| Institute of Basic Medical Sciences Academy of Military Medical SciencesCell adhesion molecules play important roles in cell-cell and cell-extracellular matrix interactions for recognition, adhesion and signaling transduction. In nervous system, neural cell adhesion molecules are very important in forming the brain framework, neuron growth, synaptogenesis and cells interactions. NB-3 was firstly discovered in 1996 as a neural adhesion molecule that is a member of contactin/F3 subfamily in the immunoglobulin superfamily. NB-3 expresses exclusively in nervous system. At present, the function of NB-3 is little. It has been reported that NB-3 is involved in the differentiation and maturation of the oligodendrocyte. However according to its expression and typical IgSF's structure characteristic, we suppose that NB-3 may play roles in neurite outgrowth, synaptic plasticity, fasciculation of neural fibers, study and memory during development of nervous system.This study aimed to detect the expression and relative functions of NB-3 in the development of nervous system. We firstly investigated the expression of NB-3 at different stages of development; subsequently we observed the effect of NB-3 deficiency on neuron growth. In addition, we detected the role of NB-3 in neuron growth under hypoxia. Hypoxia is an important physiological stimulate factor, involved in embryogenesis, embryo-development and the maintenance of normal physiologic function. However, the effect of NB-3 on the growth of neuron under physiologic hypoxia is not clear. Therefore we preliminarily studied the role of NB-3 under hypoxia.The results are as follows:1. Expression of NB-3 at the different developmental stages of CNSWe used the heterozygote of NB-3 transgenic mouse which labeled by the Lac Z gene. The expression and location of NB-3 in brain were detected using X-gal staining in E12, E14, E16, E18, P0, P7, P14 and adult mice. The results displayed that NB-3 expressed widely not only in the embryonic period but also in adulthood, although the expression intensity was different. NB-3 was first found on E14 during embryonic development. Then NB-3 was also found in the surrounding lateral cerebral ventricle, cortex, thalamus, hypothalamus and hippocampus. In the postnatal early period the expression of NB-3 abruptly increased to a high level in the cerebrum, and reached a maximum on the postnatal seventh day. Thereafter, the expression declined a little, and it maintained this level during adulthood. The distribution of NB-3 was widespread in the cerebrum of adult mouse. The regions with the highest expression of NB-3 were the layers II/III and V of cortex, piriform cortex, amygdala, thalamus, hypothalamus and CA1 of hippocampus.2. Effect of NB-3 deficiency on the growth of the cortical neuronsSince NB-3 expression was higher in cortex, we investigated the role of neural adhesion molecular NB-3 in the growth of cortical neurons. We dissociated and cultured the cortical neurons from E18 wild type (WT) and NB-3 knockout (KO) mice respectively. And then we observed the survival and growth of NB-3 deficient neurons. The results showed that:(1) NB-3 expressed in the E18 cerebrum cortex detected by X-gal staining. It mainly localized in the layer II/III of the cortex. In the cultured E18 cortical neurons in vitro, NB-3 expressed mainly in the soma and partly in the neurite of neurons. The results displayed that NB-3 expressed in cortex both in vivo and in vitro.(2) We subsequently detected the effect of NB-3 deficiency on cell survival by MTT assay after 1d or 3d culture. The result showed that the number of cells in NB-3 deficient group was obviously less than that in WT group.(3) In order to study the role of NB-3 in the outgrowth of neurite, we first counted the rate of neurite sprouting using Coomassie brilliant blue R250 staining after 1d culture. The result showed that the rate of neurite sprouting was reduced in NB-3 deficient group compared with WT group.(4) Then we measured the longest neurite of neurons after Coomassie brilliant blue R250 staining. The result displayed that the neurite length in NB-3 deficient group was shorter than that in WT group. 3. Effect of hypoxia in the growth of cortical neurons and the role of NB-3During embryogenesis, cells are in the hypoxia environment all the time. It has been reported that hypoxia can promote neural stem cells (NSC) to differentiate into neurons and promote the survival of neurons under 1%~3% O2 in vitro. So we observed the growth of cortical neurons and the role of NB-3 in the growth of neuron under 3% O2 environment in vitro. The results showed that:(1) To investigate the effect of hypoxia on the survival of neurons, we counted the number of neuronal marker MAP2 positive cells by immunochemistry staining after 3d culture in normoxia and hypoxia environment. The statistic data displayed that the number of MAP2 positive cells in hypoxia group was obviously higher than that in normoxia.(2) We also tested the survival of cortical cells by MTT assay. Compared with normoxia, hypoxia could promote the survival of cortical cells in both WT and NB-3 deficient group during the early neuronal development.(3) Then we counted the neurite sprouting positive neurons, and found that hypoxia didn't affect the rate of neurite sprouting compared with the normoxia.(4) We also measured the length of neurite, and discovered that hypoxia enhanced the neurite length of neurons after 3d culture, though it was not changed after 1d culture.(5) In order to investigate the relation between NB-3 and the growth of neuron under hypoxia, we observed the levels of NB-3 mRNA and HIF-1αmRNA which was the key factor in response to hypoxia. We extracted the total mRNA of cells, and carried out the RT-PCR. We found that hypoxia did not affect the levels of NB-3 and HIF-1αmRNA.In a word, NB-3 deficiency not only reduced the survival of neurons, but also inhibited the outgrowth of the neurite. Therefore, we can get a conclusion that NB-3 plays an important role in the nervous system.
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