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توصيف جزيئي للخلايا الجذعية المتحفزة لتكوين خلايا عصبية == Molecular Characterization of Stem Cells that Induce Neurogenesis
Author name:
مائده حسين محمد
Supervisor name:
ناهي يوسف ياسين
General topic:
Biology
Specific topic:
Animal - Molecular Biology
Degree:
Doctorate
University:
University of Baghdad - College Of Science For Girls
Language:
English
University location:
Baghdad
First pages:
24T3485 - p.pdf
Abstract:
The in vitro isolation, identification, differentiation, and neurogenesis characterization (with molecular studies by conventional and real time - PCR) of stem cells source were investigated to produce two type of stem cells. This two types including neural cells and neural stem cells in culture which use it as a successful sources for further treatments. And finally chosen the best medium formula for maintenance the produced neural stem cells, also to prove their stemness state in culture. The mouse bone marrow mesenchymal stem cells was used as the source of stem cells in this study. Two type of neural differentiation formula was used to induced neural cells and then neural stem cells. The first formula was butylated hydroxyanisole, and the second formula was β - mercaptoethanol reagents. Also three type of different neural stages markers were used; nestin as immaturation stage marker, neurofilament light - chain as early neural marker, and microtubule association protein as maturation marker. These markers were represented the different neurogenesis stages started from mesenchymal stem cells (as undifferentiated cells), neural stem cells production stages, and towards neuron cells (as differentiated cells).The results of immunocytochemistry and real time - PCR of butylated hydroxyanisole differentiation method showed that in contrast to mesenchymal stem cells (as control group), neural differentiated cells showed neural progenitor pattern, by showing stable increased in nestin gene expression significantly through differentiation process for different exposure time, with increased of protein expression significantly compared with control. While, neurofilament light - chain gene and protein expression started to increase significantly, but not - IIreplacedthe nestin expression completely even when its expression passed nestin levels compared with control group. In contrast to the maturation marker microtubule association protein which showed a very low expression during the duration of differentiation period in protein and gene expression (with significant effect) compared with control. This results proved that the cells were still progenitors and do not passed the maturation stage, therefore there was a difficult to define the exactly time of neural stem cells production stage from this formula and as a good formula to produce neuron cells in culture. In other side, the results of immunocytochemistry and real time - PCR of β - mercaptoethanol (as another differentiation formula) were showed that this formula was successful to produce both neural stem cells as well as neuron cells from this formula. This occur through increasing and over expressing of nestin protein and gene significantly within the early hours exposure time reaching to the highest expression levels. Compared with decreased and lower expressed in neurofilament light - chain protein and gene expression levels compared with control; therefore from this stage (exposure time) we can produce neural stem cells in culture. And then decreased and lower expressed of both protein and gene expression of nestin significantly compared with increasing the expression of neurofilament light - chain protein and gene levels throw the late exposure times compared with control, this results indicated that this stage was starting of maturation stage toward neuron cells. Also the proving of this results was lower expression levels of almost all exposure times of microtubule association protein for their protein and gene (with significant effect).The results of gene expression analysis of using four different media formulas to proved the stemness state and maintained the neural stem cells in culture showed that 6 h exposure to β - mercaptoethanol was enough to create - IIIneuralstem cells which maintained using MEM media with 5% FBS, with b - FGF, or with b - FGF &EGF. This study confirms that the molecular characterization study of stem cells in culture will assist to studying and clarifying the neurogenesis pathway through neural differentiation process, by clarified the mechanisms of neural genes through the in vitro differentiation of stem cells, and therefore ability to produced both neuron cells and neural stem cells by using the molecular studies.
