Sains Malaysiana 52(6)(2023): 1771-1783

http://doi.org/10.17576/jsm-2023-5206-13

Protein Rembesan Sel Stem Pulpa Gigi Susu Manusia (SHED) semasa Pembezaan kepada Sel Osteoblas

(Proteins Secretion of Human Exfoliated Deciduous Teeth Stem Cells (SHED) during Osteoblast Cell Differentiation)

SHAHRUL HISHAM ZAINAL ARIFFIN^1*, ROHAYA MEGAT ABDUL WAHAB², THANALETCHUMI MANOGARAN¹, FARINAWATI YAZID², SITI NUR ZAHIDAH ZAHARI¹, RUS DINA RUS DIN³ & INTAN ZARINA ZAINOL ABIDIN⁴

¹Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

²Centre of Family Dental Health, Faculty of Dentistry, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Wilayah Persekutuan, Malaysia

³Forensic Science Programme, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor, Malaysia

⁴Centre for Research and Graduate Studies, University of Cyberjaya, 63000 Cyberjaya, Selangor, Malaysia

Diserahkan: 26 Januari 2023/Diterima: 13 Jun 2023

Abstrak

Analisis protein rembesan adalah penting untuk mengenal pasti penglibatan protein di dalam sistem biologi seperti mengenal pasti fungsi dan interaksi protein serta tapak jalan yang terlibat semasa pembezaan sel stem. Kajian ini dilakukan untuk mengenal pasti protein rembesan dan tapak jalan dalam pembezaan osteoblas melalui perubahan keamatan pengekspresan protein rembesan hasil aruhan asid askorbik terhadap sel stem pulpa gigi susu (SHED). SHED dirawat dengan 10 µg/mL asid askorbik selama 7 dan 21 hari dan pengekpresan gen ditentukan melalui transkripsi berbalik-tindak balas rantaian polimerase (RT-PCR) menunjukkan pengekspresanGAPDH (gen endogenous dan kawalan), diikuti dengan pengekspresan gen COL1 dan BSP (penanda osteoblas). SHED (hari ke 7 dan 21) dieram dalam medium tanpa serum selama 12 jam sebelum medium dianalisis menggunakan kromatografi cecair-spektrometri jisim/spektrometri jisim (LC-MS/MS). Protein yang sama diekspreskan dalam kawalan (positif dan negatif) serta asid askorbik seterusnya ditentukan proses biologi dan sekretom berkaitan osteoblas serta mempunyai perubahan keamatan pengekspresan melalui UniProt dan PANTHER. Hubung kait antara protein-protein rembesan ini bagi mengenal pasti tapak jalan pembezaan osteoblas ditentukan melalui STRING. Sebanyak 57 protein yang sama diekspreskan semasa aruhan asid askorbik dan kawalan dengan empat protein rembesan berkaitan dengan osteoblas didapati mengalami perubahan keamatan pengekspresan. Bagi hubung kait antara protein pula hanya tiga dari empat protein rembesan iaitu gelsolin (GSN), kolagenase jenis IV 72 kDa (MMP2) dan homolog peroksidasin (PXDN) dikenal pasti terlibat dalam tiga tapak jalan. Kesimpulannya, tiga jenis protein rembesan yang melibatkan tiga tapak jalan berpotensi sebagai penanda protein rembesan semasa pembezaan SHED kepada osteoblas.

Kata kunci: Asid askorbik; pembezaan osteoblas; protein rembesan; sel stem pulpa gigi susu manusia (SHED)

Abstract

Analysis of secreted proteins is crucial in understanding proteins’ involvement in the biological system, namely the function and interactions of the proteins and pathways involved, especially during stem cell differentiation. This study is aimed to determine the secreted proteins and pathways during osteoblast differentiation of ascorbic acid-induced exfoliated deciduous teeth stem cells (SHED) via changes in the intensity of secreted proteins’ expression results. SHED was treated with 10 µg/mL ascorbic acid for 7 and 21 days, and genes expression was determined using reverse transcriptase-polymerase chain reaction (RT-PCR), which resulted in the expressed of GAPDH (endogenous gene and control), followed by the expression of COL1 and BSP genes (osteoblast marker). SHEDS (at days 7 and 21) were incubated in a serum-free basal medium for 12 h before being analysed using liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS). Based on similar proteins expressed in controls (positive and negative) and ascorbic acid; the biological processes and osteoblast-related secreted proteins that experienced changes in expression intensity were determined using UniProt and PANTHER. The interaction between secreted proteins was determined using STRING to establish pathways involved during SHED-osteoblast differentiation. A total of 57 similar proteins were identified in ascorbic acid induction and controls of which only four secreted proteins were discovered to be associated with osteoblast and experiencing changes in expression intensity. Regarding protein interaction, only three out of four proteins; gelsolin (GSN), 72 kDa type IV collagenase (MMP2), and peroxidasin homolog (PXDN) were involved in three pathways. In conclusion, the three types of secreted proteins involved in three pathways are suggested as secreted proteins markers during osteoblast differentiation of SHED.

Keywords: Ascorbic acid; osteoblast differentiation; protein secretion; stem cells from human deciduous tooth pulp (SHED)

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*Pengarang untuk surat-menyurat; email: hisham@ukm.edu.my