The Expression Changes And Significance Of Mammalian Target Of Rapamycinin The Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells Into Neurons

Background&ObjectiveMouse bone marrow-derived mesenchymal stem cells (MSCs) are adult stem cells that have the potential to undergo multi-lineage differentiation into multiple connective tissue cell types. They have a high capacity in self-renewal and proliferation. Under different conditions it can differentiate into mesoderm tissues, such as osteoblasts and cadiocytes, while it also can differentiate into the ectodermal tissues, such as neuron. Because of the convenience in obtaining, strong capacity of proliferation and large potential of differentiation, it can be widely used in clinical application. transplantation to treat neurological diseases has become an important domain of neuroscience research. The research about the differerntiation of MSCs into neurons and the application in the nervous systerm related diseases as a therapeutic tool have become an significant territory of neuroscience research. Unlike neural stem cells, MSCs differentiate into neural cells by a mechanism of transdifferentiation, but the regulatory factors involved remain poorly understood.The target of rapamycin (TOR) was originally discovered in the mutations of Brewer’s yeast. Genes of tor1and tor2encode two large (280kDa) protein TOR1and TOR2, which are highly homologous (70%). In1996, Abraham et al. also found the structure and function conservative of TOR in a mammal, and it was called mTOR. mTOR has a kinase domain of about234amino acids in the C-terminal, and thereby it is considered as a member of the phosphatidylinositol3-kinase related kinase (PIKK) protein family. In mammals, there was only one mTOR gene, which encoded2549amino acids in human, mouse and rat. So far can be determined is that the mTORCl contains at least three kinds protein mTOR, raptor and mLST8, whereas mTORC2contains mTOR, rictor and mLST8. The upstream and downstream signals of mTOR pathways are more complex. Insulin and growth factor (insulin-like growth factor, platelet-derived growth factor and epidermal growth factor, etc.) gather together in the transmembrane which contains insulin receptor and tyrosine kinase receptor (IR RTK), activated the type I phosphatidylinositol3kinase (Class I PI3K). Class I PI3K phosphoric PIP into PIP3and then PIP3bind to Akt/PKB and its activation molecule PDK1. mTOR effecters4EBP1and S6Ks promote autophagosome formation when mTOR activity is inhibited. PI3K/Akt/mTORCl signaling pathway plays a key role in the regulation of cell growth and proliferation. The pathway integrated the nutritional molecules, energy status and growth factor signals and regulated a large number of life process, such as autophagy, ribosome biosynthesis and metabolism.The aim of this study is to To investigate the distribution changes and significance of mammalian target of rapamycin (mTOR) in the differentiation of rat bone marrow mesenchymal stem cells(MSCs) into neurons and thereby to explore the potential roles of mTOR signaling pathway in neuronal differentiation of bone marrow mesenchymal stem cells (MSCs).Materials and MethodologyRat MSCs were cultured by conventional method in vitro,and were induced by β-mercaptoethanol(β-ME) to differentiate into neurons. The experiment were divided into MSCs blank control group, Dimethyl sulfoxide(DMSO) negative control group,β-ME induced group, different concentrations rapamycin intervention group, different concentrations rapamycin intervention group+P-ME induced group. The immunofluorescence was used to test the intracellular distuibution of mTOR before and after induction, and the fluorescence was observed under laser confocal microscope. Western Blot method was used to detect the expression of neurons related proteins and mTOR before and after induction.Results1. Before induced, mTOR were mainly distributed in the cell nucleus as points, and a small amount were distributed in cytoplasm; after induction, the fluorescence signal in nuclear was abate, mTOR transfered into cytoplasm. After the treatment of rapamycin, with the concentration increased of rapamycin(10to30μmol/L), the phenomenon that mTOR transfered to cytoplasm was more obvious. When the concentration of rapamycin was more than50μmol/L, the morphological of MSCs became round, and part of the cells take off from the wall (P<0.05). Meanwhile, the cell death rate was increased significantly.2. MSCs were induced by (3-ME to differetiate into neurons and the expression of Tau and MAP-2was increased significantly. After the treatment of rapamycin, with the concentration of rapamycin(10to20μmol/L) increased, the expression of Tau and MAP-2gradually was increased. When the concentration reached30μmol/L, the expression of Tau and MAP-2was down(P<0.05).3. Western blot results suggested that:the total expression of mTOR was same before and after induction; the expression of phospho-mTOR phbspho-p70S6K phosphor-4EBP1was down after induction(P<0.05); after the treatment of rapamycin, the expression of phospho-mTOR phospho-p70S6K phosphor-4EBP1declined more obviously(P<0.05).ConclusionDuring the induction of MSCs to neurons, mTOR transfers into cytoplasm from nuclear, and the expression of mTOR decline. mTOR may play important role in regulating MSCs differentiation into neurons.