تمایز سلول های بنیادی مزانشیمال با القای ذرات نانو تیوب تیتانیوم و اشعه فرابنفش به سلول های جنسی

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه زیست شناسی، واحد علوم تحقیقات، دانشگاه آزاد اسلامی، تهران، ایران

2 گروه آناتومی، دانشگاه علوم پزشکی البرز، کرج، ایران

چکیده

مقدمه و هدف: بسیاری از درمان های ناباروری به علت فقدان گامت های جنسی بیماران کارآمد نیستند. تمایز سلول های بنیادین به سلول های زایا ی جنسی می تواند گزینه مناسبی برای تولید گامت و کاربرد کلینیکی برای درمان ناباروری باشد. 
مواد و روش ها: سلول های بنیادین مزانشیمال به روش فلاشینگ از استخوان ران موشهای بالغ نژاد NMRI تهیه گردید. از ذرات نانو تیوب تیتانیوم titanium nanotube particles (TNT)، پرتو UV با فرکانس (254 ± 20nm)  و رتینوییک اسید برای تمایز استفاده شد. ویژگی ساختاری TNTو فیبرین با میکروسکوب FE- SEM و SEM ارزیابی شد. از مارکر های  CD90,CD31 برای تایید ماهیت سلول ها و برای بیان زایایی سلولهای تمایز یافته جنسی از مارکر MVH استفاده شد.  آزمون های  ANOVA و Mann-Whitney برای تحلیل آماری داده ها  بکارگرفته شد.
نتایج: ساختار یکنواخت TNT با ابعاد 10-15 نانومتر  با کمک  FE- SEM مشخص گردید. تصاویر SEM پوشیده شدن TNT روی فیبرین را نشان داد. در MTT بیشترین میزان بقای سلول ها در غلظت TNT 50 µg/ml دیده شد. ماهیت سلول های بنیادین با بیان مارکر های اختصاصی با روش فلوسایتومتری مشخص گردید. رنگ آمیزی های Alizarin red  رسوب کلسیوم  و Oil red با تایید قطرات چربی ماهیت چند توانی سلول های بنیادی مزانشیمی را اثبات کرد. با روش ایمنوهیستوشیمی بیان مارکر  MVH در سلولهای تمایز یافته در گروه های درمانی نسبت به کنترل تایید گردید.
نتیجه‌گیری: کاربرد TNT  و UV  روی سلول های بنیادی مزانشیمال موجب افزایش معنی دار تمایز سلول های زایای جنسی می شود.

کلیدواژه‌ها

عنوان مقاله [English]

Mesenchymal stem cells differentiation into gamete germ cells by titanium nanotube particles and UV radiation induction

نویسندگان [English]

  • Saman Ebrahimi 1
  • Alireza Shams 2
  • Parvaneh Maghami 1
  • Azadeh Hekmat 1
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran
2 Department of Anatomy, Alborz University of Medical Sciences, Karaj, Iran
چکیده [English]

Background and Objective: Many of traditional infertility treatment methods due to lack of gamete are ineffective.  Differentiation of stem cells to gamete germ cells may be a good choice for producing gametes for treatment of infertility in clinique.
Materials and Methods: Bone marrow mesenchymal stem cells (BMMSc) were obtained from femoral bone of adult NMRI mice by flushing method. Nano tube particles of titanium (TNT), retinoic acid and UV beam 254 ± 20 nm frequencies were used for differentiation of MSc to gamete germ cells. Ultrastructure of TNT was studied with FEM SE electron microscope. Fibrin coating of Nano tubes was evaluated by SEM. Nature and character of stem cells were measured using cluster– differentiation (CD) by flow cytometry confirmed by CD90, and CD31 markers.  Finally, MVH marker was used for assessment of differentiation of MSc to gamete germ cells with immunohistochemistry assay. Statistical data was analyzed by ANOVA and Mann-Whitney tests.
Results: Electron microscopic pictures by FE SEM showed homogenous ultrastructure of titanium nanotubes in 10-15 nm diameters. Coating of TNT by fibrin was confirmed by SEM. MTT assay showed most survival rate of MSc cells in 50 µg/ml concentration of TNT. By CD90 and CD31 markers, nature of the stem cells was controlled by flow cytometry. Finally MVH marker expression showed differentiation of MSc to gamete germ cells in treated as compared to control groups and was confirmed by immunohistochemistry.
Conclusion: Application of TNT fibrin coated and UV beam can increase differentiation rate of BMMSc to gamete germ cells significantly as a clinical protocol.

کلیدواژه‌ها [English]

  • Mesenchymal stem cells
  • Differentiation
  • Titanium nanotube
  • Ultraviolet
  • gamete germ cells

مراجع

  1. Boivin J, Bunting L, Collins JA, Nygren KG. International estimates of infertility prevalence and treatment-seeking: potential need and demand for infertility medical care. Human Reproduction 2007;22(6):1506-12.
  2. Hull MG, Glazener CM, Kelly NJ, Conway DI, Foster PA, Hinton RA, et al. Population study of causes, treatment, and outcome of infertility. British Medical Journal 1985;291(6510):1693-7.
  3. Schlegel PN. Evaluation of male infertility. Minerva Ginecologica 2009;61(4):261-83.
  4. Stukenborg J-B, Kjartansdóttir KR, Reda A, Colon E, Albersmeier JP, Söder O. Male germ cell development in humans. Hormone Research in Paediatrics 2014;81(1):2-12.
  5. Hübner K, Fuhrmann G, Christenson LK, Kehler J, Reinbold R, De La Fuente R, et al. Derivation of oocytes from mouse embryonic stem cells. Science 2003;300(5623):1251-6.
  6. Volarevic V, Ljujic B, Stojkovic P, Lukic A, Arsenijevic N, Stojkovic M. Human stem cell research and regenerative medicine—present and future. British Medical Bulletin 2011;99(1):155-158.
  7. Kolios G, Moodley Y. Introduction to stem cells and regenerative medicine. Respiration 2013;85(1):3-10.
  8. Segers S, Mertes H, de Wert G, Dondorp W, Pennings G. Balancing ethical pros and cons of stem cell derived gametes. Annals of Biomedical Engineering 2017;45(7):1620-32.
  9. Mahmoudian Sani MR, Mehri-Ghahfarrokhi A, Hashemzadeh-Chaleshtori M, Saidijam M, Jami M-S. Comparison of three types of mesenchymal stem cells (bone marrow, adipose tissue, and umbilical cord-derived) as potential sources for inner ear regeneration. The International Tinnitus Journal 2017;21(2).
  10. Danner S, Kajahn J, Geismann C, Klink E, Kruse C. Derivation of oocyte-like cells from a clonal pancreatic stem cell line. Molecular Human Reproduction 2007;13(1):11-20.
  11. Antonucci I, Di Pietro R, Alfonsi M, Centurione MA, Centurione L, Sancilio S, et al. Human second trimester amniotic fluid cells are able to create embryoid body-like structures in vitro and to show typical expression profiles of embryonic and primordial germ cells. Cell Transplantation 2014;23(12):1501-15.
  12. Hua J, Pan S, Yang C, Dong W, Dou Z, Sidhu KS. Derivation of male germ cell-like lineage from human fetal bone marrow stem cells. Reproductive Biomedicine Online 2009;19(1):99-105.
  13. Monfared MH, Minaee B, Rastegar T, Khrazinejad E, Barbarestani M. Sertoli cell condition medium can induce germ like cells from bone marrow derived mesenchymal stem cells. Iranian Journal of Basic Medical Sciences 2016;19(11):1186.
  14. Bowles J, Knight D, Smith C, Wilhelm D, Richman J, Mamiya S, et al. Retinoid signaling determines germ cell fate in mice. Science 2006;312(5773):596-600.
  15. Koshimizu U, Watanabe M, Nakatsuji N. Retinoic acid is a potent growth activator of mouse primordial germ cells in vitro. Developmental Biology 1995;168(2):683-5.
  16. Evans TRJ, Kaye SB. Retinoids: present role and future potential. British Journal of Cancer 1999;80(1):1-8.
  17. Zhou Q, Li Y, Nie R, Friel P, Mitchell D, Evanoff RM, et al. Expression of stimulated by retinoic acid gene 8 (Stra8) and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro. Biology of Reproduction 2008;78(3):537-45.
  18. Tanaka T, Satoh T, Onozawa Y, Kohroki J, Itoh N, Ishidate Jr M, et al. Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells. Cell Biology International 1999;23(8):541-50.
  19. Pozio A, Palmieri A, Girardi A, Cura F, Carinci F. Titanium nanotubes stimulate osteoblast differentiation of stem cells from pulp and adipose tissue. Dental Research Journal 2012;9(Suppl 2):S169.
  20. Task K, D'Amore A, Singh S, Candiello J, Jaramillo M, Wagner WR, et al. Systems level approach reveals the correlation of endoderm differentiation of mouse embryonic stem cells with specific microstructural cues of fibrin gels. Journal of The Royal Society Interface 2014;11(95):20140009.
  21. Birlea SA, Costin GE, Roop DR, Norris DA. Trends in regenerative medicine: repigmentation in vitiligo through melanocyte stem cell mobilization. Medicinal Research Reviews 2017;37(4):907-35.
  22. Ilie I, Ilie R, Mocan T, Bartos D, Mocan L. Influence of nanomaterials on stem cell differentiation: designing an appropriate nanobiointerface. International Journal of Nanomedicine 2012;7:2211. doi:10.2147/IJN.S29975
  23. Zhao C, Tan A, Pastorin G, Ho HK. Nanomaterial scaffolds for stem cell proliferation and differentiation in tissue engineering. Biotechnology Advances 2013;31(5):654-68.
  24. Li P, Hu H, Yang S, Tian R, Zhang Z, Zhang W, et al. Differentiation of induced pluripotent stem cells into male germ cells in vitro through embryoid body formation and retinoic acid or testosterone induction. BioMed Research International 2013(10):608728.
  25. Secretariat MA. In vitro fertilization and multiple pregnancies: an evidence-based analysis. Ontario Health Technology Assessment Series 2006;6(18):1.
  26. Miyamoto T, Minase G, Shin T, Ueda H, Okada H, Sengoku K. Human male infertility and its genetic causes. Reproductive Medicine and Biology 2017;16(2):81-8.
  27. Latifpour M, Shakiba Y, Amidi F, Mazaheri Z, Sobhani A. Differentiation of human umbilical cord matrix-derived mesenchymal stem cells into germ-like cells. Avicenna Journal of Medical Biotechnology 2014;6(4):218.
  28. Jung D, Xiong J, Ye M, Qin X, Li L, Cheng S, et al. In vitro differentiation of human embryonic stem cells into ovarian follicle-like cells. Nature Communications 2017;8(1):1-13.
  29. Shah SM, Singla SK, Palta P, Manik RS, Chauhan MS. Retinoic acid induces differentiation of buffalo (Bubalus bubalis) embryonic stem cells into germ cells. Gene 2017;631:54-67.
  30. Geijsen N, Horoschak M, Kim K, Gribnau J, Eggan K, Daley GQ. Derivation of embryonic germ cells and male gametes from embryonic stem cells. Nature 2004;427(6970):148-54.
  31. Rao M, Condic ML. Alternative sources of pluripotent stem cells: scientific solutions to an ethical dilemma. Stem Cells and Development 2008;17(1):1-10.
  32. Blum B, Benvenisty N. The tumorigenicity of diploid and aneuploid human pluripotent stem cells. Cell Cycle 2009;8(23):3822-30.
  33. Lv L, Liu Y, Zhang P, Bai X, Ma X, Wang Y, et al. The epigenetic mechanisms of nanotopography-guided osteogenic differentiation of mesenchymal stem cells via high-throughput transcriptome sequencing. International Journal of Nanomedicine 2018;13:5605.
  34. Kerativitayanan P, Carrow JK, Gaharwar AK. Nanomaterials for engineering stem cell responses. Advanced Healthcare Materials 2015;4(11):1600-27.
  35. Wu J, Zhou L, Ding X, Gao Y, Liu X. Biological effect of ultraviolet photocatalysis on nanoscale titanium with a focus on physicochemical mechanism. Langmuir 2015;31(36):10037-46.
  36. Braydich-Stolle L, Hussain S, Schlager JJ, Hofmann M-C. In vitro cytotoxicity of nanoparticles in mammalian germline stem cells. Toxicological Sciences 2005;88(2):412-9.
  37. Wong DY, Ranganath T, Kasko AM. Low-dose, long-wave UV light does not affect gene expression of human mesenchymal stem cells. PloS One 2015;10(9):e0139307.
  38. Mittelman AM, Fortner JD, Pennell KD. Effects of ultraviolet light on silver nanoparticle mobility and dissolution. Environmental Science: Nano 2015;2(6):683-91.
  39. Shbahangfar MR , Jafari E , Tarashohi A , Olyaee P , Bitaraf T.   Effect of UV-Photofunctionalization on Bioactivity of Titanium to Promote Human Mesenchymal Stem Cells. Journal of Research in Dental and Maxillofacial Sciences 2019;4(1):7-11. DOI: 10.29252/jrdms.4.2.7
  40. Aita H, Att W, Ueno T, Yamada M, Hori N, Iwasa F, et al. Ultraviolet light-mediated photofunctionalization of titanium to promote human mesenchymal stem cell migration, attachment, proliferation and differentiation. Acta Biomaterialia 2009;5(8):3247-57.
  41. Minamikawa H, Ikeda T, Att W, Hagiwara Y, Hirota M, Tabuchi M, et al. Photofunctionalization increases the bioactivity and osteoconductivity of the titanium alloy Ti6Al4V. Journal of Biomedical Materials Research - Part A 2014; 102(10):3618-30.
دانشور پزشکی
دوره 29، شماره 3 - شماره پیاپی 154
مرداد و شهریور 1400
صفحه 1-16

فایل ها

هم رسانی

ارجاع به این مقاله

آمار