The Differentiation Of NSC Induced By Astrocytes And The Repairing Effects On SCI

The study on repair of spinal cord injury (SCI) becomes a hot researching subject today. Conventional idea was that the neurons in adult mammal couldn't mitosis and the injured axon could hardly regenerate. The sensory and motor dysfunction resulting from SCI would be permanent and inreversible. But in these years, the discovery of neural stem cell (NSC) has finished the traditional conclusion. NSC not only has the ability of self-replication which can meet the quantitive requirement for transplanting cells, but also can differentiate into three different lineages: neurons, astrocytes and oligodendrocytes under some certain conditions. The differentiated neurons can replace the lost and injured neurons in spinal cord, and the differentiated astrocytes can produce many kinds of cytokines and neurotrophic factors beneficial for injured tissue to protect the neurons, improving the construction and function rehabilitation. The finding of NSC will bring us a splendid prospect and enhance our confidence in overcoming SCI. Some advanced researching proved that NSC transplanted into the damaged brain or spinal cord could survive and differentiate in vivo, recovering the loss function in some extent. However, the most of NSC (over 95 percents) differentiated into glial cells, but few into neurons, and the transplanted cells could not form the effective synaptic connection. Some exogenous cytokines such as BDNF, PDGF, FGF, TGF-βcan facilitate NSC to differentiate into more neurons, but in vivo the concentration of cytokines would decrease following the differentiation of NSC and their biological effects would be reduced, then hardly to keep the effective dosage, which limits the application of NSC in fact. Researching for the regulatory mechanisms of NSC differentiation become more and more important. Astrocyte is a very important kind of glia cells with a large number in central nervous system (CNS). Astrocytes can produce many types of neurotrophic factors incuding GDNF, NGF, BDNF, NT, CNTF, etc and several cytokines, such as EGF, FGF and CAM etc. These molecular substances will be beneficial for differentiation of NSC and improve the neurons surviving, growing and migrating. So astrocytes are playing a very important role in the development and regeneration of CNS. There are many differences in the quantities of produced cytokines and neurotrophic factors between various astrocytes, protoplasmic astrocyte (PAS) and fibrous astrocyte (FAS), so maybe they have different effects on the differentiation of NSC. The regulation by cells interaction has more virtue than the regulation by cytokines, less influenced by the enviroment and can realize the action much easier in vivo. Based on the reasons above, we designed the next researching program. At first we isolated, cultured, and identified NSC from embryonic spinal cord of E13-E14 Sprague-Dawley rats. Then we obtained purified PAS and FAS from 2d new born rats'cortex. The influences of each astrocytes as feeder cells on inducing NSC differentiation was observed and the best ratio between NSC and astrocyte for NSC differentiating into neurons was acquired. At the same time, NSC combined with astrocytes in the best ratio as donor (combination group) were transplanted into injured spinal cord. Compared with NSC group and PAS group, regeneration and repairing effects were observed by methods of pathology, immunocytochemistry, neuroelectrophisology and praxiology. We investigated NSC surviving and differentiating in vivo and concluded the repairing mechanisms of the combinated cells transplantation on injured spinal cord. The main results and conclusions are as followed: 1. We successfully isolated NSC from embryonic spinal cord. NSC could keep the ability of self-replication when cultured with bFGF and differentiate into different lineages: neurons, astrocytes and oligodendrocytes under the inducing of 5%FBS+DMEM/F12 medium. 2. By using the method of shaking in differential velocity, purified PAS and FAS was obtained, the purity over 95%. 3. We used immunocytochemisty technology to observe the differentiation of NSC cultured with PAS or FAS as feeder cells, then found that the percentage of neurons in PAS group (61%) was much more than FAS group (41%). This result suggested that PAS had more effects to induce NSC differentiating into neuron in vitro. 4. Through having cultured NSC with PAS in various ratios, we detected thepercentage of neurons differentiated from NSC and drew a conclusion that the ratio of 2:1 might have more advantages for NSC differentiating into neurons in vitro. 5. NSC combined with PAS in the ratio of 2:1 could differentiated into much more neurons than NSC single in the enviroment of SCI. 6. In vivo, we used different cells transplantation to treat SCI. By pathological methods we observed that the transplanted NSC combined with PAS in the ratio of 2:1 could survive more than 8 weeks, mend the damaged areas and spread fiber contacting with normal tissue. With NY tracing, we found the positive neurons in upper region of injured spinal cord in the combination group was much more than NSC group and PAS group, which suggested that the differentiated cells in the combination group might repair the fiber contact of injured spinal cord much better. 7. At 8 weeks after transplantation, observed by staining with AchE, the morphology of motor end-plate in the combination group repaired much better than NSC group and PAS group, which suggested that the lower limb muscle might re-acquire innervation and the degeneration of motor end-plate was inhibited effectively. Sensory and motor function in the combination group were rehabilitated much better than NSC group and PAS group detected with the technologies of neuroelectrophisology and praxiolog. The result proved that the combination group might repair the neurological function much better.