese medicine extract into a mesenchymal stem cell culture medium;
B2, resuscitating the frozen 3-generation mesenchymal stem cells, transferring to 5-generation mesenchymal stem cells, re-suspending and inoculating to a mesenchymal stem cell culture medium for culture, discarding the mesenchymal stem cell culture medium, adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract obtained in the step B1 for continuous culture,
In the step B1, the concentration of the traditional Chinese medicine extracting solution added into the culture medium is 4mL/L;
The step C comprises the following steps:
resuscitate the frozen mesenchymal stem cells for 3 generations, transfer to 5 generations, continue to culture, discard supernatant, add the mesenchymal stem cell culture medium containing the Chinese medicine extract to continue to culture,
The step of adding the mesenchymal stem cell culture medium containing the traditional Chinese medicine extract for continuous culture comprises the following steps:
c1, recovering the frozen 3-generation mesenchymal stem cells, and transferring to 5-generation mesenchymal stem cells;
c2, culturing for 24 hours, discarding supernatant, and adding a mesenchymal stem cell culture medium containing a traditional Chinese medicine extract for culturing;
and C3, continuously culturing for 72 hours, and collecting supernatant containing vascular endothelial growth factor and the traditional Chinese medicine extract to obtain the composition.
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