اثر تعداد سلولهای گرانولوزا در محیط کشت در بیان ژن آنزیمهای مؤثر در ساخت هورمونهای استروییدی

نویسنده

گروه علوم دامی، دانشکده علوم دامی و شیلات دانشگاه علوم کشاورزی و منابع طبیعی ساری، ساری-ایران

چکیده

زمینه مطالعه: سلول‌های گرانولوزا دارای نقشی کلیدی در مسیر استروئیدوژنسیز هستند. هدف: از مطالعه حاضر بررسی تراکم سلول‌های گرانولوزا کشت داده شده حاصل از فولیکول‌های mm 5-2بر تراوش استرادیول و بیان ژن‌های مرتبط با ساخت استرادیول و پروژسترون بود. روش کار: به طور خلاصه، فولیکول‌های mm 5-2 از تخمدان گاوهای بالغ از کشتارگاه جمع آوری شدند، پس از جدا سازی و شمارش سلول‌ها با استفاده از رنگ تریپان بلو 4/0‌%، سلول‌های گرانولوزا در محیط DMEM/F12 به مدت شش روز کشت داده شدند. تیمارها شامل 1) 500 هزار سلول در mL‌500 محیط کشت، 2) 500 هزار سلول در mL 200 محیط کشت، 3) 250 هزار سلول در mL‌500  محیط کشت و 4) 250 هزار سلول در mL‌200 محیط کشت بود. داده‌های مربوط به بیان ژن‌ها با استفاده از نرم افزار JMP مورد تجزیه و تحلیل قرار گرفتند. نتایج: در شرایطی که مقدار محیط کشت ثابت بود کاهش تعداد سلول‌ها (کاهش تراکم) منجر به افزایش غلظت استرادیول گردید و در شرایطی که تعداد سلول‌ها ثابت بود کاهش محیط کشت (افزایش تراکم) سبب کاهش غلظت استرادیول گردید. میزان پروتئین و RNA استخراجی تفاوتی بین تیمارها نداشت. با کاهش تراکم بیان ژن‌های FSHR، CYP19، 17βHSD افزایش و بیان ژن‌های GADD45β و StAR کاهش یافت. نتایج مطالعه حاضر نشان داد کاهش تراکم سلول سبب افزایش تولید استرادیول و همچنین افزایش mRNA کد کننده آنزیم‌های استروژنیک گردید ولی بالعکس سبب کاهش بیان mRNA کد کننده آنزیم‌های پروژستژنیک (Progestagenic) گردید. نتیجه‌گیری‌نهایی: غلظت پروتئین با کاهش تراکم سلول تغییر نکرد در نتیجه می‌توان سلول را از اثرات مضر افزایش تراکم محافظت کرد.
 

کلیدواژه‌ها


عنوان مقاله [English]

Effect of number of culture medium granulosa cells on gene expression of enzymes associated with synthesis of steroid hormones

نویسنده [English]

  • Essa Dirandeh
Sari Agricultural Sciences and Natural Resources University, Sari-Iran
چکیده [English]

BACKGROUND: Granuloca cells have a key role during estroeidogenesis. OBJECTIVES: The purpose of this study was to determine the effect of number of culture medium granulosa cells on estradiol concentrations and mRNA codding estrogenic and progestagenic enzyme. METHODS: Briefly, follicles between 2 and 5 mm diameter were dissected from the ovaries of adult cows and were collected by rinsing the follicle walls with Dulbecco Modified Eagle medium/F12 (DMEM/F12). The number of cells was counted with a haemocytometer and the viable cells were assessed by the dye exclusion method using 0.4% Trypan Blue. Treatments were 1) 500,000 cell/500 ml, 2) 250,000 cell/500 ml, 3) 500,000 cell/200 ml 4) 250,000 cell/ 200 ml.  All data were analyzed by JMP (SAS). RESULTS: Low plating density increased E2 secretion and mRNA encoding LHR, FSHR and estrogenic enzymes (17βHSD, CYP19), whereas decreased mRNA encoding GADD45β. There were no differences among treatments for RNA and protein concentration. Low plating density also decreased protein amount but there was no difference among treatments for RNA amount. In conclusion, decreased cell density cause increase in mRNA encoding codding estrogenic enzyme gene expression and decrease in mRNA encoding progestagenic enzyme gene expression. CONCLUSIONS: Protein concentrations did not changed with decreased cell density therefore we can save cells against harmful effect of increasing cell density.
 
 

کلیدواژه‌ها [English]

  • cell culture
  • granulosa cell
  • Holstein cow
  • PCR
  • RIA
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