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Title:	Analisis populasi dan keupayaan pembezaan sel stem daripada ampaian sel mononulleus darah periferi Mus musculus
Authors:	Intan Zarina Zainol Abidin (P51676)
Supervisor:	Shahrul Hisham Zainal Ariffin, Prof. Madya Dr.
Keywords:	Stem Cells Blood cells
Issue Date:	9-Nov-2012
Description:	Kesemua sel stem yang telah diasingkan dan dikenalpasti merupakan sel melekat manakala sel stem dalam bentuk ampaian masih belum pernah dilaporkan sehingga kini. Objektif kajian ini adalah untuk mengasingkan populasi ampaian sel stem darah periferi dan pengenalpastian sebagai sel stem multipoten melalui keupayaan sel untuk membeza kepada sel osteoblas, osteoklas dan kondrosit secara in vitro. Sampel kajian terdiri daripada ampaian sel mononukleus dan sel Lin- Mus musculus (mencit) daripada tiga tempoh pengkulturan yang berbeza iaitu jangkamasa pendek (5 hari), sederhana (15 hari) dan panjang (30 hari). Sel mononukleus diasingkan daripada darah periferi secara pengemparan kecerunan ketumpatan dengan menggunakan larutan Ficoll-PaqueTM Plus. Sel mononukleus dikulturkan di dalam medium proliferasi bagi tiga tempoh pengkulturan dan pengasingan sel Lin- dilakukan menggunakan butiran magnetik. Bagi menilai keupayaan pembezaan sel mononukleus kepada sel osteoblas, medium proliferasi ditambah dengan faktor pembezaan iaitu 50 g/mL asid askorbik dan -gliserofosfat 10 mM. Bagi pembezaan sel osteoklas pula, 50 ng/mL RANKL dan 25 ng/mL M-CSF ditambah ke dalam medium proliferasi. Manakala bagi membezakan sel mononukleus kepada sel kondrosit, medium pembezaan kondrosit (ZenBio, Inc) digunakan. Bagi kawalan, sel yang sama digunakan tanpa penambahan sebarang faktor pembezaan. Seterusnya analisis pembezaan yang terdiri daripada analisis viabiliti (pewarnaan tripan biru), biokimia (pengasaian enzim penanda), morfologi (pewarnaan sel) dan biologi molekul (tindakbalas rantaian polimerase transkripsi berbalik; RT-PCR) dilakukan. Aktiviti sel dilakukan terhadap kesemua populasi sel yang dikultur di dalam medium pembezaan osteoblas dan osteoklas bagi menentukan keupayaan pembezaan sel. Analisis terhadap viabiliti sel yang membeza menunjukkan sel-sel tersebut berupaya untuk bermandiri tanpa sebarang peningkatan yang signifikan sehingga 10 (osteoklas), 14 (osteoblas) dan 21 (kondrosit) hari dengan kehadiran faktor-faktor pembezaan tertentu di dalam medium pembezaan masing-masing. Analisis biokimia terhadap kesemua sel mononukleus dan Lin- menunjukkan peningkatan aktiviti enzim yang signifikan (p<0.05) apabila dikulturkan dalam medium pembezaan osteoblas (alkali fosfatase; ALP), osteoklas (asid fosfatase rintang tartarat; TRAP) dan kondrosit (ALP) dengan aktiviti enzim sel Lin- adalah lebih tinggi berbanding sel mononukleus bagi ketiga-tiga tempoh pengkulturan pada kesemua jenis pembezaan sel. Apabila dikulturkan dalam medium pembezaan masing-masing, sel mononukleus dan Lin- didapati menyerupai morfologi sel osteoblas, osteoklas dan kondrosit masing-masing selepas pewarnaan von Kossa, May-Grunwald-Giemsa dan toluidin biru. Analisis biologi molekul ke atas sel mononukleus dan Lin- daripada hasil pengkulturan jangkamasa sederhana yang dibezakan menunjukkan pengaktifan penanda molekul bagi sel osteoblas (Alp+, Opn+, Ocn+), osteoklas (Trap+, Catk+) dan kondrosit (ColII+, Agcn+). Penentuan aktiviti sel osteoblas dan osteoklas matang dalam medium pembezaan masing-masing menggunakan pewarnaan von Kossa ke atas cakera osteologik menghasilkan nodul berkalsium yang berwarna coklat kehitaman (aktiviti osteoblas) dan ruang penyerapan yang jernih (aktiviti osteoklas). Kesimpulannya, sel mononukleus dan Lin- didapati mempunyai ciri sel stem yang berkeupayaan untuk membahagi dan membeza kepada sel matang, maka hasil kajian ini telah dapat menentusahkan bahawa kedua-dua jenis sel ini merupakan sel stem multipoten.,All stem cells that have been isolated and characterized are adherent cells, whilst stem cell in suspension form still not been reported until now. The aim of this study is to isolate suspension stem cell population of peripheral blood and identification as multipotent stem cell via the potentiality of this isolated cell to in vitro differentiate into osteoblast, osteoclast and chondrocyte cells. Study samples comprise of Mus musculus (murine) suspension mononucleated cell and Lin- cell from three different culturing terms, i.e., short (5 days), medium (15 days) and long (30 days) terms. The mononucleated cells were isolated from peripheral blood by density gradient centrifugation using Ficoll-PaqueTM Plus. The mononucleated cells were cultured in proliferation medium for three culturing terms and Lin- cells were isolated using magnetic beads. For differentiation potentiality evaluation of mononucleated cells into osteoblast cells, the proliferation medium was then supplemented with differentiation factors, i.e., 50 g/mL ascorbic acid and β-glycerophosphate 10 mM. For osteoclast differentiation, 50 ng/mL RANKL and 25 ng/mL M-CSF were added into proliferation medium. Whilst for differentiation of mononucleated cells into chondrocyte cells, chondrocyte differentiation medium (ZenBio, Inc) was used. For control, the same cells were used without supplementation of respective differentiation factors. Differentiation analyses comprise of viability (trypan blue staining), biochemistry (enzyme markers assays), morphology (cells stainings) and molecular biology (reverse transcription polymerase chain reaction; RT-PCR) analyses then were done. The cells activities analyses for all cells population cultured in osteoblast and osteoclast differentiation mediums were done to identify the potentiality of cells differentiation. The viability analysis of differentiated cells showed that all cells were able to survive until 10 (osteoclast), 14 (osteoblast) and 21 (chondrocyte) days in the presence of respective differentiation factors without significant increased in the medium. Biochemical analyses for all mononucleated and Lin- cells showed the significant increment (p<0.05) of enzyme activities when cultured in osteoblast (Alkaline Phosphatase; ALP), osteoclast (Tartrate Resistant Acid Phosphatase; TRAP) and chondrocyte (ALP) differentiation mediums. Enzyme activities of Lin- cells are higher than mononucleated cells for all three culturing terms for all types of cells differentiation. When cultured in their respective differentiation mediums, the mononucleated and Lin- cells showed morphologies like osteoblast, osteoclast and chondrocyte cells after stained by von Kossa, May-Grunwald-Giemsa and toluidine blue, respectively. Besides, the molecular biology analyses towards mononucleated and Lin- cells differentiated from medium term cells population showed activation of molecular markers for osteoblast (Alp+, Opn+, Ocn+), osteoclast (Trap+, Catk+) and chondrocyte (ColII+, Agcn+) cells. Determination of mature osteoblast and osteoclast cells activities in their respective differentiation mediums using von Kossa staining towards osteologic discs produced brown black calcium nodules (osteoblast activity) and clear resorption pits (osteoclast activity). In conclusion, mononucleated and Lin- cells showed the characteristics of stem cell that able to proliferate (self-renew) and differentiate into mature cells hence this study has confirmed that both types of cells are multipotent stem cells.,PhD
Pages:	344
Call Number:	QH588.S83I555 2012 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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