اثر تمایزی اگزوزوم های مشتق شده از مونوسیت ها و ماکروفاژها در داربست های قطره ای کیتوزان آلژینات

نوع مقاله : مقاله پژوهشی

نویسندگان

1 گروه زیست شناسی، واحد مشهد، دانشگاه آزاداسلامی، مشهد، ایران

2 گروه زیست شناسی و مرکز تحقیقات بیولوژی کاربردی تکوین جانوری، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران

چکیده

آسیب­های استخوانی یکی از چالش­های علم پزشکی محسوب می­شود که هر ساله هزینه­ی زیادی را در دنیا برای درمان به خود اختصاص می­دهد. اگزوزوم­ها، نانووزیکول­هایی هستند که پروتئین­ها و ماده­ی ژنتیک به سلول هدف وارد می­کنند و به دنبال آن، تکثیر، بقای سلولی و تمایز را در سلول گیرنده القا می­نمایند. این ویژگی­ها، می­تواند اگزوزوم­ها را به عامل تمایزی مناسب تبدیل کند. هدف از انجام این مطالعه، بررسی اثر تمایزی اگزوزوم­های مشتق شده از مونوسیت­ها و ماکروفاژها در داربست­های قطره­ای کیتوزان آلژینات. داربست­های کیتوزانی- آلژیناتی به روش قطره­ای به همراه سلول­های بنیادی چربی ساخته شد. سپس سلول­های مونوسیتی کشت و مایع رویی جمع­آوری شد و اگزوزوم­ها به روش اولتراسانتریفوژ جمع شدند و توسط روش DLS  وSEM  شناسایی شدند. نمونه­ها تیمار و سپس تستMTT  و  DAPI و برای بررسی فرآیند تمایز تست آلکالین فسفاتاز و Real-time PCR انجام شد. بررسی یافته­های آزمون MTT  نشان داد میزان بقای سلول­های تمایزیافته با اگزوزوم­ها در روزهای 7، 14 و 21 نسبت به کنترل تفاوت معناداری داشت. نتایج DAPI نشان­دهنده عدم مرگ سلولی در گروهای تیماری بود و نتایج تست آلکالین فسفاتاز تفاوت معناداری با گروه کنترل نشان داد و همچنین نتایج Real-time PCR افزایش بیان ژن­هایBMP 2/6، SMAD4، ژن­های تمایزی استئوکلسین (Osc) و استئوپونتین(Opn) نسبت به نمونه کنترل را به همراه داشت. این مطالعه، نشان داد اگزوزوم مشتق از سلول­های مونوسیتی و ماکروفاژی، می­توانند سبب بقا و تمایز استئوژنیک سلول­های مزانشیمی چربی در درابست­های کیتوزان آلژینات شوند.

کلیدواژه‌ها


عنوان مقاله [English]

Differentiation Effect of Exosomes Derived from Monocytes and Macrophages on Chitosan Alginate Drip Scaffolds

نویسندگان [English]

  • Nasrin Hosseini 1
  • Javad Baharara 2
  • Khadijeh Nejad Shahrokhabadi 1
  • Saeedeh Zafar Balanezhad 1
1 Department of Biology, Mashhad Branch, Islamic Azad University, Mashhad, Iran
2 Department of Biology & Research Center for Animal Development Applied Biology, Mashhad
چکیده [English]

Bone injuries are one of the challenges of medical science which costs a lot of money in the world for the treatment every year. Exosomes are nanovesicles which carry the proteins and genetic material into the target cell, which in turn, induces the proliferation, cell survival, and differentiation in the recipient cell. These features make exosomes a proper differentiation factor. This study aimed to investigate the differentiating effect of exosomes derived from the monocytes and macrophages on chitosan alginate drip scaffolds. Chitosan-alginate scaffolds were made by drip method with fat stem cells. Monocytes were cultured, and the supernatant was collected, and the exosomes were collected by the ultracentrifugation. Exosomes were identified by DLS and SEM methods. The samples were treated, and then MTT and DAPI tests were performed. The osteogenic differentiation was examined by alkaline phosphatase and real-time PCR. MTT results showed that the survival rate of differentiated cells with exosomes on days 7, 14, and 21 was increased compared to control group. Moreover, DAPI results showed no cell death in the treatment groups, and the results of the alkaline phosphatase test showed an increase with the control group, and also the results of real-time PCR increased the expression of genes BMP2/6, SMAD4 and differentiation genes Osc and Opn in the treatment group. This study showed that the exosomes derived from the monocytes and macrophages could cause survival and osteogenic differentiation of the fat mesenchymal cells in the chitosan alginate scaffold.

کلیدواژه‌ها [English]

  • Exosome
  • Fat Stem Cell
  • cell differentiation
  • Monocyte
  • Chitosan
  • Alginate Scaffold
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