Sains
Malaysiana 51(5)(2022): 1425-1436
http://doi.org/10.17576/jsm-2022-5105-13
Characterization
of Keratinocytes, Fibroblasts and Melanocytes Isolated from Human Skin using
Gene Markers
(Pencirian
Keratinosit, Fibroblas dan Melanosit Dipencilkan daripada Kulit Manusia
menggunakan Penanda Gen)
PAU-RING
WONG1, NUR FATIN NABILAH MOHD SAHARDI1, JEN-KIT TAN1, KIEN-HUI CHUA2, WAN ZURINAH WAN NGAH1 & SUZANA MAKPOL1,*
1Department of Biochemistry, Faculty
of Medicine, Level 17, Preclinical Building, Universiti
Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif,
Bandar Tun Razak, 56000 Cheras, Kuala Lumpur,
Federal Territory, Malaysia
2Department of Physiology, Faculty
of Medicine, Level 17, Preclinical Building, Universiti
Kebangsaan Malaysia, Kuala Lumpur Campus, Jalan Yaacob Latif, Bandar
Tun Razak, 56000 Cheras, Kuala Lumpur, Federal
Territory, Malaysia
Received: 24 July 2021/Accepted: 16 September
2021
Abstract
Cells isolated from skin have wide
applications in studies of the pathogenesis of skin-related diseases and the
construction of 3D skin equivalents. This study aimed to isolate keratinocytes,
fibroblasts, and melanocytes from human foreskin and characterize the purity of
the cell types. Keratinocytes, fibroblasts, and melanocytes from human foreskin were isolated by differential trypsinization and media selection. The purity of the
cell types was characterized based on the expression
of gene markers. The assessment of gene marker expression involved RNA
extraction, primer design, quantitative polymerase chain reaction (qPCR) and
immunocytochemical staining. Our results showed that in cocultures of keratinocytes and fibroblasts
isolated from the dermis, fibroblasts
could be separated from keratinocytes by quick trypsinization and culture in
Dulbecco’s modified Eagle’s medium. The remaining
keratinocytes are cultured in Epilife
medium. Melanocytes in cocultures of melanocytes and keratinocytes
isolated from the epidermis could be
selected by changing Epilife medium to M254 medium. Gene marker results
suggested that cytokeratin 14 (CK14) is a suitable marker for keratinocytes,
elastin (ELN) is a suitable marker for fibroblasts, and tyrosinase (TYR) and tyrosinase-related proteins 1
and 2 (TYRP1 and TYRP2) are suitable markers for melanocytes. In
conclusion, keratinocytes, fibroblasts, and melanocytes can be isolated from
the same human foreskin sample by differential trypsinization and media
selection techniques and characterized by
suitable gene markers. This finding will aid in the isolation of pure skin cell types for various applications in regenerative medicine and toxicity studies.
Keywords:
Epidermis; fibroblasts; gene markers; keratinocytes; melanocytes
Abstrak
Sel yang
dipencilkan daripada kulit mempunyai kegunaan meluas dalam kajian patogenesis
penyakit berkaitan dengan kulit serta pembinaan setara kulit 3D. Kajian ini
bertujuan untuk memencilkan sel keratinosit, fibroblas dan melanosit daripada
kulit manusia serta mencirikan ketulenan jenis sel tersebut. Sel keratinosit,
fibroblas dan melanosit telah dipencilkan dengan cara pentripsinan pembezaan
dan pemilihan media. Sementara itu, ketulenan jenis sel telah dicirikan melalui
ekspresi penanda gen. Pengekspresan penanda gen melibatkan pengekstrakan RNA, mereka bentuk primer, tindak balas rantai polimerase (qPCR) kuantitatif dan
pewarnaan imunositokimia. Hasil yang diperoleh menunjukkan fibroblas di dalam
ko-kultur sel keratinosit-fibroblas yang dipencilkan daripada lapisan dermis
boleh diasingkan daripada keratinosit melalui proses pentripsinan pantas dan
dikulturkan di dalam medium Eagle Pengubahsuaian Dulbecco, manakala sel
keratinosit yang berbaki dikulturkan di dalam medium Epilife. Sel melanosit di
dalam ko-kultur melanosit-keratinosit yang dipencilkan daripada lapisan
epidermis boleh dipilih dengan menukarkan medium Epilife kepada medium M254.
Keputusan penanda gen mencadangkan bahawa gen sitokeratin 14 (CK14) adalah
penanda gen yang sesuai untuk sel keratinosit dan gen elastin (ELN) sesuai
digunakan untuk sel fibroblas. Sementara itu, gen tirosin (TYR) dan tirosinase
berkaitan protein 1 dan 2 (TYRP1 dan TYRP2) sesuai digunakan untuk sel
melanosit. Secara kesimpulannya sel keratinosit, fibroblas dan melanosit boleh
dipencilkan daripada sumber kulit manusia yang sama melalui proses pentripsinan
pembezaan dan teknik pemilihan media; serta dicirikan melalui penanda gen yang
sesuai. Hasil kajian ini boleh membantu dalam memencilkan jenis sel kulit tulen
untuk pelbagai kegunaan dalam perubatan regeneratif dan kajian kesitotoksikan.
Kata
kunci: Epidermis; fibroblas; keratinosit; melanosit; penanda gen
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*Corresponding author;
email:
suzanamakpol@ppukm.ukm.edu.my
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