Effects of umbilical cord exctracts on proliferation of human keratinocyte and expression of tyrosine kinase gene of melaninocyte in vitro

Nội dung text: Effects of umbilical cord exctracts on proliferation of human keratinocyte and expression of tyrosine kinase gene of melaninocyte in vitro

1. 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 4 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 keratinocyte. 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 microbes (bacteria, fungi, parasites, and viruses), heat, UV light or dehydration [5]. Skin aging is known to be caused mainly Once a pathogen comes to contact to skin, by exposure to light, mostly important is keratinocyte may react by secreting inflamation sunlight UV. Aging affects three main skin mediators such as chemokines CXCL10, cell types: fibroblast, keratinocyte and CCL2 to attract white blood cells to the melaninocyte. Among them, skin softness and brightness are mainly determined by invasion places. Keratinocyte plays an keratinocyte. important role in filling up the defects of injured skin. Once a wound happened, Keratinocyte is the main type of dermis, accounting for about 90% of dermis cells, keratinocytes from hair bulb will migrate in works as barrier to protect skin from harmful to fix the wound temporally the keratincytes factors from environment such as pathologic 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
2. Journal of military pharmaco-medicine 7-2013 E.mail: levandong@yahoo.com Melaninocytes are cells which help produce been standardized in our previous study. skin pigment - melanin, to form skin color. Human skin samples, which were voluntarily By melanogenesis process, melaninocyte donated after plastic surgery, were obtained produces melanin for skin, eye and hair. from Cho Ray Hospital. Skin samples were In human, melanogenesis is classified into kept in PBS supplemented with penicillin- two types: basal melanogenesis and activated streptomycin and anti-mycotic and transferred melanogenesis. In general, people who have to the laboratory. In the lab, skin samples bright skin have low basal melanogenesis. were washed with 1X antibiotic-mycotic two Exposure to UV-B radiation will increase times followed two times with D-PBS, and activated melanogenesis. The purpose of then placed on petri dish to remove fat tissue melanogenesis is to protect dermis layers with blade and scissors. Cleaned skin was cut as UV can damage DNA. Melanin absorbs into small pieces of 0.5x0.5 cm then incubated UV very well and prevents it from penetration in dispase II solution 0.5% (Sigma-Aldrich, to dermis [2]. Melanogenesis require amino St Louis, CA) at 370C for 2 hours. Finally, acid tyrosine as material and enzyme separate dermis and epidermis layers out tyrosinase, by which tyrosinase convert of each others and used them for the next tyrosine to melanin. experiments. Recently, stem cells and its extract have In order to isolate keratinocyte, the dermis been extensively studied to demonstrate layer was cut into small pieces of 2 - 3 mm2 that they have stimulating effects on skin and then placed in flask T-25 (Nunc, Denmark) regeneration and rejuvenation, especially with 5 - 10 pieces per flask. 2 mL medium skin softening and brightening. It is postulated Stemline Keratinocyte medium (Sigma-Aldrich, that stem cells and stem cell-derived factors St Louis, CA) supplemented with HKGS directly and/or indirectly affect on keratinocyte (Life Technologies, USA) was then added to o proliferation and inhibit melanogenesis the flask for culturing at 37 C, 5% CO2 in an process in melaninocyte. Since then, this incubator. The medium was replaced every research was carried out to: Test the effects four days. Sub-culture was done when the of umbilical cord extracts on proliferation of cells reached 70 - 80% confluence using human keratinocyte and activity of enzyme trypsin/EDTA 0.25% (GeneWorld, Hochiminh responsible for melaline production of human City, Vietnam). During subculturing, protease melaninocyte in vitro. inhibitor (Sigma-Aldrich, St Louis, CA) was used for neutralization of eccess trypsin. MATERIAL AND METHODS In order to isolate melininocyte, the 1. Isolation of human keratinocyte and epidermis layer was cut into small pieces of melaninocyte. 2 - 3 mm2 and then placed in flask T-25 Adult human skin keratinocytes and (Nunc, Denmark) with 5 - 10 pieces per flask. melaninocytes were isolated and cultured 2 mL medium 254CF supplemented with following routine procedures which have HMGS-2 (Invitrogen, USA) was then added 2
3. Journal of military pharmaco-medicine 7-2013 o to the flask for culturing at 37 C, 5% CO2 130 newborn umbilical cords samples that in an incubator. The medium was replaced voluntarily donated by biological mothers every four days. Sub-culture was done when were collected for research purpose. Mothers the cells reached 70 - 80% confluence using and the cords were selected following a trypsin/EDTA 0.25% (GeneWorld, Hochiminh strictly screening tests followed NetCord City, Vietnam). standards and had negative results to all Keratinocytes and melaninocytes of the HIV, HBV, HCV, CMV as described in our 3rd passage were analyzed for expression previous paper [1]. After collection in hospitals of specific markers, CD24 for keratinocyte and transferred to the lab, each cord was and CD117 for melaninocyte with the following taken a portion with the length ranging from protocol. Once the cells reached 70% confluence, 18 to 22 cm. The cords were then stored at they were treated with trypsin/EDTA 0.25% -800C till further usage. (GeneWorld, Hochiminh City, Vietnam) then * Preparation of extracellular extract: collect the single cell suspension. 106 cells Frozen cords were defrosted in the 37oC were stained with specific antibody for 30 water bath then washed with physiological minutes in dark at 40C. The antibody stained saline; sliced, add buffer (saline supplemented cells were then washed two times with with protease inhibitor, Sigma-Aldrich, USA) FACS flow to remove unbound antibody then to protect protein from hydrolysis during the resuspened in 500 µL FACSflow. The cells were then analyzed with FacsCalibur system preparation process. The mixture was milled (BD Bioscience). All data were analyzed by thoroughly then centrifuged at 3,000 rpm for o CellQuest Pro with 10.000 cells. 20 minutes at 4 C. The supernatant (namely as cord tissue extract or extracellular extract) was 2. Umbilical cord stem cell sorting. then serially diluted to different concentrations Umbilical cord tissues were processed and kept at -80oC till the next usage. mechanically to cell suspension. Stem cells * Preparation of cellular extract: were sorted by fluorescence activated cell sorting (FACS) method on FACSJazz system The pellet obtained from above was quickly (BD Bioscience, USA). Each stem cell type frozen with liquid nitrogen and defrosted was sorted as it stained with antibody to a with warm buffer (saline supplemented with specific marker: CD90 for mesenchymal protease inhibitor) then centrifuged at 3,000 stem cell; CD117 for epithelial stem cell; rpm for 20 minutes at 4oC. The supernatant CD113 for vascular endothelial stem cell. obtained after this step (namely as cord cellular 3. Preparation of cord cell and tissue extract or intracellular extract) was then serially extracts. diluted to different concentrations and kept at -80oC till the next usage. The cord tissue * Umbilical cord collection and storage: extract and cord cellular extract were mixed 59
4. Journal of military pharmaco-medicine 7-2013 together in different ratio to form various Reader DTX880, Beckman-Coulter, USA) formulas for further activity testing. 570 nm (measure wavelength) and 690 nm * Preparation of stem cell extract: (referent wavelength). Stem cell cellular extract was also prepared 5. Realtime RT-PCR analysis. as described above. Total RNA was extracted as described * Cord extracts combinations: in our previous study [6]. The Real-time 5 main preparations were formulated: cord RT-PCR analysis was performed on Eppendorf tissue extract, total cord cellular extract, cord gradient S thermal Cycler system (Eppendorf- stem cell extract, mixture of cord tissue and AG, Hamburg, Germany). cord cellular extracts, control (physiological 6. Data analysis. saline). Each preparation was diluted into five different concentrations and form 25 All tests were repeated three times. Value testing formulas coded as followed: Cord p ≤ 0.05 is considered as statistical significant. tissue extract: solution 1-1; 1-2; 1-3; 1-4; Data was analyzed by Statgraphics software 1-5; Cord cellular extract: solution 2-1; 2-2; 7.0 (Statgraphics Graphics System, Warrenton, 2-3; 2-4; 2-5; Mixture of cord tissue and VA). cord cellular extracts: solution 3-1; 3-2; 3-3; 3-4; 3-5; Cord stem cell extract: solution RESULTS AND DISCUSSION 4-1; 4-2; 4-3; 4-4; 4-5; Control: solution 5-1; 1. Effects of different extraction formulas 5-2; 5-3; 5-4; 5-5. on the proliferation of keratinocyte. 4. Cell proliferation assessment by MTT Skin softening is one of the properties in assay. the modern beauty products. While wrinkle The cell proliferation was evaluated following is results of structural breakdown of skin the instruction of manufacturer (Cell proliferating extracellular proteins, large hair bulb micro kit, GeneWorld, Hochiminh City, Vietnam) scar leading to rough skin, skin softening as followed: Take the 96 well cell culture will stimulate keratinocytes of epidermis to plates out of the incubator and move to the develop to fill up micro defects on skin. With clean cell culture area, add sterile MTT the aim to develop a product, which has equal to 10% of final volume, put the 96 well skin softening effetc, we test the effects cell culture plates back into the incubator for another 3 - 4 hours; after the incubation, of main ingredient on the proliferation of take the plates out of the incubator and keratinocyte. dissolved the MTT formazan crystals by After testing the effects of 25 formulas adding equal volume of solvent. The absorbent and identified that supplementation of 7.5% was measured within 1 hour after adding of formula 3-5 having anti-wrinkle effect in the solvent with a spectrometer (Multimode vitro. Based of that data, we continue to test 60
5. 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 1. we then selected to supplement to the OD value OD value OD value OD value OD value OD OD value OD 61
6. Journal of military pharmaco-medicine 7-2013 Figure 1: Keratinocyte proliferation when adding 25 formulas at 7.5% after (0, 2, 4, 6, 8 and 10 days from left to right). Data from figure 1 show that supplementation enzyme tyrosine kinase. Since then in of 25 formulas have different effects on evaluation of skin brightness we indirectly keratinocyte proliferation. In general, it is test the ability to inhibit enzyme tyrosine different from the effects on fibroblast, some kinase by RT-PCR. The data show that formulas has no clear effect on keratinocyte supplementation of formula 3-5 at 7.5% to in comparison to control group, esspecially melaninocyte culture medium; the expression some formulas including 1-3, 1-4, 1-5, 2-1, of tyrosine kinase gene did not decrease as 2-3, 2-5, 4-1, 4-3, 4-4 inhibite the proliferation compared to control (repeated three times, of keratinocyte. Among 25 formulas tested, p > 0,5) (Figure 2.). It is clear that formula formula 3-5 also shows most positive effects 3-5 did not inhibit the expression of tyrosine in stimulation the proliferation of human kinase gene; in the other words it did not keratinocyte. 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. 2. 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 Figure 2: Comparison of tyrosine kinase gene in some places make black spots on skin. expression between control and formula Consequently, black spots are condition 3-5 supplemented group. caused by over melanin production. In short, formula 3-5 from umbilical crod All melanin are made from polyacetylene. cells/tissue lack of factor that reduce melanin Most of them are products of tyrosine amino production. In order to produce a brightening acid. Melanin synthesis from tyrosin requires skin care product, one may think of adding 62
7. Journal of military pharmaco-medicine 7-2013 a strong antioxidant or a factor to inhibit 2. Agar N and Young AR. Melanogenesis: a melanin production or stimulation of melanin photoprotective response to DNA damage?. breakdown. Mutation Research. 2005, 571 (1-2), pp.121-132. 3. Claudinot. S, Nicolas. M, Oshima. H, CONCLUSION Rochat. A, Barrandon. Y. Long-term renewal of hair follicles from clonogenic multipotent stem This in vitro study demonstrates that cells. Proceedings of the National Academy of umbilical cord extracts have some effetcs Sciences of the United States of America. 2005, on human skin keratinocyte. Especially, formula 102 (41), pp.14677-1482. 3-5 which is mixture of cord tissue and 4. Ito. M, Liu. Y, Yang. Z, Nguyen. J, Liang. cellular extracts, keratinocyte got strongest F, Morris. RJ, Cotsarelis. G. Stem cells in the stimulation effect at 7.5%. However, at this hair follicle bulge contribute to wound repair but concentration, no inhibition on melanin not to homeostasis of the epidermis. Nature production was observed. These data suggest Medicine. 2005, 11 (12), pp.1351-1354. that umbilical cord extracts can be used for 5. McGrath JA, Eady RAJ, Pope FM. development of skin softening but not Anatomy and Organization of Human Skin. In brightening product. Burns T, Breathnach S, Cox N, Griffiths C. Rook's Textbook of Dermatology (7th ed). Blackwell ACKNOWLEDGMENT Publishing. 2004, p.4190. This research was partially financially 6. Phuc P.V. Nhung T.H. Loan D.T, Chung supported by research project “Development D.C, Ngoc P.K. Differentiating of banked human and evaluation the effects of beauty product umbilical cord blood-derived mesenchymal stem from umbilical cord stem cell” Code: 188- cells into insulin-secreting cells. In Vitro Cell Dev 2010 sponsored by Department of Science Biol Anim. 2011, 47 (1), pp.54-63. and Technology of Hochiminh City. REFERENCES 1. Phạm Thúy Trinh, Lê Văn Đông và CS. Nghiên cứu phân lập tế bào gốc trung mô từ màng dây rốn trẻ sơ sinh. Tạp chí Thông tin Y dược. Số Chuyên đề Miễn dịch học. 2010, tr1-6. 63
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