Application and preparation of MSCs and AMP in the preparation of products for intervention of atopic dermatitis

Abstract

The present invention provides an application and a preparation of MSCs and AMP in the preparation of a product for intervening in atopic dermatitis, relating to the technical field of biomedicine. The preparation for intervening in atopic dermatitis comprises mesenchymal stem cells and AMP; the concentration of the mesenchymal stem cells is (0.25-2.5)×10<supgt;7< / supgt> cells / mL; the concentration of AMP is 10-100 μmol / L. Applying the mesenchymal stem cells (MSCs) and AMP (adenosine monophosphate) to the preparation of a product for intervening in atopic dermatitis alleviates the technical problem of poor efficacy of the existing atopic dermatitis treatment products.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to an application and preparation of MSCs and AMP in the preparation of a product for intervening in atopic dermatitis. Background Art

[0002] Atopic dermatitis (AD), also known as atopic eczema, is a common chronic, pruritic, recurrent and inflammatory skin disease.

[0003] The incidence rate in children is 10-30%, and the incidence rate in adults is 2-10%. AD has serious complications, and patients often have rhinitis and allergic asthma, especially moderate and severe AD patients, who often suffer from severe sleep disorders, depression and anxiety. Currently, the commonly used means for the treatment of dermatitis in clinical practice are topical application of glucocorticoids, systemic use of antihistamines, immunosuppressants, immunomodulators, phototherapy and other treatment methods. The efficacy is not ideal, the side effects are large, the symptoms of AD cannot be well relieved, the use price is expensive, and the cost-effectiveness is low. Therefore, how to improve the products for the treatment of AD is an urgent problem to be solved.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The first purpose of the present invention is to provide an application of mesenchymal stem cells and AMP in the preparation of a product for intervening in atopic dermatitis, thereby alleviating the technical problem of poor efficacy of atopic dermatitis treatment products in the prior art.

[0006] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0007] According to one aspect of the present invention, the present invention provides a use of mesenchymal stem cells and AMP in preparing a product for intervening in atopic dermatitis.

[0008] Preferably, the concentration of the AMP does not exceed 100 μmol / L;

[0009] Preferably, the concentration of AMP is 10-100 μmol / L; preferably 30-80 μmol / L; more preferably 50 μmol / L.

[0010] Preferably, the concentration of the mesenchymal stem cells is (0.25-2.5)×10 7 Pieces / mL;

[0011] Preferably, the concentration of the mesenchymal stem cells is 2.5×10 7 Pieces / mL;

[0012] Preferably, the source of the mesenchymal stem cells includes at least one of bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue;

[0013] Preferably, the mesenchymal stem cells are selected from umbilical cord mesenchymal stem cells.

[0014] Preferably, the intervention subject for atopic dermatitis includes mammals;

[0015] Preferably, the mammal comprises a mouse, a rat, a guinea pig, a hamster, a rabbit, a ferret, a cat, a dog, a goat, a sheep, a cow, a pig, a horse, a monkey or a human;

[0016] Preferably, the mammal comprises mouse, rat or human.

[0017] According to another aspect of the present invention, the present invention also provides a preparation for intervening atopic dermatitis, the preparation comprising mesenchymal stem cells and AMP; the concentration of the mesenchymal stem cells is (0.25-2.5)×10 7 The concentration of the AMP is 10 to 100 μmol / L.

[0018] Preferably, the concentration of the mesenchymal stem cells is 2.5×10 7 Pieces / mL;

[0019] Preferably, the source of the mesenchymal stem cells includes at least one of bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue;

[0020] Preferably, the mesenchymal stem cells include umbilical cord mesenchymal stem cells;

[0021] Preferably, the umbilical cord mesenchymal stem cells express CD73, CD90 and CD105, and do not express CD14, CD19, CD34, CD45 and HLA-DR.

[0022] Preferably, the umbilical cord mesenchymal stem cells are prepared according to the following method:

[0023] The Wharton's jelly separated from the umbilical cord is cultured in a complete culture medium, and when the cell confluence reaches 70-90%, the Wharton's jelly fragments are discarded. After the culture is completed, the cells are subcultured to obtain umbilical cord mesenchymal stem cells;

[0024] Preferably, the culture conditions of the adherent culture include: a culture time of 10 to 14 days, a culture temperature of 37° C., and a carbon dioxide concentration of 5%; from the 5th to 21st day of primary culture, the primary culture medium in the culture bottle is completely replaced every 2 to 3 days;

[0025] Preferably, the culture conditions of the subculture include: a culture time of 3 to 5 days, a culture temperature of 37° C., and replacing half of the subculture medium every 2 to 3 days of subculture;

[0026] Preferably, the umbilical cord mesenchymal stem cells in the preparation for intervening atopic dermatitis are umbilical cord mesenchymal stem cells passaged 3 to 8 times.

[0027] Preferably, the concentration of AMP is 30-80 μmol / L, preferably 50 μmol / L.

[0028] Preferably, the preparation further comprises a pharmaceutically acceptable excipient;

[0029] Preferably, the formulation uses a buffer as a matrix;

[0030] Preferably, the buffer is selected from physiological saline or PBS, preferably physiological saline.

[0031] Preferably, the preparation is an injection, a spray, a gel, a liniment, a paint, a film coating, a patch, a plaster, an implant or a film, preferably an injection.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The present invention applies mesenchymal stem cells and AMP to the preparation of atopic dermatitis intervention products. Mesenchymal stem cells have been widely used in clinical treatment, with good safety and low side effects; experimental verification shows that mesenchymal stem cells can reduce the inflammation level of atopic dermatitis model animals, reduce skin damage scores, and improve immune regulation function. The mixed use of AMP and umbilical cord mesenchymal stem cells can effectively improve the anti-inflammatory properties of umbilical cord stem cells, thereby exerting their efficacy in treating atopic dermatitis.

[0034] Based on the above concept, the preparation for intervening atopic dermatitis provided by the present invention has a good effect in preventing, alleviating and treating atopic dermatitis. Compared with existing clinical treatment methods, it is more convenient and safer and has the advantage of a simple treatment process. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The morphological observation of the back skin tissue of mice in each experimental group;

[0037] Figure 2 and Figure 4 The skin lesions on the back of mice in each experimental group were scored;

[0038] Figure 3 Figure 2 is the IgE test results of the serological index of mice in each experimental group. DETAILED DESCRIPTION

[0039] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that:

[0041] In the present invention, unless otherwise specified, all the embodiments and preferred implementation methods mentioned herein can be combined with each other to form a new technical solution; all the technical features and preferred features mentioned herein can be combined with each other to form a new technical solution; the components involved or their preferred components can be combined with each other to form a new technical solution.

[0042] In the present invention, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, wherein a and b are real numbers. The "range" disclosed in the present invention is in the form of lower limit and upper limit, which can be one or more lower limits, and one or more upper limits, respectively. In the present invention, unless otherwise specified, each reaction or operation step can be carried out sequentially or not.

[0043] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0044] According to one aspect of the present invention, the present invention provides a use of mesenchymal stem cells (MSCs) and AMP in the preparation of a product for intervening atopic dermatitis. The intervening atopic dermatitis of the present invention includes preventing, alleviating and / or treating atopic dermatitis.

[0045] Stem cells are a type of undifferentiated cells with multidirectional differentiation and self-renewal potentials. They have great application value in cell and tissue repair, as well as immune regulation. Mesenchymal stem cells are an important member of the stem cell family. They are derived from the early mesoderm of development and are pluripotent stem cells. Studies have shown that MSCs have immunosuppressive effects and fewer adverse reactions. They can be used not only to treat autoimmune diseases or inflammatory skin diseases, such as GvHD, lupus erythematosus, systemic sclerosis and rheumatoid arthritis, but also allergic diseases, such as asthma, allergic rhinitis and contact dermatitis.

[0046] AMP (adenosine monophosphate) is a component of deoxyribonucleic acid and ribonucleic acid. It is involved in almost all metabolic processes in the body and plays an important role in cellular material energy metabolism and function regulation.

[0047] The present invention has found through experiments that the use of mesenchymal stem cells alone can reduce the degree of skin damage and inflammation level of atopic dermatitis model animals to a certain extent; however, the use of AMP alone has no alleviating effect on atopic dermatitis. The combined use of mesenchymal stem cells and AMP significantly improves the therapeutic effect of atopic dermatitis compared to the use of mesenchymal stem cells alone. The combined use of mesenchymal stem cells and AMP is better than the use of mesenchymal stem cells alone in reducing the level of skin damage and inflammation in atopic dermatitis, indicating that AMP improves the therapeutic effect of mesenchymal stem cells on atopic dermatitis.

[0048] Based on the above findings, the present invention provides an application of mesenchymal stem cells and AMP in the preparation of a product for intervening in atopic dermatitis.

[0049] The present invention also found through experiments that in the synergistic effect of AMP and mesenchymal stem cells in the treatment of atopic dermatitis, the therapeutic effect is related to the concentration of AMP. AMP alone has no therapeutic effect on atopic dermatitis. When AMP is used in combination with mesenchymal stem cells at an appropriate concentration, the therapeutic effect can be significantly improved; however, when the concentration of AMP is too high, the therapeutic effect of the combined use of AMP and mesenchymal stem cells is not as good as that of mesenchymal stem cells alone, indicating that the synergistic effect of AMP on mesenchymal stem cells is related to its concentration. The experiment found that when the concentration of AMP is 100 μmol / L, AMP has no synergistic effect on mesenchymal stem cells in the treatment of atopic dermatitis. When the concentration of AMP is 50 μmol / L, the therapeutic effect of the combined use of AMP and mesenchymal stem cells is significantly better than that of atopic dermatitis alone.

[0050] Therefore, in some preferred embodiments, mesenchymal stem cells and AMP are used to prepare a drug for treating atopic dermatitis, and the concentration of AMP does not exceed 100 μmol / L, for example, it can be but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μmol / L, preferably 10-100 μmol / L; more preferably 30-80 μmol / L; further preferably 50 μmol / L.

[0051] In some preferred embodiments, the concentration of the mesenchymal stem cells is (0.25-2.5)×10 7 / mL, for example, it can be, but is not limited to, 0.25, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25 or 2.5×10 7 The source of mesenchymal stem cells can be selected from sources known in the art, including but not limited to at least one of bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue, preferably umbilical cord mesenchymal stem cells.

[0052] In some preferred embodiments, the intervention subjects for atopic dermatitis include mammals, including but not limited to mice, rats, guinea pigs, hamsters, rabbits, ferrets, cats, dogs, goats, sheep, cows, pigs, horses, monkeys or humans, more preferably mice, rats or humans.

[0053] According to the concept of applying mesenchymal stem cells and AMP to prepare a product for intervening atopic dermatitis, the present invention also provides a preparation for intervening atopic dermatitis, the preparation comprising umbilical cord mesenchymal stem cells and AMP, wherein the concentration of the mesenchymal stem cells is (0.25-2.5)×10 7 / mL, for example, it can be, but is not limited to, 0.25, 0.5, 1, 1.25, 1.5, 1.75, 2, 2.25 or 2.5×10 7 / mL, preferably 2.5×10 7 The concentration of the AMP is 10 to 100 μmol / L, for example, but not limited to 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 μmol / L, preferably 30 to 80 μmol / L, more preferably 50 μmol / L.

[0054] The concentrations of mesenchymal stem cells and AMP described herein are the concentrations of mesenchymal stem cells and AMP in the preparation when the preparation for intervening atopic dermatitis is administered to a subject. It is understood that the preparation for intervening atopic dermatitis can be a high-concentration reserve preparation, which is diluted to the concentration when used.

[0055] In some preferred embodiments, the source of the mesenchymal stem cells in the preparation for intervening atopic dermatitis includes but is not limited to bone marrow, umbilical cord blood, umbilical cord tissue, placental tissue and adipose tissue, preferably umbilical cord mesenchymal stem cells.

[0056] In some preferred embodiments, the umbilical cord mesenchymal stem cells are fibroblast-like cells isolated from umbilical cord tissue, express CD73, CD90 and CD105, and do not express CD14, CD19, CD34, CD45 and HLA-DR.

[0057] In some preferred embodiments, umbilical cord mesenchymal stem cells meeting the above conditions are prepared according to the following method: Wharton's jelly separated from the umbilical cord is cultured in a complete culture medium, and when the cell confluence is 70-90%, the Wharton's jelly fragments are discarded, and after the culture is completed, the cells are subcultured to obtain umbilical cord mesenchymal stem cells.

[0058] In some preferred embodiments, the umbilical cord is first cleaned with physiological saline, cut into 2-3 cm segments, the veins and arteries are removed, and then the Wharton's jelly is peeled off and cut into pieces before being used for adherent culture.

[0059] In some preferred embodiments, the culture conditions for adherent culture include: a culture time of 10 to 14 days, a culture temperature of 37° C., and a carbon dioxide concentration of 5%; from the 5th to 21st day of primary culture, the primary culture medium in the culture bottle is completely replaced every 2 to 3 days.

[0060] In some preferred embodiments, after the primary culture is completed, the adherent cells are digested, and then the cells are filtered, dispersed and centrifuged, and subcultured to obtain umbilical cord mesenchymal stem cells.

[0061] In some preferred embodiments, the culture conditions of the subculture include: a culture time of 3 to 5 days, a culture temperature of 37° C., and replacing half of the subculture medium every 2 to 3 days of the subculture.

[0062] In some optional embodiments, the preparation further includes a pharmaceutically acceptable excipient, which includes but is not limited to at least one of a solvent, a diluent, an antibacterial agent, an osmotic pressure regulator, a pH regulator, an adsorbent, a glidant, an antioxidant, a protective agent, a matrix and a drug carrier material; the pharmaceutically acceptable excipient can also be selected from components used to cultivate and maintain the physiological and biochemical functions of mesenchymal stem cells, including but not limited to culture medium, amino acids, vitamins, minerals or serum, etc.

[0063] In some preferred embodiments, the preparation uses a buffer as a matrix, and the buffer can be selected from well-known and mature buffers in the art, such as but not limited to physiological saline or PBS, preferably physiological saline.

[0064] In some optional embodiments, the dosage form of the preparation may be, for example, but not limited to, an injection, a spray, a gel, an ointment, a paint, a film coating, a patch, a plaster, an implant or a film, preferably an injection.

[0065] The technical scheme and technical effects of the present invention are further explained below in conjunction with preferred embodiments.

[0066] Example 1

[0067] This embodiment provides a preparation for intervening atopic dermatitis, comprising umbilical cord mesenchymal stem cells and AMP.

[0068] The preparation method of mesenchymal stem cells is as follows:

[0069] After cutting the umbilical cord into 1 cm small segments, clean the surface with physiological saline, and squeeze out the blood in the tissue with sterile tweezers and discard it; after removing the venous and arterial tubes in the tissue, peel off the gelatinous Wharton's jelly, cut the Wharton's jelly into pieces, and culture it in complete culture medium (the complete culture medium is a commercial complete culture medium purchased from Youkang Biotechnology (Beijing) Co., Ltd.).

[0070] The culture conditions for primary culture are: culture time is 10 to 14 days, culture temperature is 37°C, and carbon dioxide concentration is 5%; on the 5th day of primary culture, the primary culture medium in the culture bottle is completely replaced every 2 to 3 days; when the cell confluence is 70 to 90%, the umbilical cord is discarded and the Wharton's jelly fragments are retained for continued culture.

[0071] After the primary culture, the adherent cells were digested, and then filtered, dispersed and centrifuged, and subcultured to obtain umbilical cord mesenchymal stem cells. The molecular expression of mesenchymal stem cells was detected, and the results were: CD73: 99.94%, CD90: 99.94%, CD105: 95.55%, CD34: 0.05%, CD45: 1.29%, CD19: 0.03%, CD14: 0.27%, HLA-DR: 0.01%.

[0072] The culture conditions for subculture are preferably: the culture time is 3 to 5 days, the culture temperature is 37° C., and half of the subculture medium is replaced every 2 to 3 days.

[0073] The preparation method of the preparation is:

[0074] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord mesenchymal stem cell suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 50μmol / L.

[0075] Example 2

[0076] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 100 μmol / L. The preparation method of the preparation is:

[0077] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 100μmol / L.

[0078] Example 3

[0079] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 30 μmol / L. The preparation method of the preparation is:

[0080] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 30μmol / L.

[0081] Example 4

[0082] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 70 μmol / L. The preparation method of the preparation is:

[0083] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 70μmol / L.

[0084] Example 5

[0085] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 50 μmol / L. The preparation method of the preparation is:

[0086] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 0.25×10 7 / mL, and the AMP concentration was 50μmol / L.

[0087] Example 6

[0088] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 60 μmol / L. The preparation method of the preparation is:

[0089] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 60μmol / L.

[0090] Example 7

[0091] This example provides a preparation for intervening atopic dermatitis, comprising mesenchymal stem cells and AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The concentration of AMP is 80 μmol / L. The preparation method of the preparation is:

[0092] Umbilical cord mesenchymal stem cells and AMP were mixed and suspended in physiological saline to prepare an umbilical cord stem cell preparation suspension with a total volume of 200 μL, in which the concentration of umbilical cord mesenchymal stem cells was 2.5×10 7 / mL, and the AMP concentration was 80μmol / L.

[0093] Comparative Example 1

[0094] The preparation provided in Comparative Example 1 does not contain umbilical cord mesenchymal stem cells, but only contains AMP, and the AMP concentration is 50 μM.

[0095] Comparative Example 2

[0096] The preparation provided in Comparative Example 2 does not contain AMP. The preparation method of umbilical cord mesenchymal stem cells is the same as that of Example 1. The umbilical cord mesenchymal stem cells are suspended in physiological saline to prepare an umbilical cord stem cell suspension with a total volume of 200 μL, wherein the concentration of umbilical cord mesenchymal stem cells is 2.5×10 7 Pieces / mL.

[0097] Effect example

[0098] 1. Experimental groups:

[0099] Table 1

[0100] Group Mouse Active substances preparation Group 1 Normal mice - - Group 2 Model mice - - Group 3 Model mice 50 μM AMP Comparative Example 1 Group 4 Model mice UCMSC Comparative Example 2 Group 5 Model mice UCMSC+50μM AMP Example 1 Group 6 Model mice UCMSC+100μM AMP Example 2 Group 7 Model mice UCMSC+70μM AMP Example 4 Group 8 Model mice UCMSC+60μM AMP Example 6 Group 9 Model mice UCMSC+80μM AMP Example 7

[0101] 2. Construction of mouse atopic dermatitis model:

[0102] 2.1 Mice: BALB / c female mice, 6 weeks old, weighing 17-20 g, 6 mice per group.

[0103] 2.2 The atopic dermatitis animal model was prepared by combined peritoneal and skin sensitization, including the following steps:

[0104] (1) Intraperitoneal injection:

[0105] The dosage of the sensitization mixture for each mouse was: 10 μg OVA was dissolved in 100 μl PBS, then mixed with 100 μl alum adjuvant (40 mg / ml), and after thorough mixing for 30 min, the mixture was intraperitoneally injected once a week for three weeks (days 0, 7, and 14). Each mouse was injected with 200 μl.

[0106] (2) Skin irritation:

[0107] On day 21, the mice were anesthetized, the back and neck were shaved, and the mice were disinfected. After 100 μl of OVA solution (10 μg OVA in 100 μL PBS) was added to sterile gauze, the gauze was fixed on the shaved skin surface (1 cm×1 cm), and the dressing was changed every day for one week.

[0108] 2.3 Cell therapy:

[0109] Each test group randomly selected 3 to 5 mice, of which group 1 selected normal mice, and groups 2 to 6 selected prepared atopic dermatitis animal model mice. After the model mice were induced to develop AD-like symptoms for 2-3 days, they were treated on the 31st, 33rd and 35th days of the experiment. The corresponding preparations were injected into the subcutaneous part of the back of the mouse model according to Table 1. After treatment, the skin lesions on the back of the model mice in groups 2 to 9 were stimulated again in the same way as above for a total of 7 days. After the stimulation was completed, the observation was conducted for 3 days, and venous blood was taken for serological index IgE detection; the skin condition was observed and the skin lesion score was performed; at the same time, the back skin tissue was taken and the pathological condition of the skin structure was detected by HE staining.

[0110] 3. Experimental results:

[0111] The experimental results are as follows Figure 1 , Figure 2 , Figure 3 and Figure 4 As shown, Figures 1 to 4 The groups are shown in Table 1. Figure 2 to Figure 4 * indicates P<0.05; ** indicates P<0.01.

[0112] Figure 1 The model group is an untreated mouse model. Figure 1 The skin tissue pathology results clearly show that the back skin of the mice in the model group was obviously damaged, which is a symptom of early atopic dermatitis; the different treatment groups were able to recover the damage caused by atopic dermatitis, among which the therapeutic effect of the preparation in Example 1 was the most significant.

[0113] Figure 2 According to the skin injury scoring rules, each group was scored. The score of Example 1 was significantly lower than that of other groups (**p<0.01), indicating that it had the best effect in reducing atopic skin damage. The skin injury scoring rules refer to the literature "Study on the immune intervention of Peitu Qingxin granules on atopic dermatitis-like mouse model".

[0114] Figure 3 The results show that preparation 1 can significantly reduce the IgE value (**p<0.01), indicating that it has the best anti-inflammatory effect.

[0115] Figure 4 The results show that the skin damage scoring rule was used to score Group 7, Group 8 and Group 9, respectively, and it was shown that the scores of Examples 4, 6 and 7 were all lower than those of Group 2, indicating that all of them can reduce atopic skin damage.

[0116] It can be seen from the above examples that a single umbilical cord stem cell can reduce the degree of skin damage to a certain extent, reduce the inflammation level of atopic dermatitis, and reduce the skin damage score; the use of Example 2 cannot improve the anti-inflammatory effect of umbilical cord stem cells; the use of Example 1, that is, the addition of 50 μmol / L of AMP to the umbilical cord stem cell preparation can significantly reduce the degree of skin damage, reduce the inflammation level of atopic dermatitis, and reduce the skin damage score. This shows that the preparation for intervening in atopic dermatitis prepared in Example 2 can significantly reduce the skin damage of atopic dermatitis and protect the skin structure.

[0117] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of umbilical cord mesenchymal stem cells and AMP in the preparation of a product for intervention of atopic dermatitis, wherein the concentration of the umbilical cord mesenchymal stem cells is 2.5×10 7 / mL, and the concentration of AMP is 50-80 μmol / L.

2. The use according to claim 1, characterized in that: The concentration of the AMP is 50 μmol / L.

3. The use according to claim 1, characterized in that: The intervention subjects for atopic dermatitis are mice.

4. A preparation for intervention of atopic dermatitis, characterized in that The invention comprises umbilical cord mesenchymal stem cells and AMP; the concentration of the umbilical cord mesenchymal stem cells is 2.5×10 7 / mL; the concentration of the AMP is 50-80 μmol / L.

5. The preparation according to claim 4, characterized in that The umbilical cord mesenchymal stem cells express CD73, CD90 and CD105, but do not express CD14, CD19, CD34, CD45 and HLA-DR.

6. The preparation according to claim 5, characterized in that The umbilical cord mesenchymal stem cells are prepared according to the following method: The Wharton's jelly separated from the umbilical cord is cultured in a complete culture medium, and when the cell confluence is 70-90%, the Wharton's jelly fragments are discarded. After the culture is completed, the cells are subcultured to obtain umbilical cord mesenchymal stem cells.

7. The preparation according to claim 6, characterized in that The culture conditions of the adherent culture include: a culture time of 10 to 14 days, a culture temperature of 37° C., and a carbon dioxide concentration of 5%; from the 5th to 21st day of primary culture, the primary culture medium in the culture bottle is completely replaced every 2 to 3 days.

8. The preparation according to claim 6, characterized in that The culture conditions of the subculture include: a culture time of 3 to 5 days, a culture temperature of 37° C., and replacing half of the subculture medium every 2 to 3 days of the subculture.

9. The preparation according to claim 6, characterized in that The umbilical cord mesenchymal stem cells in the preparation for intervening atopic dermatitis are umbilical cord mesenchymal stem cells passaged to the 3rd to 8th generations.

10. The preparation according to claim 4, characterized in that The concentration of the AMP is 50 μmol / L.

11. The preparation according to claim 4, characterized in that The preparation also includes pharmaceutically acceptable excipients.

12. The preparation according to claim 11, characterized in that The formulation uses a buffer as a base.

13. The preparation according to claim 12, characterized in that The buffer is selected from physiological saline or PBS.

14. The preparation according to claim 11, characterized in that The preparation is an injection, a spray, a gel, a liniment, a paint, a patch, a plaster, an implant or a film.

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