Expression Of Galanin And Its Effects On The Olfactory Ensheathing Cells

Galanin (GAL) is a 29 - 30 amino-acid neuropeptide. It is spread throughout the central nervous system. Galanin has been involved in multiple functional regulations, for example, it is involved in the regulation of learning and memory, neuroendocrine and neuronal activity functions. This experiment investigates the expressions of GAL and it's receptors in olfactory ensheathing cells (OECs), a special glial cell, and further studies the effects of GAL on these cells.To do this, four expressions were carried out. First, we selected neonatal (one- to three-day-old) SD rats and got olfactory bulbs of their brain. The olfactory nerve layer was peeled away from the rest of bulb and the OECs cultures were obtained. Then, the culture was treated with cytosine arabinoside (Ara-C) and the purified OECs were received. These cells were enriched by addition of bovine pituitary extract (BPE) to the culture medium. Whereafter, the immunohistochemical ABC method and the immunofluorescent staining were used to identify OECs. Second, to determine if OECs in vitro expressed GAL and its receptors including GalRl, GalR2 and GalR3, we detected the expression of GAL protein using immunofluorescent staining in this study, and then, the RT-PCR analysis was used to detect mRNAs for GAL and its three receptors. Third, to determine effects of GAL on neonatal OECs, 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 OECs. Then, the TUNEL method was further used to detect if GAL could result in cell apoptosis. Finally, to further study the mechanism of GAL on the proliferation of OECs, the semiquantitative RT-PCR analysis was used to detect the expressions of several neurotrophic factors that associated with the growth and survival of nerve cells, for example, LIF, CNTF, GDNF, NTN and BDNF. By these changes of their expressions, we concluded some functional mechanisms of GAL. In the experiment, the human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was carried out to be the internal standard.The followings are the results of this study: 1) By the above methods of isolation and purification, we successfully obtained OECsand the cell purity was 90-93%. What's more, OECs could grow stably in vitro.2) In vitro, OECs express mRNAs for GAL, but the GAL protein is not detected. Furthermore, OECs express mRNA for GalR2 but not for two another receptors, GalRl and GalR3.3) Except lnM GAL1-11, every concentrations of GAL and two receptor agonists, GAL1-11 and GAL2-11, can significantly inhibit the proliferation of OECs. Moreover, the effects of GAL1-11 and GAL2-11 may be stronger than GAL. But the influence can be blocked by M35, a nonspecific antagonist of GAL receptors. In addition, the result of TUNEL analysis showed that GAL didn't lead OECs to apoptosis.4) After OECs were incubated with GAL for 3 days, the expressions of LIF and GDNF decreased significantly in these cells, but there was no any change on the expression of CNTF. It was worth to saying that OECs expressed LIF, which was not reported previously.In a word, using the isolation and purification method, we obtained more pure OECs from the olfactory nerve layer of newborn rat olfactory bulb, 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. In addition, OECs only expressed the receptor GalR2, which suggested that, through the receptor GalR2, GAL aroused the corresponding biological efficacy, including decrease of LIF and GDNF expressions. Meanwhile, we concluded the decrease of these two neurotrophic factors expressions may be one of the reasons why the cell proliferation was inhibited.