The Research Of Galanin's Effects On The Neural Stem Cells, PC12 Cells And B104 Cells

Galanin (GAL) is a 29 - 30 amino-acid neuropeptide. It is spread throughout the central nervous system. Galanin is involved in the regulation of neurogenesis and neural regeneration process. In this process, the proliferation of nerve cells are maily involved. To study the effect of galanin on neural cells proliferation, this experiment investigates the expressions of GAL and it's receptors in proliferative neural cells such as neural stem cells (NSCs), PC12 cells, B104cells, and further studies were made for uncerstanding the effects of GAL on the proliferation of these cells. Four expressions were carried out as follows:First, The neural stem cells from the subventricular zone of rat embryonic day 14(E14) were cultured in the serum-free medium consisting of DMEM/F12 (1:1), B27 supplement and bFGF. Identification was carried out using Nestin antibodies for undifferentiated cells. In order to determine the phenotype of the differentiated cells, MAP₂, GFAP, CNP, distinctive marker for neuron, astrocytes and oligodendrocytes were used.Second, to determine if NSCs in vitro, PC12 cells and B104 cells expressed GAL and its receptors including GalR1, GalR2 and GalR3, in this study, we detected mRNAs for GAL and its three receptors using RT-PCR analysis, and then, the immunofluorescent staining was used to detect GAL.Third, to determine effects of GAL on NSCs, PC12 cells and B104cells, MTT analysis was used to detect the effects of different concentrations of GAL and agonist (GAL1-11 and GAL2-11), antagonist (M35) of GAL receptors on the proliferation of proliferative neural cells.The fourth, the TUNEL method was further used to detect if GAL could result in cell apoptosis. LDH was detected to see if GAL causes cell death.The followings are the results of this study:1) With the methods of isolation and purification mentioned above, we successfully obtained the cells that exhibit properties and satisfy criteria representative of stem cells.2) NSCs express mRNA of GAL and all the three GalRs. PC12 cells express mRNA for all the three GalRs but not for GAL. B104 cells express mRNA for GAL and for GalR2 and GalR3 but not for GalR1.3) GAL and its agonists can inhibit the proliferation of NSCs and PC 12 cells but can increase the proliferation of B104 cells. All the effects can be blocked by M35.4) In addition, the results of TUNEL and LDH analysis showed that GAL didn't lead NSCs to apoptosis and cell death.In a word, using the isolation and purification method, we obtained much pure NSCs and studied the effect of GAL on these cells. The result showed that GAL inhibited these cells proliferation, but the effect was realized not by leading cells to apoptosis and death. It suggest that GAL mediates the effect by slowing the cell cycle. PC12 cells and B104 cells express different GalRs and have different effect to GAL. It suggests that different GalRs can mediate different cell function..