CN110946876A - Application of mesenchymal stem cell exosome preparation in treating alopecia areata - Google Patents
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- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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Abstract
The invention relates to an application of a mesenchymal stem cell exosome preparation in treating alopecia areata, the exosome preparation comprises exosomes secreted by human umbilical cord mesenchymal stem cells and PBS, and the preparation method of the exosomes secreted by the human umbilical cord mesenchymal stem cells comprises that umbilical cord mesenchymal stem cells separated from umbilical cords of healthy fetuses born in due date are cultured at 37 ℃ to be P3; when the cells are fused to 60% -80%, starving and culturing for 48h in a DMEM/HAM' SF-12 culture medium without fetal calf serum; centrifuging the collected culture medium to remove cells and cell debris, and then extracting exosomes by an ultracentrifugation method; and (4) resuspending the precipitate with PBS and preserving at-80 ℃ to obtain the umbilical cord mesenchymal stem cell source exosome preparation, namely UC-MSCs-Exo. The UC-MSCs have the advantages of low preparation cost, rich sources, convenient collection, no traumatic operation to human bodies, simple UC-MSCs-Exo components, stable and non-degeneration cell factors, and capability of improving the skin tissue structure of alopecia areata and repairing or reconstructing damaged hair follicles.
Description
Technical Field
The invention belongs to the field of biological medicine, relates to a technology for preparing exosomes by using mesenchymal stem cells, and particularly relates to an application of a mesenchymal stem cell exosome preparation in treating alopecia areata.
Background
Alopecia has become a ubiquitous "modern disease" today. Although not serious, it brings great annoyance and inconvenience to people's life. The causes of hair loss and the types of hair loss are different, and the causes of a considerable portion of hair loss are not known. Alopecia areata is an autoimmune non-scar alopecia areata. The head is called round hair loss, round baldness or ghost shaving head. If the hair falls completely, the alopecia totalis is called alopecia universalis, and the skin of the affected area has no other abnormality except no hair. The pathological examination of alopecia areata shows that the hair follicle is reduced, the lymphocyte infiltration exists around the hair follicle, and the disease is sometimes combined with other autoimmune diseases. Despite the increasing understanding and awareness of alopecia areata and the numerous supportive or auxiliary treatment options that are developed with the current technology and diagnostic devices, there is still a need for more effective and comprehensive treatment options to treat alopecia areata in the clinical setting.
Mesenchymal Stem Cells (MSCs) are a type of pluripotent cells with the ability to self-replicate, and under certain conditions, can differentiate into a variety of functional cells. The umbilical cord mesenchymal stem cell is one of the stem cells with obvious advantages, and has some special advantages: has stronger proliferation and differentiation capability, low tumor formation rate, lower risk of generating graft-versus-host disease after allograft and does not involve more disputes in the aspects of society, morality, ethics and the like. However, since mesenchymal stem cells have a large limitation on the disease of scalp alopecia, researchers have focused on their secretions. It has been proved that the secretion of umbilical cord mesenchymal stem cells contains a large amount of cytokines, proteins and the like. The secreted factors include Platelet Derived Growth Factor (PDGF), Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (KGF), Hepatocyte Growth Factor (HGF), Transforming Growth Factor (TGF), etc., and the use of these factors in hair and skin has been generally accepted by scientists of various countries. However, the low content, unstable property and difficult storage of secreted factors have limited their application.
Exosomes (exosomes) are membrane vesicles of about 30-150 nm in diameter. Exosomes are present in cell culture fluids and body fluids, including saliva, pleural fluid, cerebrospinal fluid, ascites, urine, plasma, serum, and all other body fluids. Exosomes contain but do not fixedly express a variety of biomolecules, such as mRNA, miRNA, mtDNA, proteins, lipids, and the like. The exosome has the characteristics of good biocompatibility, no toxicity and no immunoreaction. And, the behavior of target cell-hair follicle cell is influenced by carrying biological molecules, such as proliferation, differentiation, apoptosis, recovery, reconstruction, etc. The exosome preparation is produced by using exosomes secreted by umbilical cord mesenchymal stem cells, can effectively treat scalp alopecia diseases, replaces the traditional pilatory, and has a good application effect in treating alopecia areata.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a mesenchymal stem cell exosome preparation capable of treating alopecia areata or alopecia, which replaces the traditional Chinese medicine or western medicine pilatory, and effectively avoids secondary damage and hormone resistance to the scalp when in use.
The technical problem to be solved by the invention is realized by adopting the following technical scheme:
the application of a mesenchymal stem cell exosome preparation in treating alopecia areata comprises exosomes secreted by human umbilical cord mesenchymal stem cells and PBS, and the preparation method of the exosomes secreted by the human umbilical cord mesenchymal stem cells comprises the following specific steps:
(1) umbilical cord mesenchymal stem cells isolated from umbilical cord of a full-term eutocia healthy fetus were cultured at 37 ℃ to P3;
(2) when the cells are fused to 60% -80%, starving and culturing for 48h in a DMEM/HAM' SF-12 culture medium without fetal calf serum;
(3) centrifuging the collected culture medium to remove cells and cell debris, and then extracting exosomes by an ultracentrifugation method;
(4) and (4) resuspending the precipitate with PBS and preserving at-80 ℃ to obtain the umbilical cord mesenchymal stem cell source exosome preparation, namely UC-MSCs-Exo.
Further, the concentration of the exosome preparation stored in the PBS resuspension pellet at-80 ℃ in the step (4) was 10 mg/mL.
Moreover, the prepared umbilical cord mesenchymal stem cell source exosome preparation is applied to treatment of alopecia areata or alopecia, and the specific method comprises the following steps:
(1) C3H/HeJNju mouse alopecia areata model establishment and inspection;
(2) treating a mouse alopecia areata model by UC-MSCs-Exo;
(3) the treatment of alopecia areata was tested.
And in the step (2), the concentration of the UCMSCs-Exo is 100ug/mL, the treatment mode is surface coating, and the treatment is continuously carried out for 6 weeks every 3 days.
Moreover, the method for detecting the treatment of the alopecia areata in the step (3) comprises surface observation and pathological section observation of skin.
The invention has the advantages and positive effects that:
1. the UC-MSCs-Exo is derived from mesenchymal stem cells separated from healthy umbilical cords which are produced in a normal manner in term, and the UC-MSCs have the advantages of low preparation cost, rich sources, convenience in collection and no traumatic operation on a human body.
2. The method for separating the umbilical cord mesenchymal stem cell source exosome is simple and universal, and can be used for large-scale production. The umbilical cord mesenchymal stem cell source exosome preparation has simple components and stable and uneasy degeneration of cell factors. Can be combined with medical apparatus for external application or in vivo injection.
3. The umbilical cord mesenchymal stem cell source exosome preparation (UC-MSCs-Exo) provided by the invention can improve the skin tissue structure of alopecia areata and repair or reconstruct damaged hair follicles. Animal experiments prove that new hair grows on the hair loss part of the mouse, and the new hair and the original hair have no difference in texture.
Drawings
FIG. 1 is a light mirror image of third generation (P3) umbilical cord mesenchymal stem cells (UC-MSCs);
FIG. 2 is a graph showing the results of the KGF content in the culture medium after starvation of the fourth generation UC-MSCs for 48 h;
FIG. 3 is a diagram of the result of the separation and characterization of umbilical cord mesenchymal stem cell-derived exosomes, wherein A is a Transmission Electron Microscope (TEM) image of exosomes, and B is a particle size diagram of exosomes;
FIG. 4 is a graph of the results of UC-MSCs-Exo model treatment on alopecia areata, wherein A is a graph of success in modeling alopecia areata and grouping conditions, and B is a graph of experimental results after 6W treatment;
FIG. 5 is a graph of the staining of skin HE at the treatment site of alopecia areata model by UC-MSCs-Exo.
Detailed Description
The present invention will be described in further detail with reference to the following embodiments, which are illustrative only and not limiting, and the scope of the present invention is not limited thereby.
Isolated culture and identification of umbilical cord mesenchymal stem cells (UC-MSCs)
Under aseptic conditions, the umbilical cord of a healthy fetus born in normal pregnancy at term is taken, the external mold of the umbilical cord tissue and the umbilical cord-driving vein are sufficiently washed with saline water and removed, and then the remaining tissue is cut into blocks with the diameter of about 2-3 mm. The small pieces were spread evenly on the bottom of the flask, dried at 37 ℃ for 1h, and then added slowly with the appropriate amount of DMEM/HAM' SF-12 complete medium.
When the culture is carried out for 5-7 days, a half-amount liquid changing method is adopted for liquid changing, on one hand, toxic effects generated by metabolites and floating small blocks are removed, and on the other hand, nutrients required by cell growth are supplemented. After the cells fused to 70-80%, they were digested with 0.25% trypsin and subcultured. When the UC-MSCs reach the fusion degree of 3 generations to 60%, the stem cell purity is identified by observing and photographing under an optical microscope, and the result is shown in figure 1.
KGF content detection in UC-MSCs culture medium
And replacing serum-free DMEM/HAM' SF-12 culture medium with fourth generation UC-MSCs with fusion degree of about 50-60%, and continuously culturing for 48-72 h. Collecting culture medium, and centrifuging at low speed and concentrating with ultrafiltration tube with molecular weight of 3.5K. And then carrying out KGF content determination on the concentrated culture medium by using an ELISA kit, wherein the determination groups are 4 groups in total and are respectively: 1. medium, 2, medium plus proteinase K, 3, medium plus triton100 plus proteinase K at 0.3% concentration, 4, medium plus triton100 at 0.3% concentration. Wherein proteinase K can decompose protein KGF, triton100 is membrane-permeable substance. From the analysis of FIG. 2, KGF may be present in the concentrated medium in encapsulated form. Through the query of relevant data, it is suspected that the protein KGF is probably encapsulated by exosome. Therefore, the umbilical cord mesenchymal stem cell exosome is separated and identified next.
Preparation of umbilical cord mesenchymal stem cell exosome preparation
The umbilical cord mesenchymal stem cell source exosome is separated by combining concentration and ultracentrifugation. The collected culture medium was first concentrated 10-fold using a 3.5K ultrafiltration tube, then exosomes were isolated according to an ultracentrifugation step, and finally resuspended with PBS to make umbilical cord mesenchymal stem cell exosome preparations (UC-MSCs-Exo) with a concentration of 10 mg/ml. The morphology and size were determined using transmission electron microscopy and Dynamic Light Scattering (DLS). Observed under a transmission electron microscope, the mesenchymal stem cell source exosome is elliptical, has the diameter of about 50-100nm, has a complete envelope structure and contains low-density substances, and is shown in figure 3.
Detection of treatment effect of UC-MSCs-Exo on alopecia areata
The tested mouse is C3H/HeJNju, about 20g, and the alopecia areata model is established by adopting a recognized imidazole coating method.
UC-MSCs-Exo treatment for alopecia areata model: bald model mice were randomly divided into 2 groups, one group was applied to bald with PBS, and one group was applied to bald with equal volume of UC-MSCs-Exo (concentration 100ug/mL), with 3 days/treatment of 6W.
And (3) detection of treatment effect: the detection of the treatment effect of UC-MSCs-Exo on the alopecia areata model mainly comprises the observation of hair regeneration condition and the microscopic observation of pathological sections at the alopecia areata position. In order to observe and determine the hair regeneration in alopecia areata, the test mice of each group were photographed, archived and analyzed before each application of UC-MSCs-Exo. As shown in FIG. 4, the hair was significantly more treated with UC-MSCs-Exo than PBS applied to the alopecia areata area. After 6 weeks of treatment, the mice are sacrificed by taking off the spinal column, blood and skin at the treatment position are rapidly collected, and after PFA fixation, dehydration, paraffin embedding, slicing and HE staining, PBS and UC-MSCs-Exo after alopecia areata treatment are observed, photographed and compared through a microscope. As shown in FIG. 5, the HE staining of the UC-MSCs-Exo treated group showed obvious hair follicle structure, indicating that the application of UC-MSCs-Exo restored or rebuilt the hair follicle in alopecia areata and thus promoted hair regrowth.
The experiments show that the exosome secreted by the umbilical cord mesenchymal stem cells can promote the recovery or reconstruction of damaged hair follicles, and a new treatment method can be provided for scalp alopecia diseases such as alopecia areata and seborrheic alopecia in clinic.
Although the embodiments of the present invention and the accompanying drawings are disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (5)
1. The application of a mesenchymal stem cell exosome preparation in treating alopecia areata, wherein the mesenchymal stem cell exosome preparation comprises exosomes secreted by human umbilical cord mesenchymal stem cells and PBS (phosphate buffer solution), and the preparation is characterized in that: the preparation method of the exosome secreted by the human umbilical cord mesenchymal stem cells comprises the following specific steps:
(1) umbilical cord mesenchymal stem cells isolated from umbilical cord of a full-term eutocia healthy fetus were cultured at 37 ℃ to P3;
(2) when the cells are fused to 60% -80%, starving and culturing for 48h in a DMEM/HAM' SF-12 culture medium without fetal calf serum;
(3) centrifuging the collected culture medium to remove cells and cell debris, and then extracting exosomes by an ultracentrifugation method;
(4) and (4) resuspending the precipitate with PBS and preserving at-80 ℃ to obtain the umbilical cord mesenchymal stem cell source exosome preparation, namely UC-MSCs-Exo.
2. Use of a mesenchymal stem cell exosome formulation according to claim 1 in the treatment of alopecia areata, characterised in that: the concentration of the exosome preparation in the PBS resuspension pellet stored at-80 ℃ in step (4) was 10 mg/mL.
3. Use of a mesenchymal stem cell exosome formulation according to claim 1 in the treatment of alopecia areata, characterised in that: the prepared umbilical cord mesenchymal stem cell source exosome preparation is applied to treatment of alopecia areata or alopecia, and the specific method comprises the following steps:
(1) C3H/HeJNju mouse alopecia areata model establishment and inspection;
(2) treating a mouse alopecia areata model by UC-MSCs-Exo;
(3) the treatment of alopecia areata was tested.
4. Use of a mesenchymal stem cell exosome preparation according to claim 3 in the treatment of alopecia areata, characterised in that: in the step (2), the concentration of UCMSCs-Exo is 100ug/mL, the treatment mode is surface coating, and the treatment is continuously carried out for 6 weeks every 3 days.
5. Use of a mesenchymal stem cell exosome preparation according to claim 3 in the treatment of alopecia areata, characterised in that: the method for detecting the treatment of the alopecia areata in the step (3) comprises surface observation and skin pathological section observation.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111821318A (en) * | 2020-08-28 | 2020-10-27 | 上海圣特佳健康科技发展有限公司 | Exosomes for treating or preventing white hair and uses and methods of use thereof |
| CN111956787A (en) * | 2020-07-06 | 2020-11-20 | 沃森克里克（北京）生物科技有限公司 | Hair growth promoter and hair growth promoter and application thereof |
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| CN112618572A (en) * | 2020-10-16 | 2021-04-09 | 中科细胞科技(广州)有限公司 | Composition for treating baldness |
| CN112190536A (en) * | 2020-11-13 | 2021-01-08 | 深圳玄鸟生物科技有限公司 | Exosome traditional Chinese medicine shampoo capable of growing hair and preventing alopecia and preparation method thereof |
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| CN113388575A (en) * | 2021-06-11 | 2021-09-14 | 杭州露源生物科技有限公司 | Preparation method of mesenchymal stem cell exosome for skin injury repair |
| CN114231483A (en) * | 2021-12-21 | 2022-03-25 | 广州市新甡命科技有限公司 | Stem cell exosome and application thereof in promoting hair growth |
| CN115227721A (en) * | 2022-08-24 | 2022-10-25 | 兰州大学第一医院 | Application of stem cell exosome in preparation of product for treating alopecia |
| CN115814045A (en) * | 2022-11-03 | 2023-03-21 | 山东新创生物科技有限公司 | Exosome composite preparation for improving alopecia and preparation method and application thereof |
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