KR20200118627A - Inhibition of melanin synthesis using adult stem cells culture media - Google Patents

Abstract

The present invention relates to a human mesenchymal stem cell and a novel use of a stem cell culture medium and, more specifically, to a mesenchymal stem cell extracted from human fat, placenta, cord blood, or bone marrow; a stem cell culture medium obtained by culturing the same in an appropriate medium and under an appropriate condition, resulting in the activity of inhibiting melanin synthesis; or a method for using the same as a raw material for whitening-related drugs, quasi-drugs and cosmetics.

Description

Inhibition of melanin synthesis using adult stem cells culture media

This 　invention is 　about human 　 mesenchymal 　 stem cells 　 and 　 stem cells 　 new 　 uses of culture fluid.

In more detail, 　human 　 fat, 　 placenta, 　 cord blood 　 or 　 extracted from bone marrow 　 mesenchymal 　 stem cells 　 or 　 tooth 　 appropriate 　 medium

And 　 　 cultured 　 stem cells 　 cultured 　 melanin 　 inhibits the synthesis 　 has the activity 　 　 teeth 　 whitening 　 related 　 drugs,

It is about quasi-drug and 　cosmetics 　how to use 　as raw materials.

Melanin, which determines the color of human skin, is called melanocytes, called melanocytes, and is made from cells.

It moves to 　epidermis 　cells called keratinocytes. Here, 　melanine 　 forms a 　 structure like a hat and 　 around the nucleus,

Protects 　genes 　 from ultraviolet 　 free 　 removes free 　 radicals 　 protects cells 　 in 　 proteins 　

It becomes one-of-a-kind. These 　melanin-degrading 　enzymes 　in the body 　　, but 　but keratinocytes 　in the epidermis

When it grows, it is removed from the skin as it is 　. However, if 　melanine 　more 　 than necessary 　

It causes 　 and pigmentation 　 like spots 　 freckles, 　 spots 　, etc. 　 brings 　 unfavorable results 　 cosmetically. Also, 　Leisure

With the increase of the population 　 being active 　 enjoying 　 　 increasing 　 　 by ultraviolet rays 　 melanin 　 pigmentation 　 to prevent 　

　 demand to do 　 increased 　. Therefore, 　to prevent excessive 　 melanin 　 production 　 whitening agent 　 development 　 necessary 　

Efforts have been made.

So far, the development of 　whitening agents 　mainly 　 melanin 　 in biosynthesis 　 should not be 　 basic yet 　 most 　 important 　 role 　 effect

It consists of 　 to reduce the amount of melanin 　 through the 　 activity of tyrosinase 　 inhibit 　 　 　 find a substance 　

Brought. There are 　developed 　 whitening agents 　kojic acid, 　 arbutin, 　 glutathione, 　 vitamins 　 A, 　 vitamins 　 C　, but 　 consumers only 　

It does not have satisfactory 　 　 whitening effect 　 and 　 due to side effects 　 　 it 　 usage 　 limited 　 problem 　. So 　

　 is more effective 　 than lichen 　 whitener 　 a little 　 more 　 safe 　 whitening agent 　 development 　 urgent 　 actual situation.

Accordingly, the 　this 　inventors 　provided 　excellent 　 whitening effect 　 while 　 has no side effects 　 　 new 　providing 　 to provide 　

As a result, 　 found that 　 stem cells 　 cultured 　 can effectively 　 inhibit 　 production 　 without side effects 　 　 found 　 seen 　 invention 　 completed

Was.

This 　invention is 　mesenchymal 　 obtained from stem cells 　 culture solution 　 or 　 group 　 separated 　 protein 　 using 　 safe and effective

Overloaded 　 whitening-related 　 drugs 　 or 　 to provide 　 cosmetics 　 purpose 　.

This 　invention provides 　 for 　 for whitening 　 cosmetics 　 composition containing 　 separated 　 protein 　 from 　 stem cells 　 culture solution 　 or 　 group 　 culture solution.

At this time, the above 　 stem cells include 　adult stem cells 　 and 　 mesenchymal stem cells 　 derived from mammals. Also 　 above 　 adult stem

Cells include 　 adipose tissue, 　 bone marrow tissue, 　 umbilical cord blood 　 or 　 stem cells 　 isolated from the placenta.

Stem cells are 　 undifferentiated 　 cells, 　 for a long time 　 　 dividing 　 　 self-renewal 　 　 able 　, 　 some conditions

Given this 　, it refers to the 　various 　 kinds of 　 cells 　 which can be divided into 　 　 　 cells. Stem cells are 　in accordance with the origin 　 tissue 　 embryonic stem

It is divided into cells and 　 adult stem cells 　, and 　 potential 　 ability 　 than embryonic stem cells 　 limited 　 disadvantages 　 but 　 ethical 　 question

I have no 　 and no side effects 　 　 targeting adult stem cells 　 many 　 treatments 　 have been studied 　.

Specifically, in the 　this invention, 　uses 　 isolated 　 adult stem cells 　 from 　 adipocytes of an adult 　 and 　 exists in 　 adipose tissue 　

It is possible to obtain 　 cells 　 out of 　 through a simple 　 purification process 　. The 　 process for the acquisition of local tissues 　 is usually 　 often implemented 　

Because 　 in the 　 process of liposuction 　 that was 　 adipose tissue 　 used 　, 　 additional 　 invasive 　 procedure 　 required 　 not 　

Its 　useability 　 doubles.

Human 　 fat 　 tissue 　 under local anesthesia 　 　 by liposuction 　 and 　 extracellular 　 matrix of adipose tissue

After 　 enzymatic treatment with collagenase 　, 　 centrifuged 　 separated 　 mononuclear cells, 　 erythrocytes 　 and 　 several 　 branches 　 cell debris 　 separated 　 stem cells

Get

Normally 　commonly 　in the 　 practiced 　 in the 　 process of liposuction 　 obtained 　 discarded 　 adipose tissue 　 aseptic 　

Separate only 　 pure 　 adipose tissue 　 from the obtained 　 　 and 　 separated 　 fat 　 inhalation.

Separated 　 stem cells 　 　 containing serum 　 Dulbecco's 　 Modified 　 Eagle's 　 Medium (DMEM) 　 in non-inducible 　 cultured in medium

Remove non-adhesive 　 cells 　.

　 　 isolated 　 stem cells 　 in the above 　 process 　 　 　 by centrifuging 　 and filtering the supernatant 　 　 fat according to the invention 　

Derived 　 stem cells 　 culture solution was obtained.

The process of 　isolating 　 adult stem cells 　 from adipocytes 　 　 is not limited to the 　 original 　 invention 　 method 　

It is possible to implement it by the method that is performed by the method.

　According to the present 　invention 　Adipose-derived 　 Stem cells 　Existing in the culture solution 　Whitening activity 　 Related 　To check the protein 　, 　LC-MS/MS

Timed.

The result 　Adipose-derived 　Stem cells 　In the culture solution, 　Tyrosinase, 　TRP1　 and 　TRP　2 were 　inhibited 　Mitf (microphthalmia-

associated 　transcription 　factor) 　downregulation, or 　melanosomes 　transferring 　 involved 　 protein 　

It was confirmed that it contained 　 (refer to Table 　 1　).

Therefore, 　this 　invention 　 derived from fat 　 stem cells 　 cultured 　 prevents 　 pigmentation of the skin 　 or treats 　 whitening 　 cosmetics

It is useful 　 can be used 　.

In addition, in the present invention, processing the adipose derived stem cell culture according to the adipose derived stem cell culture according to the invention inhibit the melanin synthesis and tyrosinase in order to ensure that inhibits the activity, and melanin-stimulating hormone on melanoma cell lines, the present invention One 　 next 　 produced 　 melanin and tyrosinase 　 activity was measured.

As a result, 　This 　According to the invention 　Adipose-derived 　 Stem cells 　The culture fluid 　Inhibits the 　production of melanin 　and 　Tyrosinase 　Inhibits the activity

I confirmed that

In addition, 　 followed by the invention 　 fat-derived 　 stem cells 　 culture fluid 　 with stem cells 　 administered 　 to the human body 　 skin 　 result 　 remarkable 　 skin darkening

It showed improvement 　 effect 　.

Therefore, 　this 　invention 　 fat-derived 　 stem cells 　 and 　 group 　 culture fluid 　 to prevent 　 pigmentation 　 sedimentation 　 or treat 　 whitening 　

There are 　 useful 　 　 number 　 in equipment.

According to the present invention, 　 using mesenchymal 　 adult stem cells 　 using 　 human 　 growth factor 　 containing 　 culture solution 　 production 　 melanin 　 synthesis

Inhibition 　 and 　 tyrosinase 　 activity was confirmed 　 inhibiting 　. Therefore, 　Adipose-derived 　 　 culture fluid 　 and 　 minutes from it

It is expected that 　 will be useful 　 used 　 can be used 　 by applying rihan　 protein to 　 drugs for whitening purposes, 　 quasi-drugs, cosmetics, etc.

This 　invention 　based on 　 　 　 more 　 in detail 　 is as follows 　, 　 the scope of this 　 invention is 　 following 　 　 limited by 　

It is not 　.

<Example 　1> 　Isolation 　 and 　 culture of adipose stem cells

Human 　 liposuction 　 10 ㎖ 　 (Leader's Dermatology, 　 Seoul) 　 same 　 volume of 　 phosphorylation 　 buffer 　 solution 　 washing 　 only adipose tissue 　 minutes

I did it.

　 extracellular matrix of adipose tissue 　37　℃?, 　5　%　CO2　in the incubator 　45　min 　0.075　%　 collagenase 　 enzyme treatment, 　 optimal effect

Bovine 　 treated 　 adipose tissue 　 1200 　 g 　 5 minutes 　 centrifugation 　 high density 　 stem cells 　 containing 　 stroma 　 blood vessels 　 fraction

I got it. Wash the pellet with 　phosphorylation 　 buffer 　 solution 　 70㎛ 　 nylon 　 cells 　 filter 　 remove 　 other 　 tissues

Histopaque-1077 (SIGMA) was used to separate 　cell fragments containing erythrocytes and mononuclear cells only.

Isolated 　Monocytes 　Dulbecco's Modified Eagle's Medium(DMEM), 10 % fetal　bovine　serum(FBS),　1　%

Stem cells were opened by removing 　37　℃, 　5　%　CO2, 　24　hours 　 culture 　 after 　 non-adhesive isolation 　 cells with 　 culture solution containing penicillin 　 streptomycin 　.

Transfer the isolated 　 stem cells 　10　cells/ml 　cells　suspended fluid 　10　㎖ into 　T25　 flask (area 　25　㎠, 　 capacity 　50　㎖)

It was cultured in the above conditions.

The cumulative group doubling time 　 is kept 　 in the flask 　 　 　 cells being cultured 　80　%　 until confluence 　　80

% 　 Passage culture was performed at the time of consolidation.

For subculture, 　Remove the culture solution 　Wash the flask 　PBS 　, and 　0.25 　% 　 Trypsin-EDTA (GIBCO) 　After dropping the cells 　

After 　 centrifugation 　, 　 cell count 　 and 　 viability 　 test 　 after 　 DMEM, 　10　%　FBS, 　1　%　penicillin　streptomycin

From the included 　 culture solution, it was 　 again 　 3 　 times 　 passage culture 　. The 　 process was repeated 　3　 times 　.

<Example 　 2> 　 stem cells 　 culture solution 　 production 　 isolated, 　 subcultured 　 fat-derived 　 stem cells 　 4 　 10 　 dogs 　 serum-free 　 medium 　DMEM/F12 (Invitrogen-Gibco

BRL, 　Grand　Island, 　NY) 　72　hour　after 　　　, 　300　g, 　5　min 　centrifugation　　 supernatant 　0.22

　 　 　 adipose stem cells 　 culture solution was prepared by filtration with a ㎛ 　 injection type 　 filter.

<Example 　3> 　Adipose stem cells 　 　 Protein 　 analysis of culture solution

3-1. Trypsin digestion of proteins

The 　 fat stem cells 　 culture solution prepared in <Example 　 2> was 　 freeze-dried using 　 freeze dryer 　. Dried 　 powder 　 sterilized

After 　 dissolved in distilled water 　 solid phase 　 extraction 　 using a cartridge (Waters, USA) 　 protein was recovered. Protein 　C18　reverse phase

Chromatography 　 (Chromolith 　, 　 Merck) was used to divide 　 into 6 　 groups. Each 　 fraction 　reduction buffer (50　mM

NH4HCO3, 　2　mM　DTT) was used to reduce 　 at 　20　 minutes at 　56　℃. Reduced 　 protein 　 alkylation 　 buffer solution (50　mM　NH4HCO3,

5　mM　iodoacetamide) was decomposed at 　37　℃ for 　15　 minutes 　 alkylation 　 limited 　, and 　 trypsin at 　37　℃ 　12　 hours 　.

3-2. LC-MS/MS analysis using Q-TOF

Trypsin 　 decomposed 　 each 　 group 　 peptide 　 fraction 　 Agilent 　 1100 　 LC 　 system (Agilent, 　 USA) 　Q-STAR　Excel　mass

　 was connected with a spectrometer (MDS　Sciex,　Toronto,　Canada) and 　separated 　 analyzed　. The result is 　Analyst　QS　software

Acquisition was obtained by using the information-dependent　acquisition　mode.

Polyion was selected using 　MS/MS 　. Each 　cycle was composed of 　1-s　MS and 　3-s　MS/MS, and 　acetonitrile

Using a 　linear 　LC of 12.5　%~40　% 　 for 　90　 minutes.

Each 　 bulb ion was analyzed 　 using 　tandem 　MS 　 selected 　 after 　 using LC-MS/MS.

LC-MS/MS was analyzed 3 times to determine the 　peptide 　 fraction of each 　 group.

3-3. Database search LC-MS/MS results are converted to human International by using MASCOT search engine (Matrix Science, London, United Kingdom).

Protein 　 Index (IPI) 　 protein 　 sequence 　 in the database 　 search was performed. MS's 　accuracy is 　1200　ppm, and 　MS/MS　accuracy is

0.3　Da, 　trypsin 　 decomposition 　 error 　 and 　 O-deoxy-carbamidomethylated 　 cysteine, 　 oxidation 　 of methionine 　 various 　 modifications, 　 N-horse

We searched for 　 at the level of acetylated 　 protein.

Results of database 　 search 　 total 　 112 　 proteins were identified, and 　 related 　 related to whitening 　 was selected and 　 was written.

As shown, in the adipocyte-derived stem cell culture medium, it inhibits tyrosinase, TRP1 and TRP 2, and 　downregulates Mitf (microphthalmia-associated 　transcription 　factor), or 　involves 　 metastasis of melanosomes.

It is known that 　 contains 　 many 　 　 　 　 　.

<Example 　 4> 　B16 　 Cell line 　 Melanin 　 Inhibition experiment

Rodent 　 Melanoma 　 Cell line 　 B16 　 Cell line 　 DMEM 　 In medium 　10　%　 Fetal calf serum 　100　 U/ml　 Penicillin, 　100　㎍/㎖

Streptomycin was added and cultured under the conditions of 　37　℃?, 　5　%　CO2,　.

3

Cultured 　B16 　 cells 　96-well 　 plates 　 2 　 per well 　 　 2 　 　 cells 　, and 　 melanin-stimulating hormone 　α-MSH　 respectively

100　nM　 and 　fat stem cells 　 culture solution 　0　%, 　10　%, 　50　%, 　100　% 　 sample solution 　200 µl 　 was treated at the same time. 72　 hours

After liver 　, 　CCK-8(Dojindo,　Gaithersburg,　MD)　solution　10ul was added to each 　well 　 and after 　3 　 time 　 　 was cultured and measured with microplate 　 reader (TECAN, 　 　, 450 nm). The measured 　 value was corrected by making 　 standard curve 　.

Next, 　 cultured 　 B16 　 cells in 　 plate 　1.5ⅹ10 Dogs 　 After inoculation 　 　 In Example 　 2 　 Obtained 　 Fat stem cells 　 Culture solution 　 10

　 each 　　0　%, 　10　%, 　50　%, 　100　%　 each 　 for one hour 　 　 before 　, 　α-MSH 　100　nM　 after phosphorylation 　 72 hours 　

After washing the medium with a buffer solution, add melanin extract (1NNaOH + 50% DMSO) per well, and wash it at 80°C by adding 100µl at a rate of 　 per well 　

After dissolving the fabric, the absorbance of melanin was measured at 492 nm with a microplate reader (refer to Fig. 3).

The result 　 fat-derived 　 stem cells 　 culture solution 　 50 　% 　 or 　 100 　% treated with 　 α-MSH 　 　 group 　 fat-derived 　 stem cells 　

Compared to the untreated 　 group 　 showed remarkable 　 melanin 　 production 　 inhibition 　 effect.

<Example 　5> 　Tyrosinase 　 inhibition experiment

5-1 Tyrosinase activity test

In a test tube 　0.1　M　phosphate buffer (pH　6.5)　220 　 and 　 extracted 　 from B16 　 cells 　 tyrosinase 　 20 　 and 　 mushroom 　 tyrosinaa

The first 20 µl was put in the order of 　. Add 　1.5　mM　Tyrosine　solution　40　µl to this solution and let it react at 　37　℃ for 10-15 minutes　,

The absorbance was measured at 490 nm using a microplate reader.

The 　 sample solution of Example 4 was 　 each 　200 µl 　 　 tyrosine 　 　 treated 　 before 　 　, 　 sample solution was 　 sample solution 　 instead of 　 0.1 　 　 phosphate buffer was added.

　Results 　 also 　 appearing in 1 　 bara 　, 　 results 　 fat-derived 　 stem cells 　 culture fluid 　 50 　% 　 or 　 100 　% treated with 　α-MSH 　

Compared to the 　 fat-derived 　 stem cells 　 culture solution 　 untreated 　 　 remarkable 　 tyrosinase 　 activity 　 inhibitory 　 effect

Showed.

5-2 Tyrosinase inhibition experiment

In a test tube, 　0.1　M　phosphate buffer (pH　7.0)　850　ul and 　above 　 Example 　4 　 sample solution 　50　 　 and mushroom 　 tyrosinase 　50 µl were sequentially added and reacted at 37°C for 6 minutes.

Add 0.06 mM L-DOPA (L-3,4-dihydroxyphenylalanine) solution 　50　 　 to this solution 　 and then react at 37 　℃ for 1 　 minute 　.　0.1　M　phosphoric acid

A salt buffer solution (pH 7.0) was added.

<Example 　6> 　Western 　blot 　 analysis of tyrosinase

Cultured 　B16　 cells 　1.5ⅹ10　 each in 　plate 　After 　Adipose stem cells 　 Culture solution 　10㎖　 　Each 　0　%, 　10　%, 　50%, 　50　%

After each% 　 two 　 hours 　 before 　 treatment 　, the 　 sample solution of Example 4 was treated with 10 ml_

After 48 　 hours 　　 cells 　RIPA 　 buffer solution (50　mM　Tris-HCl,　0.15　M　NaCl,　1　mM　EDTA,　1　%　Tritons　X-100,　

Decomposition of mM　NaF,　1　mM　Na3VO4,　5　mM　dithiothreitol,　1　㎍/㎖　leupeptin　and　20　㎍/㎖　PMSF,　pH　7.4)　　

Thus, a protein was obtained. 25　mg of 　protein was separated using 　8　%　SDS-polyacryamide　gel　electrophoresis.

The protein-separated gel was transferred to the PVDF membrane. PVDF membrane 　anti-tyrosinase(1:500 times 　dilution)　antibody,　α-tubulin　antibody

(1:10,000 times 　 dilution) 　 after culture 　, 　horseradish　peroxidase-conjugated　anti-goat　IgG　antibodies (1:10,000　dilutio

It was cultured as n) again. The obtained 　band was exposed to 　immunobilon 　western 　reagent 　 on X-ray 　 film 　 confirmed

All.

Claims (3)

A cosmetic composition for whitening comprising a stem cell culture solution or a protein isolated from the culture solution.
The cosmetic composition according to claim 1, wherein the stem cells are adult stem cells or mesenchymal stem cells derived from mammals.
The cosmetic composition of claim 2, wherein the adult stem cells are at least one stem cell selected from the group consisting of adipose tissue, bone marrow tissue, and cord blood.