Study To Mechanism Of GAP-43 On Differentiation Of Bone Marrow Mesenchymal Stem Cells To Neuron-like Cells

There are many diseases such as optic atrophy, ischemic oculopathy and so on in ophthalmology, which can lead to blindness in the later period of diseases. There are no effect methods to control that completely until now. The pathological changes of these diseases are alike: disfunction or degeneration of retinal ganglion cells or optic nerve for some reason. Therefore it is the key point to supply retinal neural cells and to protect optic nerve. The development of gene technique and research on stem cells in the recent years provides new pathway to undergo cell therapy and gene therapy for those eye diseases.Bone marrow mesenchymal stem cells (BMSCs) are another kind of stem cells in the marrow besides haemopoietic stem cells. They are cells belong to mesoderm in early period of embryogenesis, and they have the potentiality to multi-directional differentiation. the research to BMSCs is the hot spot in scientific research of adult stem cells because BMSCs can be isolated and cultivated easily in vitro and transplantation of BMSCs can avoid the limits from immunologic rejection and ethics .BMSCs can be differentiated into various cell tapes such as neural cells, endothelial cells, lipocytes, osteocytes, hepatocytes etc. In ophthalmology, BMSCs can be differentiated into corneal epithelioid cells, photoreceptor-like cells and ganglion-like cells in vitro, furthermore, the researches show that BMSCs of intraocular transplantation could survive and integrate into ganglion cell layer, photoreceptor cell layer, and bipolar cell layer of retina, which could provide protection to impaired retinal ganglion cells. Neuronal differentiation of BMSCs has been studyed in resent years, BMSCs can be induce to differentiated into neural cells by some inducers and growth factors, but the exact mechanism is still unknown. Scientists hope to undersand the mechanism of neuronal differentiation of BMSCs in order to produce neural cells to treat the disease by cell engineering.In the past research, we induced BMSCs to differentiate into retinal ganglion-like cells, furthermore, we screened a variable expressed gene - growth associated protein -43 (GAP-43) gene by suppression subtractive hybridization (SSH). Thus means this gene maybe play an important role in the differentiation because GAP-43 is an important factor in neuron growth and regeneration. Therefore,further understanding to function of GAP-43 in neuronal differentiation is important to clearing the mechanism of neuronal differentiation and to tissue engineering research of BMSCs.Rat BMSCs were isolated and cultivated by combination methods with density gradient centrifugation, plastic adherence method and digestion control. Cell surface markers of the third passage BMSCs were assessed to characterize the purity by flow cytometry analysis and immunocytochemistry staining. BMSCs expressed CD90, CD71, CD44, andCD29, but not CD34 and CD45. The results showed that the cultivated cell were BMSCs with high purity.In our study, we firstly detected the expression of GAP-43 on the differentiation of BMSCs to neuron-like cells in vitro by various induced methods in order to understand its effect on this process. Our results showed that BMSCs could differentiate into neuron-like cells under ether induced condition with chemical inducer, growth factor or conditional differentiated agent of retina. The induced cell expressed maker of neuron: nestin, NSE, NF andβⅢ-tubulin respectively by Immunocytochemistry and RT-PCR detection. GAP-43 were found to expressed consistently in three induced conditions, that meant it attended the differentiated process and maybe have regulative ability to the differentiation.In the next study, total RNA of retina were extracted, GAP-43 gene was amplified by RT-PCR, then GAP-43 gene fragment was constructed to the eucaryotic expression vector pcDNA3.1(+). The correctness of gene sequence was idientified by Enzyme dissection analysis and DNA sequencing, and then GAP-43 gene was transfected to cultural BMSCs in vitro by Lipofectamine 2000 agent. Immunocytochemistry, RT-PCR and Western blot were used to detect GAP-43 mRNA and protein expression. The results showed that GAP-43 gene was transfected into BMSCs successfully and the expression can be detected by forementioned methods. Some of BMSCs differentiated into neuron-like cell and expressed neuron makers after transfection by GAP-43 gene. The neuronal differentiation ability of BMSCs was obviously promoted by induced condition of retinal conditional induced agent. There were increased quantity of neuron-like cells, the positive ratio of NSE and nestin staining, and the expression level of neuron makers such as nestin, NSE, NF andβⅢ-tubulin in GAP-43 gene transfection group compare to control group, and the significant difference between two group could be found. Results indicated that GAP-43 can promote the neuronal differentiation of BMSCs.Meanwhile we designed shRNA on the basis of GAP-43 sequcence, and constructed GAP-43-RNAi vector by using pGCsilencerTM U6/Neo/GFP which had a green fluorescent protein (GFP) as a label. The reconstructed plasmid was tansfected in to BMSCs by liposome. Green fluorescence could be observed in the next day after transfection. BMSCs made a fusiform shape after transfection. G418 was used to screen the transfected BMSCs, and BMSCs without GAP-43-shRNA were eliminated after screen. Forementioned methods were used to detect the expression inhibition of GAP-43 mRNA and protein under the induced condition. The resulted showed that GAP-43 expression of BMSCs was inhibited under induced condition after successfully transfection. The ability of neuronal differentiation of BMSCs was inhibited remarkably under induced condition, which was displayed by less quantity of neuron-like cells and dendrites, less positive ratio of NSE and nestin staining, and down gradulated expression level of neuron makers such as nestin, NSE, NF andβⅢ-tubulin in GAP-43-shRNA transfection group compared with control group, the significant difference between two group could be found.All the results revealed that GAP-43 gene is one of most important genes which can promote the neuronal differentiation process. GAP-43 expression could promote BMSCs to differentiate into neuron-like cells, and the ability of neuronal differentiation was elevated remarkably under induced condition. On the contrary, inhibition of GAP-43 expression can attenuate the ability of neuronal differentiation. These results will provide useful information to explore the exacted mechanism of directional differentiation of BMSCs and to gene therapy in clinic.