CN113827618B - Use of stem cell conditioned medium in preparation of medicament for treating inflammatory skin - Google Patents
Use of stem cell conditioned medium in preparation of medicament for treating inflammatory skin Download PDFInfo
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Abstract
The invention provides an application of a stem cell conditioned medium in preparing a medicament for treating inflammatory skin, and a preparation method of the stem cell comprises the following steps of: taking amniotic membrane and umbilical cord tissues in human placenta tissues as raw materials, then obtaining amniotic epithelial stem cells and umbilical mesenchymal stem cells by shearing, digestion and other methods, and then planting in a culture medium containing platelet lysate; the primary cells obtained above were passaged to the third generation, and when the cell confluency reached 90%, the medium containing the platelet lysate was discarded; changing into a serum-free basic culture medium, and continuously culturing for 20-36h; collecting the conditioned medium of the cultured cells, and removing the cells and cell fragments by centrifugation to obtain an amniotic epithelial stem cell conditioned medium (AECM) and an umbilical mesenchymal stem cell conditioned medium (UMSCM). AECM and UMSCM of the present invention can inhibit inflammatory cells of the patient's skin from infiltrating the skin, thereby inhibiting epidermal hyperplasia and inflammatory response.
Description
Technical Field
The invention belongs to the field of biological medicine, 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 skin diseases are a series of recurrent skin diseases caused by abnormal activation of the immune system by the induction stimulation of the immune system to environmental factors, and have become one of the major health problems worldwide, and the proportion of medical care expenditures for the treatment of such diseases is rising year by year. The typical refractory inflammatory dermatosis-psoriasis, which is a common chronic inflammatory dermatosis, and is mainly characterized in that a plurality of immune cells mainly comprising T lymphocytes infiltrate a lot, and the epidermal spinous process is prolonged caused by the abnormal proliferation of epidermal keratinocytes, and the keratinocytes are not fully differentiated caused by the abnormal differentiation. The pathogenesis of psoriasis is not well understood, but there is a great deal of evidence that the combined effects of environmental factors, gene sensitivity, disruption of the skin barrier, and immune abnormalities are involved in the occurrence and development of the disease. The skin barrier is broken, allowing environmental substances to enter the skin and further inducing skin immune responses and skin inflammation. 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 vicious circle of inflammation. Wherein γδ T cells of the dermis are the major source of IL-7A.
Psoriasis is an incurable disease, often suffering from life-long. Clinical treatment aims at controlling symptoms. Topical medicine and ultraviolet phototherapy can be applied to patients with local lesions and early stage light symptoms, and common medicines include moisturizer, emollient, glucocorticoid, vitamin D3 derivative, calcineurin inhibitor, tretinoin, etc. Systemic treatment for moderately severe patients includes methotrexate, cyclosporine, tretinoin, and various biologicals. However, long-term use of these drugs causes problems of side effects, drug resistance, drug tolerance, and the like. Recently, some biological agents have been explored, such as TNF antagonists, and antibodies to IL-12, IL-23, IL-17. These biological agents are more effective than traditional drugs, but are subject to expensive medical costs for long-term use. Therefore, development of a biological agent with little side effects, high safety and good therapeutic effect is highly necessary for the treatment of skin diseases caused by immune abnormality such as psoriasis.
Adult stem cells, including mesenchymal stem cells (mesenchymal stem cells, MSCs) and amniotic epithelial stem cells (amniotic epithelial cells, AEC), are currently the most widely used stem cells, and have shown therapeutic effects in many immune and inflammation-related diseases. Wherein paracrine factors of stem cells play an important role in regulating immunity, inhibiting inflammation, tissue repair and regeneration. Paracrine factors, including growth factors (EGF, KGF, FGF, etc.), immunomodulators: antagonists of interleukin-1 (IL-ra), IL-10, IL-13, transforming growth factor beta (TGF beta), some receptors, chemokines, etc., have nutritional and immunomodulatory properties, and thus, secretions of adult stem cells, i.e., conditioned Medium (CM) of adult stem cells, may play a role in the treatment of diseases. However, stem cell-derived CM has not been reported, nor has human umbilical cord-derived MSC CM (UMSCM) and human amniotic epithelial stem cell CM (AECM). Wherein the human umbilical cord and amniotic membrane are of sufficient origin, and the cells (UMSC and AEC) from which they are derived have potent proliferative and multipotent differentiation potential, and are capable of modulating immune disorders, inhibiting inflammation, and alleviating symptoms of the disease in a number of immune-related diseases. And stem cells can secrete a variety of growth factors and immunomodulators. Therefore, the invention adopts the adult stem cell condition culture medium with wider application: including human umbilical cord mesenchymal stem cell conditioned medium (UMSCM) and human amniotic epithelial stem cell conditioned medium (AECM), may be one of the potential therapeutic approaches for inflammatory skin diseases such as psoriasis.
Disclosure of Invention
The invention provides application of a stem cell regulating culture medium in preparing a medicament for treating inflammatory skin, which aims to solve the technical problem that the medicament in the prior art has poor effect on quality of inflammatory skin.
The invention provides an application of a stem cell regulating culture medium in preparing a medicament for treating psoriasis, wherein the preparation method of stem cells comprises the following steps:
firstly, separating amniotic membrane from human placenta tissue which is medical waste after delivery of a lying-in woman, peeling off a layer of amniotic epithelial layer of the amniotic membrane close to a fetus by using tweezers, then shearing, adopting pancreatin with the mass percent concentration of 0.25% to digest for 20-40 minutes, then adopting collagenase to digest for 0.5-2 hours, then filtering, centrifuging to obtain amniotic epithelial stem cells, and planting the amniotic epithelial stem cells in a DMEM/F12 culture medium added with human platelet lysate;
or alternatively, the process may be performed,
the umbilical cord is cleaned by PBS (phosphate buffered saline) firstly, blood is removed, and then umbilical arteries and umbilical veins are removed by dental forceps. Then shearing the umbilical cord mesenchymal stem cells into tissue blocks with the diameter of 2-4 mm by scissors, attaching the tissue blocks into a culture dish, adding an alpha-MEM culture medium containing human platelet lysate, changing the liquid after 7 days, and obtaining the umbilical cord mesenchymal stem cells after 14 days;
step two, digesting and passaging the umbilical cord mesenchymal stem cells or the amniotic epithelial stem cells obtained above to the third generation;
step three, when the confluence of the third generation cells reaches 90%, discarding the culture medium containing the platelet lysate, and washing the cells with sterile PBS;
step four, replacing the culture medium with a serum-free basic culture medium to continue to culture for 20 to 36 hours; the serum-free basal medium for amniotic epithelial stem cells is DMEM/F12 medium, and the serum-free basal medium for umbilical mesenchymal stem cells is alpha-MEM medium.
And fifthly, removing cells and cell debris by centrifugation, collecting a conditioned medium of the cultured cells, and then performing sterilization and filtration to obtain the stem cell conditioned medium.
Further, the stem cell conditioned medium contains the effective components IL-1ra, IL-10, KGF and bFGF, wherein the IL-1ra content is 400-1000pg/ml, the IL-10 content is 50-200pg/ml, the KGF content and the EGF content are 50-200pg/ml, and the bFGF content is 50-200pg/ml.
The invention provides the use of two adult stem cell conditioned media (UMSCM and AECM) in the preparation of inflammatory skin medicaments. The inflammatory skin disease is suitable for psoriasis, atopic dermatitis, eczema, neurodermatitis, rosacea, seborrheic dermatitis, allergic dermatitis, hormone-dependent dermatitis, lichen planus.
The external skin preparation of the stem cell conditioned medium of the invention has the characteristic that pharmaceutically acceptable isotonic agents, bacteriostats, stabilizers, adhesion promoters, curing agents, protein protectants, medicament carriers and the like can be added to the external skin preparation of the conditioned medium.
Psoriasis vulgaris is a chronic recurrent inflammatory skin disease, and the AECM and UMSCM in the invention contain anti-inflammatory 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 response.
The stem cell Conditioned Medium (CM) in the invention comprises human amniotic epithelial stem cells CM (AECM) and human umbilical mesenchymal stem cells CM (UMSCM), and the active ingredients of the CM comprise anti-inflammatory factors IL1ra (inhibitors of interleukin IL-11 and IL-11), IL10 (interleukin 10), IL13 (interleukin 13), TGFb (transforming growth factor b) and the like, chemokines (MCP-1 and CCR), and the cell growth factors comprise Keratinocyte Growth Factor (KGF), epidermal Growth Factor (EGF), fibroblast growth factor (including bFGF, FGF9, FGF11 and FGF 13) and the like, and platelet-derived growth factor (PDGF), cell adhesion molecules (NrCAM, ICAM-1, ICAM-2, ICAM-5) and the like.
We have validated the effects of AECM and UMSCM in the mouse model of Imiquimod (IMQ) induced psoriasis on the relief of skin symptoms in the mouse model of psoriasis, including reduced psoriasis, thinning of the epidermis, reduced immune cell invasion, increased expression of skin barrier genes, etc., and the AECM has better therapeutic effects. Therefore, stem cell conditioned medium including AECM and UMSCM may have good therapeutic effects on psoriasis vulgaris; in addition, the human stem cell conditioned medium has no animal source component, no tumorigenic risk, no safety problem related to stem cell transplantation, no ethical problem, simple and convenient use (external use), moderate price and popularization and application in patients. Stem cell conditioned medium is a novel biological agent for the treatment of psoriasis vulgaris.
The application of the invention also has the following characteristics: the umbilical cord mesenchymal stem cells and the amniotic epithelial stem cells are extracted from medical wastes, namely umbilical cord tissues produced by puerpera and amniotic membranes of placenta, and have no ethical disputed problem and sufficient sources.
The adult stem cell conditioned medium of the invention has the characteristics of rich growth factors, nutrition support function, anti-inflammatory factors, chemotactic factors and the like, and immunosuppression function.
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 key secreted factors in adult stem cell conditioned medium by in vitro experiments, thereby making it easier to control the quality of stem cell conditioned medium. In addition, the stem cell culture medium can be externally applied according to the medication mode, and the medication mode is common for treating skin diseases and is easy to be accepted by patients.
Compared with the prior art, the invention has obvious technical progress. 1. The invention discovers the pharmaceutical application of the human umbilical cord mesenchymal condition culture medium and the amniotic epithelial mesenchymal stem cell condition culture medium as the external skin medicament, and discovers and proves that the condition culture medium derived from the human umbilical cord mesenchymal condition culture medium and the human amniotic epithelial stem cell can inhibit and repair the lesion of psoriasis, inhibit the skin inflammation and have better treatment effect. 2. The stem cell conditioned medium is externally used for treating psoriasis, has low immunogenicity, avoids directly using cells, and is safe, effective and high in product stability. And avoid secondary injury to patients caused by subcutaneous injection. 3. The invention provides a new thought and theoretical basis for treating immune diseases by utilizing the stem cell conditioned medium clinically, and promotes the transformation scientific research of treating psoriasis by stem cells.
Drawings
Fig. 1: phase contrast morphological characteristics of amniotic epithelial stem cells (hAEC) and human Umbilical Mesenchymal Stem Cells (UMSC) of examples 1 and 2 of the present invention.
Fig. 2 shows skin symptoms of different normal Control (CON), imiquimod-induced psoriasis model (IMQ), umbilical cord mesenchymal stem cell conditioned medium treatment (imq+umscm, UMSCM) and amniotic epithelial stem cell conditioned medium treatment (imq+aecm, AECM).
FIG. 3 shows skin Ki67 immunofluorescence staining patterns in normal control (PBS), imiquimod-induced psoriasis model (IMQ) and treatment (UMSCM), treatment (AECM), showing proliferation of keratinocytes.
FIG. 4 shows immunofluorescence staining patterns of skin neutrophil marker-Gr-1 in normal control (PBS), imiquimod-induced psoriasis model (IMQ) and treatment (UMSCM), treatment (AECM), showing the invasion of neutrophils into the skin.
FIG. 5 shows flow charts of T-positive and negative IL-7A secreting cells invading skin in normal control (PBS), imiquimod-induced psoriasis model (IMQ) and treatment (UMSCM), treatment (AECM).
Fig. 6 shows RNA expression patterns of inflammatory factors and chemotactic factors recruiting immune cells in the skin for normal control (PBS), imiquimod-induced psoriasis model (IMQ) and treatment (UMSCM), treatment (AECM).
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
In the present specification, the term "human umbilical cord mesenchymal stem cells (human umbilical cord mesenchymal stem cells)" is one of the mesenchymal stem cells derived from the Wharton's jelly of umbilical cord and perivascular tissues. And "amniotic epithelial stem cell (human amniotic epithelial cells, hAEC)" refers to an epithelial stem cell derived from the epithelial layer of the amniotic membrane of the placenta. They are derived from the placenta and umbilical cord of medical waste, 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, anti-inflammatory factors and immunoregulatory factors.
In an embodiment of the present invention, the term "human umbilical cord mesenchymal stem cell conditioned medium (UMSCM) and human amniotic epithelial stem cell part medium (AEC-CM)" refers to a conditioned medium formed by directly secreting bioactive components such as microvesicles and various proteins into a medium when human umbilical cord mesenchymal stem cells and human amniotic epithelial cells are cultured in vitro. The skin preparation is prepared by using the conditioned medium.
In the experimental materials of the following examples, the platelet lysate was UltraGROTM-Advanced (AventaCell BioMedical), which was 5% by volume. DMEM/F12 (thermosusher, 21041025), a-MEM (thermosusher, 41061029), pancreatin (thermosusher, 15090046); collagenase (thermosusher, 17018029).
Example 1: preparation of human umbilical mesenchymal Stem cell conditioned Medium skin drug (UMSCM)
1) Isolated culture of human umbilical cord mesenchymal stem cells: after the umbilical cord of a person is cleaned by sterile normal saline, umbilical artery and umbilical vein blood vessels are removed by using dental forceps, the tissue left after the epidermis is torn off is sheared into tissue blocks with the size of 3mm, then a tissue climbing sheet method is adopted, the tissue is stuck 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 changed slowly and after about 2 weeks, the cells were allowed to climb out of the tissue mass. The morphology of umbilical cord mesenchymal stem cells was shown in fig. 1 when the cells reached 90% density for subculturing.
2) Collecting stem cell conditioned medium: culturing umbilical cord mesenchymal stem cells or amniotic epithelial stem cells in a culture medium with platelet lysate until the generation of 3 is continued, discarding the culture medium when the cell confluence reaches about 90%, washing the cells for 3 times with sterile PBS, replacing the culture medium with a serum-free culture medium, continuously culturing for 24 hours, removing the cells and cell fragments by centrifugation, collecting the culture solution, and sterilizing and filtering the culture solution by using a 0.22um sterile filter to obtain the umbilical cord mesenchymal stem cells or 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) Amniotic epithelial stem cells isolated culture: separating amniotic membrane from human placenta tissue, cleaning with sterile physiological saline, separating an epithelial layer, closing to a fetus, digesting for 30 minutes with trypsin with the mass percent concentration of 0.25%, then digesting for 1 hour with collagenase with the mass percent concentration of 0.1g/L, finally filtering and centrifuging to obtain primary amniotic membrane epithelial stem cells, and carrying out subculture in a DMEM/F12 culture medium containing platelet lysate with the volume percent concentration of 5% when cell colony growth is found and adherent cells are fused to 80% -90% with trypsin with the mass percent concentration of 0.25%; the morphological characteristics of the amniotic epithelial stem cells under the mirror are shown in figure 1.
2) Collecting stem cell conditioned medium: culturing umbilical cord mesenchymal stem cells or amniotic epithelial stem cells in a culture medium with platelet lysate until the generation of 3 is continued, discarding the culture medium when the cell confluence reaches about 90%, washing the cells for 3 times with sterile PBS, replacing the culture medium with a serum-free culture medium, continuously culturing for 24 hours, removing the cells and cell fragments by centrifugation, collecting the culture solution, and sterilizing and filtering the culture solution by using a 0.22um sterile filter to obtain the umbilical cord mesenchymal stem cells or 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 sterile detection, mycoplasma detection, endotoxin detection and the like.
2) Stem cell conditioned medium was subjected to independent and allergy test experiments.
3) The BCA assay was performed on the stem cell conditioned medium to determine the total protein and protein quantification of important factors therein. Specifically, the content of the active ingredients IL-1ra, IL-10, KGF and bFGF is detected, the content of IL-1ra is 400-1000pg/ml, IL-10 is 50-200pg/ml, KGF, EGF, bFGF and the content is 50-200pg/ml.
Example 4: therapeutic effects of human umbilical cord mesenchymal stem cell conditioned medium and amniotic epithelial stem cell conditioned medium on imiquimod-induced psoriasis mouse model using the above examples.
Mouse model of imiquimod-induced psoriasis. The establishment method comprises the following steps: balb/c female mice at 8 weeks of age were randomly divided into 4 groups: a normal control group, a model group (IMQ group), a UMSC treatment group (umscm+imq group) an AECCM group, an AECM treatment group (aecm+imq group), 5 each;
the back of the mice was dehaired with a dehairing paste with a dehairing area of about 2cm by 3cm. Two days later, the imiquimod ointment IMQ is used for constructing a model and carrying out grouping administration;
normal group: the basal medium is wet-applied to the back of the mouse twice at regular time every day, once in the morning and once in the afternoon, each time for about 30 minutes, and 62.5mg of Vaseline is uniformly smeared on the skin of the back of the mouse after the wet-application is completed for about 2 hours in the afternoon;
IMQ model group: the basal medium is wet-applied to the back of the mouse twice at daily time, once in the morning and once in the afternoon for about 30 minutes each time, and 62.5mg Imiquimod (IMQ) is uniformly applied to the skin of the back of the mouse after the wet-application is completed for about 2 hours each time;
AECM group: applying AECM to the back of a mouse twice at regular time every day, once in the morning and once in the afternoon for about 30 minutes each time, and uniformly applying 62.5mg of IMQ to the back skin of the mouse after finishing the wet application for about 2 hours in the afternoon;
UMSCM group: the UMSCM is wet-applied to the back of the mouse twice at daily time, once in the morning and once in the afternoon, each time is about 30 minutes, and 62.5mg of IMQ is uniformly applied to the back skin of the mouse after the wet-application is completed for about 2 hours;
daily photographs were recorded and 6 days later, mice were sacrificed for sampling. Taking a small part of skin tissue for preparing paraffin sections, frozen sections and RNA and protein extraction, and extracting skin lymphocytes by enzymolysis and digestion of the residual skin tissue.
The degree of skin damage of the psoriasis-like mice was judged based on the psoriasis and redness and swelling of the skin, and the results are shown in fig. 2. The thickness of the epidermis was judged by paraffin section, hematoxylin eosin (H & E) staining. Fig. 2A shows that IMQ increased psoriasis and skin thickness relative to the control, but UMSCM and AECM reduced psoriasis and skin thickness, with the AECM group being better. Fig. 2B is a different set of skin HE staining, showing that the IMQ group significantly increased the thickness of the epidermis relative to the CON group, and the UMSCM and AECM treated groups reduced the thickness of the epidermis. FIG. 2C is a quantitative result of FIG. 2B.
As shown in FIG. 3, abnormal proliferation of skin was observed by Ki-67 immunostaining. Real-time PCR was used to detect gene expression in epidermis to determine abnormal differentiation of epidermis. Thereby observing the barrier function of the skin. Figure 3A shows a significant abnormal proliferation of skin keratinocytes in the IMQ group relative to the CON group. While the UMSCM and AECM inhibited the abnormal proliferation of keratinocytes in the treatment group, the AECM group had a better inhibition effect. FIG. 3B is a quantitative result of 3A. Fig. 3C is the expression of markers of stratum corneum differentiation, in psoriasis Filaggrin (FLG) and Hornerin (HRNR) decreased, involucrin (IVL) increased, and likewise FLG and HRNR were less in the skin of IMQ group, IVL increased, whereas AECM or UMSCM increased FLG and HRNR expression, while IVL expression was decreased, suggesting that these two stem cell culture media inhibited abnormal differentiation and abnormal proliferation of the stratum corneum, thereby maintaining skin barrier function. Moreover, AECM has a better therapeutic effect.
Immune cell invasion was judged by immunostaining and flow through neutrophils. As shown in fig. 4A, the number of invading neutrophils in the IMQ group was significantly increased relative to the control group, while UMSCM and AECM could reduce neutrophil invasion. And AECM has better effect of inhibiting neutrophil. FIG. 4B is a quantitative result of 4A. FIG. 5A shows that IMQ indicates an increase in both IL-7A secreting T positive and negative cells that invade the skin relative to the control, while AECM and UAECM reduced invasion of both immune cells into the skin and that AECM had better inhibition. FIG. 5B is a quantitative result of 5A.
As shown in FIG. 6, IMQ indicates that inflammatory factors TNF, IL-1, IL-17A, CCL, IL-8 are increased relative to the control group, and AECM and UAECM can reduce the expression of the inflammatory factors and chemokines, and AECM has better inhibition effect.
The above examples are only preferred embodiments of the present invention and it will be apparent to those skilled in the art that several modifications and variations can be made without departing from the principle of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (2)
1. Use of a stem cell conditioned medium in the manufacture of a medicament for the treatment of an inflammatory skin disorder, said inflammatory skin disorder being psoriasis, said stem cell preparation method comprising the steps of:
firstly, separating amniotic membrane from human placenta tissue which is medical waste after delivery of a lying-in woman, peeling off a layer of amniotic epithelial layer of the amniotic membrane close to a fetus by using tweezers, then shearing, adopting pancreatin with the mass percent concentration of 0.25% to digest for 20-40 minutes, then adopting collagenase to digest for 0.5-2 hours, then filtering, centrifuging to obtain amniotic epithelial stem cells, and planting the amniotic epithelial stem cells in a DMEM/F12 culture medium added with human platelet lysate;
step two, the primary cells of the amniotic epithelial stem cells obtained above are digested and passaged, and are continuously cultured to the third generation;
step three, when the confluence of the third generation cells reaches 90%, discarding the culture medium containing the platelet lysate, and washing the cells with sterile PBS;
step four, replacing the culture medium with a serum-free basic culture medium, and continuously culturing for 20-36 hours; the serum-free basal medium is a DMEM/F12 medium;
and fifthly, removing cells and cell debris by centrifugation, collecting a 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 stem cell conditioned medium contains the effective components of IL-1ra, IL-10, KGF, EGF and bFGF, wherein the content of IL-1ra is 400-1000pg/ml, the content of IL-10 is 50-200pg/ml, the content of KGF is 50-200pg/ml, the content of EGF is 50-200pg/ml, and the content of bFGF is 50-200pg/ml.
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