تغییرات بیان ژن سه گیرنده آدرنرژیک آلفا 1، 2 و بتا 2 در سلول‌های کومولوس تخمدان زنان نابارور مبتلا به سندرم تخمدان پلی‌کیستیک (کاندید IVF) و تأثیر کلونیدین بر آن

نوع مقاله: اصیل پژوهشی

نویسندگان

1 دانشیار مرکز تحقیقات بهداشت باروری ولیعصر (عج)، بیمارستان امام خمینی، دانشگاه علوم پزشکی تهران، تهران، ایران.

2 دانشجوی دکترای ژنتیک، مرکز علوم و تحقیقات دانشگاه آزاد اسلامی، تهران، ایران.

3 دکترای تولید مثل، مرکز تحقیقات بهداشت باروری ولیعصر (عج)، بیمارستان امام خمینی، دانشگاه علوم پزشکی تهران، تهران، ایران.

4 دانشجوی ارشد بیوشیمی، دانشگاه آزاد اسلامی، تهران، ایران.

چکیده

مقدمه: عملکرد تخمدان توسط پیام یا سیگنال‌های عصبی- هورمونی به‌طور همزمان باید رشد فولیکولی، ترشح استروئید و اوولاسیون را کنترل ‌نماید. گیرنده‌های آلفا و بتای سیستم سمپاتیک در تنظیم فعالیت سلول‌های تکا و گرانولوزا جهت فولیکوژنزیس و استروئیدوژنزیس نقش دارند. مطالعه حاضر با هدف بررسی تغییرات بیان ژن‌های سه گیرنده آدرنرژیک آلفا 1، 2 و بتا 2 در سلول‌های کومولوس تخمدان زنان نابارور مبتلا به سندرم تخمدان پلی‌کیستیک (کاندید IVF) و تأثیر کلونیدین بر آن انجام شد.
روش‌کار: این مطالعه مورد شاهدی در سال 1396 بر روی دو گروه کنترل (زنان سالم اهداء کننده تخمک) و مطالعه (مبتلایان به تخمدان پلی‌کیستیک) انجام شد. تجویز داروهای محرک تخمک‌‌گذاری در دو گروه جهت رشد تخمک تجویز شد. بعد از گرفتن تخمک یا عمل پانکچر، از مایع فولیکولی مجموعه کومولوس و تخمک جمع‌آوری شد و سلول‌های کومولوس توسط آنزیم جدا و کشت داده شدند. سپس استخراج RNA از سلول‌های کومولوس انجام و غلظت RNA مورد نظر با دستگاه Nano drop خوانده شد. cDNA سنتز و برای ژن‌های ADRB2 و ADRα1,2 پرایمر طراحی و بیان ژن توسط تکنیک Real-time PCR سنجیده شد. در نهایت اثر داروی کلونیدین در سطح پروتئین ژن‌های ADRβ2 و ADRα1,2 در سلول‌های کومولوس دو گروه توسط تکنیک ایمونوسیتوشیمی (روش وسترن بلات) بررسی شد.
یافته‌ها: نتایج این مطالعه افزایش سطح بیان ژن در زنان مبتلا به تخمدان پلی کیستیک نسبت به گروه کنترل و تأثیرگذاری کلونیدین بر بیان ژن را تأیید کرد. شدت این تأثیرگذاری بر بیان ژن گیرنده‌ها به‌ترتیب ADRα2 < ADRβ2 < ADRα1 بود (02/0p<).
نتیجه‌گیری: بر اساس نتایج بیان ژن و ایمونوسیتوشیمی، کلونیدین توانست بیان ژن سه گیرنده آدرنرژیک به‌ویژه آلفادو را به‌طور معنی‌داری در بیماران کاهش دهد، لذا می‌توان درمان فارماکوتراپی کلونیدین را پیشنهاد نمود.

کلیدواژه‌ها


عنوان مقاله [English]

Changes in expression of alpha-1, 2 and β-2 adenoidal receptor gene in cumulus cells of infertile women with polycystic ovary syndrome (IVF candidate) and effect of clonidine on it

نویسندگان [English]

  • Farideh Zafari Zangeneh 1
  • Elaheh Aboutorabi 2
  • Masoumeh Dehghan 3
  • Sara Zabihzadeh 4
1 Associate Professor, Vali-e-Asr Reproductive Health Research Center, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran.
2 PhD student in Genetics, Islamic Azad University Sciences and Research Center, Tehran, Iran.
3 PhD in Reproduction, Vali-e-Asr Reproductive Health Research Center, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran.
4 M.Sc. student in Biochemistry, Islamic Azad University, Tehran, Iran.
چکیده [English]

Introduction: Ovarian function should control follicle development, steroidogenesis, and ovulation processes by ovarian external and internal hormonal signals. Sympathetic system Alpha and beta receptors play a role in regulation of theca and granulosa cells activity for folliculogenesis and steroidogenesis. This study was performed with aim to investigate the changes in expression of alpha 1, 2 and beta 2 adrenergic receptors gene of ovary cumulus in infertile women with polycystic ovarian syndrome (IVF candidate) and effect of clonidine on it.
Methods: This case-control study was conducted in 2017 on two groups of control (healthy women donating oocytes) and case (patients with polycystic ovaries). Ovulation induction drugs were prescribed in two groups for ovulation growth. After oocyte puncture, the follicular fluid was collected with cumulus-oocyte collection.  The cumulus cells were isolated by enzyme and were cultured. RNA was then extracted from cumulus cells and its concentration was read by Nano-drop device. cDNA was synthesized and primer was designed for ADRB2 and ADRα1.2 gene expression and the gene expression was assessed by Real-time PCR technique. Finally, the effect of clonidine on protein level of ADRβ2 and ADRα1.2 genes in cumulus cells was investigated by immunocytochemistry technique (western blot) in two groups.
Results: The results of this study confirmed: 1) increasing of gene expression level in women with PCOS compared with control group and 2) efficacy of clonidine on gene expression. The severity of the drug efficacy on receptors gene expression was ADRα1 <ADRβ2 <ADRα2, respectively.
Conclusion: The results of gene expression and immunocytochemistry showed that clonidine could significantly reduce gene expression of three adrenergic receptors, especially α2 in women with PCO, and therefore clonidine pharmacotherapy could be suggested.

کلیدواژه‌ها [English]

  • Clonidine
  • Gene expression
  • Immunocytochemistry
  • Polycystic ovary syndrome
  • Sympathetic nervous system
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