مطالعه تاثیر بیش بیان ژن OCT4 و مهار همزمان ژن P53 بر بیان ژن‌های پرتوانی در سلول‌های بنیادی بافت چربی انسان

نویسندگان

پژوهشگاه ملی مهندسی ژنتیک و زیست‌فناوری، پژوهشکده بیوتکنولوژی پزشکی، گروه سلول‌های بنیادی و پزشکی بازساختی

چکیده

مقدمه و هدف: OCT4 مهمترین فاکتورهای رونویسی درگیر در حفظ پرتوانی و بازبرنامه‌ریزی سلول‌های سوماتیک است. از طرفی، مطالعات اخیر نشان داده است که فقدان یا جهش در ژن P53 بازبرنامه‌ریزی هسته را تسهیل می‌کند. مطالعه‌ی حاضر با هدف بازبرنامه‌ریزی سلول‌های بنیادی بافت چربی انسانی با استفاده از بیش بیان OCT4 و کاهش بیان P53 طراحی شد.
 
مواد و روش‌ها: سلول‌های بنیادی بافت چربی با استفاده از آنزیم کلاژناز از نمونه‌های چربی شکمی بیماران تحت عمل زیبایی ابدومینوپلاستی جداسازی شد. برای شناسایی سلول‌های بنیادی بافت چربی، بیان مارکرهای مزانشیمی با روش فلوسیتومتری بررسی شد و سلول‌ها به رده‌های استخوانی و چربی تمایز داده شدند. سلول‌های بنیادی بافت چربی در مرحله‌ی پاساژ سوم با پلاسمید بیانی حامل ژن OCT4 و shRNA مهارکننده P53 (وکتور pCXLE- hOCT4/shp53) ترنسفکت شدند. یک هفته پس از ترنسفکشن، بیان ژن‌های پرتوانی با روش qPCR بررسی شد.
 
نتایج: سلول‌های بنیادی بافت چربی در مرحله پاساژ سوم مورفولوژی فیبروبلاست‌مانند نشان دادند. نشانگرهای مزانشیمی CD90، CD73 و CD105 به ترتیب در 94، 2/80 و 1/81 درصد از سلول‌ها بیان شد. به علاوه، سلول‌های بنیادی بافت چربی به سلول‌های چربی و استخوانی تمایز یافتند. طبق نتایج qPCR، بیان ژن‌های OCT4، SOX2، LIN28، REX1، CCND1 و C-MYC در سلول‌های ترنسفکت شده با وکتور حامل ژن OCT4 و shRNA مهارکننده‌ P53 نسبت به گروه کنترل افزایش معنی‌دار نشان داد.
 
نتیجه‌گیری: بیش‌بیان ژن OCT4 و مهار بیان P53 به بازبرنامه‌ریزی سلول‌های بنیادی بافت چربی به سمت حالت پرتوانی کمک می کند. این روش ممکن است توان تمایزی سلول‌های بنیادی بافت چربی را برای کاربردهای درمانی افزایش دهد.
 

کلیدواژه‌ها


عنوان مقاله [English]

Study of the exogenous induction of OCT4 and concomitant P53 inhibition on the expression of pluripotency genes in human ADSCs

نویسندگان [English]

  • Atefeh Rashidipour
  • Arash Javeri
  • Masoumeh Fakhr Taha
چکیده [English]

Background and Objective: OCT4 is the most important transcription factor for reprogramming of somatic cells and maintenance of pluripotency. Moreover, it has recently been shown that loss of P53 or its mutations improve the efficiency of reprogramming. This study was designed to reprogram human adipose tissue-derived stem cells (ADSCs) by overexpression of OCT4 and inhibition of P53 expression.
 
Materials and Methods: The ADSCs were isolated from abdominal fat tissue of patients who underwent cosmetic abdominoplasty. For characterization of the ADSCs, the expression of some mesenchymal stem cell markers was assessed by flow cytometry and the cells were differentiated into osteogenic and adipogenic lineages. The third-passaged ADSCs were transfected with a plasmid expressing OCT4 and a P53-shRNA. One week after transfection, the expression of pluripotency genes was studied by quantitative real-time PCR.
 
Results: The third-passaged ADSCs showed a fibroblast-like morphology. Meanwhile, 94%, 80.2%, and 81.1% of the ADSCs showed positive staining for CD90, CD73 and CD105 markers, respectively. Moreover, the ADSCs showed adipogenic and osteogenic differentiation. As revealed by qPCR analysis, the expression of OCT4, SOX2, LIN28, REX1, CCND1 and C-MYC mRNAs were significantly upregulated after transfection of the ADSCs with hOCT4/shp53 vector compared to the control group.
 
Conclusion: Overexpression of OCT4 and inhibition of P53 can improve reprogramming of the ADSCs toward a pluripotent state. This method may improve the differentiation capacity of the ADSCs for clinical applications.

کلیدواژه‌ها [English]

  • Adipose tissue-derived stem cells
  • Pluripotency
  • OCT4
  • shRNA
  • P53 inhibition
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