This research was carried out to: Test the effects of umbilical cord extracts on proliferation of human keratinocyte and activity of enzyme responsible for melaline production of human melaninocyte in vitro.
Journal of military pharmaco-medicine 7-2013 EFFECTS OF UMBILICAL CORD EXCTRACTS ON PROLIFERATION OF HUMAN KERATINOCYTE AND EXPRESSION OF TYROSINE KINASE GENE OF MELANINOCYTE IN VITRO Phan Kim Ngoc*; Pham Van Phuc*; Dang Thi Tung Loan* Dinh Thanh Uyen**; Le Van Dong*** summary Skin aging is a contuinuous process influenced by many factors such as free redicals, sun exposure etc This process is characterized with changes in many skin components, primarily affected by three main cell types: fibroblast, keratinocyte and melaninocyte Decreased number of keratinocytes leads to less soften skin; while increase or decrease melanine production will lead to imbalance of skin color leading to either backhead or vitiligo This suty evaluates the effects of umbilical cord exctracts on proliferation of human keratinocyte and melaline production of melaninocyte in vitro 25 preparations made of types of extracts included: extracellular exctract of cord tissue; intracellular extract of cord cells; mixtures of these two extracts; intracellular extract of umbilical cord stem cells, were supplemented to culture media of human skin keratinocyte and melaninocyte Keratinocyte cell proliferation was evalutaetd by MTT technique; melanine production was indirectly evaluated via tyrosine kinase gene expression by Realtime RT-PCR technique The results show that supplementation of 7.5% of preparation 3-5 containing a combination of both extra- and intra-cellular extracts of umbilical cord led to the highest cell proliferation of k eratinocyte However, at this concentration, the 3-5 preparation did not inhibite the expression of tyrosine kinase gene in melaninocyte These data suggests that umbilical cord extract would be a potential source of material for skin softening product but not brightening one * Key words: Skin aging; Skin softening; Skin brightening; Stem cell; Umbilical cord extract INTRODUCTION Skin aging is known to be caused mainly by exposure to light, mostly important is sunlight UV Aging affects three main skin cell types: fibroblast, keratinocyte and melaninocyte Among them, skin softness and brightness are mainly determined by keratinocyte Keratinocyte is the main type of dermis, accounting for about 90% of dermis cells, works as barrier to protect skin from harmful factors from environment such as pathologic microbes (bacteria, fungi, parasites, and viruses), heat, UV light or dehydration [5] Once a pathogen comes to contact to skin, keratinocyte may react by secreting inflamation mediators such as chemokines CXCL10, CCL2 to attract white blood cells to the invasion places Keratinocyte plays an important role in filling up the defects of injured skin Once a wound happened, keratinocytes from hair bulb will migrate in to fix the wound temporally the keratincytes from epithelial will replace them [3, 4] * Hochiminh City Nationnal University ** Mekostem Stem Cell Bank *** Vietnam Military Medical University Address correspondence to Le Van Dong: Vietnam Military Medical University 56 Journal of military pharmaco-medicine 7-2013 E.mail: levandong@yahoo.com Melaninocytes are cells which help produce skin pigment - melanin, to form skin color By melanogenesis process, melaninocyte produces melanin for skin, eye and hair In human, melanogenesis is classified into two types: basal melanogenesis and activated melanogenesis In general, people who have bright skin have low basal melanogenesis Exposure to UV-B radiation will increase activated melanogenesis The purpose of melanogenesis is to protect dermis layers as UV can damage DNA Melanin absorbs UV very well and prevents it from penetration to dermis [2] Melanogenesis require amino acid tyrosine as material and enzyme tyrosinase, by which tyrosinase convert tyrosine to melanin been standardized in our previous study Human skin samples, which were voluntarily donated after plastic surgery, were obtained from Cho Ray Hospital Skin samples were kept in PBS supplemented with penicillinstreptomycin and anti-mycotic and transferred to the laboratory In the lab, skin samples were washed with 1X antibiotic-mycotic two times followed two times with D-PBS, and then placed on petri dish to remove fat tissue with blade and scissors Cleaned skin was cut into small pieces of 0.5x0.5 cm then incubated in dispase II solution 0.5% (Sigma-Aldrich, St Louis, CA) at 370C for hours Finally, separate dermis and epidermis layers out of each others and used them for the next experiments Recently, stem cells and its extract have been extensively studied to demonstrate that they have stimulating effects on skin regeneration and rejuvenation, especially skin softening and brightening It is postulated that stem cells and stem cell-derived factors directly and/or indirectly affect on keratinocyte proliferation and inhibit melanogenesis process in melaninocyte Since then, this research was carried out to: Test the effects of umbilical cord extracts on proliferation of human keratinocyte and activity of enzyme responsible for melaline production of human melaninocyte in vitro In order to isolate keratinocyte, the dermis layer was cut into small pieces of - mm2 and then placed in flask T-25 (Nunc, Denmark) with - 10 pieces per flask mL medium Stemline Keratinocyte medium (Sigma-Aldrich, St Louis, CA) supplemented with HKGS (Life Technologies, USA) was then added to the flask for culturing at 37oC, 5% CO2 in an incubator The medium was replaced every four days Sub-culture was done when the cells reached 70 - 80% confluence using trypsin/EDTA 0.25% (GeneWorld, Hochiminh City, Vietnam) During subculturing, protease inhibitor (Sigma-Aldrich, St Louis, CA) was used for neutralization of eccess trypsin MATERIAL AND METHODS Isolation of human keratinocyte and melaninocyte Adult human skin keratinocytes and melaninocytes were isolated and cultured following routine procedures which have In order to isolate melininocyte, the epidermis layer was cut into small pieces of - mm2 and then placed in flask T-25 (Nunc, Denmark) with - 10 pieces per flask mL medium 254CF supplemented with HMGS-2 (Invitrogen, USA) was then added Journal of military pharmaco-medicine 7-2013 to the flask for culturing at 37 oC, 5% CO2 in an incubator The medium was replaced every four days Sub-culture was done when the cells reached 70 - 80% confluence using trypsin/EDTA 0.25% (GeneWorld, Hochiminh City, Vietnam) Keratinocytes and melaninocytes of the passage were analyzed for expression of specific markers, CD24 for keratinocyte and CD117 for melaninocyte with the following protocol Once the cells reached 70% confluence, they were treated with trypsin/EDTA 0.25% (GeneWorld, Hochiminh City, Vietnam) then collect the single cell suspension 106 cells were stained with specific antibody for 30 minutes in dark at 40C The antibody stained cells were then washed two times with FACS flow to remove unbound antibody then resuspened in 500 µL FACSflow The cells were then analyzed with FacsCalibur system (BD Bioscience) All data were analyzed by CellQuest Pro with 10.000 cells rd 130 newborn umbilical cords samples that voluntarily donated by biological mothers were collected for research purpose Mothers and the cords were selected following a strictly screening tests followed NetCord standards and had negative results to all HIV, HBV, HCV, CMV as described in our previous paper [1] After collection in hospitals and transferred to the lab, each cord was taken a portion with the length ranging from 18 to 22 cm The cords were then stored at -800C till further usage * Preparation of extracellular extract: Frozen cords were defrosted in the 37oC water bath then washed with physiological saline; sliced, add buffer (saline supplemented with protease inhibitor, Sigma-Aldrich, USA) to protect protein from hydrolysis during the preparation process The mixture was milled thoroughly then centrifuged at 3,000 rpm for 20 minutes at 4oC The supernatant (namely as cord tissue extract or extracellular extract) was Umbilical cord stem cell sorting then serially diluted to different concentrations Umbilical cord tissues were processed mechanically to cell suspension Stem cells were sorted by fluorescence activated cell sorting (FACS) method on FACSJazz system (BD Bioscience, USA) Each stem cell type was sorted as it stained with antibody to a specific marker: CD90 for mesenchymal stem cell; CD117 for epithelial stem cell; CD113 for vascular endothelial stem cell and kept at -80oC till the next usage Preparation of cord cell and tissue extracts extract or intracellular extract) was then serially * Umbilical cord collection and storage: * Preparation of cellular extract: The pellet obtained from above was quickly frozen with liquid nitrogen and defrosted with warm buffer (saline supplemented with protease inhibitor) then centrifuged at 3,000 rpm for 20 minutes at 4oC The supernatant obtained after this step (namely as cord cellular diluted to different concentrations and kept at -80oC till the next usage The cord tissue extract and cord cellular extract were mixed 59 Journal of military pharmaco-medicine 7-2013 together in different ratio to form various formulas for further activity testing * Preparation of stem cell extract: Stem cell cellular extract was also prepared as described above * Cord extracts combinations: main preparations were formulated: cord tissue extract, total cord cellular extract, cord stem cell extract, mixture of cord tissue and cord cellular extracts, control (physiological saline) Each preparation was diluted into five different concentrations and form 25 testing formulas coded as followed: Cord tissue extract: solution 1-1; 1-2; 1-3; 1-4; 1-5; Cord cellular extract: solution 2-1; 2-2; 2-3; 2-4; 2-5; Mixture of cord tissue and cord cellular extracts: solution 3-1; 3-2; 3-3; 3-4; 3-5; Cord stem cell extract: solution 4-1; 4-2; 4-3; 4-4; 4-5; Control: solution 5-1; 5-2; 5-3; 5-4; 5-5 Cell proliferation assessment by MTT assay The cell proliferation was evaluated following the instruction of manufacturer (Cell proliferating kit, GeneWorld, Hochiminh City, Vietnam) as followed: Take the 96 well cell culture plates out of the incubator and move to the clean cell culture area, add sterile MTT equal to 10% of final volume, put the 96 well cell culture plates back into the incubator for another - hours; after the incubation, take the plates out of the incubator and dissolved the MTT formazan crystals by adding equal volume of solvent The absorbent was measured within hour after adding the solvent with a spectrometer (Multimode Reader DTX880, Beckman-Coulter, USA) 570 nm (measure wavelength) and 690 nm (referent wavelength) Realtime RT-PCR analysis Total RNA was extracted as described in our previous study [6] The Real-time RT-PCR analysis was performed on Eppendorf gradient S thermal Cycler system (EppendorfAG, Hamburg, Germany) Data analysis All tests were repeated three times Value p ≤ 0.05 is considered as statistical significant Data was analyzed by Statgraphics software 7.0 (Statgraphics Graphics System, Warrenton, VA) RESULTS AND DISCUSSION Effects of different extraction formulas on the proliferation of keratinocyte Skin softening is one of the properties in the modern beauty products While wrinkle is results of structural breakdown of skin extracellular proteins, large hair bulb micro scar leading to rough skin, skin softening will stimulate keratinocytes of epidermis to develop to fill up micro defects on skin With the aim to develop a product, which has skin softening effetc, we test the effects of main ingredient on the proliferation of keratinocyte After testing the effects of 25 formulas and identified that supplementation of 7.5% of formula 3-5 having anti-wrinkle effect in vitro Based of that data, we continue to test 60 Journal of military pharmaco-medicine 7-2013 if formula 3-5 also can increase skin softness culture medium 7.5% of each of all 25 Targeting for a skin care product that has formulas Data on keratinocyte proliferation both anti-wrinkle and skin softening effects, are shown in figure OD value OD value OD value OD value we then selected to supplement to the 61 Journal of military pharmaco-medicine 7-2013 Figure 1: Keratinocyte proliferation when adding 25 formulas at 7.5% after (0, 2, 4, 6, and 10 days from left to right) Data from figure show that supplementation of 25 formulas have different effects on keratinocyte proliferation In general, it is different from the effects on fibroblast, some formulas has no clear effect on keratinocyte in comparison to control group, esspecially some formulas including 1-3, 1-4, 1-5, 2-1, 2-3, 2-5, 4-1, 4-3, 4-4 inhibite the proliferation of keratinocyte Among 25 formulas tested, formula 3-5 also shows most positive effects in stimulation the proliferation of human keratinocyte enzyme tyrosine kinase Since then in evaluation of skin brightness we indirectly test the ability to inhibit enzyme tyrosine kinase by RT-PCR The data show that supplementation of formula 3-5 at 7.5% to melaninocyte culture medium; the expression of tyrosine kinase gene did not decrease as compared to control (repeated three times, p > 0,5) (Figure 2.) It is clear that formula 3-5 did not inhibit the expression of tyrosine kinase gene; in the other words it did not reduce melanin production in melaninocyte Similar to the effects of umbilical cord extracts on fibroblast, keratinocytes also requires both extracellular and intracellular proteins in order to get optimum proliferation rate in comparison to just adding either extracellular or intracellular extract Evaluation of concentration of formula 3-5 melanin production in melaninocyte Melanin is natural pigment of skin, produced by melaninocyte Skin pigments make skin darker; increasement of melanin production in some places make black spots on skin Consequently, black spots are condition caused by over melanin production All melanin are made from polyacetylene Most of them are products of tyrosine amino acid Melanin synthesis from tyrosin requires Figure 2: Comparison of tyrosine kinase gene expression between control and formula 3-5 supplemented group In short, formula 3-5 from umbilical crod cells/tissue lack of factor that reduce melanin production In order to produce a brightening skin care product, one may think of adding 62 Journal of military pharmaco-medicine 7-2013 a strong antioxidant or a factor to inhibit melanin production or stimulation of melanin breakdown CONCLUSION This in vitro study demonstrates that umbilical cord extracts have some effetcs on human skin keratinocyte Especially, formula 3-5 which is mixture of cord tissue and cellular extracts, keratinocyte got strongest stimulation effect at 7.5% However, at this concentration, no inhibition on melanin production was observed These data suggest that umbilical cord extracts can be used for development of skin softening but not brightening product ACKNOWLEDGMENT This research was partially financially supported by research project “Development and evaluation the effects of beauty product from umbilical cord stem cell” Code: 1882010 sponsored by Department of Science and Technology of Hochiminh City Agar N and Young AR Melanogenesis: a photoprotective response to DNA damage? Mutation Research 2005, 571 (1-2), pp.121-132 Claudinot S, Nicolas M, Oshima H, Rochat A, Barrandon Y Long-term renewal of hair follicles from clonogenic multipotent stem cells Proceedings of the National Academy of Sciences of the United States of America 2005, 102 (41), pp.14677-1482 Ito M, Liu Y, Yang Z, Nguyen J, Liang F, Morris RJ, Cotsarelis G Stem cells in the hair follicle bulge contribute to wound repair but not to homeostasis of the epidermis Nature Medicine 2005, 11 (12), pp.1351-1354 McGrath JA, Eady RAJ, Pope FM Anatomy and Organization of Human Skin In Burns T, Breathnach S, Cox N, Griffiths C Rook's Textbook of Dermatology (7th ed) Blackwell Publishing 2004, p.4190 Phuc P.V Nhung T.H Loan D.T, Chung D.C, Ngoc P.K Differentiating of banked human umbilical cord blood-derived mesenchymal stem cells into insulin-secreting cells In Vitro Cell Dev Biol Anim 2011, 47 (1), pp.54-63 REFERENCES Phạm Thúy Trinh, Lê Văn Đông CS Nghiên cứu phân lập tế bào gốc trung mô từ màng dây rốn trẻ sơ sinh Tạp chí Thơng tin Y dược Số Chun đề Miễn dịch học 2010, tr1-6 63 Journal of military pharmaco-medicine 7-2013 64 ... the effects of umbilical cord extracts on proliferation of human keratinocyte and activity of enzyme responsible for melaline production of human melaninocyte in vitro In order to isolate keratinocyte, ... positive effects in stimulation the proliferation of human keratinocyte enzyme tyrosine kinase Since then in evaluation of skin brightness we indirectly test the ability to inhibit enzyme tyrosine kinase. .. inhibit the expression of tyrosine kinase gene; in the other words it did not reduce melanin production in melaninocyte Similar to the effects of umbilical cord extracts on fibroblast, keratinocytes