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Long Term Exposure to Nitric Oxide Caused Cell Cycle Arrest Due to Down-Regulation of Cdk۲ and CDK۴

عنوان مقاله: Long Term Exposure to Nitric Oxide Caused Cell Cycle Arrest Due to Down-Regulation of Cdk۲ and CDK۴
شناسه ملی مقاله: RROYAN21_036
منتشر شده در بیست و یکمین کنگره پزشکی تولید مثل و شانزدهمین کنگره زیست شناسی و فناوری سلول های بنیادی در سال 1399
مشخصات نویسندگان مقاله:

M Maleklou - Department of Biology, Arak University, Arak, Iran
MH Abnosi - Department of Biology (Cell Culture), Arak University, Arak, Iran

خلاصه مقاله:
Objective: Nitric oxide (NO) is a free radical and a signal-ing molecule which controls many cellular and physiological mechanisms such as; blood vessel contraction, blood pres-sure and immunological response. In previous studies it was shown that the short time treatment of mesenchymal stem cells (MSCs) with sodium nitroprusside (SNP) as an NO releasing agent at low concentration did not affect the cell viability, but caused metabolic imbalance. The aim of this study was to in-vestigate the effect of low concentration of SNP for long time on cell viability, proliferation, cell cycle and expression of the genes involved in cell cycle.Materials and Methods: MSCs after the third passage was treated every ۱ hour in each ۷۲ hours with ۱۰۰ μM of SNP. After ۵, ۱۰, ۱۵ and ۲۰ days, the viability and proliferation of the cells was estimated using MTT, Tripan blue and PDN, CFA method. In addition, we used flow cytometry to study the cell cycle whereas the profile of the proteins was evaluated using electrophoresis. The expression of Raf۱, Cdk۲, Cdk۴, P۵۳ and GAPDH genes involves in the cell cycle was determined using quantitative real-time PCR. Also, we checked the effect of the repeated passages on the control and treated of MSCs growth. Results: Cell treated with SNP caused the reduction of viability at ۵, ۱۰, ۱۵ and ۲۰ days. The same treatment caused the number of the colonies to reduce significantly at all treatment times, but no changes were observed in diameter of the colonies. In addition, the cell cycle was ۹۳% arrested at G۱ stage follow-ing SNP treatment at day ۲۰. In the MSCs treated with SNP, the expression of the Cdk۲ and Cdk۴ was reduced; whereas the expression of P۵۳ was elevated and the Raf۱ expression remained the same. In the treated cells we observed changes in the density of some polypeptides, where two polypeptides with the molecular weight of ۱۶.۷۳ and ۱۳.۴۰ were expressed only in the treated group, the cell treated with SNP tolerated up to ۱۱ passages and the changes included morphological abnor-malities, enlargement continuously from the ۱۰ passage in both groups with increasing passage. Finally, it showed a decrease in growth and ultimately proliferation arrest.Conclusion: This study showed that the long time treatment with NO at low concentration may cause the reduction of MSCs resources on one side and the cell cycle arrest at G۱ stage due to elevation of P۵۳ expression and reduction of Cdk۲ and Cdk۴ expression on the other side. Also NO period at low concentra-tion in long time might cause the stem cell reservoir to reduce. In addition, this compound in long time causes replicative se-nescence in bone marrow MSCs.

کلمات کلیدی:
Cell Cycle, Mesenchymal Stem Cells, Cell Viability,Cell Proliferation, Nitric Oxide

صفحه اختصاصی مقاله و دریافت فایل کامل: https://civilica.com/doc/1421317/