CN104450606B - Sweat gland cells inducing culture and its application - Google Patents

Abstract

The invention discloses a sweat gland cell induction medium and its application. The sweat gland cell induction medium comprises DMEM/F12 cell culture medium, keratinocyte culture medium, fetal bovine serum, epidermal growth factor and triiodothyronine , hydrocortisone, insulin-transferrin-sodium selenite, L-glutamine, penicillin, streptomycin, hepatocyte growth factor, and bone morphogenetic protein 4. The sweat gland cell induction medium of the present invention can successfully induce stem cells to differentiate into sweat gland-like cells, provide sufficient sweat gland cells for the treatment of patients with extensive burns, and greatly improve the patient's quality of life. It also provides a theoretical basis for studying the differentiation and development of sweat glands, and has potential clinical application prospects.

Description

Sweat gland cell induction medium and its application

technical field

The invention relates to a sweat gland cell induction medium and its application in inducing stem cells to differentiate into sweat gland-like cells, belonging to the technical field of sweat gland cell culture.

Background technique

The annual incidence of burns in my country is about 1.5% to 2%, that is, about 10 million people suffer from different degrees of burns every year, and nearly 10,000 people suffer from severe burns, and about 10% of the patients cannot save their lives due to lack of skin sources. The rescued patients with extensive burns lost their normal skin function for life due to non-functional scar tissue replacing the skin, which seriously affected the quality of life of the patients. The treatment of severe burns on large areas of skin is a medical and social problem to be solved urgently.

The skin is the largest organ in the human body and has functions such as protecting the body, perspiration, and sensing heat, cold, and pressure. Among them, sweat glands, as important functional skin appendages, play an important role in the process of regulating body temperature, secreting sweat and excreting some metabolites of the human body. There are two types of sweat glands in the human body, apocrine sweat glands and eccrine sweat glands. Apocrine sweat glands are larger, have larger cavities, and are not involved in thermoregulation. Eccrine sweat glands are small, distributed throughout the body and involved in temperature regulation.

Eccrine sweat glands are divided into ductal and secretory parts. The secretory part is located in the dermis or subcutaneous tissue and is a coiled tubule, while the ductal part opens in the epidermis and connects the secretory part with the external environment.

For mild burns, the ductal cells of the sweat glands can be completely repaired by using the deep uninjured part as a template to form its unique three-dimensional structure through the differentiation of stem cells. However, in the case of deep burns on a large area of full-thickness skin, sweat glands cannot rely on the division, proliferation and terminal differentiation of stem cells to self-renew and rebuild their complex structures.

The number of sweat glands in the human body is certain. Since its development is a complicated process, once a large area is damaged, it will not be possible to quickly and effectively restore functional sweat glands. Therefore, although the wound surface is covered, it has no perspiration function, which seriously affects the patient's quality of life.

Skin tissue repair and functional reconstruction are the core and hotspots of biomedical research both domestically and internationally. At present, the development mechanism of sweat gland tissue is still unclear, and it is impossible to achieve clinical mass transplantation. Studies have shown that stem cells can be directed to differentiate into and construct epidermis tissue with the ability to cover and protect; however, for functional skin appendages in dermis tissue, the directed differentiation of stem cells and sweat gland cells has not been reported so far.

The skin is digested with collagenase, and the individual sweat glands are freed, sucked and separated under an inverted microscope, and cultured in a petri dish to form sweat gland cells. However, there are some problems in this research at present, such as the difficulty in the separation and purification of sweat glands and the limited passage in vitro culture, which limits its application; at the same time, the medium used for culturing sweat glands in vitro has not yet been determined, and the effect of culturing sweat gland cells in vitro is not ideal.

Existing studies have found that stem cells co-cultured with sweat gland cells can differentiate into sweat gland cells. Sweat gland cells were subjected to heat shock treatment, and the culture supernatant of sweat gland cells after heat shock was collected and co-cultured with mesenchymal stem cells. It was found that mesenchymal stem cells could differentiate into cells with sweat gland phenotype. However, this method also has problems. Due to the low efficiency of sweat gland cell separation, purification and subculture, it is difficult to obtain a large amount of culture supernatant; and the composition of the culture supernatant is not clear, and its specific mechanism of action is also unclear; The sweat gland cells are not yet identified.

Therefore, it is necessary to develop a new induction differentiation medium and establish a new and efficient method for the directed differentiation of stem cells and sweat gland cells.

Contents of the invention

The purpose of the present invention is to provide a kind of induction differentiation medium that can directionally induce and differentiate stem cells into sweat gland-like cells; and disclose a method for using it to induce the differentiation of stem cells into sweat gland-like cells, thereby solving the problem in the treatment of patients with large area burns The needs of the sweat glands.

In order to achieve the purpose of the above invention, the technical scheme adopted in the present invention is: a sweat gland cell induction medium, which includes DMEM/F12 cell culture medium and keratinized Cell culture medium; also includes the following ingredients in amounts of:

Fetal bovine serum 3～6%

Epidermal growth factor 40～55μg/L

Triiodothyronine 0.5～1.5mmol/L

Hydrocortisone 0.1～0.3mg/L

Insulin-transferrin-sodium selenite 0.5～2%

L-Glutamine 0.5～2mmol/L

Penicillin 0.02～0.08U/L

Streptomycin 0.02～0.09μg/L

Hepatocyte growth factor 20～30μg/L

Bone morphogenetic protein 4 8～12μg/L.

Preferably, the sweat gland cell induction medium includes DMEM/F12 cell culture medium and keratinocyte culture medium with a volume ratio of 0.9:1; it also includes the following components, and the consumption of each component is:

Fetal bovine serum 5%

Epidermal Growth Factor 50μg/L

Triiodothyronine 1mmol/L

Hydrocortisone 0.2mg/L

Insulin-Transferrin-Sodium Selenite 1%

L-Glutamine 1mmol/L

Penicillin 0.05U/L

Streptomycin 0.05μg/L

Hepatocyte Growth Factor 25μg/L

Bone morphogenetic protein 4 10 μg/L.

In the above technical scheme, the keratinocyte culture medium is KGM2 cell culture medium.

The invention also discloses the application of the sweat gland cell induction medium in inducing stem cells to differentiate into sweat gland-like cells.

In the above technical solution, the stem cells are amniotic fluid stem cells or pluripotent stem cells.

The method for inducing stem cells to differentiate into sweat gland-like cells using the above-mentioned sweat gland cell induction medium, comprising taking stem cells and laying them on a culture dish, and culturing them on the wall; after the stem cells are attached to the wall, adding sweat gland cell induction medium to start inducing the stem cells; Change the sweat gland cell induction medium every day; the stem cells to be induced grow to a confluence rate of 70-80%, digest and passage, inoculate into a new culture dish at a ratio of 1 pass to 3, add sweat gland directional induction differentiation medium; induce After a period of differentiation, sweat gland-like cells were obtained, and sweat gland indicators were detected on them. The number of stem cells is related to the culture equipment. If too many cells grow too fast, the number of digestion passages will increase. For example, 1×10 ⁵ stem cells are planted in a 6-well plate, and the density is more appropriate.

In the present invention, amniotic fluid stem cells (Amniotic Fluid Stem Cell, AFS) have good proliferation efficiency and low immunogenicity, and are a new type of stem cells.

Owing to above-mentioned technical scheme uses, the present invention has following advantage compared with prior art:

1. The invention discloses a new sweat gland induction medium, which can effectively induce stem cells to differentiate into sweat gland-like cells; using the sweat gland induction medium of the invention can well induce and differentiate stem cells into sweat glands with sweat gland cell phenotype in vitro Like cells, and the sweat gland-like cells can form a tubular structure similar to the structure of sweat glands in the gel.

2. The present invention uses DMEM/F12 cell culture medium and keratinocyte culture medium as the base medium to configure a new sweat gland induction medium, and the culture equipment for inducing stem cells to transform into sweat gland cells is simple, the operation steps are simple and fast, and the cost is low. It is suitable for inducing differentiation of a large number of stem cells and is easy to popularize.

Description of drawings

Figure 1 is a morphological diagram of human amniotic fluid stem cells, induced sweat gland-like cells, and human sweat gland cells in Example 1;

Fig. 2 is the sweat gland indicator diagram of the sweat gland-like cells induced by detection at the mRNA level in Example 1;

Figure 3 is a diagram of the differentiation ratio of sweat gland-like cells induced in Example 1;

4 is a transmission electron microscope image of human amniotic fluid stem cells, induced sweat gland-like cells, and human sweat gland cells in Example 1;

5 is a structural diagram of human sweat gland tissue, induced sweat gland-like cells, and sweat gland-like ducts of human sweat gland cells in Example 1;

Fig. 6 is a diagram of the process of forming a lumen in the gel of the sweat gland-like cells obtained after the induction of human amniotic fluid stem cells in Example 1;

7 is a morphological diagram of pluripotent stem cells, induced sweat gland-like cells, and human sweat gland cells in Example 2;

Fig. 8 is a sweat gland index diagram of the sweat gland-like cells induced by detection at the mRNA level in Example 2;

Figure 9 is a diagram of the differentiation ratio of sweat gland-like cells induced in Example 2;

10 is a transmission electron microscope image of pluripotent stem cells, induced sweat gland-like cells, and human sweat gland cells in Example 2;

Figure 11 is a structural diagram of human sweat gland tissue, induced sweat gland-like cells, and sweat gland-like ducts of human sweat gland cells in Example 2;

FIG. 12 is a diagram of the process of forming a lumen in the gel of the sweat gland-like cells obtained from the induced pluripotent stem cells in Example 2. FIG.

detailed description

Below in conjunction with embodiment and accompanying drawing, the present invention will be further described:

Example 1: Inducing human amniotic fluid stem cells to differentiate into sweat gland-like cells

Sweat gland cell induction medium, including 450mL DMEM/F12 cell culture medium (Hyclone, SH30023.01B), 500mL keratinocyte medium (KGM2) (Lonza, CC-3107), additionally add 50mL special grade fetal bovine serum, 1mmol/L Triiodothyronine, 0.2 mg/L hydrocortisone, 1% insulin-transferrin-sodium selenite, 1 mmol/L L-glutamine, 0.05 U/L penicillin and 0.05 μg/L streptavidin Add 50 μg/L epidermal growth factor (EGF), 25 μg/L hepatocyte growth factor (HGF) and 10 μg/L bone morphogenetic protein 4 (BMP4).

Inducing amniotic fluid stem cells to differentiate into sweat gland-like cells, including the following steps:

(1) Take ⁵ ×104 cells/well of human amniotic fluid stem cells and spread them in a 6-well plate, and culture them with amniotic fluid stem cell medium;

Amniotic fluid stem cell medium: α-MEM medium supplemented with 15% fetal bovine serum (FBS), 1 mM L-glutamine and 0.05U/L penicillin, 0.05μg/L streptomycin, 18% Chang B and 2% Chang C medium;

(2) After 20 hours, after the amniotic fluid stem cells adhere to the wall, add sweat gland cell induction medium; then change the culture sweat gland cell induction medium every day;

(3) After the induced amniotic fluid stem cells were grown to a confluence rate of 80%, the culture medium was aspirated, washed with phosphate buffered saline (PBS), and cells were digested with 1 mL of 0.25% trypsin for 2 minutes, and the culture medium containing serum was used Terminate the action of trypsin, suck the digested cells into a centrifuge tube and centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, add sweat gland cell induction medium, mix the cell pellet, and inoculate the cells into a new culture dish at a ratio of 1 to 3;

(4) Repeat step (3) to obtain sweat gland-like cells after 28 days of induced differentiation, and detect sweat gland indicators on them.

Accompanying drawing 1 is the morphological diagram of human amniotic fluid stem cells cultured in vitro, sweat gland-like cells induced and differentiated from human amniotic fluid stem cells, and human sweat gland cells under an optical microscope. It can be seen that human amniotic fluid stem cells are small, spindle-shaped, and arranged irregularly; sweat gland-like cells induced and differentiated from human amniotic fluid stem cells are enlarged, epithelial-like, and arranged regularly; human sweat gland cells are larger , with a distinct epithelial cell morphology, showing paving stone-like growth with neatly arranged cells. The sweat gland-like cells obtained after the differentiation of human amniotic fluid stem cells by sweat gland induction medium are similar in morphology to human sweat gland cells cultured in vitro.

Accompanying drawing 2 is the sweat gland indicator map of the induced sweat gland-like cells detected at the mRNA level. Realtime-PCR results showed that human amniotic fluid stem cells gradually expressed sweat gland cell indicators EDA, EDAR, K8 and CEA during the induction process, and gradually approached sweat gland cells during the differentiation process. Human amniotic fluid stem cells (hAFS) were used as a negative control, and human sweat gland cells (hSG) were used as a positive control.

Accompanying drawing 3 is the graph of the differentiation ratio of the induced sweat gland-like cells. After 28 days of induction, the expression of the induced cells on sweat gland cell indicators was detected by flow cytometry. After analysis, the sweat gland-like cells induced by human amniotic fluid stem cells expressed about 36% of EDA, about 43% of EDAR, about 45% of K8, and about 32% of CEA. Human amniotic fluid stem cells can be well induced to differentiate into sweat gland trophoblasts.

Accompanying drawing 4 is the human amniotic fluid stem cell detected at the organelle level by the transmission electron microscope, the sweat gland-like cells induced and differentiated by the human amniotic fluid stem cell, and the sweat gland index diagram of the human sweat gland cell. The sweat gland-like cells induced by human amniotic fluid stem cells are close to human sweat gland cells at the organelle level. Specifically, the induced sweat gland-like cells have microvilli (black arrows) that human sweat gland cells have, while human amniotic fluid stem cells do not have microvilli.

Accompanying drawing 5 is the human sweat gland tissue, the sweat gland-like cell induced differentiation of human amniotic fluid stem cell and the sweat gland-like duct structure diagram of human sweat gland cell, the cross-section of the sweat gland tubular structure can be seen in the figure, and the sweat gland lumen can be observed; The sweat gland tubular structure formed by human sweat gland-like cells in the gel, the tubular structure cross-section and lumen can be seen; the tubular structure formed by human sweat gland cells in the glue, the tubular structure cross-section and lumen can be seen.

Accompanying drawing 6 is the process of forming tube lumens of sweat gland-like cells obtained after human amniotic fluid stem cells are induced. Sweat gland-like cells proliferated into clusters in the gel, and the cells in the middle gradually apoptotic to form a lumen.

Example 2: Inducing Pluripotent Stem Cells to Differentiate into Sweat Gland-like Cells

Sweat gland cell induction medium, including 450mL DMEM/F12 cell culture medium, 450mL keratinocyte medium (KGM2), additionally added 55mL special grade fetal bovine serum, 1.5mmol/L triiodothyronine, 0.3mg/L hydrocortisone Pine, 2% insulin-transferrin-sodium selenite, 1.5mmol/L L-glutamine, 0.08U/L penicillin and 0.09μg/L streptomycin; then add 55μg/L epidermal growth factor (EGF) , 30 μg/L hepatocyte growth factor (HGF) and 8 μg/L bone morphogenetic protein 4 (BMP4).

Inducing pluripotent stem cells to differentiate into sweat gland-like cells includes the following steps:

(1) Spread 1×10 ⁵ pluripotent stem cells/well in a 6-well plate and culture in pluripotent stem cell medium;

Pluripotent stem cell medium: containing 20% KnockOut serum, 1% non-essential amino acids, 0.1mmol/L β-2-mercaptoethanol, 4μg/L FGF-based, 2mmol/L L-glutamine, 0.1U/L penicillin and DMEDF12 cell culture medium with 0.1ug/L streptomycin.

(2) After 24 hours, after the pluripotent stem cells adhered to the wall, add sweat gland cell induction medium; then replace the culture sweat gland cell induction medium every day;

(3) The induced pluripotent stem cells were grown to a confluence rate of 70%, the culture medium was aspirated, washed with phosphate buffered saline (PBS), and the cells were digested with 0.8mL 0.25% trypsin for 2 minutes. The medium stops the action of trypsin, and the digested cells are sucked into the centrifuge tube and centrifuged at 1200 rpm for 5 minutes. Discard the supernatant, add sweat gland cell induction medium, mix the cell pellet, and inoculate the cells into a new culture dish at a ratio of 1 to 4;

(4) Repeat step (3) to obtain sweat gland-like cells after 21 days of induction and differentiation, and detect sweat gland indicators on them. The pluripotent stem cells can be successfully induced into sweat gland-like cells by using the sweat gland cell induction medium.

Fig. 7 is a morphological diagram of pluripotent stem cells cultured in vitro, sweat gland-like cells induced and differentiated from pluripotent stem cells, and human sweat gland cells under an optical microscope. It can be seen that the pluripotent stem cells are small, grow in a clone-like manner, and have a high nuclear-cytoplasmic ratio; the sweat gland-like cells induced and differentiated from pluripotent stem cells, the cells become larger, epithelial-like, and the nuclear-cytoplasmic ratio becomes smaller; human sweat glands Cells, large cells, with obvious epithelial cell morphology, paving stone-like growth, cells arranged neatly. The sweat gland-like cells obtained after the differentiation of pluripotent stem cells by sweat gland induction medium are similar in morphology to human sweat gland cells cultured in vitro.

Accompanying drawing 8 is the sweat gland indicator diagram of the induced sweat gland-like cells detected at the mRNA level. Realtime-PCR results showed that pluripotent stem cells gradually expressed sweat gland cell indicators EDA, EDAR, K8 and CEA during the induction process, and gradually approached sweat gland cells during the differentiation process. Pluripotent stem cells (SG-iPS) were used as a negative control, and human sweat gland cells (SG) were used as a positive control.

Figure 9 is a diagram of the differentiation ratio of the induced sweat gland-like cells. After 21 days of induction, the expression of the induced cells on sweat gland cell indicators was detected by flow cytometry. After analysis, the sweat gland-like cells induced by pluripotent stem cells expressed about 63% of EDA, about 65% of EDAR, about 76% of K8, and about 56% of CEA. Pluripotent stem cells can be induced to differentiate into sweat gland trophoblasts at high yields.

Fig. 10 is a transmission electron microscope detection of pluripotent stem cells at the organelle level, sweat gland-like cells induced and differentiated from pluripotent stem cells, and sweat gland index diagrams of human sweat gland cells. The sweat gland-like cells induced by pluripotent stem cells are close to human sweat gland cells at the organelle level, specifically, the induced sweat gland-like cells have the microvilli (black arrows) that human sweat gland cells have, while the pluripotent stem cells do not have microvilli.

Accompanying drawing 11 is the structure diagram of human sweat gland tissue, sweat gland-like cells induced and differentiated by pluripotent stem cells, and sweat gland-like ducts of human sweat gland cells. In the figure, the cross-section of the sweat gland tubular structure can be seen, and the sweat gland lumen can be observed; after induction of pluripotent stem cells, The sweat gland tubular structure formed by human sweat gland-like cells in the gel, the tubular structure cross-section and lumen can be seen; the tubular structure formed by human sweat gland cells in the glue, the tubular structure cross-section and lumen can be seen.

Figure 12 shows the process of forming tube lumens of sweat gland-like cells obtained from induced pluripotent stem cells in the gel. Sweat gland-like cells proliferated into clusters in the gel, and the cells in the middle gradually apoptotic to form a lumen.

To sum up, the sweat gland cell induction medium of the present invention can be used to successfully induce the directional differentiation of stem cells into sweat gland-like cells, provide a sufficient number of sweat gland cells for the treatment of patients with extensive burns, and greatly improve the quality of life of patients. It also provides a theoretical basis for studying the differentiation and development of sweat glands, and has potential clinical application prospects.

Claims (5)

1. a kind of sweat gland cells inducing culture, it is characterised in that：The sweat gland cells inducing culture is including volume ratio （0.8~1）: 1 DMEM/F12 cell culture mediums and keratinocyte culture medium；Also include following component, the consumption of each composition For：

Hyclone 3~6%

The μ g/L of EGF 40~55

0.5~1.5mmol/L of trilute

0.1~0.3mg/L of hydrocortisone

Insulin-Transferrin-sodium selenite 0.5~2%

0.5~2mmol/L of Glu

0.02~0.08U/L of penicillin

The μ g/L of streptomysin 0.02~0.09

The μ g/L of HGF 20~30

The μ g/L of bone morphogenetic protein 4 8~12.

2. sweat gland cells inducing culture according to claim 1, it is characterised in that：The sweat gland cells inducing culture bag Include DMEM/F12 cell culture mediums and keratinocyte culture medium that volume ratio is 0.9: 1；Also include following component, each composition Consumption is：

Hyclone 5%

The μ g/L of EGF 50

Trilute 1mmol/L

Hydrocortisone 0.2mg/L

Insulin-Transferrin-sodium selenite 1%

Glu 1mmol/L

Penicillin 0.05U/L

The μ g/L of streptomysin 0.05

The μ g/L of HGF 25

The μ g/L of bone morphogenetic protein 4 10.

3. the sweat gland cells inducing culture according to claims 1 or 2, it is characterised in that：The keratinocyte culture Base is KGM2 cell culture mediums.

4. the answering in differentiation of stem cells is sweat gland like cell of sweat gland cells inducing culture described in claims 1 or 2 With.

5. application according to claim 4, it is characterised in that：The stem cell is that amniotic fluid stem cell or pluripotency are dry thin Born of the same parents.