PEMBAHASAN UMUM
SIMPULAN DAN SARAN
Simpulan
1. Hasil purifikasi dan kultur in vitro memperlihatkan bahwa Nycodenz II (5 kolom) efektif untuk purifikasi sel Leydig.
2. Kombinasi penambahan ITS dan hCG meningkatkan konsentrasi testosteron di dalam medium kultur sel Leydig
3. Penggunaan CM sel Leydig sebagai medium kultur stem cell mesenkimal sumsum tulang dapat mengarahkan diferensiasi menjadi sel Leydig (transdiferensiasi).
Saran
1. Untuk meningkatkan perolehan jumlah sel Leydig yang dipurifikasi dengan gradien Nycodenz diperlukan kombinasi rentang konsentrasi gradien Nycodenz yang lebih kecil.
2. Peningkatan jumlah sel hidup pada produksi galur sel Leydig memerlukan modifikasi medium kultur dengan menambahkan faktor pertumbuhan lainnya seperti Transforming Growth Factor (TGF-α), Insulin-like Growth Factor (IGF-I) dan Platelet-derived Growth Factor (PDGF-A).
3. Produk conditioned medium kultur sel Leydig memerlukan standarisasi produksi dan analisis kandungan bahan bioaktif yang lebih spesifik sehingga dapat digunakan sebagai medium standar untuk pengarahan stem cell mesenkimal menjadi sel Leydig.
4. Perlu dilakukan kajian lanjutan untuk membuktikan adanya transdiferensiasi stem cell mesenkimal sumsum tulang menjadi sel Leydig dengan menggunakan karakterisasi molekul protein permukaan stem cell (CD) dan juga dengan bantuan marka molekuler.
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DAFTAR PUSTAKA
Amersham Bioscience. 2010. Percoll : Methodology and Applications. Anonimous 2012. Blue Histology: Male Reproductive System.
m#Testes
Ariyaratne HBS, Mendis-Handagama SMLC, Hales DB, Mason JI. 2000. Studies on the onset of Leydig precursor cell differentiation in the prepubertal rat testis. Biol Reprod 63 : 165-171.
Avallet O, Vigier M, Chatelain PG, Saez JM. 1991. Regulation by growth factor of Leydig cell differentiated functions. J Steroid Biochem. Molec Biol 40(1-3) : 453 – 464.
Behre HM, Kliesch S, Leifke E, Link TM, Nieschlag E. 1997. Long-term effect of testosterone therapy on bone mineral density in hypogonadal men. J Clin Endocrinol Metab 82 : 2386-2390.
Bernier M, Gibb W, Haour F, Collu R, Saez JM, Ducharme JR.1983. Studies with purified immature porcine Leydig cells in primary culture. Biol Reprod 29 : 1172 – 1178.
Bhasin S, Singh AB, Mac RP, Carter B, Lee MI, Cunningham GR. 2003. Managing the risk of prostate disease during testosterone replacement therapy in older men : recommendations for a standardized monitoring plan. J Androl 24(3): 299-311.
Bilinska B, Genissel C, Carreau S. 1997. Paracrine effect of seminiferous tubule factors on rat Leydig cell testosterone production: Role of cytoskeleton. Biol Cell 89:435-442.
Bilinska B, Kotula-Balak M,Sadowska J. 2009. Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological. European J Histochem 53 (1) : 35-42.
Browning JY, Heindel JJ, Grotjan Jr HE. 1983. Method for primary culture of purified Leydig cells isolated from adult rat testes. J Tissue Culture Methods 7(2) : 55-58.
Butler M. 2004. Animal Cell Culture & Technology 2nd ed. London: Bios Scientific Publisher, Taylor & Francis Group.
Caplan AI, Bruder SP. 2001. Mesenchymal stem cells: building block for molecular medicine in the 21st century. Trends Mol Med 7(6) : 259-264. Chemes H, Cigorraga S, Bergada C, Schteingart H, Rey R, Pellizzari E. 1992.
Isolation of human Leydig cell mesenchymal precursors from patients with the androgen sensitivity syndrome: Testosteron Production and response to human chorionic gonadotrophin stimulation in culture. Biol Reprod 46: 793-801.
Chen G-R, Ge R-S, Lin H, Dong L, Sottas CM, Hardy MP. 2007. Development of a cryopreservation protocol for Leydig cells. Hum Reprod 22(8) : 2160- 2168.
Cudicini C, Lejeune H, Gomez E, Bosmans E, Ballet F, Saez J, Jegou B.1997. Human Leydigs cells and Sertoli cells are producers of Interleukin-1 and -6. J Clin Endocrin Metab 82 (5) : 1426-1433.
Djuwita I, Mohamad K, Prasetyaningtyas WE, Nurhidayat. 2010. In vitro differentiation of adult rat bone marrow stromal cells into neurons using conditioned medium of newborn rat neuron primary culture. The Proceedings of the first Congress of South East Asia Veterinary School Association. Bogor, Indonesia. Hal 51-52.
Eguizabal C, Montserrat N, Veiga A, Belmonte JCI. 2013. Dedifferentiation, transdifferentiation and reprogramming: Future direction in regenerative medicine. Semin Reprod Med 31: 82-94.
El-Sherbiny AM, Amin SO, Hernandez C, Carreau S. 1994. The immature rabbit testis : presence of two distinct populations of Leydig cells. World Rabbit Sci 2(4) : 141-146.
Evans WH, Flint N. 1985. Subfraction of hepatic endosomes in Nycodenz gradients and by free electrophoresis. Biochem J 232 :25-32. Abstract Freshney 1987. Animal Cell Culture : A practical approach. Washington: IRL
Press.
Freshney RI. 2005. Culture of animal cells: A manual of basic technique. John Wiley & Sons Inc. Publication.
Ge R-S, Dong Q, Sottas CM, Chen H, Zirkin BR, Hardy MP. 2005. Gene expression in rat Leydig cells during development from the progenitor to adult stage : a cluster analysis. Biol Reprod 72 : 1405-1415.
Gruenewald DA, Matsumoto AM. 2003. Testosterone Supplementation Therapy for Older Men: Potential Benefits and Risks. J American Geriatrics Soc 51(1):101-115
Habert R, Lejeune H, Saez JM. 2001. Origin, differentiation and regulation of fetal and adult Leydig cells. Mol Cell Endocrin 179 : 47-74
Hardy MP, Kelce WR, Klinefelter GR, Ewing LL. 1990. Differentiation of Leydig cell precursors in vitro : a role for androgen. Endocrin 127: 488-490
Halim D, Murti H, Sandra F, Boediono A, Djuwantono, dan Setiawan B. 2010. Stem Cell: Dasar teori dan aplikasi klinis. Jakarta. Erlangga
Heller CG, Leach DR. 1971.Quantification of Leydig cells and measurement of Leydig-cell size following administration of human Chorionic Gonadotrophin to normal men. J Reprod Fert 25 : 185-192.
Hendlund TE dan Miller GJ. 1994. A serum-free defined medium capable of supporting growth of four established human prostatic carcinoma cell lines. Prostate 24(5) : 221-228.
Hu J, You S, Li W, Wang D, Nagpal ML, Mi Y, Liang P, Lin T. 1998. Expression and regulation of interferon-γ-inducible protein 10 genes in rat Leydig cells. Endocrin 139 : 3637-3645.
Kaiin EM, Djuwita I, Yusuf TL, Setiadi MA. 2013. Konsentrasi, kemurnian dan viabilitas sel Leydig hasil purifikasi dengan gradien Nycodenz dan kultur in vitro. J K H Unsyiah Vol.7(1) : 75-80.
Kaiin EM, Djuwita I, Yusuf TL, Setiadi MA. 2013. Development of in vitro culture of rat Leydig cells after purification with Nycodenz gradient. Open J Anim Sci 3 (4) : 299-304.
Klinefelter GR, Hall PF dan Ewing LL. 1987. Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure. Biol Reprod 36 : 769-783.
50
Kropf J, Schurek JO, Wollner A, AM Gressner. 1997. Imunological measurement of transforming growth factor- beta I (TGF-βI) in blood, assay, development and comparison. Clin Chem 43(10) : 1965-1975.
Lakshmanan J, Salido EC, Lam R, Barrjas L, Fisher DA. 1990. Identification of pro-epidermal growth factor and high molecular weight EGF in adult mouse urine. Biochem Biophys Res Commun 173 (3) : 902-911.
Mader SS. 2000. Human Biology. Iowa: McGraw Hill.
Manayagi T, Kurosawa R, Ohnuma K, Ueyama A, Ito K, Takahashi J.2003. Purification of mouse primordial germ cells by Nycodenz. Reprod 125: 667-675.
Mendis-Handagama SMIC, Ariyaratne HBS. 2001. Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod 65 : 660-671. Miller SR. 2006. Assessment of Nycodenz gradient on enrichment and culture of
perinatal porcine spermatogonial stem cell. Thesis (pdf). The Graduate Faculty of North Carolina State University.
Nakayama Y, Yamamoto T, Abe S. 1999. IGF-I, IGF-II and insulin promote differentiation of spermatogonia to primary spermatocytes in organ culture of newt testes. Int J DevBiol 43 : 343-347.
Phinney DG, Prockop DJ. 2007. Concise Review: Mesenchymal stem/multipotent stromal cells : The state of transdifferentiation and modes of tissue repair-Current views. Stem Cells 25: 2896-2902.
Prentice DA.2003.Stem Cells and Cloning. San Francisco. Pearson Education Inc. Pointis G, Rao B, Latreille MT, Cedard L. 1984. Hormonal regulation of
testosteron in short term primary culture of fetal mouse Leydig cells. J Steroid Biochem 20(1) : 525-528.
Renlund N. 2006. Hormonal and paracrine influences on Leydig cell steroidogenesis [dissertation]. Stockholm: Karolinska Institutet.
Risbridger GP, Kerr JB, Peake R, Rich KA, de Kretser DM. 1981. Temporal changes in rat Leydig cell function after the induction of bilateral cryptorchidism. J Reprod Fert 63 : 415-423.
Risbridger GP, Hedger MP. 1992. Adult rat Leydig cell cultures: minimum requirement for maintenance of luteinizing hormone responsiveness and testosterone production. Mol Cell Endocrin 83: 125-132.
Saez JM. 1994. Leydig cells: endocrine, paracrine, and autocrine regulation. Endocrin Rev 15(5): 574-626.
Salva A, Klinefelter GR, Hardy MP. 2001. Purification of rat Leydig cells: Increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells. J Androl 22: 665-671
Senger PL. 2005. Pathway to Pregnancy and Parturition. Washington: Current Conception Inc.
Sedelaar JPM, Isaacs JT. 2009. Tissue culture media supplemented with 10% Fetal Calf Serum contains a castrate level of testosterone. Prostate 69 (16) : 1724-1729.
Sharpe PT. 1988. Methods of Cell Separation Vol. 18.http://books.google.co.id/ Song L, Tuan RS. 2004. Transdifferentiation potential of human mesenchymal
stem cell derived from bone marrow. FASEBJ 18 : 980-982.
Suarez-Quian, Niklinski.1990. Immunocytochemical localization of EGF receptor in mouse testis. Biol Reprod 43 : 1087-1097.
Sun J, Xi Y-B, Zhang Z-D, Shen P, Li H-Y, Yin M-Z, Li W-Y, Shi C-R. 2009. Leydig cell transplantation restores androgen production in surgically castrated prepubertal rats. Asian J Androl 11: 405-409.
Thermo. 2012. Basic centrifugation.
Wakefield J St John, Gale JS, Berridge MV, Jordan TW ,Ford HC. 1982. Is Percoll innocuous cells? Biochem J 202 : 795-797.
Woodbury D, Schwarz EJ, Prockop DJ, Black IB. 2000. Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61: 364-371
Wu YJ, Dong Q, Li SF, Wei X, Long D, Zeng Y. 2012. Differentiation of human bone marrow mesenchymal stem cells into Leydig or steroidogenic cells in
vitro
Yang J-M, Arnush M, Chen Q-Y, Wu X-D, Pang B, Jiang X-Z.2003. Cadmium- induced damaged to primary cultures of rat Leydig cells. Reprod Toxicol 17 : 553-560.
Yazawa T, Mizutani T, Yamada K, Kawata H, Sekiguchi T, Yoshino M, Kajitani T, Shou Z, Umezawa A, Miyamoto K. 2006. Differentiation of adults stem cells derived from bone marrow stroma into Leydig or adrenocortical cells. Endocrin 147(9) : 4104-4111.
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