CN102552323B - Medicine for accelerating skin repair and regeneration, preparation method thereof and application thereof - Google Patents

Abstract

The invention discloses a drug for accelerating skin layer repair and skin accessory organ regeneration, which is composed of epidermal stem cells, bone marrow mesenchymal stem cells and a drug carrier, and both the epidermal stem cells and bone marrow mesenchymal stem cells are adhered to the drug carrier. The number of epidermal stem cells on the drug carrier of mm is (0.3-1)×10 ⁴ , and the number of bone marrow mesenchymal stem cells is (0.3-1)×10 ⁴ . The application of the medicine of the present invention has significant effects in promoting the regeneration of the epidermis, promoting the regeneration of the dermis, enhancing the mechanical strength of the regenerated skin, promoting the content of collagen in the regenerated skin, promoting the regeneration of blood vessels, and promoting the regeneration of hair follicles, etc. Repair in a short period of time, and can accelerate the regeneration of skin appendages. The invention also discloses a preparation method of a medicine for accelerating skin layer repair and regeneration of skin appendages, which has the advantages of simple preparation, good controllability, strong operability and good reproducibility.

Description

Medicine for accelerating skin repair and regeneration, its preparation method and application

technical field

The invention relates to a medicine for accelerating skin layer repair and skin accessory organ regeneration, in particular to a medicine composed of epidermal stem cells, bone marrow mesenchymal stem cells and carriers.

Background technique

The skin is the largest organ of the human body, covering the whole body, and it can protect various tissues and organs in the body from physical damage, mechanical damage, chemical damage and biological damage. Skin regeneration mainly includes: 1) regeneration of various skin cells; 2) regeneration and remodeling of skin extracellular matrix, including various collagen and other components; 3) regeneration and functional recovery of skin appendages. With the continuous in-depth understanding of the mechanism of skin regeneration, the promotion of skin regeneration has become an important guide for skin repair and beauty, breaking the traditional old concept of "scars will inevitably be produced after wound wounds and surgical incisions heal", and replaced by "old scars". The fact that sexual scars can regenerate new skin without skin pigmentation and marks". Therefore, finding and developing drugs and their preparations that can promote skin regeneration and reduce scars to achieve non-traumatic skin repair has important application value for skin regeneration and beauty, and is a topic of common concern in the medical and cosmetic circles.

Under normal circumstances, the stratum corneum cells of the epidermis are continuously shed and replenished by the proliferation of basal cells, which is physiological regeneration. The repair and healing of the skin after injury is called compensatory regeneration. The regeneration process and repair time depend on the area and depth of the injury. When the skin injury area is small, it can heal within a few days without leaving scars; when the skin defect When it is larger and deeper, due to the excessive stress response of the skin to the injury, it will lead to excessive proliferation of cells and excessive secretion of extracellular matrix, resulting in the formation of scars. Compared with the normal healing mode after skin trauma, the characteristics of scar formation during skin regeneration are significantly weakened, and the phenomenon of excessive secretion of extracellular matrix such as collagen is significantly weakened. In order to achieve skin regeneration and reduce the formation of scars, one of the core links is to directly activate the regeneration potential of the skin, quickly realize the re-epithelialization of epidermal tissue in the early stage of healing, and at the same time inhibit the effect of fibrin and collagen on new injured wounds and surgical incisions. Synthetic deposition, to avoid scarring and its sequelae.

Stem cell research is a research hotspot in the field of life science in recent years. Since stem cells are primitive cells with self-renewal ability and multi-directional differentiation potential, under certain conditions, they can also proliferate and differentiate into different functional cells. Therefore, the research on stem cells plays a very important role in the renewal and damage repair of various tissues and organs in the living body, and has become the hope for many incurable diseases, especially the diseases of cell and tissue loss or damage. As stem cell drug research has obtained some research results in vitro and animal experiments, and cell transplantation has begun to explore the application of clinical treatment. More and more basic researchers and pharmaceutical researchers believe that the use of stem cell drugs to treat diseases caused by cell loss or damage has great potential and attractive prospects. Although so far, stem cell drugs are only a class of conceptual drugs, and no stem cell drug in the true traditional sense has been developed and put into the market, but with the deepening of stem cell biology and clinical application research, the concept of stem cell drugs is gradually becoming more and more important. deeply rooted in the hearts of the people. "Stem cell drug" refers to a class of therapeutic and preventive drugs that can prevent and treat diseases caused by cell loss or damage by regulating the proliferation and differentiation of stem cells in organisms. At present, a variety of stem cells have been used in the repair and treatment of various tissue wounds. It is believed that in the near future, stem cell medicine will become an important part of basic research and clinical prevention and treatment just like gene medicine. The research and development of stem cell drugs will become a new hotspot in the research and development of innovative drugs, and will eventually serve as a class of therapeutic and preventive drugs to serve patients and benefit mankind.

In recent years, many studies have initially proved that bone marrow mesenchymal stem cells (BMSCs) have special multipotential differentiation potential and can be transformed into various tissue cells such as bone, cartilage, fat, muscle, tendon, and skin. It has functional characteristics such as the ability to repair damaged functional tissue and immune regulation, and has broad therapeutic prospects. Krause et al. (Krause DS et al. 2001. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105: 369-377) found through experiments that BMSCs can also differentiate into epidermal cells in vivo, and can exist for a long time. Satoh et al. (Satoh H. et al. 2004. Transplanted mesenchymal stem cells are effective for skin regeneration in acute cutaneous wounds. Cell Transplant. 13(4): 405-12) showed that wound granulation tissue after bone marrow mesenchymal stem cells treatment Rich, strong function, high blood vessel density, obvious stratification. BMSCs not only have the cell differentiation potential to repair tissue, but also have the potential to induce angiogenesis by paracrine cytokines. A variety of experiments have proved that BMSCs can improve tissue blood supply through various channels, such as secreting pro-angiogenic factors and having the potential to differentiate into vascular endothelial cells, which are important mechanisms for promoting angiogenesis.

On the other hand, the repair of skin damage not only involves the participation of bone marrow mesenchymal stem cells, but also depends on the proliferation, differentiation and fusion of skin stem cells. Among them, the number of epidermal stem cells is considerable, and it has been proved that they are the progenitor cells of various skin cells, and are important elements for maintaining normal skin physiological functions and regulating wound skin regeneration. Compared with bone marrow mesenchymal stem cells, epidermal stem cells theoretically have a greater tendency to differentiate into the skin and its appendages. Therefore, epidermal stem cells undoubtedly have important research significance for the regeneration of wounded skin and its appendages, and the targeted delivery of epidermal stem cells to the local area is a key step in its therapeutic effect. Epidermal stem cells have strong plasticity, retain the pluripotency similar to embryonic cells, and are potential pluripotent stem cells. Epidermal stem cells can be used to construct artificial skin with complete epidermis, dermis and skin appendages, and functional integrity, so as to meet the needs of the treatment of large-area skin defects such as burns and trauma. In recent years, with the continuous improvement of the separation, purification and culture technology of epidermal stem cells, in order to achieve the purpose of free construction of the epidermis. By precisely inducing the differentiation of epidermal stem cells to realize the construction of skin appendages such as hair follicles and sweat glands, it has become a research hotspot in the field of skin wound regeneration therapy.

At present, there are few products that are reported to directly promote the regeneration of the skin and its appendages. Therefore, there is an urgent need for a drug that accelerates the repair of the skin layer and the regeneration of the skin appendages to meet the market demand.

Contents of the invention

The invention provides a drug for accelerating skin layer repair and regeneration of skin appendages. Epidermal stem cells and bone marrow mesenchymal stem cells are compounded on the drug carrier and applied to animal skin full-thickness defect models, so that the prepared drug can accelerate skin regeneration. Rapid repair of damage and rapid regeneration of various skin appendages.

A drug that accelerates the repair of the skin layer and the regeneration of skin appendages, consisting of epidermal stem cells, bone marrow mesenchymal stem cells and a drug carrier, wherein both the epidermal stem cells and the bone marrow mesenchymal stem cells adhere to the drug carrier, and each cubic millimeter The number of epidermal stem cells on the drug carrier is (0.3-1)×10 ⁴ , and the number of bone marrow mesenchymal stem cells on the drug carrier per cubic millimeter is (0.3-1)×10 ⁴ .

The epidermal stem cells and bone marrow mesenchymal stem cells used in the present invention are self-made cells, wherein the purity of epidermal stem cells is greater than 80%, and the purity of bone marrow mesenchymal stem cells is greater than 90%. Through systematic animal pharmacodynamic tests, it was discovered and proved The combined application of epidermal stem cells and bone marrow mesenchymal stem cells can effectively promote the repair of traumatic skin and the regeneration of skin appendages such as hair follicles and blood vessels.

In order to achieve better invention effects, the following are preferred as the present invention:

The number of epidermal stem cells per cubic millimeter of drug carrier is (0.6-0.75)×10 ⁴ , and the number of bone marrow mesenchymal stem cells per cubic millimeter of drug carrier is (0.6-0.75)×10 ⁴ . From the experimental results, the drugs that accelerate skin layer repair and skin accessory organ regeneration under the optimal conditions are very good in promoting epidermal layer regeneration and dermis layer regeneration, enhancing the mechanical strength of regenerated skin, promoting blood vessel regeneration, and promoting hair follicle regeneration. efficacy.

The epidermal stem cells are prepared by the following preparation method: the isolated epidermis obtained after pretreatment is added to trypsin cell digestion solution containing 0.03% to 0.08% by weight of trypsin to digest the isolated epidermis for 3 to 8 minutes , to obtain epidermal stem cells after isolation. More preferably, the epidermal stem cells are prepared by the following preparation method: add the separated epidermis obtained after pretreatment to trypsin cell digestion solution containing 0.05% by weight of trypsin to digest and separate the epidermis for 5 minutes, separate obtained epidermal stem cells.

During the in vitro culture of epidermal stem cells, how to obtain purer epidermal stem cells and how to maintain the phenotype and in vitro proliferation characteristics of epidermal stem cells are urgent problems to be solved in current research. The concentration of trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) and the digestion time used in the preparation of epidermal stem cells have an important impact on the biological characteristics (such as proliferation ability) of epidermal stem cells. On the one hand, a high concentration of Trypsin-EDTA is beneficial to the extraction and separation of epidermal stem cells from the basal layer of the epidermis. On the other hand, if the concentration of Trypsin-EDTA is too high, it will inhibit the biological activity of the cells, such as inhibiting their proliferation characteristics. . The epidermal stem cells obtained under the preferred conditions of the present invention have better proliferative ability, and through molecular identification by flow cytometry, the purity of the cells is over 80% (cumulative expression of molecular markers > 70%).

The drug carrier is gelatin sponge/β-tricalcium phosphate (β-TCP) scaffold; the mass ratio of gelatin sponge to β-tricalcium phosphate in the gelatin sponge/β-tricalcium phosphate scaffold is 1:1～ 100. For the preparation method, refer to the literature (Yoshitake Takahashi et al. 2005. Osteogenic differentiation of mesenchymal stem cells in biodegradable sponges composed of gelatin andb-tricalcium phosphate. Biomaterials 26.3587-3596).

Tests have shown that the used gelatin sponge/β-tricalcium phosphate ratio is higher than that of two commercially available cell scaffolds, i.e. collagen sponge scaffolds and polyethylene terephthalate (PET) scaffolds. Gelatin sponge/β- The adhesion of tricalcium phosphate to epidermal stem cells is beneficial to the growth and reproduction of epidermal stem cells in vitro. The commercially available collagen sponge scaffolds are often poor in hardness, and the pores of the scaffold are easy to collapse, which affects the supply of nutrients and oxygen, and cannot meet the mechanical strength required for local application on skin wounds. However, because of the excessive hardness and lack of flexibility of PET stents, it is difficult to produce ideal therapeutic effects in the application of skin wounds. Gelatin (with an isoelectric point of 9) is a collagen protein purified from pig skin collagen, which can be completely degraded, has high biological safety, and is low in cost. It has been widely used in medicine. On the other hand, biodegradable ceramic materials such as beta(β)-calcium triphosphate (beta(β)-TCP) have also been gradually developed as cell scaffold materials, but these ceramic materials have low degree of degradation and poor flexibility when used alone. and other shortcomings. Therefore, the combined application of biodegradable materials such as gelatin and bioceramic materials is conducive to optimizing the hardness and flexibility of cell scaffolds, maintaining the porous structure of cell scaffolds, ensuring the supply of nutrients and oxygen to cells in the scaffold carrier, and promoting cell growth in vitro. Proliferate and maintain their biological properties. In the present invention, gelatin combined with β-tricalcium phosphate is used to prepare the cell scaffold carrier of the stem cell medicine. And, when β-TCP is 50% (the mass ratio of gelatin and β-tricalcium phosphate in the gelatin sponge/β-tricalcium phosphate scaffold is 1: 1), the aperture of gelatin/β-tricalcium phosphate scaffold is 180mm ~200mm, which is most conducive to the adhesion, growth and reproduction of bone marrow mesenchymal stem cells on the cell scaffold. Therefore, the use of gelatin/β-tricalcium phosphate (β-TCP) scaffold as a stem cell drug carrier is beneficial to the adhesion, growth and reproduction of epidermal stem cells and bone marrow mesenchymal stem cells in vitro.

The invention also provides a preparation method of a drug for accelerating skin layer repair and regeneration of skin appendages, which is simple in preparation, good in controllability, strong in operability and good in reproducibility.

1) The epidermal stem cells are first washed with buffer solution, and then digested with trypsin cell digestion solution containing 0.03% to 0.08% trypsin by weight for 6 to 8 minutes, and then added to the digested epidermal stem cells Serum and penicillin-streptomycin DMEM low-sugar/F12 culture medium were centrifuged, the supernatant was discarded, and then DMEM low-sugar/F12 culture medium was added to obtain epidermal stem cell suspension;

The bone marrow mesenchymal stem cells are first washed with buffer, and then digested with trypsin cell digestion solution containing 0.03% to 0.08% trypsin by weight for 2 to 4 minutes, and then added to the digested bone marrow mesenchymal stem cells Add DMEM low-sugar/F12 culture medium containing fetal bovine serum and penicillin-streptomycin, centrifuge, discard the supernatant, then add DMEM low-sugar/F12 culture medium to obtain bone marrow mesenchymal stem cell suspension;

The DMEM low-sugar/F12 culture solution is composed of DMEM low-sugar culture solution and F12 culture solution with a weight ratio of 1.25 to 3.5:1;

Described DMEM low-sugar/F12 culture solution containing fetal bovine serum and penicillin consists of the following components in weight percentage:

DMEM low-sugar culture medium 50%～70%;

F12 culture medium 20%～40%;

Fetal bovine serum 3% to 9%;

Penicillin and Streptomycin 0.5%～1.5%;

Both DMEM low-sugar culture medium and F12 culture medium can use the products of Gibico Brl in the United States, and the identification of DMEM low-sugar culture medium can adopt the unified standard of the biomedical industry in the United States.

2) Mixing the drug carrier, the epidermal stem cell suspension and the bone marrow mesenchymal stem cell suspension in step 1), and incubating to obtain a drug that accelerates skin layer repair and skin accessory organ regeneration.

The invention provides the application of a drug for accelerating skin layer repair and skin accessory organ regeneration, and the drug for accelerating skin layer repair and skin accessory organ regeneration can be used as a drug for promoting epidermal layer regeneration and dermis layer regeneration. The skin is divided into three main layers from the inside to the outside, namely the subcutaneous tissue, dermis, and epidermis. The epidermis is the outermost layer of the skin and is composed of two types of cells: one is keratinocytes, which account for the vast majority of epidermal cells, and they can synthesize a large amount of keratin during differentiation, causing the cells to keratinize and shed; The cells are non-keratinocytes, with a small number, scattered among keratinocytes, including melanocytes, Langerhans cells and Merkel cells, each of which has a special function, but they are not related to the epidermis. Keratosis is not directly related. The lower layer of the epidermis, which occupies most of the structure is the dermis, with a thickness of about 2 mm, and can be divided into three layers, namely the papillary layer, sub-papillary layer and reticular layer. The normal dermis contains fibroblasts, mast cells, histiocytes, lymphocytes, a small amount of dendritic cells, a small amount of melanocytes, and a small amount of Langerhans cells. Fibroblasts can secrete and produce collagen fibers, elastic fibers, reticular fibers and matrix; at the same time, they are the main tissue repair cells after deep damage to skin tissue. Therefore, the proliferation and migration test of fibroblasts has become an important technical means to simulate the repair and regeneration of the dermis in vitro and to measure the acceleration of drugs to repair and regenerate the dermis. During wound healing, the migration, proliferation and differentiation of keratinocytes to form a new epidermis is one of the necessary processes to cover the wound. At the same time, dermal fibroblasts proliferate, migrate to form a new dermis, and secrete collagen And other extracellular matrix, together with the epidermis to form a complete skin tissue is the main process of wound skin repair. Through experiments, compared with the stent control group, after using the medicine of the present invention to accelerate skin layer repair and skin appendage regeneration, the wound healing speed is about 1.4 times that of the stent control group, which proves that the present invention accelerates skin layer repair and skin appendage regeneration. Drugs for organ regeneration have significant effects in promoting the regeneration of the epidermis and the dermis, and accelerate the regeneration of the skin at the wound site.

The invention provides the application of a drug for accelerating skin layer repair and skin accessory organ regeneration, and the drug for accelerating skin layer repair and skin accessory organ regeneration can be used as a drug for enhancing the mechanical strength of regenerated skin. As the body's barrier against adverse external environments, the skin has multiple functions, and its biomechanical properties ensure its ability to resist the influence of physical factors such as external tension and pressure. The skin has a certain degree of elasticity and toughness, and its tension mainly depends on the mechanical properties and interweaving mode of dermal collagen fibers. The recovery of wound tissue biomechanics is an important measure of wound healing. Through experiments, it is proved that the medicine for accelerating skin layer repair and regeneration of skin appendages of the present invention tends to enhance the tensile strength of regenerated skin at wound sites, and makes the biomechanical properties of skin tend towards normal tissues.

The invention provides an application of a medicine for accelerating skin layer repair and regeneration of skin appendages. The medicine for accelerating skin layer repair and regeneration of skin appendages can be used as a medicine for promoting the collagen content of the regenerated skin, and accelerates the regeneration of collagen in the skin protein production. Collagen is an extremely important structural protein and extracellular matrix component of vertebrate connective tissue. It widely exists in animal skin, bones, tendons, ligaments and other tissues, accounting for about 1/3 of the total protein in mammals. Collagen, as the attachment and scaffold for cell growth, can induce the proliferation, differentiation and transplantation of epithelial cells, etc., and play an important role in supporting organs and protecting the human body. Collagen and other components are arranged and combined in a specific form to form a network structure of the extracellular matrix. This structure anchors and supports cells, and provides an appropriate microenvironment for cell proliferation and growth. As can be seen from the Masson staining results of the experiment, the medicine for accelerating skin layer repair and regeneration of skin appendages of the present invention significantly promotes the content of collagen in the regenerated skin at the wound site, which is similar to the morphology and arrangement of collagen in normal tissues, improving Improve the quality of regenerated skin at the wound site.

The invention provides the application of a drug for accelerating skin layer repair and skin accessory organ regeneration, and the drug for accelerating skin layer repair and skin accessory organ regeneration can be used as a drug for promoting blood vessel regeneration. Angiogenesis is one of the key links in the process of wound repair, which directly affects the effect of wound healing and the outcome of diseases. Wound angiogenesis refers to the process of angiogenesis on the basis of the original microvascular bed under the stimulation of various factors after skin tissue injury, such as bleeding, ischemia, hypoxia, and inflammatory reactions. Human blood vessels supply oxygen, nutrients and bioactive substances to the wound site. Under normal physiological conditions, the doubling time of human vascular endothelial cells is about 1 year. Angiogenesis occurs in the human body only in special circumstances, such as wound healing and embryogenesis. One of the key links in tissue repair is angiogenesis. The quality of angiogenesis directly affects the speed of tissue repair. Angiogenesis-promoting agents can improve the healing of almost all types of wounds. At the same time, regenerated blood vessels are an important element in accelerating the return of the regenerated skin to its normal function. Therefore, the regulation of angiogenesis has always been one of the focuses in the field of tissue repair research. The development of wound-promoting local angiogenesis has been one of the most challenging research areas in trauma drug therapy. Through experiments, it is proved that the medicine for accelerating skin layer repair and regeneration of skin appendages of the present invention can promote blood vessel regeneration.

The invention provides the application of a drug for accelerating skin layer repair and skin accessory organ regeneration, and the drug for accelerating skin layer repair and skin accessory organ regeneration can be used as a drug for promoting hair follicle regeneration. The hair follicle is an accessory organ of the skin. Hair follicle stem cells exist around the hair and participate in the periodic growth of the hair follicle. The hair follicle is a periodic organ. In addition to maintaining the periodic growth of the hair, it can also participate in the repair of skin and soft tissue trauma. There is a large amount of observational evidence that the participation of hair follicles in wound repair can improve the quality of wound healing and reduce scarring. The hair follicle structure is also an important sub-organ in the hair, and its absence will cause the skin to lose important physiological functions, which will bring great pain to the patient. As an important accessory organ of the skin, the hair follicle has a unique structure and the ability to periodically regenerate, so it has important functions in physiology, pathology, immunity, and beauty. research hotspot. Promoting the regeneration of hair follicles in regenerated skin is one of the main therapeutic goals in the field of skin regeneration therapy. Through experiments, it is proved that the medicine for accelerating skin layer repair and regeneration of skin appendages of the present invention can promote hair follicle regeneration.

Compared with prior art, the present invention has following advantage:

According to the mechanism of skin regeneration, the present invention systematically designed the evaluation test of skin wound healing and accessory organ regeneration, and carried out the compound application of epidermal stem cells, bone marrow mesenchymal stem cells and drug carriers to prepare drugs that accelerate skin layer repair and skin accessory organ regeneration. Comprehensive and scientific evaluation, including proof that the medicine for accelerating skin layer repair and regeneration of skin appendages of the present invention can promote epidermis regeneration, promote dermis layer regeneration, enhance the mechanical strength of regenerated skin, promote the content of regenerated skin collagen, and promote angiogenesis And promote new functions such as hair follicle regeneration. The invention is scientific and rigorous in design, reasonable and practical in method, and the experimental results obtained are systematic and comprehensive, and the results will provide important scientific basis and technical support for formulating a new local treatment plan for skin regeneration.

The invention accelerates the repair of the skin layer and the regeneration of the skin appendages, so that the injured wound can be repaired in a short period of time, and can accelerate the regeneration of the skin appendages, and can satisfy the patient's desire for rapid wound healing and recovery of normal skin functions to the greatest extent. Vision, has a good application prospect and broad market demand.

The invention has the advantages of simple preparation, good controllability, strong operability and good reproducibility.

Description of drawings

Fig. 1 is the microscopic morphological observation, cell immunohistochemical identification results and flow cytometric identification results of epidermal stem cells prepared under preferred conditions in Example 1;

Fig. 2 is the measurement result figure of skin wound healing of different test groups in embodiment 4;

Fig. 3 is the measurement result figure of different test groups in embodiment 5 to skin regeneration skin regeneration mechanics;

Fig. 4 is the measurement result figure of different test groups to skin collagen content, angiogenesis and hair follicle regeneration in embodiment 6;

FIG. 5 is a graph showing the measurement results of immunohistochemical identification of regenerated blood vessels in the skin of different test groups in Example 7. FIG.

Detailed ways

Example 1: Preparation method, optimization of preparation conditions and identification of epidermal stem cells

1. Preparation method of epidermal stem cells

At present, the preparation method of epidermal stem cells mainly includes two steps: 1) Firstly, a single skin epidermis is obtained through the functional separation of the junction between the epidermis and the dermis by neutral protease, which avoids the pollution of dermal cells; and, because trypsin damages the cells Therefore, the neutral protease digestion method is firstly used to separate the epidermal layer of the skin by using its function of hydrolyzing the junction between the epidermis and the dermis, and then the epidermal layer of the skin is digested with trypsin to obtain a single cell. It is beneficial to the protection of the activity of the digested cells and ensures that a sufficient number of active target cells are obtained; 2) the epidermal stem cells are then separated by the specific combination of type IV collagen and epidermal stem cells, or flow cytometry is used to sort This technique separates epidermal stem cells from other extracted epidermal cells. Type IV collagen is a macromolecular protein composed of three polypeptide chains, which are connected by disulfide bonds. Compared with other collagen polypeptide chains, each polypeptide chain has three helical regions, glycine, lysine , The proportion of proline is also higher than other types of collagen. The adhesion of type IV collagen to epidermal stem cells and transiently expanded cells is different. The adhesion of epidermal stem cells can be completed within 20 minutes, while the adhesion of transiently expanded cells takes more than 60 minutes. The sorted epidermal stem cells and transiently amplified cells were separated by the adhesion of type IV collagen, and the epidermal stem cells could be obtained by harvesting the cells adhered to type IV collagen within <20 min. The method of isolating epidermal stem cells from epidermal cells through the rapid adhesion of epidermal stem cells to type IV collagen is simpler than other cell separation and purification methods, such as flow cytometry, and does not require special instruments and equipment, and the cost is lower. It is beneficial to its industrialization and clinical application. Therefore, the present invention selects rapid enzymatic hydrolysis combined with rapid adhesion of type IV collagen, and separates and prepares epidermal stem cells.

2. Optimization of preparation conditions

Although the development of epidermal stem cell technology has brought new impetus to skin regeneration and other fields. However, epidermal stem cells are located in the basal layer of the epidermis, the number is very small, accounting for only 1% to 10% of basal cells, and their growth cycle is slow. It is difficult to obtain a large amount in a short period of time, which greatly limits its clinical application. How to improve the efficiency of the isolation and culture of epidermal stem cells in vitro is an urgent problem to be solved in clinical application of epidermal stem cells. During the preparation of epidermal stem cells, the concentration of type IV collagen, cell density and the concentration of Typsin-EDTA are the three main factors affecting the yield and proliferation activity of epidermal stem cells. Therefore, in the present invention, the concentration of type IV collagen, the cell density and the concentration of Typsin-EDTA during cell fast adhesion are set as three investigation factors, and three investigation levels are set, and an orthogonal design method is adopted to set Experimental arrangements, using the extraction rate of epidermal stem cells as the index, compared the effects of different preparation conditions of epidermal stem cells on the extraction rate of epidermal stem cells (see Table 1 for the factors and levels of investigation, and Table 2 for the experimental arrangement).

Table 1

Table 2

3. Preparation of epidermal stem cells, taking test group 1# as an example

1) Remove the back and head skin of the rat, remove fat and blood streaks, and cut into rat strips with a length of 1.5cm and a width of 2mm;

2) Wash 3 times with ordinary D-Hanks (phosphate buffered saline solution without calcium and magnesium ions, Beijing Hongbokang Pharmaceutical Technology Co., Ltd.), 20 mL/time, 3 minutes each time;

3) Then use high anti-D-Hanks (high anti-D-Hanks is obtained by mixing ordinary D-Hanks solution and double antibody solution, wherein, when mixing, the volume ratio of ordinary D-Hanks solution and double antibody solution is 100:4, The double-antibody is penicillin and streptomycin, and the double-antibody solution is purchased from Gibico, Brl, USA) and soaked for 150 minutes;

4) put into the neutral protease aqueous solution that contains dispase II neutral protease (purchased from Gibico, Brl, USA) weight percent and be 0.25%, wherein, the volume of neutral protease aqueous solution is that immersion rat pimp is appropriate, at 4 Place overnight at ℃ for 15 hours;

5) After leaving overnight, gently peel off the epidermis with tweezers;

6) DMEM low-sugar/F12 culture fluid containing fetal bovine serum (the DMEM low-sugar/F12 culture fluid containing fetal bovine serum is cultivated by the DMEM low-sugar culture fluid of 60% by weight and the F12 culture of 30% by weight liquid and 10% fetal bovine serum by weight, wherein DMEM low-sugar medium, F12 medium and fetal bovine serum were all purchased from Gibico, Brl, USA) containing trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) is 0.25% by weight of trypsin cell digestion solution (trypsin cell digestion solution is obtained by dissolving trypsin-ethylenediaminetetraacetic acid in phosphate buffer solution of pH=7.4) diluted to 0.02%, to obtain Trypsin cell digestion solution containing 0.02% by weight of Trypsin-EDTA;

Add the epidermis peeled off in step 5) to the trypsin cell digestion solution containing 0.02% by weight of Trypsin-EDTA, and the trypsin cell digestion solution containing 0.02% by weight of Trypsin-EDTA is peeled off by immersion An appropriate amount of epidermis was digested and separated at 37°C for 5 minutes;

7) Use a 15ml centrifuge tube to shake gently for 5 minutes, and repeatedly blow and beat;

8) Repeatedly wash 4 times with PBS solution, pass through 200 mesh and 300 mesh successively, and centrifuge at 1000rpm for 3 minutes;

9) Count the cell density with a cell counter (Beijing Zhuochuan Electronic Technology Co., Ltd.);

10) Use DMEM low-sugar/F12 culture fluid (DMEM low-sugar culture fluid/F12 culture fluid is made up of DMEM low-sugar culture fluid and F12 culture fluid with a weight ratio of 2:1, DMEM low-sugar culture fluid and F12 culture fluid are all purchased from Gibico, Brl, USA) resuspend the cells, adjust the cell density to 5×10 ⁵ cells/mL, and pour into the culture bottle (the culture bottle is pre-coated with an aqueous solution containing type IV collagen and acetic acid, and the water solution containing type IV collagen and acetic acid The weight percentage of type IV collagen is 0.01%, and the weight percentage of acetic acid in the aqueous solution containing type IV collagen and acetic acid is 0.1%); after standing for 10 minutes, discard the supernatant, and change fresh DMEM low sugar /F12 culture solution, cultured in a cell culture box, changing the solution once every three days, on the 8th day, the cells reached 80% confluence, digested cells, used to prepare medicines for accelerating skin layer repair and regeneration of skin appendages.

Experimental groups 2#, 3#, 4#, 5#, 6#, 7#, 8#, and 9# were prepared with epidermal stem cells according to the above steps, and the preparation conditions used the specific conditions in Table 2, and the rest of the conditions were the same as above.

The morphology, number and clone formation of epidermal stem cells prepared by each experimental group are shown in Table 3. As can be seen from Table 3, the number of epidermal stem cells prepared by experimental group 7# is the largest, and the ability to form clones is relatively strong (indicating that the cell viability is relatively high Strong), the time for forming clones is shortened from 7 to 10 days to 3 to 5 days, which is determined to be the most optimal condition for preparing epidermal stem cells in the present invention.

table 3

4. Identification of Epidermal Stem Cells

I. Cell immunohistochemistry:

1) Place the sterilized 20mm cover glass in a 90mm petri dish, inoculate the epidermal stem cells prepared by test group 7# in the petri dish at a cell density of 2× ¹⁰⁴ /ml for cell climbing, 5 Immunocytochemical staining was carried out for identification two days later;

2) Wash the cells with PBS (phosphate buffer solution with pH=7.4) for 3 times, 2 min each time;

3) Submerging the washed cells in an aqueous paraformaldehyde solution containing 4% by weight of paraformaldehyde and fixing them for 15 minutes, and the fixing process was carried out in a refrigerator at 4 degrees Celsius;

4) Dry the fixed cells in air for 5 minutes;

5) Wash the cells on the coverslip 3 times with PBS, 2min/time;

6) Dissolving polyethylene glycol octylphenyl ether (Triton X-100) in PBS to obtain a solution containing 0.5% by weight of TritonX-100, and incubating in the solution for 20 minutes;

7) After being incubated with a solution containing 0.5% by weight of Triton X-100, wash with PBS for 3 times, 2min/time;

8) Dissolving H ₂ O ₂ in PBS to obtain a solution containing 3% by weight of H ₂ O ₂ , and incubating in the solution for 15 minutes;

9) After incubation with a solution containing 3% H ₂ O ₂ by weight, wash with PBS for 3 times, 2 min each time;

10) Incubate the cells on the coverslip in the blocking serum for 20 min;

11) After incubation with the blocking serum, incubate at 37°C for 60 min in the primary antibody (p63) solution (the primary antibody solution is prepared from the primary antibody p63 and PBS, wherein the volume ratio of the primary antibody p63 and PBS is 1:25) ;

The negative control was incubated in PBS at 37°C for 60min;

12) Wash the incubated cells with PBS 3 times, 5min/time;

13) Incubate in PV6001 (wet box) with secondary antibody working solution at 37°C for 30min;

14) Wash the incubated cells with PBS 5 times, 2min/time;

15) The cells on the coverslip were developed with DAB for about 10-15 minutes;

16) Wash with distilled water for 5 minutes;

17) counterstain with hematoxylin for 10 minutes;

18) Wash with distilled water for 5 minutes;

19) Seal the slides with gum.

II. Flow detection:

1) Add 100ul of epidermal stem cells (ESCs) prepared by test group 7# to 100ul of Percp-CD34 (Santa Cruze, USA) antibody connected with fluorescent dye, and incubate in ice water bath for 30 minutes;

2) Add PBS solution again, 1.5ml/time, centrifuge for 5 minutes (2000rpm), discard the supernatant, wash twice to remove excess antibody;

3) Resuspend the cells with 500 ul of paraformaldehyde aqueous solution containing 1% by weight of paraformaldehyde, fix in an ice bath for 30 minutes, centrifuge for 5 minutes (2000 rpm), and discard the supernatant;

4) Wash twice with PBS solution, 1.5ml/time, centrifuge for 5 minutes (2000rpm), discard the supernatant;

5) adding a PBS solution containing fetal bovine serum (the PBS solution containing fetal bovine serum is obtained by mixing fetal bovine serum and PBS, wherein the weight percentage of fetal bovine serum in the PBS solution containing fetal bovine serum is 5%), and incubated in an ice bath for 10 minutes;

6) Add 1 ml of paraformaldehyde aqueous solution containing 4% by weight of paraformaldehyde, and fix at room temperature 25°C for 30 minutes;

7) Replace the antibody solution in step 1) with PBS solution, repeat steps 1) to 6), and process the sample in the same steps as a negative control.

8) The positive expression rates of two cell surface antigens, CD34 and CD71, in the sample were determined by flow cytometry (BD, USA).

Figure 1 is the molecular marker flow identification results of epidermal stem cells prepared under the condition of experimental group 7#. Among them, Figures a and b are the microscopic morphology of epidermal stem cells, and Figure a is the microscopic morphology of the epidermal stem cells prepared under the condition of experimental group 7# on the 3rd day, as shown in Figure a, the separated cells are dispersed Uniform, relatively round, but with different sizes; Figure b is the microscopic morphology of the epidermal stem cells prepared under the condition of experimental group 7# on the 11th day, as shown in Figure b, the cells are connected into sheets, forming a pavement Stone-like, individual areas will grow in multiple layers. Figure c is the immunohistochemical identification of p63 positive staining of epidermal stem cells under the condition of experimental group 7#; Figure g is the positive rate of epidermal stem cell positive marker p63 under the condition of flow cytometry experimental group 7# is 49.2%, and Figure d is the positive rate of the experimental group Under the condition of 7#, the positive rate of p63 in the negative control of epidermal stem cells was 1.2%; Figure h shows that the positive rate of CD3, a positive marker of epidermal stem cells in the experimental group 7#, was 31.1%, and Figure e shows the negative control CD34 in the experimental group under the condition of 7# The positive rate was 1.1%; Figure i shows that the negative marker CD71 positive rate of epidermal stem cells in the experimental group 7# was determined by flow cytometry, and the negative marker CD71 positive rate was 2.7%, and Figure f shows that the negative control CD71 positive rate was 1.5% under the experimental group 7# condition. Epidermal stem cells are derived from the basal layer of the epidermis and the bulge of the hair follicle, among which p63 is a specific cell surface antigen highly expressed by the epidermal stem cells derived from the basal layer of the epidermis, and CD34 is the specific cell surface of the epidermal stem cells derived from the bulge of the hair follicle antigen. Figure 1 proves that the purity of epidermal stem cells prepared under the condition of experimental group 7# is relatively ideal (the sum of the expression rates of p63 and CD34 cell surface antigens is as high as 80.3%), and the cells have strong proliferation ability, which can be used as a therapeutic drug for wound tissue regeneration .

Example 2: Preparation of bone marrow mesenchymal stem cells

The preparation of bone marrow mesenchymal stem cells can refer to the literature (Cai-Xia He, Ni Li, Yu-Lan Hu, Xiu-Mei Zhu, Hai-Jie Li, Min Han, Pei-Hong Miao, Zhong-Jie Hu, Gang Wang, Wen-Quan Liang, Yasuhiko Tabata, Jian-Qing Gao*, Effective gene delivery tomesenchymal stem cells based on the novel reverse transfection and three-dimensional cell culture system, Pharm Res, 2011, 28(7): 1577-1590)

1) Preparation and in vitro culture of primary rat bone marrow mesenchymal stem cells (BMSCs): 3-week-old SD rats were sacrificed by neck pulling, the tibia and femur were separated, and the containing The tibia and femur were repeatedly washed with serum culture solution, and the obtained bone marrow was passed through a 200-mesh cell sieve (Shanghai Ruigu Biotechnology Co., Ltd.), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. After centrifugation, add the DMEM low-sugar/F12 medium containing fetal bovine serum and double antibody (the DMEM low-sugar medium containing fetal bovine serum and double-antibody consists of 62% DMEM low-sugar medium by weight, 32% by weight F12 culture solution, 5% fetal bovine serum by weight and 1% double antibody solution by weight, wherein, fetal bovine serum, double antibody solution, DMEM low-sugar culture medium and F12 culture medium were all purchased from In Gibico, Brl, USA, double antibodies refer to penicillin and streptomycin). On the 7th day, use trypsin cell digestion solution containing trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) at a weight percentage of 0.25% (trypsin cell digestion solution is dissolved by trypsin-ethylenediaminetetraacetic acid at pH= The phosphate buffer solution obtained in 7.4) digested the primary cells, subcultured at 1:3～1:4, changed the fresh culture medium once after the first generation and subsequent cells were subcultured, and digested and subcultured when the cells grew to 80% full of the culture dish ( About the fifth day after passage), the second to fifth passage cells were used for experiments.

2) Change the medium: use a pipette gun to discard the culture medium in the culture dish, then add 10 mL of fresh DMEM low-sugar/F12 culture medium containing fetal bovine serum and double antibody, and shake in a horizontal 8 shape. When the newly prepared BMSCs are replaced for the first time, they can be washed once with phosphate buffer saline (PBS) at pH=7.4 before adding fresh DMEM low-sugar/F12 culture medium containing fetal bovine serum and double antibody;

3) Subculture: After discarding the culture medium in the culture dish with a pipette gun, wash it twice with PBS, and add 2.5 mL of trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) containing 0.25% by weight Digest the cells with trypsin and observe under a light microscope. When the cells curl up into a round shape, immediately add 5 mL of low-sugar DMEM/F12 medium containing 10% fetal bovine serum (DMEM low-sugar/F12 medium containing 10% fetal bovine serum) It is obtained by mixing 5% fetal bovine serum by weight, 63.33% DMEM low-sugar culture medium by weight and F12 culture medium with 31.67% by weight. Fetal bovine serum, DMEM low-sugar culture medium and F12 The culture medium was purchased from Gibico, Brl, USA) to terminate the digestion. Use a pipette gun to blow repeatedly into a cell suspension, put it in a centrifuge tube, and centrifuge at 1200rpm for 5 minutes, discard the supernatant, and add DMEM low-sugar/F12 culture solution containing 15% fetal bovine serum (containing 15% fetal bovine serum) The DMEM low-sugar/F12 culture fluid of serum is obtained by mixing 15% fetal bovine serum by weight, 56.67% DMEM low-sugar culture fluid and 28.33% F12 culture fluid by weight. Serum, DMEM low-sugar culture medium and F12 culture medium were all purchased from Gibico, Brl, USA), mixed by pipetting, and subcultured at 1:3-1:4.

Embodiment 3: the preparation of the medicine that accelerates skin layer repair and skin accessory organ regeneration

1) The epidermal stem cells (ESCs) prepared by experimental group 7# in Example 1 were first washed twice with PBS, and then washed with trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) containing 0.05% by weight of pancreatic Enzyme cell digestion solution (trypsin cell digestion solution obtained by dissolving trypsin-ethylenediaminetetraacetic acid in phosphate buffer at pH = 7.4) was digested for 7 minutes, and then added to the epidermal stem cells digested by trypsin cell digestion solution containing The DMEM low-sugar/F12 culture fluid of fetal bovine serum and penicillin streptomycin (the DMEM low-sugar/F12 culture fluid that contains fetal bovine serum and penicillin streptomycin is made of 10% fetal bovine serum by weight percentage, and the weight percentage is 1% penicillin, streptomycin, DMEM low-sugar culture medium of 59.33% by weight and F12 culture medium of 29.67% by weight are mixed, fetal bovine serum, penicillin, DMEM low-sugar culture medium and F12 The culture solution was purchased from Gibico, Brl, USA), centrifuged at 1200rpm for 3 minutes, discarded the supernatant, and added an appropriate volume of DMEM low-sugar/F12 culture solution (DMEM low-sugar/F12 culture solution was prepared from DMEM low-sugar solution with a weight ratio of 2:1 Composition of culture medium and F12 culture medium, DMEM low-sugar culture medium and F12 culture medium are all purchased from Gibico, Brl, USA) to obtain epidermal stem cell suspension, the concentration of epidermal stem cells in the epidermal stem cell suspension is 1.3334×10 ⁷ /ml ;

Wash the bone marrow mesenchymal stem cells (BMSCs) prepared in Example 2 twice with PBS, and then use trypsin cell digestion solution containing 0.05% by weight of trypsin-ethylenediaminetetraacetic acid (Trypsin-EDTA) (Treatin cell digestion solution is obtained by dissolving trypsin-ethylenediaminetetraacetic acid in phosphate buffer at pH=7.4) to digest for 3 minutes, and then add fetus-containing bone marrow mesenchymal stem cells digested by trypsin cell digestion solution The DMEM low-sugar/F12 culture fluid of bovine serum and penicillin streptomycin (the DMEM low-sugar/F12 culture fluid that contains fetal bovine serum and penicillin streptomycin is made of 10% fetal bovine serum by weight percentage, 1% by weight % penicillin streptomycin, 59.33% by weight of DMEM low-sugar culture fluid and 29.67% by weight of F12 culture fluid are mixed to obtain fetal bovine serum, penicillin and streptomycin, DMEM low-sugar culture fluid and F12 culture All liquids were purchased from Gibico, Brl, USA), centrifuged at 1200rpm for 3 minutes, discarded the supernatant, and added an appropriate volume of DMEM low-sugar/F12 culture solution (DMEM low-sugar/F12 culture solution was cultured from DMEM low-sugar with a weight ratio of 2:1) DMEM low-sugar culture medium and F12 culture medium were both purchased from Gibico, Brl, USA) to obtain the bone marrow mesenchymal stem cell suspension, and the concentration of bone marrow mesenchymal stem cells in the bone marrow mesenchymal stem cell suspension 1.3334×10 ⁷ /ml;

2) Equal volumes of the epidermal stem cell suspension and the bone marrow mesenchymal stem cell suspension were mixed to obtain a mixed solution, and the concentrations of the epidermal stem cells and bone marrow mesenchymal stem cells in the mixed solution were both 6.6667×10 ⁶ cells/ml;

3) The drug carrier gelatin sponge/β-tricalcium phosphate (β-TCP) scaffold (volume is 1cm×1cm×3mm, the mass ratio of gelatin sponge and β-tricalcium phosphate in the gelatin sponge/β-tricalcium phosphate scaffold is Add 1:1) to a 24-well plate, add 300 μl of the mixture in step 2) to each well, and incubate for half an hour to obtain a drug that accelerates the repair of the skin layer and the regeneration of skin appendages, which can be used as a drug. Mix The epidermal stem cell suspension and bone marrow mesenchymal stem cells in the solution almost all adhered to the drug carrier, and the number of epidermal stem cells contained on the 1cm×1cm×3mm drug carrier was (1.8～2)×10 ⁶ , 1cm×1cm×3mm The number of bone marrow mesenchymal stem cells contained on the drug carrier of 1 cm×3 mm is (1.8-2)×10 ⁶ . That is, the number of epidermal stem cells per cubic millimeter of drug carrier is (0.6-0.67)×10 ⁴ , and the number of bone marrow mesenchymal stem cells per cubic millimeter of drug carrier is (0.6-0.67)×10 ⁴ indivual.

Embodiment 4: animal wound skin regeneration test

1) 25 6-week-old female SD rats of clean grade, weighing about 140 g, were divided into 5 groups, 5 rats in each group. The five groups are: blank control group, scaffold control group, BMSCs (bone marrow mesenchymal stem cells) administration group, ESCs (epidermal stem cells) administration group, and BMSCs+ESCs compound drug group;

2) Anesthetize the animal with amobarbital sodium (6 mg/100 g), cut off the back hair, measure an area of 1 cm × 1 cm with an electric vernier caliper, mark the area with a marker pen, and cut out a 1 cm × 1 cm area with scissors The area of 1cm is disinfected with iodine;

3) Half an hour after the wound, apply drugs locally on the wound surface respectively, that is, the scaffold control group (without stem cells, the drug carrier is gelatin sponge/β-tricalcium phosphate scaffold, the volume is 1cm×1cm×3mm, gelatin The mass ratio of gelatin sponge and β-tricalcium phosphate in the sponge/β-tricalcium phosphate scaffold is 1:1), BMSCs administration group (the gelatin sponge/β-tricalcium phosphate scaffold with a volume of 1cm×1cm×3mm contains The number of BMSCs is 4× ¹⁰⁶ , the mass ratio of gelatin sponge to β-tricalcium phosphate in the gelatin sponge/β-tricalcium phosphate scaffold is 1:1), the ESCs administration group (gelatin sponge with a volume of 1cm×1cm×3mm /β-tricalcium phosphate scaffold contains 4× ¹⁰⁶ ESCs, the mass ratio of gelatin sponge to β-tricalcium phosphate scaffold in gelatin sponge/β-tricalcium phosphate scaffold is 1:1), BMSCs+ESCs administration group (Adopt the medicine of accelerating skin layer repair and skin accessory organ regeneration prepared in Example 3), after the wound surface is pasted with medicine, fix and protect the wound with a transparent biological dressing, and the blank control group does not do any treatment;

4) After that, except for the blank control group, the dressings of the other groups were changed every three days. The wound healing and scar formation of animals in each group were observed and photographed every day, and the experiment period was 10 days. Rats were sacrificed, and the skin of the area circled at the beginning of the experiment was cut off (1 cm×1 cm area) for future investigation.

Fig. 2 is the skin wound healing rate of each test group in 10 days. As shown in Figure 2, compared with the blank control group, the stent control group only showed a promoting effect on wound healing on the 8th day. The BMSCs-administered group showed significant healing-promoting effects on days 7 and 8; the ESCs-administered group showed significant promoting effects on days 5-9. The BMSCs+ESCs compound drug group showed a significant or extremely significant accelerated healing effect in 4 to 10 days (*p<0.05, **p<0.01), which proved that both BMSCs and ESCs could accelerate skin healing. The efficacy of wound healing (wound skin regeneration), on the other hand, also proves the synergistic effect of the compound application of two kinds of stem cells in accelerating skin wound healing (wound skin regeneration).

Embodiment 5: wound skin biomechanical strength test

Use a tensiometer to measure the biomechanical strength of the wound regenerated skin of each group on the 10th day in Example 4, test the tension of the regenerated skin on the tensiometer, take healthy skin as a positive control, and as a normal skin group, pull the skin tissue The speed is 25mm/min, and the specific mechanical data are shown in Figure 3.

Figure 3 shows the biomechanical strength of the regenerated skin at the wound site on the 10th day of each experimental group. As shown in Figure 3, compared with the blank control group, only the BMSCs+ESCs compound drug group can significantly increase the biomechanical strength of the skin group (* p<0.05, **p<0.01), which proves that the combination of epidermal stem cells and bone marrow mesenchymal stem cells can enhance the biomechanical strength of newborn skin, making the tensile strength of regenerated skin approach to that of normal skin .

Embodiment 6: Staining test of animal wound newborn skin tissue

On the 10th day, the regenerated wound skin and normal skin in each test group in Example 4 were taken for tissue section staining, and the specific implementation contents included:

1) Material collection: the whole skin was removed, with an area of 1cm×1cm, and the subcutaneous fat was removed to obtain new skin tissue from the wound in each test group;

2) Fix: fix the skin tissue in step (1) in an aqueous paraformaldehyde solution containing 4% by weight of paraformaldehyde for 24 hours, the volume of the fixative is 15 times the volume of the skin, and the fixation process is carried out in a refrigerator at 4 degrees Celsius in progress;

3) Water washing: put the fixed skin tissues of the blank control group, stent control group, BMSCs administration group, ESCs administration group, and BMSCs+ESCs compound drug group into PBS buffer (pH=7.4), and refresh every 10 minutes Washing solution once, accumulatively 1 hour;

4) dehydration: (mass percentage is 0～95% ethanol aqueous solution dehydration 1～10 hours) specific dehydration conditions are as follows:

70% ethanol, 5 hours,

85% ethanol, 2 hours,

95% ethanol, 2 hours,

100% ethanol, 2 hours,

100% ethanol, 2 hours;

5) Transparency: the skin tissues of the blank control group, stent control group, BMSCs administration group, ESCs administration group, and BMSCs+ESCs compound drug group after ethanol dehydration were blotted with absorbent paper and then put into an anhydrous The mixture of ethanol and xylene mixed at 1:5 (volume ratio) was transparent for 5 minutes, then placed in xylene and soaked twice, each time for 0.5 hours;

6) Waxing and embedding: dipping in liquid paraffin (45°C) for 5 hours, then dipping in liquid paraffin (60°C) for 5 hours, and embedding;

7) Slicing: the thickness of the slice is 8 μm, and a paraffin slice is obtained;

8) Dyeing (the steps are as follows):

a. Paraffin sections were soaked twice in xylene for 5 minutes each time. Then soak in absolute ethanol twice, 3 minutes each time. Place again in 95%, 85%, and 75% ethanol for 3 minutes; wash with tap water, wash with distilled water, and then transfer to 0.5% toluidine blue solution (Aladdin Company) in mass percent;

b. In hematoxylin-ferric chloride mixed solution (hematoxylin 3g, be dissolved in 100ml 95% ethanol aqueous solution, record as stock solution A; 19% ferric chloride solution 4ml, double distilled water 95ml, hydrochloric acid 3ml, Recorded as stock solution B; A and B were mixed in equal volumes) (hematoxylin was purchased from Sigma Company, and ferric chloride solution was purchased from Sinopharm Group) and stained for 10 minutes;

c. Rinse with double distilled water for 5 minutes;

d. 1% by mass percentage of Bibreci Scarlet-1% by mass percentage of acid fuchsin mixture (volume ratio 9:1) (Shanghai Jiexing Biotechnology Co., Ltd.) for 5 minutes;

e. Distilled water washing;

f. In phosphomolybdic acid phosphotungstic acid mixed solution (the mass percent is 5% phosphotungstic acid and the mass percent is 5% phosphotungstic acid mixed with volume ratio as 1: 1) (phosphotungstic acid and phosphomolybdic acid are purchased from Aladdin Company) for 15 minutes or until the collagen is no longer red;

g. directly transfer to 2.5% by mass percent aniline blue solution, dye for 5 minutes, simply wash in water, and distinguish in 1% glacial acetic acid solution for 3 minutes by mass percent;

h. Wash and dry the water;

i. dehydration in sequence (95% ethanol, 100% ethanol, dehydration twice in xylene, 1 minute each time), and seal with neutral resin;

1. Take pictures, the results are as shown in Figure 4, the masson's trichrome stained sections of the wounded parts of each test group and the normal skin parts on the tenth day under the 10 times eyepiece, wherein, the blank control group is shown in A in Figure 4, The stent control group is shown in Figure 4 B, the normal skin group is shown in Figure 4 C, the BMSCs-administered group is shown in Figure 4 D, and the ESCs-administered group is shown in Figure 4 E, BMSCs+ESCs composite The administration group is shown as F in Fig. 4 .

As shown in Figure 4, compared with the blank control group A, the stent control group B can promote the synthesis of some collagen; compared with the blank control group A and the stent control group B, the BMSCs administration group can significantly promote angiogenesis ( Solid line arrows are blood vessels), ESCs administration group significantly promoted the regeneration of hair follicles (dashed arrows are hair follicles), in the BMSCs+ESCs compound administration group, both blood vessel regeneration and hair follicle regeneration were found in the skin tissue, more Close to the structure of normal skin tissue. On the one hand, it proves that BMSCs and ESCs have the function of accelerating the regeneration of blood vessels and hair follicles in the skin layer. It has great potential to be developed into a new treatment protocol to accelerate the regeneration of skin and its appendages in the clinic.

Example 7: Molecular immunohistochemical identification test of animal wound regenerated blood vessels

Immunohistochemical staining, the specific steps are as follows:

1) On the 10th day, remove the wound site skin slices and normal skin slices in the blank control group, stent control group, BMSCs administration group, ESCs administration group, BMSCs+ESCs compound drug group in Example 4, and place Skin sections were routinely dewaxed to water;

2) wash with pH=7.4 phosphate buffered saline (PBS) 3 times, 5 minutes each time;

3) Incubate for 20 minutes at a room temperature of 25° C. with 3% methanol aqueous solution containing methanol mass percentage and an appropriate amount of hydrogen peroxide;

4) Wash with distilled water for 3 times, 5 minutes each time;

5) Repair in a 1mol/L citric acid buffer (pH=6.0) incubator, maintain at 98°C for 20 minutes, then cool to room temperature;

6) In the PBS solution containing fetal bovine serum (the PBS solution containing fetal bovine serum is obtained by mixing fetal bovine serum and PBS, wherein the weight percentage of fetal bovine serum in the PBS solution containing fetal bovine serum is 10%) , incubate for 30 minutes in a 37°C incubator humid box:

7) Absorb excess serum with filter paper, add CD31 primary antibody solution (the CD31 primary antibody solution is prepared by primary antibody CD31 and PBS, wherein the volume ratio of primary antibody to PBS is 1:20), overnight at 4°C for 15 hours;

8) wash with PBS (phosphate buffer saline) 3 times, 5 minutes each time;

9) Add an appropriate amount of biotinylated secondary antibody (goat anti-mouse secondary antibody) working solution dropwise, and incubate in a humid box at 37°C for 30 minutes;

10) Wash with PBS 3 times, 5 minutes each time;

11) Add an appropriate amount of horseradish peroxidase-labeled streptavidin working solution dropwise, and incubate for 30 minutes in a humid box at 37°C:

12) wash with PBS 3 times, 5 minutes each time;

13) Appropriate amount of DAB chromogen, develop color for 10 minutes;

14) Fully rinse with tap water to stop color development;

15) Hematoxylin counterstaining for 60 minutes;

16) Gradient alcohol dehydration: elute with ethanol aqueous solution containing 70%, 80%, and 90% ethanol mass percentage for 5 minutes respectively; Twice, 60 minutes each time;

17) Elute with xylene three times, each time for 60 minutes, until transparent;

18) Seal with neutral gum.

19) For the negative control, replace the primary antibody in step 7) with PBS, and the rest of the steps are the same as above.

Figure 5 shows the results of immunohistochemical identification of CD31 in the skin of the wound site on the tenth day under a 10x eyepiece, wherein the blank control group is shown in Figure 5 A, the stent control group is shown in Figure 5 B, and the normal skin group is shown in Figure 4 As shown in C, the BMSCs administration group is shown in Figure 5 D, the ESCs administration group is shown in Figure 5 E, and the BMSCs+ESCs compound administration group is shown in Figure 5 F.

As shown in Figure 5, in the blank control group and the stent control group, no obvious vascular endothelial cell molecular marker CD31 was identified; CD31, which is close to the normal skin identification results, on the one hand, further verified the function of BMSCs in accelerating the regeneration of blood vessels in the skin layer, on the other hand, it also proved that the combination of BMSCs and ESCs, two kinds of stem cells, also has the effect of accelerating skin wound angiogenesis.

Claims (5)

1. a preparation method of accelerating the medicine of skin layer reparation and skin accessory organ regeneration comprises the following steps:

1) epidermal stem cells is first used to buffer solution for cleaning, again with the pancreatin cell dissociation buffer digestion that is 0.03%～0.08% containing the trypsin weight percentage 6～8 minutes, then to adding DMEM low sugar containing hyclone and mycillin/F12 culture fluid in postdigestive epidermal stem cells, centrifugal, abandon supernatant, add again DMEM low sugar/F12 culture fluid, obtain the epidermal stem cells suspension;

Mesenchymal stem cells MSCs is first used to buffer solution for cleaning, again with the pancreatin cell dissociation buffer digestion that is 0.03%～0.08% containing the trypsin weight percentage 2～4 minutes, then to adding DMEM low sugar containing hyclone and mycillin/F12 culture fluid in postdigestive mesenchymal stem cells MSCs, centrifugal, abandon supernatant, add again DMEM low sugar/F12 culture fluid, obtain the mesenchymal stem cells MSCs suspension;

DMEM low sugar culture fluid and F12 culture fluid that described DMEM low sugar/the F12 culture fluid is 1.25～3.5:1 by weight ratio form;

The described DMEM low sugar containing hyclone and mycillin/F12 culture fluid is comprised of the component of following weight percentage:

2) the epidermal stem cells suspension in pharmaceutical carrier, step 1) and mesenchymal stem cells MSCs suspension are mixed, hatch the medicine that obtains accelerating skin layer reparation and skin accessory organ's regeneration;

Described pharmaceutical carrier is gelfoam/bata-tricalcium phosphate support;

The medicine of the reparation of described acceleration skin layer and skin accessory organ's regeneration is comprised of epidermal stem cells, mesenchymal stem cells MSCs and pharmaceutical carrier, wherein, epidermal stem cells and mesenchymal stem cells MSCs all stick on pharmaceutical carrier, and the number that contains epidermal stem cells on the pharmaceutical carrier of every cubic millimeter is (0.3～1) * 10 ⁴Individual, the number that contains mesenchymal stem cells MSCs on the pharmaceutical carrier of every cubic millimeter is (0.3～1) * 10 ⁴Individual.

2. acceleration skin layer according to claim 1 is repaired the preparation method of the medicine of regenerating with the skin accessory organ, it is characterized in that, the number that contains epidermal stem cells on the pharmaceutical carrier of every cubic millimeter is (0.6～0.75) * 10 ⁴Individual, the number that contains mesenchymal stem cells MSCs on the pharmaceutical carrier of every cubic millimeter is (0.6～0.75) * 10 ⁴Individual.

3. acceleration skin layer according to claim 1 and 2 is repaired the preparation method of the medicine of regenerating with the skin accessory organ, it is characterized in that, by following preparation method, prepared by described epidermal stem cells: in the pancreatin cell dissociation buffer that will to join containing the trypsin weight percentage by the separation epidermis obtained after pretreatment be 0.03%～0.08%, digestion separates epidermis 3 minutes～8 minutes, obtains epidermal stem cells after separation.

4. acceleration skin layer according to claim 3 is repaired the preparation method of the medicine of regenerating with the skin accessory organ, it is characterized in that, by following preparation method, prepared by described epidermal stem cells: in the pancreatin cell dissociation buffer that will to join containing the trypsin weight percentage by the separation epidermis obtained after pretreatment be 0.05%, digestion separates epidermis 5 minutes, obtains epidermal stem cells after separation.

5. acceleration skin layer according to claim 1 and 2 is repaired the preparation method of the medicine of regenerating with the skin accessory organ, it is characterized in that, in described gelfoam/bata-tricalcium phosphate support, the mass ratio of gelfoam and bata-tricalcium phosphate is 1:1～100.

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