CN113201481B - Skin microsphere and preparation method and application thereof - Google Patents
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Abstract
The application discloses a skin microsphere 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 dermal cells having a first signaling pathway activity reporter system; and the epidermis layer is coated on the surface of the dermis cell microsphere, the epidermis layer comprises epidermis cells, and the epidermis cells are provided with a second signal path activity reporting system. The skin microsphere provided by the embodiment of the application has at least the following beneficial effects: the skin microsphere adopts a composite double-layer structure formed by wrapping inner core and outer layer epidermis cells formed by dermis cells, so that the skin structure of a human body is better simulated. Meanwhile, signal channel activity reporting systems are respectively carried on the inner core and the outer shell, so that the signal channel activity reporting systems 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 a skin microsphere and a preparation method and application thereof.
Background
The skin is the largest organ of human body, mainly comprises epidermis, dermis, subcutaneous tissue and skin attachment, has the functions of physical barrier, sensing the outside, maintaining the body temperature and moisture, etc. Skin health is closely related to the state of cellular and matrix components in the epidermis and dermis. If one of the components is dysfunctional, it will lead to skin diseases, for example overactivated fibroblasts will lead to scar formation. At present, skin medicines and skin care products in the market can often act on different components in skin to achieve the purposes of treating diseases and improving skin conditions. The discovery of the effective components is greatly dependent on an in vitro model and a screening technology, and the in vitro model has a plurality of advantages of low cost, low ethical risk and the like compared with the human body and animal test. Therefore, a reliable and advanced skin in vitro model is of great significance for skin drug screening.
In vitro models used prior to drug screening platforms are often single cell lines, such as immortalized epidermal cells HaCaT, whose effectiveness is judged by detecting the effect of a compound on the proliferation, apoptosis, etc. cell behavior of such cell lines. However, because the cellular components are single and are far from the normal skin structure and function, the components really effective for skin health are difficult to find, and the requirements of high-flux drug screening are not met. Currently, attempts on tissue engineering skin of various cells have been used for cytotoxicity detection of cosmetics and partial replacement of animal experiments. Such tissue engineering skin products using various cells are composed of keratinocytes at the upper layer, which are keratinized to be used for testing the barrier function of the epidermis, and a simulated skin structure at the lower layer. However, it is difficult to confirm the actual effect of the drug in the epidermis and dermis to achieve the corresponding screening.
Disclosure of Invention
The present application aims to solve at least one of the technical problems existing in the prior art. Therefore, the application provides a skin microsphere capable of realizing detection of the effect of a drug in an epidermis or dermis layer, 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 dermal cells having a first signaling pathway activity reporter system;
and the epidermis layer is coated on the surface of the dermis cell microsphere, the epidermis layer comprises epidermis cells, and the epidermis cells are provided with a second signal path activity reporting system.
The skin microsphere provided by the embodiment of the application has at least the following beneficial effects:
the skin microsphere adopts a composite double-layer structure formed by wrapping inner core and outer layer epidermis cells formed by dermis cells, so that the skin structure of a human body is better simulated. Meanwhile, signal channel activity reporting systems are respectively carried on the inner core and the outer shell, so that the signal channel activity reporting systems 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 path activity reporting system refers to a system capable of detecting the activity of a specific signal path and making a qualitative or quantitative response according to the detection result, and a non-limiting manner of implementation of the signal path activity reporting system includes a reporter gene capable of detecting the specific signal path. The first signaling pathway activity reporter system and the second signaling pathway activity reporter system are used only to distinguish that the signaling 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 to specifically limit the kind and number of the signaling pathway activity reporter systems.
In some embodiments of the application, the first signaling pathway activity reporting system comprises at least one of a Shh signaling pathway activity reporting system, a MAPK/ERK signaling pathway activity reporting system. Shh plays an important role in regulating the development of various tissues and organs involved in cell structure, proliferation and survival, and in particular, can promote proliferation of papilla cells leading to hair follicle regeneration and hair growth, and signal pathways having similar regulatory effects thereto also include MAPK/ERK and the like. Therefore, the interaction between the drug and the cell can be detected by the signal channel reporting system carried by the dermal cell, so as to judge whether the drug can activate the cell channel in the dermal cell after penetrating the epidermis barrier, and further judge the activity of the signal channel after being activated.
In some embodiments of the application, the dermal cells are transfected with an SRE reporter gene. The MAPK/ERK signaling pathway is a major participant in cell growth and differentiation regulation, and during activation, transcription factor substrates form complexes with Serum Response Factors (SRF) and bind with Serum Response Elements (SRE) to realize gene expression regulation. Thus, selection of SRE as a reporter gene in a signaling pathway activity reporter system is effective in detecting MAPK/ERK signaling pathway activity.
In some embodiments of the application, the second signaling pathway activity reporting system comprises at least one of a canonical Wnt signaling pathway activity reporting system, an Akt signaling pathway activity reporting system. The Wnt signaling pathway is a complex regulatory network that includes three branches of canonical Wnt signaling pathway. The canonical Wnt signaling pathway is primarily involved in regulating multipotent differentiation of stem cells, development and regeneration of organs, and in particular in promoting cell proliferation and regeneration. And the Akt signal pathway has similar regulation and control effects with the classical Wnt signal pathway. Therefore, the interaction between the drug and the cell can be detected by carrying the signal channel activity reporting systems on the epidermal cells, and the effect of the interaction on cell proliferation and regeneration can be judged.
In some embodiments of the application, the 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 signaling, and participates in feedback regulation of Wnt signaling pathway, and a stable beta-catenin complex combined with the transcription factor will cause activation of canonical Wnt signaling pathway. Therefore, TCF/LEF1 is selected as a reporter gene in a signal path activity reporting system, so that the activity of a classical Wnt signal path can be detected more sensitively.
Thus, by setting the specific signaling pathway activity reporting system described above, the epidermal cell reporting system includes key pathways that promote cell proliferation and regeneration, such as canonical Wnt signaling pathway, akt pathway, and the like. The dermal cell reporting system selects signal pathways promoting hair follicle papilla formation and hair follicle regeneration, such as Shh signal pathway, MAPK/ERK signal pathway, etc., so that it can be used to observe whether or not the test article can activate the cell signal pathway and how much to activate it to find the effective components of the drugs promoting skin and hair follicle regeneration.
In some embodiments of the application, the skin microspheres have a diameter of 0.02 to 10mm. The actual size of the skin microspheres can be determined according to research requirements, but the diameter of the microspheres is preferably 0.02-10 mm, preferably within 1 mm, in consideration of the requirements of freezing and resuscitating the product.
In some embodiments of the application, the epidermal cells are skin or mucosal derived keratinocytes, which may be primary cells, or keratinocytes after culture expansion, or immortalized keratinocytes such as HaCaT. The keratinocytes in the epidermis layer may further be a monolayer or a plurality of layers according to the application requirements of the skin microsphere, for example, when detecting the effect of the drug on the proliferation of keratinocytes, the skin microsphere may employ the epidermis layer containing a monolayer of keratinocytes; when it is desired to examine the barrier function of the epidermis and the transdermal ability of the drug, keratinocytes can be further differentiated and matured to form the stratum corneum. In addition, other cells or substances besides keratinocytes can be doped into the epidermis layer according to different application requirements, for example, a certain proportion of melanocytes can be doped to test the influence of drugs on the melanocytes in the epidermis layer of the skin; the effect of drugs on skin defense can also be studied by infiltration of a proportion of immune cells (e.g., langerhans cells).
In some embodiments of the application, the dermal cells are fibroblasts isolated from the dermis of the skin, which may be primary cells, or fibroblasts after culture expansion, or immortalized fibroblasts; in addition, the fibroblast may be an embryonic stem cell or an iPS cell, which is formed by induced differentiation. The dermal cell microspheres containing fibroblasts can be used for detection and screening of drugs or cosmetics for dermal cells, in addition to functional support of the epidermal layer coated thereon. According to different application requirements of the skin microsphere, besides the fibroblast, a certain proportion of endothelial cells can be further added into the dermal cell microsphere to detect the angiopoiesis medicine, or a certain proportion of immune cells can be added into the dermal cell microsphere to carry out immune related detection or research.
In some embodiments of the application, the dermal cell microspheres may contain, in addition to dermal cells, an extracellular matrix macromolecule, such as collagen, by way of non-limiting example, and may further contain other extracellular matrix molecules, such as glycosaminoglycans, which may be hyaluronic acid. Alternatively, these extracellular matrix molecules may be replaced in part or in whole by synthetic materials.
In a second aspect of the present application, there is provided a method for preparing the skin microsphere described above, the method comprising the steps of:
mixing dermal cells with hydrogel uniformly to obtain dermal cell mixed solution;
dropwise adding the dermal cell mixture into an oil phase, and incubating to form dermal cell microspheres;
mixing dermal cell microspheres with epidermal cells, and performing 3D culture to obtain the skin microspheres.
The preparation method of the skin microsphere provided by the embodiment of the application has at least the following beneficial effects:
by adopting the method, the microspheres of the dermal cells are formed in the oil phase firstly, and then the microspheres are mixed with the epidermal cells for 3D culture, compared with the existing product, the epidermal shell formed on the outer side can form tight connection with the pore wall, so that the microspheres can be more completely covered on the surface of the dermal cell microspheres, and when the dermal microspheres are adopted for detecting and screening medicines, the medicines cannot directly enter the internal dermal cell microspheres from the pore wall, so that the barrier effect of the epidermal shell and the transdermal capability of the medicines can be reflected more. In addition, the morphology of the finally obtained skin microsphere also makes the skin microsphere easier to sample and analyze, and is more convenient to store and transport.
In some embodiments of the application, the dermal cell microspheres are resuspended in a medium and cultured for 12-36 hours prior to mixing with the epidermal cells, during which time the dermal cell microspheres are slightly reduced in size as they are spread, thereby achieving a more stable structure that facilitates subsequent adherence of the epidermal cells to their surfaces.
In a third aspect of the application, there is provided the use of a skin microsphere as described above for detecting a drug and/or cosmetic. The skin microsphere carries corresponding signal channel activity reporting systems on the inner core and the outer shell respectively, and can timely respond to the action effect of the medicine and/or the cosmetic to be detected, so that the skin microsphere can be used as a better detection model to realize the detection of the action of the medicine and/or the cosmetic on the epidermis layer and the dermis layer.
In a fourth aspect of the present application, there is provided a method for detecting an article to be detected, the method comprising the steps of:
and (3) contacting the skin microsphere with a to-be-detected product, and detecting the change of the skin microsphere before and after contact.
The skin microsphere used in the detection method carries corresponding signal channel activity reporting systems on the inner core and the outer shell respectively, and when the to-be-detected product is contacted with the skin microsphere, the skin microsphere can be used as a more accurate and visual detection model to reflect the effects of medicines and/or cosmetics on the epidermis layer and the dermis layer.
Additional aspects and advantages of the 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 application.
Drawings
FIG. 1 is a photograph of skin microspheres prepared in example 1 of the present application.
FIG. 2 shows the results of the reaction of skin microspheres to drugs in example 2 of the present application.
FIG. 3 is a schematic diagram of a plasmid carrying a signaling pathway activity reporter system and a photograph of a skin microsphere carrying a signaling pathway activity reporter system in example 3 of this application.
Detailed Description
The conception and the technical effects produced by the present application will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present application. It is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present application based on the embodiments of the present application.
The following detailed description of embodiments of the application is exemplary and is provided merely to illustrate the application and is not to be construed as limiting the application.
In the description of the present application, the meaning of a number is one or more, the meaning of a number is two or more, and greater than, less than, exceeding, etc. are understood to exclude the present number, and the meaning of a number is understood to include the present number. The description of the first and second is for the purpose of distinguishing between technical features only and should not be construed 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, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean 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, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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.
The experimental materials used in the preparation method are as follows:
rat tail type I collagen (Corning corporation, usa, product number 354236); f3100 oil phase (american 3M company); cisplatin (mikrin); DMEM (Corning corporation, usa); FBS (American BI Co.).
The preparation method comprises the following specific preparation steps:
(1) Taking rat tail type I collagen to prepare hydrogel with the concentration of 2mg/ml, uniformly mixing, and then placing the hydrogel into an ice box without regulating the pH.
(2) Human skin fibroblasts were digested, counted, and a certain amount of fibroblast suspension was taken and centrifuged.
(3) Adjusting the pH of the hydrogel to 7.0 with 0.1M NaOH, fully mixing, removing supernatant from the fibroblast suspension, mixing with the hydrogel with the pH of 7.0 to obtain a fibroblast mixed solution, evenly distributing the fibroblast mixed solution into eight connecting tubes, and placing the eight connecting tubes into an ice box.
(4) The F3100 oil phase was filter sterilized and added to 200ul of Teflon-96 well plates, respectively. Then, 4. Mu.l of the fibroblast mixture was sucked up in an eight-joint tube by a 10. Mu.l range of a row gun, dropped into the oil phase of a Teflon-96 well plate, then the Teflon-96 well plate was covered with Teflon-lad, and placed in an incubator for 15-20min to form dermal cell microspheres.
(5) After the dermal cell microspheres were formed, the well plate was removed from the incubator, 200. Mu.l of DMEM was added to each well, allowed to stand for 5min, and after the Teflon-lad was capped, the microspheres were collected by inversion, the upper liquid in Teflon-lad was poured into a 50ml beaker, the oil phase was prevented from being poured, washed 3 times with PBS, all the microspheres were resuspended in complete medium (DMEM+10% FBS), and all the liquid was transferred to a normal petri dish (non-treated) and placed into the incubator for cultivation for 24 hours.
(6) Pouring the dermal Cell microspheres and the culture medium into a Cell Spinner rotating bottle for 3D culture, digesting the immortalized epidermal cells HaCaT at the same time, taking a certain amount of epidermal Cell suspension, adding the epidermal Cell suspension into the rotating bottle which is running, fully mixing the dermal Cell microspheres and the epidermal cells, and co-culturing for 7 days to form the complete skin microspheres.
The photograph of the skin microsphere is shown in FIG. 1, A is a picture of the skin microsphere at a scale of 1000 μm, and B is a picture after bright field, different color channels (GFP and mCherry) and merger at a scale of 200 μm. As can be seen from fig. 1, the skin microsphere after 7 days of co-culture can be completely formed, has a regular shape and uniform size, has a double-layer skin structure, comprises dermal cell microspheres (mCherry marks) composed of fibroblasts and extracellular matrix (collagen), and has a composite structure formed by epidermal layers (green fluorescent protein GFP marks) wrapped by epidermal cells on the outer layer of the dermal cell microspheres, and the structure is quite similar to the skin structure of a human body, and can be well used as a detection model to simulate the skin of the human body.
Example 2
Reaction experiment of skin microsphere to drug
The skin microspheres prepared in example 1 were treated with drugs to test the proliferation and apoptosis changes of the skin microspheres before and after drug administration.
Experimental materials: bFGF (Sigma usa); mouse anti-E cadherin antibody (thermosusher, product No. 13-1700); rabbit anti-Ki 67 antibody (CST, product number 9662); goat anti-rabbit 555 antibody (CST, product number 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 placed in a 24-well plate without adherence for culture, 2ml of medium (containing 2% serum) was added to each well, then the drug was added, and the culture was continued in a 37-degree incubator for 48 hours.
(2) The skin microspheres are collected into a centrifuge tube by a thick mouth pipette, and after the microspheres are precipitated by a natural sedimentation method, the upper liquid is removed. After washing 2 times with PBS, 4% paraformaldehyde was added and the mixture was fixed at 4℃overnight according to the same method.
(3) Then, after washing with PBS for 2 times, the mixture was dehydrated with 10%, 20% and 30% sucrose solutions in sequence, and each step was dehydrated for 2 hours.
(4) The skin microspheres were then transferred to a tissue embedding cassette and excess water was removed by capillary siphoning with filter paper to minimize contact with the cells. Adding OCT embedding agent into the embedding box to immerse the skin microsphere, and rapidly freezing into blocks at-80deg.C. And (3) putting the frozen sample embedded blocks into a constant temperature frozen microtome of Leica for frozen section, wherein the section thickness is 8-10 microns. The temperature of the case of the frozen microtome is set to be minus 20 ℃, the temperature of the machine head is set to be minus 18 ℃, and the cut sample is put at minus 20 ℃ for standby.
(5) The frozen sections were taken out of the freezer at-20℃and left at room temperature for 15min, washed with PBS for 2 times, 10min each. After the slide was wiped dry, the samples were treated with an immunohistochemical pen for 30min with 0.25% Triton X-100 permeabilization along the edges of the samples. After absorbing Triton X-100 and washing 2 times with PBS, the mixture was blocked with PBS solution containing 3% Bovine Serum Albumin (BSA) or 10% goat serum at room temperature for 30min. After adding the primary antibody and incubating overnight at 4 ℃, the primary antibody was blotted off, washed 2 times with PBS, and the secondary antibody with fluorescent label added at the appropriate dilution and incubated for one hour at room temperature. Sucking the secondary antibody, washing for 2 times by using PBS, adding DAPI dye solution, dyeing for 20 minutes at room temperature, washing for 2 times by using PBS, sealing the tablet by using a sealing tablet, airing, and observing and photographing by using 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.
As a result, as shown in fig. 2, it can be seen from the graph that the skin microspheres in the experimental group showed more cells expressing Ki67 than the control group, i.e., the number of proliferated cells was significantly increased, after 48 hours of treatment of the skin microspheres with the fibroblast growth factor bFGF.
Example 3
Referring to FIG. 3A, luciferase reporter plasmids 1 and 2 are constructed, wherein reporter plasmid 1 has thereon a TCF/LEF reporter gene comprising a TCF/LEF binding sequence and a corresponding luciferase Eluc sequence; and on reporter plasmid 2 there is an SRE reporter gene comprising an SRE binding sequence and a luciferase Fluc sequence. In addition, the reporter plasmids 1 and 2 were further provided with a green Renilla luciferase reporter gene (GrRenilla) and a Renilla luciferase reporter gene (Renilla), respectively, as internal controls. The reporter plasmid 1 and the reporter plasmid 2 are packaged by slow viruses and then are respectively infected with epidermis cells and dermis cells, and high-expression cell strains are separated through flow. Then, a skin microsphere having a dual luciferase reporter system was constructed according to the preparation method of example 1. The skin microsphere obtained by construction is shown as B in FIG. 3, the epidermis layer comprises epidermis cells with a Wnt signal channel activity reporting system, and the dermis microsphere comprises dermis cells with a MAPK/ERK signal channel activity reporting system.
After the skin microsphere is treated by the drug, the specific effect of the drug can be determined by detecting the luciferase activity. The specific detection process is as follows:
adding substrate D-luciferin for reaction 30s, measuring fluorescence spectrum in a corresponding wavelength region by using a filter disc (2 s) in the corresponding wavelength region to obtain the activity intensity of luciferase FLuc and ELuc, adding a quencher and substrate coelenterazine for reaction 7s, measuring the activity intensity of luciferase GrRenilla and Renilla by using a filter disc (1 s), and finally calculating the activation degree of a classical Wnt signal path and a MAPK/ERK signal path. The classical Wnt signal pathway is a key pathway related to promoting cell proliferation and regeneration, the MAPK/ERK signal pathway is a signal pathway related to promoting hair follicle papilla formation and hair follicle regeneration, and the activation of the two cell signal pathways by observing the drug can be used for discovering the active ingredients of the drug capable of promoting skin and hair follicle regeneration, so that the detection or screening purpose of the to-be-detected product is realized.
The present application has been described in detail with reference to the embodiments, but the present application is not limited to the embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. Furthermore, embodiments of the application and features of the embodiments may be combined with each other without conflict.
Claims (5)
1. The preparation method of the skin microsphere is characterized by comprising the following steps:
mixing dermal cells with hydrogel uniformly to obtain dermal cell mixed solution, wherein the hydrogel is prepared from type I collagen;
dropwise adding the dermal cell mixture into an F3100 oil phase, and incubating for 15-20min to form dermal cell microspheres;
mixing the dermal cell microspheres with epidermal cells, and culturing in a rotary bottle for 7 days in a 3D mode to obtain the skin microspheres;
the dermal cells are fibroblasts separated from dermis of skin, the epidermal cells are immortalized keratinocytes HaCaT derived from skin or mucosa, the dermal cell microspheres are resuspended and cultured for 12-36 hours by adopting a culture medium before being mixed with the epidermal cells, and the diameter of the dermal microspheres is 0.02-10 mm.
2. The method of claim 1, wherein the dermal cells have a first signaling pathway activity reporter system, the first signaling pathway activity reporter system being a MAPK/ERK signaling pathway activity reporter system; the epidermal cells have a second signaling pathway activity reporting system that is a canonical Wnt signaling pathway activity reporting system.
3. The method of claim 2, wherein the dermal cells are transfected with an SRE reporter gene.
4. The method of claim 2, wherein the epidermal cells are transfected with a TCF/LEF1 reporter gene.
5. Use of the skin microsphere prepared by the preparation method of any one of claims 1 to 4 for detecting bFGF, for diagnosis or treatment of a non-disease.
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