Study And Comparison Of The Mechanism Of GAP-43 On Orientational Differentiation Of Retinal Stem Cells And Neural Stem Cells

There are many refractory diseases in ophthalmology such as age-related macular degeneration, ischemic optic neuropathy, glaucoma, etc. These diseases can lead to blindness due to the demage or loss of the retinal neurons. The discovery of retinal stem cells (RSCs) provides a new method for treatment of these refractory eye diseases.RSCs have the ability to undergo multiple potential differentiation, and researches have shown that RSCs can differentriate into retinal photoreceptors, retinal ganglion cells and other types of retinal cells. Recent studies have shown that RSCs can differentiate into specific type of retinal cells after modification of some specific genes, but the definite mechanism of this phenomenon is not yet known.Growth associated protein-43 (GAP-43) is a type of phosphoprotein located on the cell membrane of neurons and always distributes to the soma and axon of the neuron. It has been shown that the GAP-43 gene plays an important role in the development of retina and nerve system, regeneration of axon and construction of synapse. However, the mechanism of GAP-43 in the neuronal differentiation of neural stem cell (NSCs) or other stem cells is not known, we take the research of GAP-43 and the differentiation of RSCs and NSCs together, we hope to elucidate the specific mechanism of GAP-43 in different stem cells and make a comparison.Expression of GAP-43 in the retina of neonate rats was detected by immunocytochemistry staining and RT-PCR, which proved that the gene is involved in the retinal development of neonate rat. Falling which, isolation, cultivation and amplification of RSCs and NSCs by serum-free cultivation was carried out in vitro. Both RSCs and NSCs of the second generation expressed marker of neural stem cells such as nestin, and the marker of proliferation such as BrdU. The expression of Pax6 can be detected by RT-PCR in RSCs. We can find that the characteristics of cell cultivation and proliferation between RSCs and NSCs are similar from growth curve of stem cells, compared to NSCs, the ability of self renewal of RSCs was lower. The characteristic of cell differentiation between RSCs and NSCs was also similar, both stem cells can differentiate into neural cells with typical morphology by induction with serum, and express the makers of neuron and glial cell such as NSE, NF,βⅢ-tubulin and GFAP. Thy1.1, a marker of retinal ganglion cells can be detected by RT-PCR in differentiated cells of RSCs, which indicated that some of the differentiated cells showed characteristics of retinal ganglion cells. Through immunocytochemistry staining, we found that nestin, a marker of stem cells, decreased after differentiation in both stem cells, which indicated most stem cells entered the status of differentiation. The ratio of NSE-positive cells and the ratio of GFAP-positive cells in the differentiated RSCs were significantly lower than those in the differentiated NSCs, which indicated that the neuronal differentiation of RSCs were lower compared with NSCs. The expression of GAP-43 can be detected by immunocytochemistry staining and RT-PCR on the third day and the seventh day of differentiation of RSCs and NSCs, which indicated that the GAP-43 gene played a role in the neuronal differentiation of both stem cells.Next, the sequence of GAP-43 gene was identified by DNA sequencing, then we introduced GAP-43 gene into RSCs and NSCs by gene transfection with Lipofectamine 2000, gene expression was then proven by immunocytochemistry staining, RT-PCR and western blot after G418 selection. Both stem cells were induced by 5% FBS after a week of G418 selection. GAP-43 gene modified stem cells showed more facility in differentiation, which was proved by more processes of the neural cells and the more extensity, furthermore, the ratio of NSE-positive cells significantly increased in differentiation of gene modified stem cells (P<0.05), The ratio of GFAP-positive cells showed indistinctive change(P>0.05), which was also shown by the mRNA level by real-time fluorescent quantitative PCR(FQ-PCR), the makers of the neuron increased in differentiation(partial P<0.05), the marker of glial cell showed indistinctive change(P>0.05). The results indicated that GAP-43 gene can promote the neuronal differentiation of RSCs and NSCs. Compared to RSCs, the effect of GAP-43 gene on NSCs is more obvious in morphology.To sudy the role of GAP-43, we transfected the stem cells with pGCsi-U6/Neo/GFP/ GAP-43 shRNA successfully, the silence of gene in differentiation was proved by immunocytochemistry staining, RT-PCR and western blot after G418 selection. After inhibiting the expression of GAP-43, both stem cells showed obstruction in the development of processes in the bodies of differentiated neurons, the number of NSE staining positive cells and the relative mRNA level of NSE andβⅢ-tubulin decreased in gene modified stem cells(partial P<0.05), the number of GFAP staining positive cells and the relative mRNA level showed indistinctive change respectively (P>0.05). The results indicated the silence of GAP-43 gene inhibited the neuronal differentiation of RSCs and NSCs. The effect of GAP-43 gene silence on NSCs is more obvious than RSCs in morphology. In summary, expression of GAP-43 gene can promote neuronal differentiation of RSCs and NSCs. On the contrary, silencing of GAP-43 gene inhibited the neuronal differentiation of both stem cells. GAP-43 gene played an important role of guidance in neuronal differentiation of RSCs and NSCs, with the effect of GAP-43 gene on NSCs more obvious than the effect on RSCs. The research constructs the basis to further exploration of the mechanism of neuronal differentiation of both stem cells.