KR101859500B1 - 3-dimensional matrix - Google Patents
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- KR101859500B1 KR101859500B1 KR1020100121220A KR20100121220A KR101859500B1 KR 101859500 B1 KR101859500 B1 KR 101859500B1 KR 1020100121220 A KR1020100121220 A KR 1020100121220A KR 20100121220 A KR20100121220 A KR 20100121220A KR 101859500 B1 KR101859500 B1 KR 101859500B1
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Abstract
The present invention discloses a composition for a three-dimensional matrix comprising collagen 4, laminin, nidogen and proteoglycan, and a three-dimensional matrix prepared using the composition. The present invention also discloses a method for culturing cells using the three-dimensional matrix.
Description
The present invention relates to a three-dimensional matrix for degenerating cells, microorganisms and the like.
Melanoma is a malignant tumor of melanocyte that produces melanin pigment, which can occur anywhere in the area where melanocytes are present, but it occurs most often in the skin and is the most malignant of the skin cancer. It is necessary to study the mechanisms and treatment methods of pigment cells related pigment cells including melanoma, which are known to be differentiated from human normal skin pigment cells.
The present invention provides a composition for a three-dimensional matrix and a three-dimensional matrix. The present invention also provides a cell culture method using a three-dimensional matrix.
One aspect of the present invention provides a composition for a three dimensional matrix comprising Collagen IV, Laminin, Nidogen and Proteoglycan.
Another aspect of the present invention provides a three-dimensional matrix produced using the composition for a three-dimensional matrix.
Another aspect of the present invention provides a cell culture method comprising culturing a cell using the three-dimensional matrix.
A three-dimensional matrix can be prepared using a composition for a three-dimensional matrix comprising collagen IV, laminin, nidogen and proteoglycan according to the present invention. When cells are cultured on the 3-dimensional matrix, cell division is inhibited and dedifferentiation occurs. Based on this, it is possible to study the mechanism of skin cell differentiation, the mechanism of skin cancer including melanoma (mealnoma), treatment method such as skin hypercholesterolemia such as nevi, surplus or black spot, pigment deficiency such as vitiligo or white moth can do.
FIG. 1 is a photograph showing cytomorphological changes when human normal skin pigment cells were cultured on Comparative Examples 1 and 2 and Examples. FIG.
Fig. 2 is a still cut of a moving picture of a cell cultured on the example. Fig.
FIG. 3 is a photograph showing morphological changes of cells when the cells cultured for 3 days on the example were cultured again on Comparative Example 1. FIG.
FIG. 4 is a graph showing the degree of expression of tyrosinase, tyrosinase related proteins 1 and 2 (TRP 1, 2) and GAPDH by electrophoresis when skin pigment cells were cultured on Comparative Examples 1, 2, and Examples.
FIG. 5 is a graph comparing melanin contents when skin pigment cells were cultured on Comparative Examples 1 and 2 and Examples. FIG.
FIG. 6 shows the results of electrophoresis of the expression levels of PAX 3, SOX 10 and PCNA when skin pigment cells were cultured on Comparative Examples 1, 2 and Example.
The skin pigment cell (melanocyte) is a cell that produces melanin that determines skin color. It is located on the basement membrane, which acts as a boundary between the skin's dermis and epidermis. From the developmental point of view, skin pigment cells originate from neural-crest cells and move to skin through melanocyte stem cell, melanoblast precursor cell (melanoblast), and eventually produce melanin It is known to differentiate into pigment cells that can be made. However, these are the results mostly derived from mouse embryological studies, and since human embryos can not be studied in humans, how the skin pigment cells are differentiated has not been studied precisely. In experiments using completely differentiated skin pigment cells, there are experiments in which skin pigment cells are reverse-differentiated into melanoblasts by methods such as genetic manipulation or growth hormone treatment. However, this experiment also shows that there is no experiment using human skin pigment cells, Are experiments using pigment cells.
Skin pigment hyperpigmentation such as nevi, blackness or black spot, pigment deficiency such as vitiligo or white moth, cancer including mealnoma is considered to be human-specific when abnormalities in the differentiation and development of skin pigment cells occur Because of the phenomenon that occurs, experiments on the differentiation and development of human skin pigment cells are important. In particular, in the case of melanoma, mutation occurs in skin pigment cells, and cell division is promoted, and the expression of protein appearing in the pre-differentiation stage is increased. In comparison with normal skin pigment cells, A method of degenerating pigment cells will be very useful. In addition, the point is similar to melanoma, but at the same time, cell division stops, and development of cancer cell does not develop. Such a difference study will be helpful in the treatment of melanoma. It has been known that white mosaic leukaemia is an aging phenomenon caused by depletion of stem cells from skin pigment cells. Therefore, the method of differentiation, de-differentiation and generation of human skin pigment cells is very important in itself.
However, human skin pigment cells do not exist in the skin alone but on a specific mattress called the basement membrane, which is dominated by the keratinocyte that occupies most of the epidermis and produces melanin. The major components of the basement membrane are Collagen IV, Laminin, Nidogen, and Proteoglycan. It is not known how each of these components plays a role in the differentiation and activity of skin pigment cells. In particular, there is no report on what happens when human skin pigment cells are cultured on a three-dimensional mattress containing these components.
However, in melanoma, it passes through the basement membrane of the skin and moves to the dermis, which is a very important step in cancer metastasis. To study this, the four major components are coated on the bottom of the cell culture, There are examples, but this is different from three-dimensional culture. In addition, since the basement membrane is present in blood vessels, brains, and other living tissues as well as skin, it forms a boundary with other tissues. Therefore, vascular endothelial cells are cultured on the basement membrane, and in There is an example in which blood vessels are formed in vitro . However, there is no example in which fully differentiated normal human skin pigment cells are cultured three-dimensionally on the basement membrane.
As used herein, the term "matrix" refers to a support which grows with cells, microorganisms, etc., and is used as a support for providing a physical or chemical characteristic of a real environment, Means a mixture made by adding a structural protein or various components.
As used herein, the term "skin" refers to a tissue covering the body surface of an animal, and is a concept of the broadest notion including a scalp and hair as well as a tissue covering a body surface such as a face or a body.
Hereinafter, the present invention will be described in detail.
One aspect of the present invention provides a composition for a three dimensional matrix comprising Collagen IV, Laminin, Nidogen and Proteoglycan. Another aspect of the present invention provides a three-dimensional matrix produced using the composition for a three-dimensional matrix.
When cells, specifically completely normal human skin pigment cells, are cultured in the 3-dimensional matrix, they are attracted to each other like stem cells, and melanin production, which is a final differentiation product of pigment cells, is inhibited and melanin is produced Tyrosinase and related proteins (tyrosinase related proteins 1 and 2) are decreased. In addition, the expression of PAX 3, a degenerative marker known to be expressed in fully differentiated human normal skin pigment cells, is increased as an important transcription factor that is expressed in neural-crest cells and transformed into melanoblasts do. In contrast to PAX 3, the expression of SOX 10, which is a differentiation marker that is continuously expressed in fully differentiated human skin pigment cells, is a transcription factor that is expressed with PAX 3 from the early differentiation stage. SOX 10 is also known to be inhibited in the hair bulge of hair known to contain melanocyte stem cells. In addition, the expression of PCNA, a marker of cell proliferation, is inhibited and cell division is reduced. This is an effect that can not be obtained when a composition for a matrix is two-dimensionally coated with a skin pigment cell, or when a composition for a matrix comprising other components is used.
Based on these results, it was found that when cells were cultured in a three-dimensional matrix prepared using a composition for a three-dimensional matrix containing collagen 4, laminin, nidogen and proteoglycans, cell division was inhibited and degenerated Able to know. That is, the three-dimensional matrix can cause cell differentiation inhibition or de-differentiation to occur.
Melanoma is known to be differentiated from human normal skin pigment cells, and it is a typical skin symptom that PAX 3 expression increases with point. It is known that the reduction of cell division is one of the features of hyperpigmentation and stellate cells. Therefore, when the cells are cultured, the three-dimensional matrix according to the present invention in which the cleavage is inhibited and the differentiation is exhibited, can be used to treat skin hyperpigmentation such as nevi, black or black, pigment deficiency such as vitiligo or white moth, The mechanisms of cancer, including mealnoma, and the treatment methods. More specifically, the three-dimensional matrix according to the present invention can determine whether any substance is a carcinogenic substance by checking whether melanoma or dots appear when ultraviolet rays or an arbitrary substance are treated in a state where cells are degenerated.
In one aspect of the present invention, the composition for a three-dimensional matrix may contain collagen 4, laminin, nidogen, and proteoglycan in an amount of 0.001 wt% to 30 wt%, respectively, based on the total weight of the composition. In another aspect of the present invention, the composition for a three-dimensional matrix may contain collagen 4, laminin, nidogen and proteoglycan in an amount of 0.01 to 20% by weight based on the total weight of the composition, respectively. In another aspect of the present invention, the composition for a three-dimensional matrix may contain collagen 4, laminin, nidogen and proteoglycan in an amount of 0.1 wt% to 10 wt%, respectively, based on the total weight of the composition. When it is contained in the above weight percentage, it is not only suitable for exhibiting the intended effect of the present invention but also can satisfy both the stability and safety of the composition and may be suitably included in the above range in terms of cost effectiveness.
In one aspect of the invention, the composition for a three-dimensional matrix may further comprise a component contained in a basement membrane or cell of a vertebrate animal such as a human, mouse or algae. In another aspect of the present invention, the composition for a three-dimensional matrix may further comprise an extract extracted from the basement membrane or cells of a vertebrate animal. This can provide conditions more similar to basement membrane or cell.
In one aspect of the invention, the composition for a three-dimensional matrix may be a gelatinous protein mixture extracted from Engelbreth-Holm-Swarm mouse sarcoma. In another aspect of the invention, the composition for a three-dimensional matrix may have a protein concentration of 2-14 mg / ml, more specifically 7-10 mg / ml. The composition for a three-dimensional matrix according to one aspect of the present invention may need to be cryopreserved below -20 < 0 > C. In another aspect of the present invention, Matrigel (TM) (manufactured by BD Sciences) can be used as a composition for a three-dimensional matrix.
In one aspect of the invention, the three-dimensional matrix may be for inhibition or reverse differentiation of vertebrate animal skin cells, skin pigment cells or normal skin pigment cells. In another aspect of the invention, the three-dimensional matrix may be for the inhibition or de-differentiation of one or more of human, skin, and pigment cells. In the above, human cells include all the cells of a black, white, yellow, or mixed-blooded human, including all human cells. In yet another aspect of the present invention, the three-dimensional matrix may be for inhibition or reverse division of human skin cells, human pigment cells, human skin pigment cells or normal human skin pigment cells. All of the cells include those completely differentiated. In particular, when fully differentiated normal human skin cells are cultured in a three-dimensional matrix according to one aspect of the present invention, they are useful because they are excellent in the effect of inhibiting cell division and degeneration. When the normal skin cells of the black skin with the highest number of differentiation of skin pigment cells are cultured in a three dimensional matrix according to one aspect of the present invention, the effect can be most clearly observed and can be more useful.
One aspect of the present invention provides a cell culture method comprising culturing a cell using the three-dimensional matrix. Since the cell division is suppressed and regenerated cells can be obtained through the above cell culture method, it is possible to obtain cells with a skin color hyperpigmentation such as nevi, black or black pigment, pigment deficiency such as vitiligo or white moth, melanoma mealnoma) and the treatment methods of cancer.
Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Examples, Comparative Examples and Experimental Examples. However, the following examples, comparative examples, and experimental examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, but the scope and scope of the present invention are not limited thereto.
[Examples and Comparative Examples]
A mixed gelatinous composition (Matrigel ™ ) containing collagen IV, laminin, Nidogen and proteoglycan in an amount of 7-10 mg / ml, an essential component of the basement membrane, , BD Biosciences) (frozen at -20 ° C) were dissolved in ice to make them liquid. This was added to a 6-well plate in an amount of 1.5 ml per well, followed by hardening in a 37 ° C incubator for 1 hour or more to prepare a three-dimensional matrix. A 6-well plate coated with gelatin was prepared and used as Comparative Example 1. A 3-dimensional matrix made of collagen 1 (collagen I, 1 mg / ml), which is a constituent of the dermis, was prepared and used as Comparative Example 2.
[Experimental Example 1] Morphological characteristics of cultured cells
Human normal skin pigment cells were placed on the 3-dimensional matrix of the plate of Comparative Example 1 and the Comparative Example 2 and the Example so as to be 2 x 10 5 per well. The human normal skin pigment cells used in the experiment were cultured on the epidermis of a black newborn baby. The melanin production was high and the expression of the differentiation-related gene was high, so that the effect of the experiment can be clearly seen. Thereafter, human melanocyte growth supplements (HMGs, human) containing 10 ng / ml of phorbol 12-myristate 13-acetate (PMA) were added to Medium 254 medium sold by Cascade Biologics melanocyte growth supplement) was added to the culture medium, and 2 ml of the culture medium was added per well. The cells were cultured in a 37 ° C, 5% CO 2 incubator, and after 1 day and 3 days, the cells were photographed with an optical microscope to confirm morphological changes. The results are shown in FIG. A and D respectively show the 1st and 3rd days of the culture of Comparative Example 1, B and E respectively show the 1st and 3rd days of the culture of Comparative Example 2, and C and F show the 1st and 3rd days of the culture of the Example, respectively. After the cells were cultured using a time lapse microscope, the morphological changes between one day and two days were photographed. A photographing still cut is shown in Fig.
As shown in FIG. 1, in Comparative Examples 1 and 2, the cells grow dispersed evenly while spreading a large number of cell dendrites. In contrast, in the example, cell clusters disappeared and clustered to form a cell cluster . In addition, in the case of the video recording using time-lapse, in the case of the embodiment, it was confirmed that the cells pulled each other while gathering each other's branches. In Fig. 2, we can see the appearance of the cells stretching the branches and attracting each other.
In the case of stem cells, since the cells do not lose their stem cell properties in order to maintain their stem cell properties, it is necessary to artificially allow the cells to grow together. In this experiment, when human skin pigment cells completely differentiated in a three-dimensional matrix containing collagen 4, laminin, nidogen and proteoglycan in the examples were cultured, aggregation phenomena like stem cells were observed to be observed In the example, when the cells are cultured, the properties of the stem cells can be confirmed.
[Experimental Example 2] Cultivation in a general medium
As in Experimental Example 1, the human normal skin pigment cells growing on the three-dimensional matrix of the Example for 3 days were poured out and then placed in a 6-well plate in which nothing was coated and cultured for 9 days. The photographs are shown in Fig. 3 after 1, 3, 4, 5 and 9 days of culture.
As can be seen from FIG. 3, when the cells were cultured again in a 6-well plate in which nothing was coated, the human normal skin pigment cell mass was loosened, and the cell branches were dispersed while growing again. Also, it was confirmed that the cell mass was again formed when this was again taken out and grown on the three-dimensional matrix of the example.
[Experimental Example 3] Evaluation of the degree of differentiation protein and melanin production in cultured cells
Cells cultured for 3 days on the 3-dimensional matrix of the example were removed, proteins were extracted, and proteins were separated by size by electrophoresis. The tyrosinase and tyrosinase related proteins 1 and 2 (tyrosinase-related proteins 1 and 2) in the proteins separated by electrophoresis were confirmed by Western blotting using antibodies. The results are shown in Fig. 4 (A: Comparative Example 1, B: Comparative Example 2, and C: Examples).
The melanin content of each cell was measured, and the relative melanin content (%) of the remaining melanin when the melanin content of the cells cultured in Comparative Example 1 was taken as 100 is shown in FIG.
As can be seen from the GAPDH in Fig. 4, it can be seen that the proteins were electrophoresed in the same amounts in both Comparative Examples 1 and 2 and the Examples. It can be seen that the expression of the differentiated protein of the cells cultured in the three-dimensional matrix of the example is significantly reduced as compared with the cells cultured in the matrix of the comparative example 1 or the matrix of the comparative example 2. That is, it can be confirmed that when the cells are cultured in the three-dimensional matrix of the embodiment, their differentiation is inhibited.
As can be seen from FIG. 5, the amount of melanin in the cells cultured on the three-dimensional matrix of the example is reduced by about 50% as compared with the comparative example. That is, when the cells are cultured in the three-dimensional matrix of the embodiment, the differentiation is inhibited and melanin production is not observed. On the other hand, although the expression of the differentiated proteins was almost suppressed within 3 days after the incubation, the amount of melanin was reduced only by half. Since the melanin produced is not decomposed in the cells, This is because only the amount of melanin that has been created for 3 days has changed.
[Experimental Example 4] Observation of change of related protein expression
PAX 3 and SOX 10, which are known to increase or decrease expression in melanoblasts, melanocyte stem cells and melanomas at pre-differentiation stages, and PCNA, a marker of cell proliferation, And confirmed by Western blotting. The results are shown in Fig. 6 (A: Comparative Example 1, B: Comparative Example 2, and C: Examples).
As can be seen from the GAPDH in FIG. 6, it can be seen that the proteins were electrophoresed in the same amounts in both Comparative Examples 1 and 2 and the Examples. In contrast, in Comparative Examples 1 and 2, the expression of PAX 3 was decreased and the expression of SOX 10 and PCNA was increased. On the other hand, in the case of cells cultured on the 3-dimensional matrix of Example, PAX 3 expression was increased and SOX 10 and PCNA It can be confirmed that the expression decreases.
From the above results, culturing human normal skin pigment cells completely differentiated on a three-dimensional matrix containing collagen 4, laminin, nidogen and proteoglycan inhibits differentiation of cells, degeneration occurs, and cell division is reduced Such as stem cells, dots, and melanomas. Therefore, it is possible to know the occurrence and physiological mechanisms of various pigment - related skin diseases and to develop the technology necessary to prevent and control them.
Claims (8)
Wherein the collagen 4, laminin, nidogen, and proteoglycan are each contained in an amount of 0.001 wt% to 30 wt% relative to the total weight of the composition.
Collagen 4, laminin, nidogen, and proteoglycan are contained in an amount of 0.001 to 20 wt% based on the total weight of the composition, respectively.
Wherein the normal skin pigment cells are human normal skin pigment cells.
Wherein the collagen 4, laminin, nidogen and proteoglycan are each contained in an amount of 0.001 to 30 wt% based on the total weight of the composition.
Collagen 4, laminin, nidogen, and proteoglycan are each contained in an amount of 0.001 wt% to 20 wt% based on the total weight of the composition.
Wherein the normal skin pigment cells are human normal skin pigment cells.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003051419A1 (en) | 2001-12-19 | 2003-06-26 | Henkel Kommanditgesellschaft Auf Aktien | Skin/hair equivalent with reconstructed papillae |
| JP2008541734A (en) | 2005-05-30 | 2008-11-27 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション | Production and use of basement membrane particles |
| JP7040932B2 (en) * | 2017-12-19 | 2022-03-23 | 株式会社ダイヘン | Welding position detection device, welding position detection method and welding robot system |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003051419A1 (en) | 2001-12-19 | 2003-06-26 | Henkel Kommanditgesellschaft Auf Aktien | Skin/hair equivalent with reconstructed papillae |
| JP2008541734A (en) | 2005-05-30 | 2008-11-27 | コモンウェルス サイエンティフィック アンド インダストリアル リサーチ オーガニゼイション | Production and use of basement membrane particles |
| JP7040932B2 (en) * | 2017-12-19 | 2022-03-23 | 株式会社ダイヘン | Welding position detection device, welding position detection method and welding robot system |
Non-Patent Citations (3)
| Title |
|---|
| J. Cell. Physiol. 221: 18-25, 2009 |
| Journal of Investigative Dermatology 89(2): 156-163 (1987.8) |
| Molecular and Cellular Biochemistry 283: 181-189, 2006 |
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