مقايسه بيان ژن اگريكان در كندروژنز سلول‌هاي بنيادي مشتق از چربي در دو سيستم كشت Pellet و Micromass

Comparison of aggrecan gene expression in chondrogenesis of adiposederived stem cells in pellet and micromass culture systems

زمينه و هدف: امروزه سلول‌هاي بنيادي مشتق از چربي (ADSC) به‌دليل خصوصيات منحصر به فرد مانند راحتي و عدم استفاده از روش‌هاي تهاجمي حين استخراج به‌طور گسترده در مهندسي بافت استفاده مي‌شوند. به منظور افزايش تعاملات بين سلولي مشابه با تراكم‌هاي پيش غضروفي جنيني، استفاده از سيستم‌هاي كشت سه بعدي همچون Pellet و Micromass ضروري مي‌باشد. اگريكان يكي از مهمترين اجزاي ماتريكس خارج سلولي است، بنابراين در مطالعه حاضر بيان اگريكان در طي روند كندروژنز ADSC هاي انساني در دو سيستم كشت Pellet و Micromass بررسي شد. روش بررسي: اين مطالعه تجربي در گروه آناتومي دانشكده پزشكي دانشگاه علوم پزشكي اصفهان از فروردين 1392 تا بهمن ماه 1393 انجام شد. بافت چربي شكمي از سه بيمار در طي جراحي ليپوساكشن جدا شد. سلول‌هاي بنيادي با روش‌هاي مكانيكي و آنزيمي استخراج و در كشت تك لايه كشت داده شدند. سپس به منظور القاي روند كندروژنز، تعداد 105×5 سلول‌هاي پاساژ سوم (P3) به سيستم‌هاي كشت Pellet و Micromass حاوي مديوم كندروژنيك در گروه‌هاي آزمايش 7 و 14 روز منتقل شدند. ارزيابي بيان ژن اگريكان توسط تكنيك Real-time PCR انجام شد. يافته‌ها: بررسي نتايج Real-time PCR نشان داد كه بيان اگريكان در Micromass روز 14 در مقايسه با Pellet روزهاي 14 و 7 افزايش معناداري دارد (P=0/008). همچنين بيان اگريكان در Micromass روز هفتم در مقايسه با Pellet روز هفتم نيز افزايش معناداري نشان داد (0/03=P). نتيجه‌گيري: با توجه به بيان بالاي ژن اگريكان در سيستم كشت Micromass نسبت به كشت Pellet شايد بتوان نتيجه گرفت كه كشت Micromassدر مقايسه با كشت Pellet كارايي بيشتري در بيان ژن اگريكان در كندروژنز سلول‌هاي بنيادي مشتق از چربي داشته است.

Background: Nowadays, Human adipocyte-derived stem cells (hADSCs) has been widely used in tissue engineering because of its unique features such as extraction from more sources, more easily and non-invasive extraction methods. In order to increase cell-cell interactions, similar to embryonic pre-cartilage condensation, the use of three dimensional (3D) high-density cell culture systems such as Pellet and Micromass that simulates optimal condensation in chondrogenesis in vivo is necessary. Also, these culture systems provide the proper diffusion of nutrients. Aggrecan is a proteoglycan and one of the important components of extracellular matrix of cartilage tissue that plays an important role in the organization of the extracellular matrix. The high concentrations of aggrecan produces the osmotic properties that is necessary to normal tissue function of cartilage. In current study, Aggrecan gene expression was investigated during chondrogenesis of hADSCs in two Pellet and Micromass culture systems. Methods: This experimental study was done in Department of Anatomical Sciences Department of Faculty Medical in Isfahan University of Medical Sciences, Iran, from April 2013 to January 2015. First, the abdominal adipose tissue was obtained from three patients after obtaining written consent during their liposuction surgeries. ADSCs were extracted by mechanical and enzymatic methods and were cultured in monolayer culture. Then, in order to induction of chondrogenic differentiation, 5×105 cells of third passage (P3) were transferred to three-dimensional culture systems Pellet and Micromass containing chondrogenic mediums in experimental groups of 7 and 14 days. The evaluation of aggrecan gene expression was performed by real-time PCR technique. Results: Gene expression analysis revealed that aggrecan was significantly increased in micromass culture at day 14 compared to Pellet culture at days 14 and 7 (P≤0.01). Also, aggrecan was significantly increased in Micromass culture at day 7 compared to Pellet culture at day 7 (P≤0.05). Conclusion: Due to higher expression of aggrecan gene in Micromass culture compared to Pellet culture, this system may be more efficient than Pellet culture in synthesis of aggrecan in chondrogenic differentiation of ADSCs.