WO2016086527A1

WO2016086527A1 - Sebaceous gland-containing skin tissue, formation method and usage thereof

Info

Publication number: WO2016086527A1
Application number: PCT/CN2015/071777
Authority: WO
Inventors: 吴耀炯; 王潇潇; 王旭升
Original assignee: 清华大学深圳研究生院
Priority date: 2014-12-02
Filing date: 2015-01-28
Publication date: 2016-06-09
Also published as: CN104491931B; CN104491931A

Abstract

Disclosed are a sebaceous gland-containing skin tissue, formation method and usage thereof, the method for forming sebaceous gland comprising: co-culturing hypodermal cells and epidermal stem cells in a gel.

Description

Skin tissue containing sebaceous glands and method and use thereof

Priority information

Priority is claimed on Japanese Patent Application No. 201410723513.5, filed on Dec. 2, 2014, the entire entire entire entire entire entire entire entire content

Technical field

The present invention relates to the field of cell biology and regenerative medicine, and in particular to a method of forming a sebaceous gland and its use, and more particularly to a method of forming a sebaceous gland, a skin tissue containing a sebaceous gland, and a method of forming a sebaceous gland for use in skin regeneration.

Background technique

As the largest tissue of the human body, the skin is a barrier to contact with the external environment. When external skin damage or diseases cause skin defects, skin damage and infection are often caused. China has a large population, with 15 million burns and ulcers per year, of which 3.5 million are required for skin grafts and more than 400 million square centimeters. Therefore, finding an ideal skin substitute has always been a clinically urgent problem. With the rapid development of cell biology, materials science, biochemistry, bioengineering, and transplantation, people have a deeper understanding of the biological characteristics of epidermal keratinocytes, the processing and synthesis of artificial materials, and so on. It is possible to build tissue engineering skin.

The sebaceous gland is the most important accessory organ for maintaining the stability of skin structure. Sebaceous glands and sweat and exfoliated epithelial cells form a layer of sebum sweat latex film on the surface of the skin. Its thickness is 7-10μm, which is a three-dimensional lipid structure on the skin surface. Promotes the integrity of the skin barrier. In the absence of this three-dimensional cortical structure, rough skin and dry hair will appear. Since some skins of the palms, ankles, fingers, and toes do not have sebaceous glands, skin dryness often occurs. Although the tissue engineering skin has a dermis and epidermis similar to normal skin, it lacks sebaceous glands and cannot provide nutrient and moisturizing, antibacterial and anti-oxidant protection and moisture retention. After skin transplantation, skin shrinkage and deformation are serious and appearance is easy. Problems such as chapped and susceptible to infection, while the performance of tissue-engineered skin is significantly lower than that of natural normal skin.

However, existing tissue engineered skin remains to be improved.

Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a method of forming a sebaceous gland.

It should be noted that the present invention has been completed based on the following work of the inventors:

The inventors have found that epidermal stem cells derived from the epidermis of the skin and dermal cells derived from the dermis of the skin have a large potential for expansion, and after isolation and culture in vitro, epidermal stem cells are characterized by high expression of integrin CD29, CD49f and K15 ( Keratin 15), and the characteristic marker molecule expression of two stem cells in subsequent passages stable. The inventors amplified the above two cells in different culture systems, and then transplanted the two cells to the skin of nude mice in proportion, and histological analysis revealed a newly formed sebaceous gland structure.

Thus, in accordance with one aspect of the invention, the invention provides a method of forming a sebaceous gland. According to an embodiment of the invention, the method comprises co-culturing dermal cells and epidermal stem cells in a gel. The inventors have surprisingly found that the method can form skin tissue with sebaceous glands. According to an embodiment of the present invention, the present application forms a method of skin tissue having sebaceous glands, which is simple, convenient, quick, easy to control, and easy to achieve large-scale production.

Further, the method of sebaceous gland according to the above embodiment of the present invention may further have the following additional technical features:

According to an embodiment of the invention, the co-cultivation is carried out subcutaneously in the animal. Thereby, the regeneration of the skin tissue of the sebaceous glands is promoted, and the structure of the regenerated skin tissue is similar to that of normal skin.

According to an embodiment of the invention, the dermal cells comprise at least one of dermal stem cells and fibroblasts.

According to an embodiment of the invention, the dermal cells are fibroblasts, and the gel contains insulin, dexamethasone, glitazone and XAV939 (3,5,7,8-tetrahydro-2-[ 4-(Trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one). Thus, the chemical agent induces differentiation of epidermal stem cells into free sebaceous glands, thereby forming a sebaceous gland structure similar to normal skin.

The final concentration of the insulin is 50-150 μg/mL, the final concentration of the dexamethasone is 10 ^-4 -10 ^-6 M, and the final concentration of the glitazone is 150-250 μM, the end of the XAV939 The concentration is 5-15 μM. Thus, the chemical agent induces the differentiation of epidermal stem cells and fibroblasts into free sebaceous glands.

According to an embodiment of the present invention, the final concentration of the insulin is 100 μg / mL, the final concentration of the dexamethasone is 10 ^-5 M, the final concentration of the glitazone is 200 μM, and the final concentration of the XAV939 is 10 μM. Thus, the chemical agent induces the differentiation of epidermal stem cells and fibroblasts into free sebaceous glands.

In addition, it should be noted that the term "final concentration" as used in the present application refers to the concentration of the chemical agent in the gel after adding various chemical agents to the gel containing the mixed cells.

According to an embodiment of the invention, the ratio of the dermal cells to the epidermal stem cells is 10:1 to 1:10.

According to an embodiment of the present invention, the number of mixed cells is not less than 10 ⁴ per 3 mm ^{2 of} skin. Thus, the number of mixed cells satisfies the need for regeneration of sebaceous gland skin tissue.

According to an embodiment of the invention, the gel is at least one selected from the group consisting of Matrigel, collagen or hydrogel.

According to an embodiment of the invention, the dermal cells and the epidermal stem cells are all derived from autologous, allogeneic or xenogeneic cells. It should be noted that the xenogenic-derived cells are used for the regeneration of sebaceous glands in immunodeficient animals. Thereby, the regenerated sebaceous gland skin tissue has a small rejection effect.

According to an embodiment of the present invention, the dermal cells and the epidermal stem cells are independently cultured in a liquid medium for 0 to 30 days, wherein the dermal stem cells are suspension culture, and the cell culture medium is DMEM/F12. (3:1), 2% B27, 40 ng/μL bFGF and 20 ng/μL EGF; epidermal stem cells are adherent culture, and the cell culture medium is CnT-07 medium; wherein the dermal stem cells further contain liquid medium a cell active factor and an extracellular matrix molecule, wherein the cell active factor is at least one selected from the group consisting of bFGF and EGF, and the extracellular matrix molecule is at least one selected from the group consisting of hyaluronic acid and fibronectin. Thus, dermal cells and epidermal stem cells have high activity and small cell variation.

According to an embodiment of the invention, the method further comprises: identifying the oil secretion of the surface of the skin tissue containing the regenerated sebaceous glands using in situ mass spectrometry. Thereby, the secretion of oil on the surface of the skin tissue containing the regenerated sebaceous glands can be more accurately identified.

According to another aspect of the present invention, the present invention also provides a skin tissue containing a sebaceous gland. According to an embodiment of the invention, the sebaceous glands are formed by the method described in the foregoing embodiments of the invention. The inventors have surprisingly found that the skin tissue has a structure of sebaceous glands similar to normal skin tissue.

According to a further aspect of the invention, the invention also provides for the use of the aforementioned method in skin regeneration. According to an embodiment of the invention, the method is for regenerating a sebaceous gland or a skin tissue containing a sebaceous gland. The inventors have surprisingly found that by applying this method in skin regeneration, sebaceous glands similar to normal skin can be obtained.

The additional aspects and advantages of the invention will be set forth in part in the description which follows.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 shows a photograph of the expression result of the cell surface marker molecule CD49f of epidermal stem cells according to an embodiment of the present invention;

2 shows a photograph of the expression result of the cell surface marker Nestin of dermal stem cells according to an embodiment of the present invention;

Figure 3 shows a photograph of confocal microscopy of normal skin tissue sections in accordance with one embodiment of the present invention;

4 is a photograph showing confocal microscopic observation of a skin tissue section formed by co-transplantation of epidermal stem cells and dermal stem cells according to an embodiment of the present invention;

Figure 5 is a photograph showing confocal microscopic observation of skin tissue sections formed by co-transplantation of epidermal stem cells and fibroblasts after induction of co-transplantation with chemical agents according to an embodiment of the present invention;

Figure 6 is a graph showing the results of sebaceous gland secretion detected by MALDI mass spectrometry in skin tissue formed after transplantation according to one embodiment of the present invention.

Detailed description of the invention

Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are intended to be illustrative of the invention and are not to be construed as limiting.

Where specific techniques or conditions are not indicated in the examples of the present application, they are carried out according to the techniques or conditions described in the literature in the field or in accordance with the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are conventional products that can be obtained commercially.

The embodiments of the present invention are described in detail below. Unless otherwise specified, ex vivo skin stem cells are isolated and cultured from the skin of human C57 mice or human scalp tissues in the following examples; the dermal stem cell culture medium is composed of DMEM/F12 (3: 1) (purchased from Invitrogen) containing 2% B27 (purchased from Invitrogen), 40 ng/μL bFGF (Peprotech) and 20 ng/μL EGF (Peprotech); fibroblast culture medium is DMEM containing 10% fetal bovine serum Accutase was purchased from sigma; 3M membrane was Tegaderm ^TM Film; suspension cell culture dish was purchased from Guangzhou Jiete Biofiltration Products Co., Ltd., and the product number was MCD-000-090. Adherent cell culture dish, purchased from Corning. Matrigel was purchased from BD. The experimental animals were purchased from the Guangdong Medical Laboratory Animal Center.

Example 1

The process of isolation, culture and identification of epidermal stem cells is as follows:

(1) The whole skin of the fetus was taken and added with a mass concentration of 0.25% dispase II enzyme at 4 ° C overnight.

(2) The skin epidermis of the fetal rat was peeled off, the epidermis was cut, and the mass concentration of 0.25% collagenase was added, and the reaction was terminated after digestion at 37 ° C for 30 min to obtain primary cells of the differentiated epidermal stem cells.

(3) The obtained primary cells were passed through an 80-mesh sieve, and then centrifuged at 300 × g for 10 min, and the supernatant was discarded, and the cells were suspended in a medium of CnT-07 (manufactured by CELLnTEC Co., Ltd.).

(4) The primary cells suspended in CnT-07 were inoculated on a 10 cm cell culture plate plated with mouse type I collagen. After incubation for 60 min, the cells were washed twice with 0.01 mol/L HBSS, and then cultured with CnT-07.

(5) When the primary cells are fused to 60% to 80%, they are digested with Accutase and then passaged at a ratio of 1:2.

(6) Identification of epidermal stem cells: The identification of cell surface markers is as follows: Take the first, third, and fifth representative skin cells for cell climbing, and perform immunofluorescence staining of integrin CD29, CD49f and K15. The coloration of the cells. It was observed that the integrin CD29, CD49f and K15 of epidermal stem cells were highly expressed, and the specific results are shown in Fig. 1.

Example 2

The process of isolation, culture and identification of dermal stem cells is as follows:

(1) The whole skin of the fetus was taken, and after the skin was cut, a mass concentration of 0.25% dispase was added at 4 ° C overnight.

(2) The epidermis of the fetal rat was peeled off, the dermis was separated, and the dermis was cut, and the concentration was 0.25% trypsin, and the reaction was terminated after digestion at 37 ° C for 20-40 min to obtain primary cells of the digested dermal stem cells.

(3) The primary cells of the dermal stem cells were passed through a 200 mesh sieve, and then centrifuged at 300 × g for 10 min, and the supernatant was discarded, and the primary cells were suspended in the dermal stem cell culture medium.

(4) The dermal stem cell culture medium suspension primary cells were seeded in an non-adherent culture dish.

(5) After more cell spheres appeared in the non-adherent culture dish, the dermal stem cells were passaged at a ratio of 1:2.

(6) Identification of dermal stem cells: the identification of cell surface markers, the specific methods are as follows: Take the first, third and fifth generation dermal stem cells as cell bracts, immunofluorescence staining of Nestin and Fibronectin of dermal stem cells, observe the cells Coloring situation. Immunofluorescence analysis revealed that Nestin and Fibronectin were highly expressed in dermal stem cells, and the results of Nestin positive were shown in Fig. 2.

Example 3

The use of dermal stem cells to induce epidermal stem cells to form sebaceous gland structures, the specific process is as follows:

(1) The epidermal stem cells obtained in Example 1 were cultured, and the third representative skin stem cells were digested with Accutase, and a suspension of epidermal stem cells having a total cell number of 1 million was prepared in PBS.

(2) The dermal stem cells obtained in Example 2 were cultured, and a total of 1 million cell suspensions of the third generation dermal stem cells were prepared in PBS.

(3) The two cell suspensions in (1) and (2) were mixed and centrifuged at 300 g for 5 min.

(4) Take the centrifuged cells, discard the supernatant, add 10 μl of Matrigel to the cell pellet, mix and set aside.

(5) BALB/c nude mice were anesthetized with 1% sodium pentobarbital, and a wound having an area of about 7 mm ² was made on the skin of the back with a puncher.

(6) The Matrigel mixed with the skin stem cells prepared in the step (4) was transplanted to the surface of the wound surface of the nude mouse.

(7) The surface of the wound of the nude mouse was covered with a 3M membrane, and then the wound of the nude mouse was bandaged with a bandage.

(8) Normally, the nude mice were reared. After 3 weeks, the bandage was removed to remove the 3M membrane. The skin tissue of the transplanted cells was excised, and after staining, the sebaceous gland structure was observed, and a large amount of sebaceous gland structure was found.

Example 4

Using chemical reagents to induce epidermal stem cells to form sebaceous gland structures, the specific process is as follows:

(1) The epidermal stem cells obtained in Example 1 were cultured, and the third representative skin stem cells were digested with Accutase, and a cell suspension of 2 million was prepared in PBS.

(2) After fibroblasts were digested with Accutase, 2 million cell suspensions were prepared in PBS.

(3) The two cell suspensions in (1) and (2) were mixed and divided equally into 2 portions, one serving as an experimental group and the other serving as a control group, 300 g, and centrifuged for 5 min.

(4) The above 2 centrifuged cells were discarded, and 10 μl of Matrigel was added to the cell pellet and mixed.

(5) Add appropriate amount of insulin, dexamethasone, glitazone and XAV939 to the experimental group's Matrixgel to give a final concentration of: insulin 100 μg / mL, dexamethasone 10 ^-5 M, glitazone 200 μM, XAV939 10 μM, fully aspirate and mix the above chemical reagents. The above chemical reagents were not added to the control group.

(6) BALB/c nude mice were anesthetized with 1% sodium pentobarbital, and a wound having an area of about 7 mm ² was made on the skin of the back with a puncher.

(7) Two sets of Matrigel mixed with skin stem cells obtained in the step (5) were transplanted to the wound surface of the nude mouse skin, respectively.

(8) The surface of the wound of the nude mouse was covered with a 3M membrane, and then the wound of the nude mouse was bandaged with a bandage.

(9) Normally, the nude mice were reared. After 3 weeks, the bandage was removed to remove the 3M membrane, and the skin tissue of the transplanted cells was excised, and after staining, the structure of the sebaceous glands was observed. It was found that the experimental group had a large amount of sebaceous gland formation and the control group had only a small amount of sebaceous gland formation.

Example 5

The histomorphological identification of the skin tissues of the stem cells transplanted in the experimental group and the control group in Example 3 and Example 4 was as follows:

1. Preparation of epidermal stem cells to regenerate sebaceous gland tissue tissue sections:

(1) The skin tissue regions of the stem cells transplanted in the experimental group and the control group in Example 3 and Example 4 were respectively cut out, fixed in 4% paraformaldehyde overnight, immersed in PBS for 3 hours, and dehydrated in 30% sucrose for 8 hours.

(2) OCT embedded tissue, frozen sections, and tissue sections of 10 μm thickness were taken.

(3) Dry the tissue section naturally.

(4) Tissue sections were washed three times with PBS for 5 minutes each time.

(5) Tissue sections were permeabilized for 10 minutes with 0.2-0.5% triton X-100 (in PBS).

(6) The tissue sections after the membrane treatment were washed three times with PBS for 5 minutes each time.

(7) Tissue sections were blocked with 2% BSA for 30 minutes and then washed twice with PBS.

(8) The blocked tissue sections were added to the avidin Cy3 labeled 1 antibody and incubated for 3 hours at room temperature.

(9) The tissue sections to which the primary antibody was ligated were washed three times with PBS for 5 minutes each time.

(10) Dyeing was carried out by adding 0.5 μg/ml DAPI (in PBS preparation) for 10 minutes.

(11) The stained tissue sections were washed three times with PBS to remove excess DAPI.

(12) Tissue sections from which excess DAPI was removed were added to a 20 μl capsule seal.

(13) The tissue dyeing sample with good sealing is placed in a 4 degree refrigerator and can be stored for more than 2 weeks.

2. Histomorphological identification of tissue sections:

The tissue sections were observed by confocal microscopy. It was observed that the sebaceous glands of normal skin showed immunohistochemical staining showing a large amount of biotin synthesis, and the cells exhibited a vacuolar structure; the regenerated skin tissue of Example 3 had a sebaceous gland structure. Forming, its structural morphology is similar to that of sebaceous glands in normal skin; in the chemical-induced tissue of Example 4 (ie, the experimental group), there is a large amount of biotin-synthesized sebaceous gland structure, while the control group has almost no similar structure formation, and the specific results are as follows. Figure 3-5 shows.

Example 6

The method for identifying the oil secretion ability of the sebaceous glands of the skin tissue after transplantation in Example 4 by in situ mass spectrometry is specifically as follows:

(1) The BALB/c nude mice transplanted with the stem cell skin of Example 4 were anesthetized with 1% sodium pentobarbital, and the back skin was scrubbed with 100% ethanol to wash off all the oily substances on the skin surface.

(2) The skin tissues after transplantation of the experimental group and the control group were respectively taken out, and the skin tissue of 2 mm diameter was cut out with a skin biopsy, including normal skin and stem cell transplanted skin, and the tissue piece was placed in an incubator at 37 ° C. Incubate for 2 hours to secrete sebum in the tissue to the surface of the skin.

(3) Before in situ mass spectrometry was used to identify the secretion of oil on the surface of the skin tissue, the tissue sections were pretreated by a 50% ACN aqueous solution containing 0.1% TFA to prepare a 15 g/L CHCA matrix solution. The pump was used to spray 100 μl of the substrate solution evenly on the surface of the tissue at 5 μl/s. When the surface solution of the tissue was dry, the substrate solution was sprayed again and circulated twice.

(4) The tissue section of the sprayed substrate was placed at room temperature, and the solvent was evaporated to precipitate crystals, which were then dried in a desiccator for 2 hours.

(5) The dried tissue sections were attached to a MALDI stainless steel target plate with a conductive tape and sent to a mass spectrometer for analysis.

(6) Mass spectrometry conditions Randomly take 3 points of each tissue section, add 1000 scan data to each point to obtain the mass spectrum, and add 3 points to obtain the total mass spectrum. Detection by positive ion reflection mode, mass scanning range m/z 1 00-1 000; positive ion linear mode detection, mass scanning range m/z 100-2000.

The results are shown in Fig. 6. The red line represents the oil secreted by the normal sebaceous gland, and the green line represents the oil secreted by the regenerated sebaceous gland. The mass spectrometry results show that the regenerated sebaceous gland has a similar oil secretion capacity as the normal sebaceous gland.

Industrial applicability

The method for forming sebaceous glands of the present invention can effectively form skin tissue having sebaceous glands, and the method is simple, convenient, quick, easy to control, and easy to achieve large-scale production.

Although specific embodiments of the invention have been described in detail, those skilled in the art will understand. Various modifications and alterations of the details are possible in light of the teachings of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiment", "example", "specific example", or "some examples", etc. Particular features, structures, materials or features described in the examples or examples are included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

Claims

A method of forming a sebaceous gland, comprising:

The dermal cells and epidermal stem cells are co-cultured in a gel.
The method of claim 1 wherein said co-cultivation is carried out subcutaneously in the animal.
The method of claim 1 wherein said dermal cells comprise at least one of dermal stem cells and fibroblasts.
The method according to claim 3, wherein the dermal cells are fibroblasts, and insulin, dexamethasone, glitazone and XAV939 are contained in the gel.
The method according to claim 4, wherein said insulin has a final concentration of 50-150 μg/mL and said dexamethasone has a final concentration of 10 -4 -10 -6 M, said glitazone The final concentration is 150-250 [mu]M and the final concentration of XAV939 is 5-15 [mu]M.
The method according to claim 5, wherein the final concentration of the insulin is 100 μg/mL, the final concentration of the dexamethasone is 10 -5 M, and the final concentration of the glitazone is 200 μM. The final concentration of the XAV939 was 10 μM.
The method according to claim 1, wherein the ratio of the dermal cells to the epidermal stem cells is 10:1 to 1:10.
The method according to claim 2, wherein the number of mixed cells is not less than 10 4 per 3 mm 2 of skin.
The method according to claim 1, wherein the gel is at least one selected from the group consisting of Matrigel, collagen or hydrogel.
The method according to claim 1, wherein the dermal cells and the epidermal stem cells are all derived from autologous, allogeneic or xenogeneic cells.
The method according to claim 1, wherein the dermal cells and the epidermal stem cells are independently cultured in a liquid medium for 0 to 30 generations, wherein the dermal stem cells are cultured in suspension, and the cell culture medium is composed of DMEM/F12 (3:1), 2% B27, 40 ng/μL bFGF and 20 ng/μL EGF; epidermal stem cells are adherent culture, cell culture medium is CnT-07 medium; wherein the dermal medium of dermal stem cells Further comprising a cell active factor and an extracellular matrix molecule, wherein the cell active factor is at least one selected from the group consisting of bFGF and EGF, and the extracellular matrix molecule is at least one selected from the group consisting of hyaluronic acid and fibronectin.
The method of claim 1 further comprising: identifying the secretion of oil from the surface of the skin tissue containing the regenerated sebaceous glands using in situ mass spectrometry.
A skin tissue comprising a sebaceous gland, characterized in that the sebaceous gland is formed by the method of any one of claims 1-12.
Use of the method of any of claims 1-12 in skin regeneration.