Sains Malaysiana 51(7)(2022): 2173-2186

http://doi.org/10.17576/jsm-2022-5107-19

 Profil Protein Sekretom Sel Stem Pulpa Gigi Manusia semasa Aruhan Asid Askorbik

 (Secretome Protein Profile of Human Dental Pulp Stem Cells during Ascorbic Acid Induction)

SHAHRUL HISHAM ZAINAL ARIFFIN^1,*, THANALETCHUMI MANOGARAN¹, ROHAYA MEGAT ABDUL WAHAB², SAIFUL ANUAR KARSANI³, INTAN ZARINA ZAINOL ABIDIN⁴, FARINAWATI YAZID², MUHAMMAD DAIN YAZID⁵ & ZAIDAH ZAINAL ARIFFIN⁶

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

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

³Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Wilayah Persekutuan, Malaysia

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

⁵Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras, Kuala Lumpur, Wilayah Persekutuan, Malaysia

⁶School of Biology, Faculty of Applied Sciences, Universiti Teknologi MARA, 40450 Shah Alam, Selangor Darul Ehsan, Malaysia

Received: 10 June 2021/Accepted: 4 January 2022

Abstrak

Pembezaan sel stem secara in vitro kepada osteoblas secara umumnya melibatkan gabungan aruhan asid askorbik dan β-gliserofosfat. Namun, β-gliserofosfat didapati menyebabkan penurunan keviabelan sel. Maka, kajian ini adalah untuk menentukan potensi asid askorbik tanpa kehadiran β-gliserofosfat dalam mengaruh pembezaan osteoblas serta mengenal pasti perubahan keamatan pengekspresan sekretom bagi protein osteoblas. Sel stem pulpa gigi kekal (DPSC) yang dipencilkan; diaruh dengan 10 µg/mL asid askorbik selama 21 hari. Pengekspresan gen osteoblas pada hari ke-7 dan 21 ditentukan melalui transkripsi berbalik-tindak balas rantaian polimerase (RT-PCR). Selepas 21 hari aruhan, sel dieram dengan medium basal tanpa serum selama 12 jam dan dianalisis melalui pendekatan kromatografi cecair-spektrometri jisim/spektrometri jisim (LC-MS/MS). Kehadiran protein sekretom pada kedua-dua aruhan asid askorbik dan kawalan positif (gabungan asid askorbik + β-gliserofosfat) digunakan untuk menentukan proses biologi yang terlibat. Seterusnya, protein yang terlibat dalam pembezaan osteoblas serta mengalami perubahan terhadap keamatan pengekspresannya ditentukan melalui analisis UniProt dan PANTHER. Hubung kait antara protein sekretom ini ditentukan menggunakan STRING bagi penentuan tapak jalan pembezaan DPSC kepada osteoblas. Hasilnya, gen BSP didapati diekspreskan semasa perlakuan asid askorbik serta sebanyak 57 protein sekretom dikenal pasti terlibat semasa aruhan asid askorbik dan kawalan, dengan hanya tiga protein didapati berkaitan dengan pembezaan osteoblas serta mengalami perubahan keamatan pengekspresan melalui penggunaan UniProt dan PANTHER. Seterusnya, analisis menggunakan STRING mendapati hanya dua sekretom bagi DPSC dikenal pasti melibatkan tiga tapak jalan. Kesimpulannya, asid askorbik sahaja walaupun tanpa β-gliserofosfat berupaya mengaruh pengekspresan protein sekretom yang berkaitan dengan osteoblas. Inhibin beta rantai A dan fibrilin-2 dicadangkan sebagai penanda sekretom semasa pembezaan DPSC kepada osteoblas.

Kata kunci: Asid askorbik; pembezaan osteoblas; penanda sekretom; sel stem pulpa gigi kekal (DPSC)

Abstract

In vitro stem cell differentiation into osteoblast involved a combination of ascorbic acid and β-glycerophosphate. However, β-glycerophosphate reduced cell viability in the culture.  Therefore, this study was conducted to determine the potential of ascorbic acid to induce osteoblast differentiation by identifying differentially expressed osteoblast-related secretome. Stem cells isolated from permanent (DPSC) tooth pulp was induced with 10 µg/mL ascorbic acid for 21 days, and osteoblast gene expression was determined via reverse transcriptase-polymerase chain reaction (RT-PCR). After 21 days of induction, cells were incubated in a serum-free basal medium for 12 h and analyzed via liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) approach. The biological processes and differentially expressed osteoblast-related secretome were determined using UniProt and PANTHER based on similar proteins expressed in ascorbic acid induction and positive control. Interaction between secretome was determined using STRING for DPSC differentiation pathways determination. As a result, the BSP gene was expressed in ascorbic acid induction. A total of 57 secretome proteins were identified in ascorbic acid induction and control, where three differentially expressed osteoblast-related secretome were determined in DPSC. For secretome interaction, only two secretomes were involved in three pathways of DPSC after STRING analysis. In conclusion, ascorbic acid alone without β-glycerophosphate successfully induced osteoblast-related secretome. Inhibin beta A chain and fibrillin-2 were suggested as secretome markers for DPSC during osteoblast differentiation.

Keywords: Ascorbic acid; osteoblast differentiation; secretome; stem cells from human permanent tooth pulp (DPSC)

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^*Corresponding author; email: hisham@ukm.edu.my