| The nerve growth factor ( NGF ), a neurotrophic polypeptide, belongs to a closely related family of neurotrophins. In the past few years, the mechanisms by which neurotrophins, such as nerve growth factor, promote the survival and development of sympathetic and sensory neurons through its high affinity receptor-pp140c-trk have been the focus of much inquiry. Although a wide variety of signaling processes may be involved, protein phosphorylation appears to play a prominent role. The NGF receptor, ppl40c-trk is a tyrosine kinase, that undergoes autophosphorylation in response to NGF. Activation of this kinase initiates a cascade of molecular interactions, ultimately resulting in stimulation of the activity of a number of serine/threonine kinases, including the extracellular signal-regulated kinase (ERK). The rat adrenal pheochromocytoma cells (PC 12) which express the NGF receptors are useful model systems for studying the role of NGF. To elucidate the precise function of the ERK pathway and other proteins in NGF action, the present study was performed in several parts as follows:1. Establishing the NGF-induced PC 12 cell differentiation model. Phase-contrast microscopy and microscopy planometer were used to observe cell differentiation.The results showed that the normal PC 12 cells were round or elliptical and had a diameter of approximate 6-8 um. After treatment with NGF, cells produced very little neurite outgrowth at 12 h, and the processes were more obvious at 24 h. There were elongated and polarized PC 12 cells and the number of outgrowths was added. At 48 h, cell neurite outgrowths extended continually and the longest outgrowth could reach 80 um. PC 12 cells were treated with NGF in different concentration (5 μg/Lx 10 μg/L, 20 μg/ and 50 ug/L), then our study showed that along with the increase of NGF concentration, the diameter, the length and the number of axon-like processes of PC 12 cells all added or extended obviously. So we choosed 50 ug/L as the concentration ofNGF used in the study.2. Observing the effect of NGF on protein expression of ERK and pp-ERK and using the MEK inhibitor, U0126, to more clearly define the role of ERK.After treatment with 50 ug/L NGF, cell extracts were subjected to immunobblott-ing with antibodies against ERK and pp-ERK. The results showed that there were not distinct difference of ERK protein between the normal cells and the treated cells. However, NGF maximally increased the pp-ERK protein within 5 min, and the activation of pp-ERK remained for at least 1 h. On the other hand, our study showed that along with the increase of NGF concentration, the protein level of pp-ERK in PC 12 cells increased too.To confirm whether U0126 can block ERK activation via inhibition of MEK, we treated PC12 cells with 50 ug/L NGF at different point (10 mim 30 min> 1 h and 2 h )in the presence or absence of 10 umol/L U0126. Cell extracts were subjected to immunobblotting with antibodies against pp-ERK, and the results showed that U0126 blocked the activation of ERK completely and instantly. Using phase-contrast microscopy and microscopy planometer to observe the effect of U0126 on NGF-induced neurite outgrowth, we found that PC 12 cells incubating with U0126 before addition of NGF were round or elliptical. Compared with the normal PC 12 cells, there was not clear difference. Along with the delay of the adding point of U0126, the diameter, the length and the number of axon-like processes of PC 12 cells all increased and extended.3. Compared with the PC 12 cells treated with 1% FBS, we observed the protein expresses of P53 and P21 induced by NGF, and used U0126 to more clearly define the relation between ERK, P53 and P21.PC 12 cells were treated with both NGF and 1% FBS, respectively, then the flow cytometer was used to mensurate the cell cycle. Experimental results showed that we could observe GI cycle arrest in both of two groups' cells. NGF and 1% FBS both increased the protein expression of P53 and P21, but with different kinetics. In NGF-treated groups, P53 increased star...
|
PDF Full Text Request