CN113201481A - Skin microsphere and preparation method and application thereof - Google Patents
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Abstract
The invention discloses skin microspheres and a preparation method and application thereof. In a first aspect of the present application, there is provided a skin microsphere comprising: a dermal cell microsphere comprising a dermal cell, the dermal cell having a first signaling pathway activity reporter system; and the epidermal layer is coated on the surface of the dermal cell microsphere and comprises epidermal cells, and the epidermal cells have a second signal pathway activity reporter system. The skin microspheres according to the embodiment of the application have at least the following beneficial effects: the skin microsphere adopts a composite double-layer structure formed by wrapping inner core and outer layer epidermal cells formed by dermal cells, so that the skin structure of a human body can be better simulated. Meanwhile, the inner core and the outer shell are respectively provided with a signal path activity reporting system, so that the signal path activity reporting system can timely respond to the action effect of the to-be-detected product, and the detection of the action of the to-be-detected product on the epidermis layer and/or the dermis layer is realized.
Description
Technical Field
The application relates to the technical field of tissue engineering, in particular to skin microspheres and a preparation method and application thereof.
Background
The skin is the largest organ of the human body, mainly consists of epidermis, dermis, subcutaneous tissue and skin appendages, and has the functions of physical barrier, external perception, body temperature and moisture maintenance and the like. The health of the skin is closely related to the state of cellular and matrix components in the epidermis and dermis. If one of the components is dysfunctional, skin diseases can occur, for example, over-activated fibroblasts can cause scar formation. At present, skin medicaments and skin care products on the market can act on different components in the skin to achieve the aims of treating diseases and improving the skin state. The discovery of the effective components depends on an in vitro model and a screening technology to a great extent, and the in vitro model has the advantages of low cost, low ethical risk and the like compared with human and animal tests. Therefore, the reliable and advanced skin in vitro model has important significance for skin drug screening.
The in vitro model used before the drug screening platform is often a single cell line, such as immortalized epidermal cell HaCaT, and the effectiveness of the in vitro model is judged by detecting the influence of a compound on cell behaviors such as proliferation and apoptosis of the cell line. However, because the cell components are single and are far from the structure and the function of normal skin, the components which are really effective to the health of the skin are difficult to find, and the method is not suitable for the requirement of high-flux drug screening. Currently, attempts on tissue engineered skin of various cells have been used for cytotoxicity detection of cosmetics and partially replacing animal experiments. Such tissue engineering skin products using various cells are composed of the upper layer of keratinocytes and the lower layer of a cuticle-like structure, and the keratinocyte on the upper layer can be used for testing the barrier function of the epidermis after keratinization. But it is difficult to confirm the actual action of the drugs in the epidermis layer and the dermis layer so as to achieve the corresponding drug screening purpose.
Disclosure of Invention
The present application is directed to solving at least one of the problems in the prior art. Therefore, the application provides a skin microsphere capable of detecting the effect of the medicament in the epidermis or the dermis, and a preparation method and application thereof.
In a first aspect of the present application, there is provided a skin microsphere comprising:
a dermal cell microsphere comprising a dermal cell, the dermal cell having a first signaling pathway activity reporter system;
and the epidermal layer is coated on the surface of the dermal cell microsphere and comprises epidermal cells, and the epidermal cells have a second signal pathway activity reporter system.
The skin microspheres according to the embodiment of the application have at least the following beneficial effects:
the skin microsphere adopts a composite double-layer structure formed by wrapping inner core and outer layer epidermal cells formed by dermal cells, so that the skin structure of a human body can be better simulated. Meanwhile, the inner core and the outer shell are respectively provided with a signal path activity reporting system, so that the signal path activity reporting system can timely respond to the action effect of the to-be-detected product, and the detection of the action of the to-be-detected product on the epidermis layer and/or the dermis layer is realized.
The signal pathway activity reporter system is a system capable of detecting the activity of a specific signal pathway and making a qualitative or quantitative response according to the detection result, and can be realized in a non-limiting manner by including a reporter gene capable of detecting a specific signal pathway. It should be noted that the first signal pathway activity reporter system and the second signal pathway activity reporter system are only used for distinguishing whether the signal pathway activity reporter system is carried by dermal cells in the dermal cell microsphere or epidermal cells in the epidermal layer, and are not used for specifically limiting the kind and number of the signal pathway activity reporter systems.
In some embodiments of the present application, the first signaling pathway activity reporter system comprises at least one of an Shh signaling pathway activity reporter system, a MAPK/ERK signaling pathway activity reporter system. Shh plays an important role in regulating the development of various tissue organs involved in cell structure, proliferation and survival, and particularly, can promote the proliferation of hair papilla cells leading to hair follicle regeneration and hair growth, and signaling pathways having similar regulating effects thereto also include MAPK/ERK and the like. Therefore, the interaction between the drug and the cell can be detected by the signaling pathway reporter system carried by the dermal cell, and it can be determined whether or not the cell pathway in the dermal cell can be activated after the penetration of the epidermal barrier, and further, it can be determined how the activity of the signaling pathway is activated.
In some embodiments of the present application, the dermal cells are transfected with an SRE reporter gene. MAPK/ERK signaling pathway is the main participant in cell growth and differentiation regulation, and during activation, transcription factor substrates and Serum Response Factors (SRF) form a complex and are combined with Serum Response Elements (SRE) to realize gene expression regulation. Therefore, selection of SRE as a reporter gene in a signaling pathway activity reporter system can effectively detect the activity of the MAPK/ERK signaling pathway.
In some embodiments of the present application, the second signaling pathway activity reporter system comprises at least one of a canonical Wnt signaling pathway activity reporter system, an Akt signaling pathway activity reporter system. The Wnt signaling pathway is a complex regulatory network comprising three branches including the canonical Wnt signaling pathway. The canonical Wnt signaling pathway is mainly involved in regulating the pluripotent differentiation of stem cells, the development and regeneration of organs, and particularly promoting cell proliferation and regeneration. And Akt signal channel with similar regulation function to classical Wnt signal channel. Therefore, the epidermal cells carry these signaling pathway activity reporter systems to detect the interaction between the drug and the cells and determine how the interaction acts on the proliferation and regeneration of the cells.
In some embodiments of the present application, epidermal cells are transfected with a TCF/LEF1 reporter gene. TCF/LEF is a HMG (high Mobility group) transcription factor which is used as a target gene of Wnt signals and is involved in the feedback regulation of Wnt signal pathways, and a stable beta-catenin complex combined with the transcription factor can cause the activation of classical Wnt signal pathways. Therefore, TCF/LEF1 is selected as a reporter gene in a signaling pathway activity reporter system, so that the activity of the canonical Wnt signaling pathway can be detected more sensitively.
Thus, through the provision of the above-mentioned specific signaling pathway activity reporter system, the epidermal cell reporter system includes key pathways promoting cell proliferation and regeneration, such as canonical Wnt signaling pathway, Akt pathway, and the like. The dermal cell reporter system selects signal channels for promoting hair follicle and hair follicle papilla formation and hair follicle regeneration, such as an Shh signal channel, an MAPK/ERK signal channel and the like, so that whether a sample to be tested can activate the cell signal channels or not and how much the cell signal channels are activated can be observed to find out the effective ingredients of the medicine for promoting the regeneration of skin and hair follicles.
In some embodiments of the present application, the skin microspheres have a diameter of 0.02 to 10 mm. The actual size of the skin microspheres can be determined according to research needs, but considering the needs of freezing and recovering the product, the diameter of the microspheres is preferably 0.02-10 mm, and preferably within 1 mm.
In some embodiments of the present application, the epidermal cells are keratinocytes derived from the skin or mucosa, which may be primary cells, or keratinocytes expanded in culture, or immortalized keratinocytes such as HaCaT. Depending on the application requirements of the skin microspheres, the keratinocytes in the epidermal layer may further be a single layer or multiple layers, for example, in the case of detecting the effect of drugs on the proliferation of keratinocytes, the epidermal layer containing a single layer of keratinocytes may be used as the skin microspheres; when the barrier function of the epidermal layer and the transdermal capacity of the medicament need to be detected, the keratinocyte can be further differentiated and matured to form the stratum corneum. In addition, besides keratinocytes in the epidermis layer, other cells or substances can be incorporated according to different application requirements, for example, a certain proportion of melanocytes can be incorporated to test the effect of the drug on melanocytes in the epidermis layer of the skin; a proportion of immune cells (e.g.Langerhans cells) may also be infiltrated to study the effect of the drug on skin defense function.
In some embodiments of the present application, the dermal cells are fibroblasts isolated from the dermis of the skin, and the dermal cells may be primary cells, or fibroblasts expanded by culture, or immortalized fibroblasts; in addition, the fibroblast can also be formed by inducing differentiation of embryonic stem cells or iPS cells. Besides the function support of the epidermis layer wrapped on the dermal cell microsphere containing the fibroblast, the dermal cell microsphere can also be used for detecting and screening drugs or cosmetics aiming at the dermal cell. According to different application requirements of the skin microsphere, besides the fibroblast, endothelial cells with a certain proportion can be further added into the dermal cell microsphere to detect the angioblasts, or immune cells with a certain proportion can be added to perform immune-related detection or research.
In some embodiments of the present application, the dermal cell microspheres may contain, in addition to dermal cells, extracellular matrix macromolecules, such as collagen, as a non-limiting example, and further may contain other extracellular matrix molecules, such as mucopolysaccharides, such as hyaluronic acid. In addition, these extracellular matrix molecules may also be partially or completely replaced with synthetic materials.
In a second aspect of the present application, a method for preparing the skin microsphere is provided, which comprises the following steps:
uniformly mixing the dermal cells and the hydrogel to obtain a dermal cell mixed solution;
dripping the dermal cell mixture into an oil phase, and incubating to form dermal cell microspheres;
mixing the dermal cell microspheres with epidermal cells, and performing 3D culture to obtain the skin microspheres.
The preparation method of the skin microsphere according to the embodiment of the application has at least the following beneficial effects:
by adopting the method, the dermal cell microspheres are formed in the oil phase, and then the dermal cells are mixed with the epidermal cells and cultured in a 3D manner to obtain the skin microspheres, compared with the existing product, the epidermal shell layer formed on the outer side can be tightly connected with the hole walls, so that the surface of the dermal cell microspheres is more completely covered, when the skin microspheres are used for detecting and screening drugs, the drugs cannot directly enter the dermal cell microspheres in the inner part from the hole walls, and the barrier effect of the epidermal shell layer and the transdermal capacity of the drugs can be more accurately reflected. In addition, the shape of the finally obtained skin microspheres also enables the skin microspheres to be easier to sample and analyze and more convenient to store and transport.
In some embodiments of the present application, the dermal cell microspheres are resuspended and cultured in a culture medium for 12-36 hours before being mixed with the epidermal cells, during which the size of the dermal cell microspheres is slightly reduced along with the spreading of the dermal cells, so as to obtain a more stable structure for facilitating the subsequent adhesion of the epidermal cells on the surface thereof.
In a third aspect of the present application, there is provided the use of the above-described skin microspheres for the detection of drugs and/or cosmetics. The skin microspheres respectively carry corresponding signal channel activity reporting systems on the inner core and the outer shell, and can timely respond to the action effect of the drugs and/or cosmetics to be detected, so that the skin microspheres can be used as a better detection model to realize the detection of the action of the drugs and/or cosmetics on the epidermis layer and the dermis layer.
In a fourth aspect of the present application, a method for detecting an object to be detected is provided, the method comprising the steps of:
and contacting the skin microspheres with a product to be detected, and detecting the change of the skin microspheres before and after the contact.
The skin microspheres used in the detection method respectively carry corresponding signal path activity reporting systems on the inner core and the outer shell, and when a product to be detected is contacted with the skin microspheres, the skin microspheres can be used as a more accurate and visual detection model to reflect the action of medicines and/or cosmetics on the epidermis layer and the dermis layer.
Additional aspects and advantages of the present application will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the present application.
Drawings
Fig. 1 is a picture of skin microspheres prepared in example 1 of the present application.
FIG. 2 is the results of the reaction of skin microspheres to the drug in example 2 of the present application.
FIG. 3 is a schematic diagram of a plasmid loaded with a signaling pathway activity reporter system and a picture of skin microspheres carrying a signaling pathway activity reporter system in example 3 of the present application.
Detailed Description
The conception and the resulting technical effects of the present application will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all embodiments, and other embodiments obtained by those skilled in the art without inventive efforts based on the embodiments of the present application belong to the protection scope of the present application.
The following detailed description of embodiments of the present application is provided for the purpose of illustration only and is not intended to be construed as a limitation of the application.
In the description of the present application, the meaning of a plurality is one or more, the meaning of a plurality is two or more, and the above, below, exceeding, etc. are understood as excluding the present number, and the above, below, within, etc. are understood as including the present number. If the first and second are described for the purpose of distinguishing technical features, they are not to be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated or implicitly indicating the precedence of the technical features indicated.
In the description of the present application, reference to the description of the terms "one embodiment," "some embodiments," "an illustrative embodiment," "an example," "a specific example," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Example 1
The embodiment provides a skin microsphere and a preparation method of the skin microsphere.
Some experimental materials used in the preparation method are as follows:
type I collagen from rat tail (Corning, usa, product No. 354236); f3100 oil phase (us 3M); cisplatin (mclin corporation); DMEM (Corning, usa); FBS (American BI Co.).
The preparation method comprises the following specific preparation steps:
(1) preparing 2mg/ml hydrogel from rat tail type I collagen, mixing, placing into ice box, and adjusting pH.
(2) Digesting human skin fibroblasts, counting, taking a certain amount of fibroblast suspension, and centrifuging.
(3) Adjusting pH of the hydrogel to 7.0 with 0.1M NaOH, mixing, removing supernatant from fibroblast suspension, mixing with hydrogel with pH of 7.0 to obtain fibroblast mixture, uniformly distributing into eight tubes, and placing into ice box.
(4) The F3100 oil phase was sterile filtered and added to Teflon-96 well plates at 200ul per well. Then, 4. mu.l of fibroblast mixture was aspirated in an eight-piece tube using a 10. mu.l range of a discharge gun, and dropped into the oil phase of a Teflon-96 well plate, and then the Teflon-96 well plate was covered with Teflon-lad and placed in an incubator to incubate for 15-20min to form dermal cell microspheres.
(5) After the dermal cell microspheres are formed, taking out the pore plate from the incubator, adding 200 μ l of DMEM into each pore, standing for 5min, covering the Teflon-lad tightly, turning over to collect the microspheres, pouring the upper layer liquid in the Teflon-lad into a 50ml beaker, avoiding pouring the oil phase, washing the microspheres for 3 times by PBS, then re-suspending all the microspheres by using a complete culture medium (DMEM + 10% FBS), transferring all the liquid into a common type culture dish (non-cultured), and putting the culture dish into the incubator to culture for 24 h.
(6) Pouring the dermal Cell microspheres and the culture medium into a Cell Spinner flask for 3D culture, digesting the immortalized epidermal cells HaCaT simultaneously, adding a certain amount of epidermal Cell suspension into the running Spinner flask, fully mixing the dermal Cell microspheres and the epidermal cells, and culturing for 7 days to form complete skin microspheres.
The photographs of the skin microspheres are shown in FIG. 1, where A is the photograph of the skin microspheres at the 1000 μm scale and B is the photograph of the bright field, different color channels (GFP and mCherry) and merged (merge) at the 200 μm scale. As can be seen from fig. 1, the skin microspheres after 7 days of co-culture can be completely molded, have regular shapes and uniform sizes, have a double-layer skin structure, and include dermal cell microspheres (mCherry label) composed of fibroblasts and extracellular matrix (collagen), and a composite structure formed by an epidermal layer (green fluorescent protein GFP label) wrapped by epidermal cells on the outer layer of the dermal cell microspheres, and the structure is very similar to the skin structure of a human body, and can be used as a detection model to simulate the skin of the human body.
Example 2
Reaction experiment of skin microspheres to drug
The skin microspheres prepared in example 1 were treated with the drug to test the proliferation and apoptosis changes of the skin microspheres before and after the drug administration.
Experimental materials: bFGF (Sigma company, usa); mouse anti-E cadherin antibody (Thermofisiher, product No. 13-1700); rabbit anti-Ki 67 antibody (CST, product No. 9662); goat anti-rabbit 555 antibody (CST, product No. 3452); goat anti-mouse 647 antibody (CST, product number 4410).
The experimental procedure was as follows:
(1) the skin microspheres prepared in example 1 were cultured in a non-adherent 24-well plate, 2ml of a culture medium (containing 2% serum) was added to each well, and then the drug was added, and the culture was continued for 48 hours in a 37-degree incubator.
(2) Collecting the skin microspheres into a centrifuge tube by using a pipette with a wide opening, and removing the upper layer of liquid after the microspheres are precipitated by a natural sedimentation method. Following the same procedure, wash 2 times with PBS, add 4% paraformaldehyde and fix overnight at 4 degrees.
(3) After washing with PBS 2 times, dehydration was performed by gradient with 10%, 20%, 30% sucrose solution for 2h each step.
(4) The skin microspheres were then transferred to a tissue embedding cassette and excess water was removed by capillary suction using filter paper, as far as possible without touching the cells. Adding OCT embedding medium into the embedding box to immerse the skin microspheres, and quickly freezing in a refrigerator at-80 deg.C to obtain blocks. The frozen solid sample embedded block is placed into a Leica constant temperature freezing microtome for frozen sectioning, and the section thickness is 8-10 microns. The case temperature of the freezing microtome is set to-20 ℃, the head temperature is set to-18 ℃, and the cut sample is stored at-20 ℃ for later use.
(5) The frozen sections were removed from the freezer at-20 deg.C, left at room temperature for 15min, and washed 2 times with PBS for 10min each time. After wiping the slide dry, an immunohistochemical pen was used to draw a circle along the edge of the sample, and the sample was permeabilized with 0.25% TritonX-100 for 30 min. TritonX-100 was aspirated, washed 2 times with PBS, and then blocked for 30min at room temperature by adding PBS containing 3% Bovine Serum Albumin (BSA) or 10% goat serum. After adding primary antibody and incubating overnight at 4 ℃, the primary antibody is aspirated, washed 2 times with PBS, added with appropriate dilution of secondary antibody with fluorescent label, and incubated for one hour at room temperature. Absorbing the secondary antibody, washing with PBS for 2 times, adding DAPI dye solution, dyeing at room temperature for 20min, washing with PBS for 2 times, sealing with a sealing agent, drying, and taking pictures by observation of an inverted fluorescence microscope or a confocal microscope. Wherein the primary antibody is a mouse anti-E cadherin antibody and a rabbit anti-Ki 67 antibody, and the secondary antibody is a sheep anti-mouse 647 antibody and a sheep anti-rabbit 555 antibody.
The results are shown in fig. 2, from which it can be seen that the skin microspheres in the experimental group showed more cells expressing Ki67 than the control group 48 hours after the treatment of the skin microspheres with fibroblast growth factor bFGF, i.e. the number of proliferating cells was significantly increased.
Example 3
Referring to a of fig. 3, luciferase reporter plasmids 1 and 2 were constructed, wherein reporter plasmid 1 has a TCF/LEF reporter gene thereon, the TCF/LEF reporter gene including a TCF/LEF binding sequence and a corresponding luciferase Eluc sequence; and an SRE reporter gene comprising an SRE binding sequence and a luciferase Fluc sequence on reporter plasmid 2. In addition, the reporter plasmids 1 and 2 further have a green Renilla luciferase reporter gene (GrRenilla) and a Renilla luciferase reporter gene (Renilla), respectively, as internal references. The report plasmid 1 and the report plasmid 2 are packaged by lentivirus and then respectively infect epidermal cells and dermal cells, and high-expression cell strains are separated by flow type. Then, a skin microsphere with a dual luciferase reporter system was constructed according to the preparation method in example 1. The constructed skin microspheres are shown in fig. 3B, wherein the epidermis layer comprises epidermal cells with Wnt signaling pathway activity reporter system, and the dermis microsphere comprises dermal cells with MAPK/ERK signaling pathway activity reporter system.
After the skin microspheres are treated by the medicine, the specific action of the medicine can be determined by detecting the activity of luciferase. The specific detection process is as follows:
adding a substrate D-luciferin for reaction for 30s, measuring a fluorescence spectrum in a corresponding wavelength interval by using a filter disc (2s) in the corresponding wavelength interval to obtain the activity intensity of luciferase FLuc and ELuc, adding a quencher and a substrate coelenterazine for reaction for 7s, measuring the activity intensity of luciferase GrRenilla and Renilla by using a filter disc (1s), and finally calculating the activation degree of a classical Wnt signal path and a MAPK/ERK signal path. The method comprises the following steps of detecting the active ingredients of a medicine capable of promoting the regeneration of skin and hair follicle by observing the activation of the medicine on the two cell signal paths, and then detecting or screening a to-be-detected product.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments described above, and various changes can be made within the knowledge of those skilled in the art without departing from the gist of the present application. Furthermore, the embodiments and features of the embodiments of the present application may be combined with each other without conflict.
Claims (10)
1. A skin microsphere, comprising:
a dermal cell microsphere comprising a dermal cell having a first signaling pathway activity reporter system;
an epidermal layer coated on the surface of the dermal cell microsphere, the epidermal layer comprising epidermal cells having a second signaling pathway activity reporter system.
2. The skin microsphere of claim 1, wherein the first signaling pathway activity reporter system comprises at least one of an Shh signaling pathway activity reporter system, a MAPK/ERK signaling pathway activity reporter system.
3. The skin microsphere of claim 2, wherein the dermal cells are transfected with an SRE reporter gene.
4. The skin microsphere of claim 1, wherein the second signaling pathway activity reporter system comprises at least one of a canonical Wnt signaling pathway activity reporter system, an Akt signaling pathway activity reporter system.
5. The skin microsphere of claim 4, wherein the epidermal cells are transfected with a TCF/LEF1 reporter gene.
6. Skin microspheres according to any one of claims 1 to 5, wherein the skin microspheres have a diameter of 0.02-10 mm.
7. A method of preparing skin microspheres according to any one of claims 1 to 6, comprising the steps of:
uniformly mixing the dermal cells and the hydrogel to obtain a dermal cell mixed solution;
dripping the dermal cell mixed solution into an oil phase, and incubating to form dermal cell microspheres;
and mixing the dermal cell microspheres and epidermal cells, and performing 3D culture to obtain the skin microspheres.
8. The method according to claim 7, wherein the dermal cell microspheres are resuspended and cultured in a culture medium for 12-36 hours before being mixed with the epidermal cells.
9. Use of skin microspheres according to any one of claims 1 to 6 for the detection of drugs and/or cosmetics.
10. The detection method of the to-be-detected product is characterized by comprising the following steps of: contacting the skin microspheres of any one of claims 1 to 6 with an article to be tested and detecting a change in said skin microspheres before and after said contacting.
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