Fare ovaryumu kök hücrelerinin Real Time-PCR ve immünhistokimyasal yöntemlerle araştırılması
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Date
2010
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Biyoteknoloji Enstitüsü
Abstract
The knowledge of formation and development of human oocyte which was acceptable until recent years has changed completely. As claimed, possible presence of oocyte and granulosa cells formation from stem cells will lead to major changes on reproductive biology and infertility treatments. Purpose of this research is to demonstrate possible existence and location of cells that have a potential of stem cells in mouse ovaries and to be used in autologous treatment of ovarian related infertility in future researches. Together with infertility treatments under in vitro circumstances, ovarian stem cells (OSCs) show unique totipotent features that have not been found in other adult stem cell types. They differentiate into fibroblasts, epithelial, and neural cells, as well as new eggs and parthenogenetic embryos producing embryonic stem cells (ESC). Because of these features, OSCs could be utilized for autologous treatment of premature ovarian failure and also for autologous stem cell therapy of neurodegenerative diseases without use of allogeneic embryonic stem cells or somatic cell nuclear transfer.This research was done on two weeks (pre adulthood) and eight weeks (adult) BALB-C type of mice ovaries. For histological analysis in cortex of ovarian tissue, general structures such as follicles, oocytes and epithelial germinativum after fixation with BOUIN solution and well-known tissue processing methods, were embedded in paraffin. Sections were stained with Hematoxylin Eosin (HE) and Periodic Acid Shift (PAS) for evaluation of follicles. In addition, by taking frozen sections from ovarium, these sections by using of antibody were investigated with stem cell markers (Sox2, Nanog and Oct-4). Possible expression and differences between expression of Oct-4, Nanog and Sox2 genes in two age groups of ovarian tissue samples were determined with Real Time-PCR.According to the results of immunohistochemical staining for both age groups, presence of Oct-4 (cytoplasm of ovarian epithelial cells, granulose cells, oocytes and theca cells), Nanog (nucleus of oocyte) and Sox2 (cytoplasm of ovarian epithelial cells, granulose cells, oocytes and theca cells) proteins have been detected.By using of RT-PCR, expression of Oct-4, Nanog and Sox2 genes were found in two and eight week mice ovaries. Moreover, according to the results of Real Time-PCR, Oct-4 and Nanog genes expression levels between two and eight week groups had significant differences.Remarkable differences in expression levels of Sox2 between two age groups were not observed. Expression level of Oct-4 in two week samples were more than eight week samples, while in case of Nanog expression level was higher in eight week samples. Our research is the first study that shows expression differences of these genes by quantitative RT-PCR.Because of these markers are ESC determiner, we considered that both of prepuberty and adult mice ovaries have had cells that sustain stem cells features.
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Fare ovaryumu, Real Time-PCR