Sains Malaysiana 39(2)(2010): 305–313

Keupayaan Pembezaan Tiga Jenis Sel Primitif daripada Hasil Perbezaan Tempoh Proliferasi Darah Mencit

(Potential Differentiation of Three Types of Primitive Cells Originated from Different Proliferation Terms of Mouse Blood)

Intan Zarina Zainol Abidin & Shahrul Hisham Zainal Ariffin*

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

Zaidah Zainal Ariffin

Jabatan Mikrobiologi, Jakulti Sains Gunaan, Universiti Teknologi MARA

40450 Shah Alam, Selangor, Malaysia

Rohaya Megat Abdul Wahab

Jabatan Ortodontik, Fakulti Pergigian, Universiti Kebangsaan Malaysia

Jalan Raja Muda Aziz, 50300 Kuala Lumpur, Malaysia

Received: 24 March 2009 / Accepted: 13 July 2009

ABSTRAK

Kajian ini dilakukan bagi melihat keupayaan sel mononukleus sistem darah pusat membeza kepada sel osteoblas dan osteoklas secara in vitro bagi tiga tempoh proliferasi yang berbeza. Sel mononukleus sistem darah pusat dikulturkan di dalam medium pemilihan proliferasi bagi tiga tempoh proliferasi yang berbeza iaitu jangkamasa pendek (5 hari), sederhana (15 hari) dan panjang (30 hari). Keupayaan sel mononukleus untuk membeza kepada sel osteoblas dan osteoklas seterusnya diperhatikan pada setiap jenis sel ini. Medium proliferasi ditambah dengan faktor pembezaan asid askorbik dan β-gliserofosfat bagi membezakan sel mononukleus kepada sel osteoblas. Bagi pembezaan sel osteoklas pula, RANKL dan M-CSF ditambah ke dalam medium proliferasi. Bagi kawalan, sel yang sama digunakan tanpa penambahan faktor pembezaan. Viabiliti sel yang membeza daripada sel jangkamasa pendek, sederhana dan panjang menunjukkan sel-sel tersebut berupaya untuk bermandiri tanpa sebarang peningkatan yang signifikan sehingga 10 dan 14 hari dengan kehadiran faktor-faktor pembezaan tertentu di dalam medium pembezaan masing-masing. Analisis biokimia ke atas aktiviti alkali fosfatase (ALP) dan asid fosfatase rintang tartarat (TRAP) menunjukkan peningkatan yang signifikan (p<0.05) apabila dikulturkan di dalam medium pembezaan masing-masing. Kesimpulannya, keupayaan sel primitif untuk membeza kepada sel osteoblas dan osteoklas matang adalah hampir sama bagi ketiga-tiga jenis jangkamasa proliferasi tetapi mempunyai kadar proliferasi yang berlainan iaitu 0.37, 0.55 dan 0.72 pembahagian/hari masing-masing bagi sel jangkamasa pendek, sederhana dan panjang. Sel mononukleus yang diasingkan daripada darah periferi ini sangat primitif kerana berpotensi untuk membeza kepada dua jenis sel matang yang berasal daripada sel stem yang berbeza, justeru boleh dikategorikan sebagai sel stem multipoten.

Kata kunci: Darah periferi; mencit; osteoblas; osteoklas; sel stem

                                                              ABSTRACT           

The aim of this study was to differentiate central blood system mononucleated cells in vitro into osteoblast and osteoclast cells for three different proliferation terms of cells. The mononucleated cells were cultured in a selective proliferation medium for three different proliferation terms, i.e., short (5 days), medium (15 days) and long term (30 days) prior to analysis of osteoblast and osteoclast cells’ differentiation potentialities. The proliferation medium was then supplemented with differentiation factors, i.e., ascorbic acid and β-glycerophosphate to differentiate mononucleated cells into osteoblast cells. For osteoclast assay, RANKL and M-CSF were added into proliferation medium. For control, the same cells were used without supplementation of respective differentiation factors. The viability of differentiated cells from short, medium and long types of cells showed that they were able to survive until 10 to 14 days in the presence of respective differentiation factors without significant increased in the specific differentiation medium. Biochemical analyses on both alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP) activities were significantly increased (p<0.05) once cultured in their respective differentiation medium. In conclusion, the three types of primitive cells have the same potentiality to differentiate into mature osteoblast and osteoclast cells even though the proliferation rates are different, i.e. 0.37, 0.55 and 0.72 division/day for short, medium and long term cells respectively. Mononucleated cells isolated from peripheral blood are primitive enough to differentiate into two distinct types of mature cells which originated from two different stem cells lineage hence can be categorized as multipotent stem cells.

Keywords: Mice; osteoblast; osteoclast; peripheral blood; stem cells

REFERENCES

Athanasou, N.A. 1996.  Cellular Biology of Bone-Resorbing Cells. The Journal of Bone and Joint Surgery (American) 78-A(7): 1096-1112.

Faccio, R., Takeshita, S., Zallone, A., Ross F.P. & Teitelbaum, S.L. 2003. c-FMS and the αvβ3 integrin collaborate during osteoclast differentiation. The Journal of Clinical Investigation 111(5): 749-758.

Halleen, J.M., Alatalo, S.L., Suominen, H., Cheng, S., Janckila, A.J. & VŠŠnŠnen, H.K. 2000. Tartrate-resistant acid phosphatase 5b: a novel serum marker of bone resorption. Journal of Bone and Mineral Research 15(7): 1337-1345.

Honig, A., Rieger, Kapp, M., Krockenberger, M., Eck, M., Dietl, J. & KŠmmerer, U. 2006. Increased tartrate-resistant acid phosphatase (TRAP) expression in malignant breast, ovarian and melanoma tissue: an investigational study. BMC Cancer 6:199 doi:10.1186/1471-2407-6-199.

Janckila, A.J., Nakasato, Y.R., Neustadt, D.H. & Yam, L.T. 2003. Disease-specific expression of tartrate-resistant acid phosphatase isoforms. Journal of Bone and Mineral Research 18(10): 1916-1919.

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.

Morrison, S.J., Wandycz, A.M., Hemmati, H.D., Wright, D.E. & Weissman, I.L. 1997. Identification of a lineage of multipotent hematopoietic progenitors. Development 124(10): 1929-1939.

Nakasato, Y.R., Janckila, J., Halleen, J.M., Vaananen, H.K., Walton, S.P. & Yam, L.T. 1999. Clinical Significance of Immunoassays for Type-5 Tartrate-resistant Acid Phosphatase. Clinical Chemistry 45(12): 2150-2157.

Passier, R. & Mummery, C. 2003. Origin and use of embryonic and adult stem cells in differentiation and tissue repair. Cardiovascular Research 58(2): 324-335.

Perez-Amodio, S., Vogels, I.M.C., Schoenmaker, T., Jansen, D.C., Alatalo, S.L., Halleen, J.M., Beertsen, W. & Everts, V. 2005. Endogenous expression and endocytosis of tartrate-resistant acid phosphatase (TRACP) by osteoblast-like cells. Bone 36(6): 1065-1077.

Rivollier, A., Mazzorana, M., Tebib, J., Piperno, M., Aitsiselmi, T., Rabourdin-Combe, C., Jurdic, P. & Servet-Delprat, C. 2004. Immature dendritic cell transdifferentiation into osteoclasts: a novel pathway sustained by the rheumatoid arthritis microenvironment. Blood 104(13): 4029-4037.

Shahrul Hisham Zainal Ariffin, Rohaya Megat Abdul Wahab, Intan Zarina Zainol Abidin, Sahidan Senafi, Nor Muhammad Mahadi & Zaidah Zainal Ariffin. 2005a. Sel Stem Dalam Perkembangan Darah. Sains Malaysiana 34(1): 21-26.

Shahrul Hisham Zainal Ariffin, Rohaya Megat Abdul Wahab, Ismanizan Ismail, Nor Muhammad Mahadi & Zaidah Zainal Ariffin. 2005b. Stem Cells, Cytokines And Their Receptors. Asia Pacific Journal of Molecular Biology and Biotechnology 13(1): 1-13.

Shanthly, N., Aruva, M.R., Zhang, K., Mathew, B. & Thakur, M.L. 2006. Stem cells: a regenerative pharmaceutical. The Quarterly Journal of Nuclear Medicine and Molecular Imaging 50(3): 205-216.

Swaminathan, R. 2001. Biochemical markers of bone turnover. Clinica Chimica Acta 313(1-2): 95-105.

Tšgel, F. & Westenfelder, C. 2007. Adult Bone Marrow-Derived Stem Cells for Organ Regeneration and Repair. Development Dynamics 236(12): 3321-3331.

Wang, D., Christensen, K., Chawla, K., Xiao, G., Krebsbach, P.H. & Franceschi, T. 1999. Isolation and Characterization of MC3T3-E1 Preosteoblast Subclones with distinct In Vitro and In Vivo Differentiation/Mineralization Potential. Journal of Bone and Mineral Research 14(6): 893-903.

Weissman, I.L. 2000. Translating stem and progenitor cell biology to the clinic: barriers and opportunities. Science 287: 1442-1446.

Yamane, T., Okuyama, H., Tsuneto, M., Hemmi, H., Yamazaki, H. & Hayashi, S.I. 2004. Osteoclast lineage. Dlm. Handbook of stem cells, Lanza, R., Gearhart, J., Hogan, B., Melton, D., Pedersen, Thomson, J. & West., M. London: Elsevier Academic Press.

Zhao, Y., Guan, H., Liu, S.F., Wu, R.C. & Wang, Z. 2005. Overexpression of QM induces cell differentiation and mineralization in MC3T3-E1. Biological and Pharmaceutical Bulletin 28(8): 1371-1376.

*Corresponding author; email: hisham@ukm.my