Sains Malaysiana 40(6)(2011): 613–621

 

Kesan ß-gliserofosfat Terhadap Ampaian Sel Mononukleus daripada Darah Periferi Manusia

(Effect of ß-glycerophosphate on Suspension Mononucleus Cell from Human Peripheral Blood)

 

Nurul Atikah Ahmad1, Shahrul Hisham Zainal Ariffin* 1, Rohaya Megat Abdul Wahab2, Muhamad Abdul Razak3 & Sahidan Senafi1

 

1Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600 UKM, Bangi, Selangor D.E., Malaysia

 

2Jabatan Ortodontik, Fakulti Pergigian, Universiti Kebangsaan Malaysia

Jalan Raja Muda Abdul Aziz, 53000 Kuala Lumpur, Malaysia

 

3Jabatan Ortopedik, Pusat Perubatan Universiti Kebangsaan Malaysia

Jalan Yaacob Latif, Bandar Tun Razak, 56000, Cheras, Kuala Lumpur, Malaysia

 

Diserahkan: 15 Julai 2010 / Diterima: 11 Ogos 2010

 

ABSTRAK

 

Sel osteoblas merupakan sel mononukleus yang bertanggungjawab untuk pembentukan tulang. Sel mononukleus telah terbukti mampu membeza kepada sel osteoblas setelah diaruh oleh kombinasi asid askorbik dan ß-gliserofosfat sebagai faktor pembezaan. Tujuan kajian ini adalah bagi melihat kesan aruhan ß-gliserofosfat terhadap ampaian sel mononukleus daripada darah periferi manusia secara in vitro. Sel mononukleus dipencilkan daripada darah periferi manusia dengan menggunakan larutan Ficoll-Paque™ Plus melalui kaedah pengemparan kecerunan ketumpatan. Sel mononukleus kemudian dikultur selama 7 hari di dalam medium proliferasi sebelum diaruh dengan ß-gliserofosfat pada kepekatan 1 mM, 5 mM, 10 mM, 20 mM, dan 100 mM. Pada hari 0, 1, 3, 7, dan 14, penentuan profil aktiviti enzim alkalin fosfatase (ALP) dan analisis morfologi bagi sel osteoblas dilakukan dalam medium masing-masing. Aktiviti enzim ALP dan analisis morfologi menunjukkan peningkatan yang signifikan (p<0.05) antara sel yang diaruh dengan sel kawalan negatif melalui statistik ujian-t berpasangan. Kesimpulannya, kehadiran ß-gliserofosfat sahaja mampu untuk mengaruh pembezaan sel mononukleus kepada sel osteoblas. Kesan ß-gliserofosfat terhadap pembezaan sel mononukleus kepada sel osteoblas menunjukkan profil enzimologi (aktiviti enzim ALP) dan morfologi yang hampir sama dengan kawalan positif (asid askorbik dan ß-gliserofosfat). Berdasarkan kepada analisis enzimologi dan morfologi 1 mM ß-gliserofosfat adalah kepekatan yang paling sesuai untuk pembezaan sel osteoblas secara in vitro.

 

Kata kunci: ß-gliserofosfat; ampaian sel mononukleus

 

ABSTRACT

 

Osteoblast cell is a mononucleus cell that is responsible for bone formation. Mononucleus cell have been proven to differentiate into osteoblast cell after being induced with combinations of ascorbic acid and ß-glycerophosphate as differentiation factors. This study aimed to observe the in vitro effect of a single ß-glycerophosphate induction towards primary mononucleated cells suspension that have been isolated from human peripheral blood. Mononucleus cells were isolated from human peripheral blood using density gradient centrifugation method in the present of Ficoll-Paque™ Plus. Mononucleus cells were cultured for 7 days in proliferation medium before being induced by 1 mM, 5 mM, 10 mM, 20 mM, and 100 mM of ß-glycerophosphate. Alkaline phosphatase (ALP) activity profile and morphological analysis were done at day 0, 1, 3, 7 and 14 in their respective medium. The activity of ALP enzyme and morphology analysis showed a significant increase (p<0.05) between cells that have been induced with the negative control cells by using paired t-test statistical analysis. In conclusion, the presence of only ß-glycerophosphate is able to induce mononucleated cell differentiation into osteoblast cells. The effect of ß-glycerophosphate towards mononucleated cells differentiation showed similar enzymological (ALP activities) and morphological profile as positif control (ascorbic acid and ß-glycerophosphate). Based on enzymological and morphological analyses, 1 mM of ß-glycerophosphate is the best concentration for in vitro osteoblast differentiation.

 

Keywords: ß-glycerophosphate; suspension mononucleus cell

 

RUJUKAN

 

Bellantuono, I. 2004. Haematopoietic stem cells. The International Journal of Biochemistry and Cell Biology 36(4): 607-620.

Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72: 248-254.

Cerovic, A., Miletic, I., Sobajic, S., Blagojevic, D. Radusinovic, M. & El-Sohemy, A. 2007. Effect of zinc on the mineralization of bone nodules from human osteoblas-like cells. Biological Trace Element Research 116(1): 61-71.

Clyton, S.M.D. 2003. Hematopoietic stem cells and hematopoiesis: cancer control. Journal of the Moffit Cancer Centre 10(1): 9-16.

Coelho, M.J. & Fernandes, M.H. 1999. Human bone cell cultures in biocompatibility testing. Part II: effect of ascorbic acid, ß-glycerophosphate and dexamethasone on osteoblastic differentiation. Biomaterials 21(11): 1095-1102.

Fratzl-Zelman, N., Fratzl, P., Hörandner, H., Grabner, B., Ellinger, A. & Klaushofer, K. 1998. Matrix mineralization in MC3T3-E1 cell cultures initiate by ß-glycerophosphate pulse. Bone 23(6): 511-520.

Fratzl-Zelman, N., Fratzl, P., Hörandner, H., Leugmayr, E., Varga, F., Ellinger, A., Erlee, M.P.M. & Khaushofer, K. 1997. Effects of thiodothyronine on the morphology of cells and matrix, the location of alkaline phosphatase and the the location of alkaline phosphatase and the frequency of apoptosis in long-term cultures of MC3T3-E1 cells. Bone 20(3): 225-236.

Intan Zarina, Z. A., Shahrul Hisham, Z.A., Rohaya, M.A.W., Sahidan, S. & Zaidah Z.A. 2008 Osteoclast and Osteoblast Development of Mus musculus Haemopoietic Mononucleated Cells. Journal of Biological Sciences 8(3): 506-516.

Intan Zarina, Z.A., Shahrul Hisham, Z.A., Zaidah Z.A. & Rohaya, M.A.W. 2010. Keupayaan pembezaan tiga jenis sel primitif daripada hasil perbezaan tempoh proliferasi darah mencit. Sains Malaysiana 39(2): 305-313.

Kassem, M., Abdullah, B.M. & Saeed, H. 2008. Osteoblastic cells: Diffrentiation and trans-differentiation. Archives of Biochemistry and Biophysics 473(2): 183-187.

Kartsogiannis, V. & Ng, K.W. 2004. Cell lines and primary cultures in the study of bone cell biology. Molecular and Cellular Endocrinology 228(1-2): 79-102.

Kim, Y.J., Lee, M.H., Wozney, J.M., Cho, J.Y. & Ryoo, H.M. 2004. Bone morphologenetic protein-2-induced alkaline phosphatase expression is stimulated by D1x5 and repressed by Msx2. The Journal of Biological Chemistry 279(49): 50773-50780.

Shahrul Hisham, Z.A., Rohaya, M.A.W., Intan Zarina, Z.A., Shahidan, S., Nor Muhamad, M. & Zaidah, Z.A. 2005. Sel stem dalam perkembangan darah. Sains Malaysiana 34(1): 21-26.

Vali, B., Rao, L.G. & El-Sohemy, A. 2007. Epigallocatechin-3-gallate increases the formation of mineralized bone nodules by human osteoblast-like cells. Journal of Nutritional Biochemistry 18(5): 341-347.

zur Niedan, N.I., Kempka, G. & Ahr, H.J. 2003. In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation 71(1): 18-27.

 

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

 

 

 

sebelumnya