1. UKM Learning and Research Repository
2. PHD Thesis / Tesis PHD
3. Faculty of Medicine / Fakulti Perubatan

Title:	Pencirian, pembentukan dan perkembangan kulit gantian daripada sel stem amnion manusia bagi tujuan regenerasi kulit
Authors:	Siti Fatimah Simat (P42704)
Supervisor:	Hayati Abdul Rahman, Prof. Dr.
Keywords:	Kulit gantian Sel stem Regenerasi kulit Universiti Kebangsaan Malaysia -- Dissertations
Issue Date:	9-Jun-2012
Description:	Sel daripada amnion manusia boleh menjadi sumber sel stem alternatif untuk tujuan regenerasi kulit kerana ciri-cirinya yang menyerupai sel stem, mempunyai antigenisiti yang rendah, tanpa isu etika dan boleh didapati dengan banyak. Oleh itu, objektif kajian ialah untuk mengasingkan, mengembang-biakkan dan mencirikan sel epitelial amnion manusia (HAECs) dan sel mesenkima amnion manusia (HAMCs) untuk pembentukan substitut yang menyerupai kulit. Kinetik pertumbuhan, keberkesanan pembentukan koloni (CFE), analisa penanda CD, keupayaan pembezaan in vitro dan pengekspresan gen stemness, epitelial, kitaran sel dan angiogenik dikenalpasti ke atas HAECs dan HAMCs untuk pencirian. Kesan penambahan Faktor Pertumbuhan Epidermal (EGF) dan Faktor Pertumbuhan Keratinosit (KGF) ke dalam media kultur HAECs juga dinilai. Dalam bahagian kedua kajian, HAMCs dicampurkan ke dalam fibrin dan HAECs disemai di atas lapisan HAMCs-fibrin untuk pembentukan substitut yang menyerupai kulit. Model kultur dalam media dan interfasa cecair-udara dikaji untuk perkembangan tisu pada 1, 2 dan 3 minggu. Keratinosit kulit dan fibroblas dermal digunakan sebagai kawalan. Untuk kajian in vivo, substitut yang menyerupai kulit diimplankan pada bahagian dorsal tikus atimik selama 3 minggu sebelum dianalisa. Kesemua substitut tersebut dinilai menggunakan immunohistokimia dan elektron mikroskopi. Hasil kajian menunjukkan HAECs dan HAMCs mempunyai klonogenisiti yang tinggi (CFE 5.7% dan 0.88%, mengikut turutan) dan boleh dibezakan kepada adiposit, osteosit dan neuron in vitro. Analisa penanda CD menunjukkan HAECs dan HAMCs positif terhadap CD9, CD44, CD73, CD90 dan HLA-ABC; dan negatif terhadap penanda hematopoietik dan endotelial: CD31, CD34, CD45, CD117 dan HLA-DR,DP,DQ. HAECs dan HAMCs mengekspreskan gen stemness, epitelial dan angiogenik pada aras yang berbeza dan menunjukkan penurunan selepas pasaj bersiri. Proliferasi HAECs meningkat dengan penambahan EGF dan kekal dalam keadaan diploid (2N). Ekspresi gen stemness, epitelial dan pengawalaturan kitaran sel menurun secara signifikan dengan penambahan EGF. Begitu juga dengan penambahan KGF dalam kultur HAECs di mana pengekspresan gen stemness dan epitelia menurun. Immunositokimia menunjukkan KGF-teraruh HAECs dan kumpulan kawalan mengekspreskan sitokeratin 14 dan sitokeratin 18. Dalam pembentukan substitut yang menyerupai kulit, HAECs mengekalkan ciri epitelium selapis selepas dikulturkan dalam media dan interfasa cecair-udara selama 3 minggu. Untuk kumpulan kawalan (sel daripada kulit manusia), interfasa cecair-udara mengaruhkan keratinosit untuk berstrata dan membeza kepada epidermis sebagaimana yang ditunjukkan oleh analisa histologi dan elektron mikroskop. Substitut HAECs yang menyerupai kulit mampu membentuk 2-5 lapisan selepas digrafkan ke atas tikus atimik. Walaubagaimanapun, ia menunjukkkan ciri-ciri degenerasi secara histologi. Kajian ini mencadangkan bahawa HAECs mempunyai potensi untuk membeza dan berstrata untuk membentuk epidermis kulit. Namun, kajian lanjutan perlu dijalankan untuk merangsang HAECs berstrata dalam kultur in vitro untuk menambah-baikkan dan mempercepatkan proses penyembuhan luka untuk lesi kulit.,Human amnion derived cells could be an alternative source of cells for skin regeneration due to their stem cell-like characteristics, low antigenicity, least ethical conflict and are highly abundant. Hence, the aims of this study were to isolate, cultureexpand and characterize human amnion epithelial cells (HAECs) and amnion mesenchymal cells (HAMCs) for the development of skin-like substitutes. The growth kinetics, colony forming efficiency (CFE), CD markers expression, in vitro differentiation ability and differential expression of stemness, epithelial, cell cycle and angiogenic genes were evaluated on cultured HAECs and HAMCs. The effects of epidermal growth factor (EGF) and keratinocyte growth factor (KGF) supplementation on HAECs were also evaluated. In the second part of the study, HAMCs were incorporated with fibrin and HAECs were seeded onto this HAMCsfibrin layer for skin-like substitute formation. Both submerged and air liquid interface constructs were tested for skin development at 1, 2 and 3 weeks. Skin keratinocytes and dermal fibroblasts were used as controls. For in vivo study, the skin-like substitutes were implanted onto the dorsal part of the athymic mice for 3 weeks before analysis. All formed skin-like substitutes were assessed using immunohistochemistry and electron microscopic studies. The results showed HAECs and HAMCs had high clonogenic properties (CFE of 5.7% and 0.88%, respectively) and could differentiate into adipocytes, osteocytes and neurons. CD markers analysis showed that HAECs and HAMCs were positive for CD9, CD44, CD73, CD90 and HLA-ABC; and were negative for hematopoietic and endothelial markers (CD31, CD34, CD45, CD117 and HLA-DR,DP,DQ). HAECs and HAMCs expressed different levels of stemness, epithelial and angiogenic genes with most of the genes being decreased in expression after serial passages. Proliferation of HAECs increased with EGF supplementation and the cells remained in diploid state (2N) after being cultured. The stemness, epithelial and cell cycle control genes expression were significantly decreased with the EGF supplementation. Similarly, KGF supplementation also decreased the stemness and epithelial gene expression levels. Immunocytochemistry showed that both the KGFtreated HAECs and the control groups expressed cytokeratin 14 and cytokeratin 18. In skin-like substitute formation, HAECs cultured in submerged and air liquid interface for 3 weeks remained as a monolayer epithelium. For the skin cells control group, air liquid interface markedly induced the keratinocytes to stratify and differentiate to epidermis as shown by histological and electron microscopic analysis. HAECs skinlike substitute was able to stratify to 2-5 layers cells after grafting on nude mice. However they showed degenerative features histologically. This study suggested that HAECs have the potential to differentiate and stratify into epidermis of the skin. However, further studies are needed to stimulate HAECs to stratify in vitro for the purpose of improving and enhancing wound healing in skin lesions.,Ph.D.
Pages:	227
Call Number:	QU20 .S622p 2012 9HUKM
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Medicine / Fakulti Perubatan

Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.