CN113827618A - Application of stem cell conditioned medium in preparation of medicine for treating inflammatory skin - Google Patents
Application of stem cell conditioned medium in preparation of medicine for treating inflammatory skin Download PDFInfo
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- CN113827618A CN113827618A CN202111222487.4A CN202111222487A CN113827618A CN 113827618 A CN113827618 A CN 113827618A CN 202111222487 A CN202111222487 A CN 202111222487A CN 113827618 A CN113827618 A CN 113827618A
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- conditioned medium
- umbilical cord
- skin
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Abstract
The invention provides an application of a stem cell conditioned medium in preparing a medicament for treating inflammatory skin, wherein the preparation method of stem cells comprises the following steps: the method comprises the following steps of taking an amniotic membrane and an umbilical cord tissue in a human placenta tissue as raw materials, obtaining amniotic epithelial stem cells and umbilical cord mesenchymal stem cells by methods of shearing, digestion and the like, and then inoculating the cells in a culture medium containing platelet lysate; passaging the primary cells to the third generation, and discarding a culture medium containing platelet lysate when the confluence degree of the cells reaches 90%; replacing with a serum-free basic culture medium, and continuing to culture for 20-36 h; collecting the conditioned medium of cultured cells, and centrifuging to remove cells and cell debris to obtain conditioned medium of amniotic epithelial stem cells (AECM) and conditioned medium of umbilical cord mesenchymal stem cells (UMSCM). The AECM and the UMSCM of the present invention can inhibit inflammatory cells of the skin of a patient from infiltrating the skin, thereby inhibiting epidermal hyperplasia and inflammatory response.
Description
Technical Field
The invention belongs to the field of biological medicines, and relates to a medicine for treating psoriasis, in particular to application of a stem cell conditioned medium in preparing a medicine for treating inflammatory skin.
Background
Inflammatory dermatoses are a series of repeated skin diseases caused by the stimulation of the immune system to environmental factors induced by the immune system and the abnormal activation of the immune system, which have become one of the major global health problems, and the proportion of medical health care expenditure for the treatment of the diseases is increasing year by year. Psoriasis, a typical refractory inflammatory skin disease, is a common chronic inflammatory skin disease and is mainly characterized in that a plurality of types of immune cells mainly comprising T lymphocytes are infiltrated in a large quantity, and epidermal spinous processes are prolonged and keratinocyte differentiation is incomplete due to abnormal proliferation and differentiation of epidermal keratinocytes and the like. The pathogenesis of psoriasis is not well understood, but there is a large body of evidence that the co-action of environmental factors, gene sensitivity, disruption of the skin barrier and immune abnormalities are involved in the development and progression of the disease. The skin barrier is broken, allowing substances in the environment to enter the skin, which in turn further induces an immune response and skin inflammation of the skin. Among them, the inflammatory factor IL-17A plays a key role in the pathogenesis of psoriasis, IL-17A induces abnormal proliferation and differentiation of keratinocytes, and keratinocytes together with other immune cells participate in maintaining the malignant cycle of inflammation. Among them, the dermal γ δ T cells are the main source of IL-7A.
Psoriasis is an incurable disease and is often afflicted with life-long. Clinical treatment is targeted at controlling symptoms. Topical medicine and ultraviolet phototherapy can be applied to patients with local diseases and early stage mild disease, and the common medicines include humectant, emollient, glucocorticoid, vitamin D3 derivative, calcineurin inhibitor, tretinoin, etc. Systemic treatment drugs for moderately severe patients include methotrexate, cyclosporine, tretinoin, and various biologics. However, long-term use of these drugs causes problems such as side effects, drug resistance, and drug tolerance. Recently, several biologies have been explored, such as TNF antagonists, and antibodies to IL-12, IL-23, and IL-17. These biological agents are more effective than conventional drugs, but face the problem of expensive medical expenses for long-term use. Therefore, development of a biological agent having a small side effect, high safety and a good therapeutic effect is urgently necessary for treatment of skin diseases caused by immune abnormality such as psoriasis.
Adult stem cells, including Mesenchymal Stem Cells (MSCs) and amniotic epithelial stem cells (AECs), have been shown to have therapeutic effects in a number of immune and inflammation-related diseases as the most widely used stem cells at present. Among them, the paracrine factor of stem cells plays an important role in regulating immunity, suppressing inflammation, repairing and regenerating tissues. Paracrine factors, including growth factors (EGF, KGF, FGF, etc.), immune modulators: antagonists of interleukin-1 (IL-ra), IL-10, IL-13, transforming growth factor beta (TGF beta), some receptors, chemokines, etc., which have nutritional and immunoregulatory properties, and therefore, the secretion of adult stem cells, i.e., the Conditioned Medium (CM) of adult stem cells, may have a role in the treatment of disease. However, no stem cell-derived CM has been reported, and human umbilical cord-derived MSC CM (umscm) and human amniotic membrane-derived amniotic epithelial stem cell CM (aecm) have not been reported. Wherein the human umbilical cord and amniotic membrane are sufficiently accessible, and the cells (UMSC and AEC) extracted from them have high proliferation and multipotential differentiation potential, and can regulate immune dysregulation, inhibit inflammation, and relieve symptoms of diseases in many immune-related diseases. And the stem cells can secrete various growth factors and immune regulatory factors. Therefore, the invention adopts adult stem cell conditioned medium with wider application: including human umbilical cord mesenchymal stem cell conditioned medium (UMSCM) and human amniotic epithelial stem cell conditioned medium (AECM), they may become one of the potential treatments for inflammatory skin diseases such as psoriasis.
Disclosure of Invention
In view of the above technical problems in the prior art, the present invention provides an application of a stem cell regulating medium in the preparation of a medicament for treating inflammatory skin, wherein the application aims to solve the technical problem that the effect of the medicament in the prior art on quality of inflammatory skin is poor.
The invention provides an application of a stem cell regulating culture medium in preparing a medicament for treating psoriasis, wherein the preparation method of the stem cell comprises the following steps:
taking separated amnion from medical waste human placenta tissue after parturition of a parturient as a raw material, peeling a layer of amnion epithelial layer of the amnion close to a fetus by using a pair of tweezers, shearing, digesting for 20-40 minutes by adopting pancreatin with the mass percentage concentration of 0.25%, digesting for 0.5-2 hours by using collagenase, filtering, centrifuging to obtain amnion epithelial stem cells, and planting the amnion epithelial stem cells in a DMEM/F12 culture medium added with human platelet lysate;
or,
the medical waste umbilical cord after parturition of a parturient is taken as a raw material, the umbilical cord is washed clean by PBS (phosphate buffer solution), blood is removed, and then the umbilical artery and umbilical vein are removed by using tooth forceps. Then, shearing into tissue blocks with the diameter of 2-4 mm by using scissors, pasting the tissue blocks into a culture dish, adding an alpha-MEM culture medium containing human platelet lysate, changing the culture medium after 7 days, and obtaining umbilical cord mesenchymal stem cells after 14 days;
digesting and passaging the umbilical cord mesenchymal stem cells or the primary amniotic epithelial stem cells obtained above, and continuously culturing to a third generation;
step three, when the confluency of the third generation cells reaches 90%, discarding a culture medium containing platelet lysate, and washing the cells by sterile PBS;
replacing the basic culture medium with a serum-free basic culture medium, and continuously culturing for 20-36 hours; the serum-free basal medium for the amniotic epithelial stem cells is DMEM/F12 medium, and the serum-free basal medium for the umbilical cord mesenchymal stem cells is alpha-MEM medium.
And fifthly, removing the cells and cell debris through centrifugation, collecting the conditioned medium of the cultured cells, and then performing sterilization and filtration to obtain the stem cell conditioned medium.
Furthermore, the effective components of the stem cell conditioned medium are IL-1ra, IL-10, KGF and bFGF, the content of IL-1ra is 400-1000pg/ml, the content of IL-10 is 50-200pg/ml, the content of KGF and EGF is 50-200pg/ml, and the content of bFGF is 50-200pg/ml.
The invention provides application of two adult stem cell conditioned media (UMSCM and AECM) in preparation of a drug for inflammatory skin. The inflammatory dermatosis is suitable for psoriasis, atopic dermatitis, eczema, neurodermatitis, acne rosacea, seborrheic dermatitis, allergic dermatitis, hormone-dependent dermatitis, and lichen planus.
The stem cell conditioned medium skin external application of the invention is also characterized in that pharmaceutically acceptable isotonic agent, bacteriostatic agent, stabilizer, tackifier, curing agent, protein protective agent, medicament carrier and the like can be added into the conditioned medium skin external application.
Psoriasis vulgaris is a chronic recurrent inflammatory skin disease, and AECM and UMSCM of the invention contain inflammation-inhibiting factors (including IL-ra, IL10, IL13 and TGF beta) which can inhibit inflammatory cells of the skin of a patient from infiltrating the skin, thereby inhibiting epidermal hyperplasia and inflammatory reaction.
The Conditioned Medium (CM) of stem cells in the present invention includes human amniotic epithelial stem cells CM (AECM) and human umbilical cord mesenchymal stem cells CM (UMSCM), and its effective components include anti-inflammatory factors IL1ra (inhibitors of interleukins IL-11 and IL-11), IL10 (interleukin 10), IL13 (interleukin 13), TGFb (transforming growth factor b), etc., chemokines (MCP-1, CCR), cell growth factors Keratinocyte Growth Factor (KGF), Epidermal Growth Factor (EGF), fibroblast growth factor (including bFGF, FGF9, FGF11, FGF13, etc.), (platelet-derived growth factor (PDGF), cell adhesion molecules (NrCAM, ICAM-1, ICAM-2, ICAM-5), etc., anti-inflammatory factors (IL-1ra and IL10, etc.) which can inhibit the infiltration of inflammatory cells into the skin of a patient, thereby inhibiting the proliferation of epidermal growth factors (KGF, FGF, EGF, etc.) can promote repair of a damaged skin barrier in a patient.
We have demonstrated in an Imiquimod (IMQ) -induced psoriasis mouse model that AECM and UMSCM have a relief effect on skin symptoms of the psoriasis mouse model, including reduction of psoriasis, thinning of the epidermis, reduction of immune cell invasion, increased skin barrier gene expression, etc., and that AECM has a better therapeutic effect. Therefore, the stem cell conditioned medium comprising AECM and UMSCM may have good therapeutic effects on psoriasis vulgaris; moreover, the human stem cell conditioned medium has no animal-derived components, no risk of causing tumor, no safety problem related to stem cell transplantation, no ethical problem, simple and convenient use (external use), moderate price and capability of being popularized and used in a large number of patients. The stem cell conditioned medium is a new biological agent for treating psoriasis vulgaris.
The application of the invention also has the following characteristics: the umbilical cord mesenchymal stem cells and amniotic epithelial stem cells are extracted from medical wastes, namely umbilical cord tissues produced by lying-in women and amniotic membrane of placenta, and have no ethical disputed problems and sufficient sources.
The adult stem cell conditioned medium of the present invention is also characterized by containing abundant growth factors and playing a role in supporting nutrition, and also containing anti-inflammatory factors, chemokines, etc. and playing a role in immunosuppression.
Compared with stem cell products, the adult stem cell conditioned medium has better characteristics in medicine preparation, is not a cell product, and has better safety. In addition, we can determine the key secretion factor in the adult stem cell conditioned medium through in vitro experiments, thereby controlling the quality of the stem cell conditioned medium more easily. In addition, the stem cell culture medium can be applied in a mode of external application, and the mode of application is common for treating skin diseases and is easy to accept by patients.
Compared with the prior art, the invention has remarkable technical progress. 1. The invention discovers the pharmaceutical application of the human umbilical cord mesenchymal conditioned medium and the amniotic epithelial mesenchymal stem cell conditioned medium as the skin external-use drug, discovers and proves that the conditioned medium derived from the human umbilical cord mesenchymal conditioned medium and the human amniotic epithelial stem cell can inhibit and repair the lesion of psoriasis and inhibit skin inflammation for the first time, and the conditioned medium of the human amniotic epithelial stem cell has better treatment effect. 2. The stem cell conditioned medium is used for treating psoriasis, has low immunogenicity, avoids directly using cells, is safe and effective, and has high product stability. And avoids the secondary damage to the patient caused by hypodermic injection. 3. The invention provides a new thought and theoretical basis for clinically treating immune diseases by using the stem cell conditioned medium, and promotes the conversion scientific research of treating psoriasis by using stem cells.
Drawings
FIG. 1: the phase difference morphological characteristics of the amniotic epithelial stem cells (hAECs) and human umbilical cord mesenchymal stem cells (UMSCs) of examples 1 and 2 of the present invention.
Figure 2 shows skin symptoms for different normal control groups (CON), imiquimod-induced psoriasis model group (IMQ), umbilical cord mesenchymal stem cell conditioned medium treated group (IMQ + UMSCM, UMSCM group), amniotic epithelial stem cell conditioned medium treated group (IMQ + AECM, AECM group).
Figure 3 shows graphs of skin Ki67 immunofluorescent staining in normal control group (PBS), imiquimod-induced psoriasis model group (IMQ) and treatment group (UMSCM), treatment group (AECM), indicating the proliferation of stratum corneum cells.
FIG. 4 shows immunofluorescence staining patterns of skin neutrophil marker-Gr-1 in normal control group (PBS), imiquimod-induced psoriasis model group (IMQ), and treatment group (UMSCM), treatment group (AECM), indicating the invasion of neutrophils into the skin.
Figure 5 shows a flow analysis of invading IL-7A secreting T positive and negative cells in the skin in the normal control group (PBS), imiquimod induced psoriasis model group (IMQ) and treatment group (UMSCM), treatment group (AECM).
Figure 6 shows RNA expression profiles of inflammatory factors and chemokines recruited to immune cells in skin in normal control group (PBS), imiquimod-induced psoriasis model group (IMQ) and treatment group (UMSCM), treatment group (AECM).
Detailed Description
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto.
In the present specification, the term "human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells)" is a kind of mesenchymal stem cells derived from Wharton's jelly and perivascular tissues of the umbilical cord. And "amniotic epithelial stem cells (hAEC)" refers to an epithelial stem cell derived from the epithelial layer of placental amniotic membrane. They are derived from the medical waste placenta and umbilical cord, and therefore, have no ethical problems, low immunogenicity, and no tumorigenicity. In addition, the two stem cells have high proliferation and multidirectional differentiation potential, have the function of immunoregulation, and can secrete various cytokines, chemokines, growth factors, inflammation-inhibiting factors and immunoregulatory factors.
In the present embodiment, "human umbilical cord mesenchymal stem cell conditioned medium (UMSCM) and human amniotic epithelial stem cell culture medium (AEC-CM)" refer to conditioned medium in which bioactive components such as microvesicles and various proteins are directly secreted into the medium when human umbilical cord mesenchymal stem cells and human amniotic epithelial cells are cultured in vitro. The conditioned medium is used for preparing skin medicines.
In the experimental materials of the following examples, the platelet lysate was UltraGROTM-advanced (Aventacell Biomedical), and its volume ratio was 5%. DMEM/F12 (Thermofisiher, 21041025), a-MEM (Thermofisiher, 41061029), pancreatin (Thermofisiher, 15090046); collagenase (thermolsurfer, 17018029).
Example 1: preparation of human umbilical cord mesenchymal Stem cell conditioned Medium dermal drug (UMSCM)
1) Separating and culturing human umbilical cord mesenchymal stem cells: after the human umbilical cord is cleaned by sterile normal saline, umbilical artery and umbilical vein blood vessels are removed by using a tooth forceps, the remaining tissue after epidermis is torn off is cut into tissue blocks with the size of 3mm, then, a tissue climbing method is adopted, the tissue is pasted in a culture dish, and a culture medium alpha-MEM containing platelet lysate with the volume percentage concentration of 5% is added. After 7 days the fluid was slowly changed and after about 2 weeks the cells climbed out of the tissue mass. When the cells reach 90% density, the umbilical cord mesenchymal stem cells are subcultured, and the morphology of the umbilical cord mesenchymal stem cells is shown in fig. 1.
2) Collecting stem cell conditioned medium: culturing the umbilical cord mesenchymal stem cells or the amniotic epithelial stem cells in a culture medium continuously added with a platelet lysate to the 3 rd generation, discarding the culture medium when the confluence degree of the cells reaches about 90%, washing the cells for 3 times by using sterile PBS, continuously culturing for 24 hours by replacing the sterile culture medium with a serum-free culture medium, removing the cells and cell fragments by centrifugation, collecting the culture medium, and performing sterile filtration by using a 0.22um sterile filter to obtain the umbilical cord mesenchymal stem cells or the amniotic epithelial cell conditioned medium.
2) Adding antiseptic and protein stabilizer into the above stem cell conditioned medium, and making into skin topical preparation.
Example 2: preparation of human amniotic epithelial cell conditioned Medium for dermal Administration (AECM)
1) Separating and culturing the amniotic epithelial stem cells: separating amnion from human placenta tissue, cleaning with sterile physiological saline, separating out epithelial layer, a layer close to fetus, digesting with trypsin with mass percent concentration of 0.25% for 30 minutes, then digesting with collagenase with 0.1g/L for 1 hour, finally filtering and centrifuging to obtain primary amnion epithelial stem cells, digesting with trypsin with mass percent concentration of 0.25% in a DMEM/F12 culture medium containing platelet lysate with volume percent concentration of 5%, and performing subculture when cell colony growth is found and adherent cells are fused to 80% -90%; the microscopic morphological characteristics of the amniotic epithelial stem cells are shown in fig. 1.
2) Collecting stem cell conditioned medium: culturing the umbilical cord mesenchymal stem cells or the amniotic epithelial stem cells in a culture medium continuously added with a platelet lysate to the 3 rd generation, discarding the culture medium when the confluence degree of the cells reaches about 90%, washing the cells for 3 times by using sterile PBS, continuously culturing for 24 hours by replacing the sterile culture medium with a serum-free culture medium, removing the cells and cell fragments by centrifugation, collecting the culture medium, and performing sterile filtration by using a 0.22um sterile filter to obtain the umbilical cord mesenchymal stem cells or the amniotic epithelial cell conditioned medium.
3) Adding antiseptic and protein stabilizer into the above stem cell conditioned medium, and making into skin topical preparation.
Example 3: quality identification of human umbilical cord mesenchymal stem cells and human amniotic epithelial cell conditioned medium
1) The stem cell conditioned medium is subjected to sterility test, mycoplasma test, endotoxin test, and the like.
2) The stem cell conditioned media were subjected to independent and allergy testing experiments.
3) The stem cell conditioned medium was subjected to BCA quantification, and the total protein amount and protein quantification of factors important therein were determined. Specifically, the content detection of the effective components IL-1ra, IL-10, KGF and bFGF shows that the content of IL-1ra is 400-1000pg/ml, the content of IL-10 is 50-200pg/ml, and the content of KGF, EGF, bFGF and bFGF is 50-200pg/ml.
Example 4: the therapeutic effect of the human umbilical cord mesenchymal stem cell conditioned medium and the amniotic epithelial stem cell conditioned medium on the imiquimod-induced psoriasis mouse model in the above examples was adopted.
An imiquimod-induced psoriasis mouse model. The establishment method comprises the following steps: balb/c female mice, 8 weeks old, were randomly divided into 4 groups: normal control group, model group (IMQ group), UMSC-treated group (UMSCM + IMQ group) AECCM group, AECM-treated group (AECM + IMQ group), 5 per group;
the back of the mouse was depilated with depilatory cream, having a depilatory area of about 2cm by 3 cm. After two days, constructing a model by using imiquimod ointment IMQ and performing grouped drug delivery;
normal group: wet-applying a basic culture medium on the back of a mouse twice every day, wherein the basic culture medium is applied once in the morning and once in the afternoon for about 30 minutes every time, and after wet-applying for about 2 hours in the afternoon, uniformly applying 62.5mg of vaseline on the skin on the back of the mouse;
IMQ model set: the basal culture medium is wet-coated on the back of the mouse twice every day, once in the morning and once in the afternoon for about 30 minutes every time, and 62.5mg of Imiquimod (IMQ) is uniformly coated on the back skin of the mouse after wet-coating is finished for about 2 hours in the afternoon;
AECM group: the AECM was applied to the back of the mouse twice a day, once in the morning and once in the afternoon, each time for about 30 minutes, and 62.5mg of IMQ was applied to the back skin of the mouse after the wet application was completed for about 2 hours in the afternoon;
UMSCM group: the UMSCM is applied to the back of the mouse twice at regular time every day, once in the morning and once in the afternoon for about 30 minutes every time, and 62.5mg of IMQ is uniformly applied to the back skin of the mouse after the wet application is finished for about 2 hours in the afternoon;
daily photographs were recorded and after 6 days, mice were sacrificed for sampling. Taking a small part of skin tissue to be used for preparing paraffin section and frozen section and extracting RNA and protein, and carrying out enzymolysis and digestion on the residual skin tissue to extract skin lymphocytes.
The degree of skin lesions was judged from the psoriasis and redness of the skin, and the results are shown in fig. 2. The thickness of the epidermis was judged by paraffin sectioning, hematoxylin eosin (H & E) staining. Figure 2A shows that IMQ increased psoriasis and skin thickness relative to the control group, but UMSCM and AECM decreased psoriasis and skin thickness, with the AECM group being more effective. Fig. 2B is a HE staining of skin from different groups, showing that the IMQ group significantly increased the thickness of the epidermis and the UMSCM and AECM treated groups decreased the thickness of the epidermis relative to the CON group. FIG. 2C is the quantification of 2B.
As shown in fig. 3, abnormal hyperplasia of the skin was observed by Ki-67 immunostaining. Real-time PCR is used for detecting the gene expression of the epidermis to judge the abnormal differentiation of the epidermis. Thus observing the barrier function of the skin. Fig. 3A shows that the skin stratum corneum cells of the IMQ group significantly proliferated abnormally relative to the CON group. Whereas the treatment groups UMSCM and AECM inhibited the abnormal proliferation of stratum corneum cells, the AECM group had a better inhibitory effect. FIG. 3B is the quantification of 3A. Fig. 3C is the expression of marker genes for stratum corneum differentiation, in psoriasis, Filggrin (FLG) and Hornerin (HRNR) decreased and Involucrin (IVL) increased, as well as less FLG and HRNR and increased IVL in the IMQ group skin, however AECM or UMSCM increased the expression of FLG and HRNR and decreased the expression of IVL, indicating that these two stem cell culture media inhibit the abnormal differentiation and abnormal proliferation of stratum corneum, thereby maintaining the function of the skin barrier. Moreover, AECM has a better therapeutic effect.
The invasion of immune cells was judged by immunostaining and flow of neutrophils. As shown in fig. 4A, it is shown that the number of invading neutrophils was significantly increased in the IMQ group relative to the control group, whereas UMSCM and AECM decreased the invasion of neutrophils. And AECM has better effect of inhibiting neutrophils. FIG. 4B shows the quantitative results of FIG. 4A. FIG. 5A shows that IMQ indicates an increase in both IL-7A secreting T positive and negative cells invading the skin relative to the control, whereas AECM and UAECM reduce invasion of both immune cells into the skin and AECM has a better inhibitory effect. FIG. 5B shows the results of quantification of 5A.
The secretion of inflammatory factors and chemokines in the skin was measured by Real-time PCR, as shown in FIG. 6, IMQ showed that inflammatory factors TNF, IL-1, IL-17A, CCL20, and IL-8 were increased, and AECM and UAECM decreased the expression of inflammatory factors and chemokines, and AECM had a better inhibitory effect, compared to the control group.
The above examples are only preferred embodiments of the present invention, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should be construed as the protection scope of the present invention.
Claims (3)
1. Use of a stem cell conditioned medium in the manufacture of a medicament for the treatment of an inflammatory skin disorder, said stem cell prepared by a process comprising the steps of:
taking separated amnion from medical waste human placenta tissue after parturition of a parturient as a raw material, peeling a layer of amnion epithelial layer of the amnion close to a fetus by using a pair of tweezers, shearing, digesting for 20-40 minutes by adopting pancreatin with the mass percentage concentration of 0.25%, digesting for 0.5-2 hours by using collagenase, filtering, centrifuging to obtain amnion epithelial stem cells, and planting the amnion epithelial stem cells in a DMEM/F12 culture medium added with human platelet lysate;
or,
the medical waste umbilical cord after parturition of a parturient is taken as a raw material, the umbilical cord is washed clean by PBS (phosphate buffer solution), blood is removed, and then the umbilical artery and umbilical vein are removed by using tooth forceps. Then, shearing into tissue blocks with the diameter of 2-4 mm by using scissors, pasting the tissue blocks into a culture dish, adding an alpha-MEM culture medium containing human platelet lysate, changing the culture medium after 7 days, and obtaining umbilical cord mesenchymal stem cells after 14 days;
digesting and passaging the umbilical cord mesenchymal stem cells or the primary amniotic epithelial stem cells obtained above, and continuously culturing to a third generation;
step three, when the confluency of the third generation cells reaches 90%, discarding a culture medium containing platelet lysate, and washing the cells by sterile PBS;
replacing the basic culture medium with a serum-free basic culture medium, and continuously culturing for 20-36 hours; the serum-free basal medium for the amniotic epithelial stem cells is DMEM/F12 medium, and the serum-free basal medium for the umbilical cord mesenchymal stem cells is alpha-MEM medium.
And fifthly, removing the cells and cell debris through centrifugation, collecting the conditioned medium of the cultured cells, and then performing sterilization and filtration to obtain the stem cell conditioned medium.
2. Use according to claim 1, characterized in that: the effective components of the stem cell conditioned medium are IL-1ra, IL-10, KGF and bFGF, the content of IL-1ra is 400-one 1000pg/ml, and IL-10 is
50-200pg/ml, KGF, EGF content is 50-200pg/ml, bFGF content is 50-200pg/ml.
3. Use according to claim 1, characterized in that: the inflammatory dermatosis is psoriasis, atopic dermatitis, eczema, neurodermatitis, acne rosacea, seborrheic dermatitis, allergic dermatitis, hormone-dependent dermatitis or lichen planus.
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