| ObjectiveSpinal cord injury is a certain kind of central nervous disease which was caused by high energy trauma, constantly going with the fracture and dislocation of spine. The therapeutic research has always been the focus of national and a-broad scientific organization. The key problem is how to reconstruct the disconnection of spinal cord. Neural stem cells is a kind of cells which can differentiated to neurons, astrocytes and oligodendrocytes. There have been experimental evidences indicated that the neural stem cells can survive , differentiate and integrate in the injuried spinal cord. But the further neurobiologic understanding and mechanisms of neural stem cells transplantation remain incompletely understood up to now. The investigation followed consists of three parts, so as to investigate the signification of prominency in the differentiation and the mechanisms of neural stem cell transplantation on the spinal cord injury.1. We separated and cultured the neural stem cells derived of hippocampus of rat born within 24 hours. Add basic fibroblast growth factor,Epidermal growth factor,Insulin like growth factor 1 into the Dulbecco' s modified Eagle medium. Then differentiated the neural stem cells to neurons in vitro, and investigate the signification of growth associated protein 43.2. The model of spinal cord injury was made, 7 days later we transplant the neural stem cells labeled with bromodeoxyuridine into the injuried spinal cord, and observe the axis plasm transportation by the tracker of horseradish peroxi-dase 36 hours before sacrifice.3. On different time point after the neural stem cell transplantation, the morphological measurement such as immunohistochemical and reverse transcription polymerase chain reaction ( RT - PCR) were used to detect the expressions of growth associated protein 43 and glial cell line derived neurotrophic factor,so as to investigate to mechanism of neural stem cells transplantation on the spinal cord injury of rats.Materials and Methods1. Isolation > culture and differentiation of neural stem cells derived of hippocampus of wistar rat. Neural stem cells were identified by immunohistochemical of nestin, and the neurons were identified by immunohistochemical of neuron specific enolase ( NSE).2. The neural stem cells were cultured for 3 passages, then growth in the six - hole cultivate utensil. Cells were divided into experimental group and contrastgroup. The experimental group was mainly made up of Dulbecco' s modified Eagle medium which consisted of nerve growth factor and insulin like growth factor 1. The contrast group was made up of Dulbecco s modified Eagle medium which consisted of fetal bovine serum only. The immunohistochemical was made to i-dentify the neuron by NSE staining. On the 5 th day after differentiation choose ten slices randomly, and took count of the cells after HE staining, calculated the whole number and the NSE positived cells, then draw the statistical rate.3. Under the condition of experimental group above, on the lth,,3th,,5th and 7th day after the differentiation, the immunohistochemical staining was used to detect the expressions of growth associated protein 43.4. After several passages of neural stem cells cultivation, kept the estate of original clones, added bromodeoxyuridine into the Dulbecco' s modified Eagle medium to label the neural stem cells, adjusted the density of cells, and prepared for the transplantation.5. Wistar rats were randomly divided into cells transplantation group x Dulbecco ' s modified Eagle medium injection group and vacant group, under the state of asepsis the T10 level spinal cord injury model of rats were made in nar-cosis. Seven days after the spinal cord injury, the neural stem cells were transplanted by minim injector. And the Dulbeccos modified Eagle medium injection group received the same quantum liquid. 24 hours before the sacrifice the sciatic was exposed, the horseradish peroxidase was injected into it slowly by the minim injection.6. The rats were sacrificed on different time point after the transplantation, immunohistochemical was made to detecte if the neural stem cells still survived, and if the transportation of axes plasm was resume on a certain extent.7. Immunohistochemical, concomitanted with the Reverse transcription pol-ymerase chain reaction ( RT - PCR) were used to detect the expressions of growth associated protein 43 and glial cell line derived neurotrophic factor.8. The photographs were digitized with a video image analysis system (Metamorph image system 4. 6) in conjunction with a computer. The mean of grey levels for all slide were automatically detected. P <0.05 means the difference has statistics significance.Results1. Cultivating and evaluating the hippocampus derived neural stem cells. 24hours after the cultivation,, we could see 4-8 cell spheres, later the cell spheres would proliferate unceasingly. On the 5th day, many floating cell spheres could be observed, looked like mulberry, refraction well. After passage , there could be seen that each single sphere proliferated gradually and generated new cell sphere. Immunohistochemical showed that the Nestin positive re-actant located at the cytoplast of round - shaped neural stem cells, and the nucleus showed negative, the nucleus is large and cytoplasm can be seen. After pasted on the cliff the neural stem cells began to differentiate, the prominencies could be seen, immunohistochemical showed that the NSE positive reactant located at the cytoplast of neuron cells.2. After the differentiation of neural stem cells, both the experimental and control group sticked to the cliff swiftly and grow processus. The experimental cells proliferated within the first 24 hours. On the lst>3rd and 5th day after dif-ferentiation, both the experimental group and control group could observe the NSE positive cells. The brown reaction products were averagely distributed in the cytoplasm, and the nucleus was not stained. The controlled group has a total of 612 cells, NSE positive cells, ratio was 25.49%. The experimental group has a total of 673 cells, and the number of NSE positive cells is 233, ratio was 34.62%.3. During the course of differentiation, the cells adhibited cliff quickly, and emitted the prominency. Growth associated protein 43 appeared on the 1st day, and highly expressed in the prominency on the 3rd day, turned to the membrane on the 5th day, on the 7th day, the expressions of growth associated protein began lower and disappear, and the neural stem cell differentiated to neurons.4. Make the thorax 10 - level spinal cord injury model, the trail convulsively swing and two lower limbs paralysis as the standard. Immunohistochemical measurement was used, the neural stem cells labeled by bromodeoxyuridine were detected on the 7th day after transplantation. The cells survived and migrated rostrally and caudally from the injection sites. With the time processed, the cells labeled with bromodeoxyuridine decreased, 28 days after the transplantation , the horseradish peroxiclase positive cells could be detected at the rostral of the spinal cord injury site of treated rats, but not be found in the control rats.5. On the lstN3rd and 5th day after cell transplantation, Reverse transcription polymerase chain reaction ( RT - PCR) was used to measure the mRNA level of glial cell line derived neurotrophic factor and Growth associated protein 43 in the injuried spinal cord. We examined the similar time - dependent change in mRNA expression of them. The expressions of glial cell line derived neurotrophic factor and Growth associated protein 43 began to increase at 1 day, ascended with the time going on, and declined on about the 5 th day. The expressions of Growth associated protein 43 mRNA and glial cell line derived neurotrophic factor mRNA were highly expressed than the Dulbeccos modified Eagle medium group. The HE staining showed that the structure of spinal cord was destroyed, with bleedings inflamed cells invading x the putrescence of neurons and demyeli-nation. With the time going on, the structure of spinal cord was rehabilitation.Immunohistochemical measurement of the spinal cord at different time after the neural stem cells transplantation showed that the immunostaining for glial cell line derived neurotrophic factor and Growth associated protein 43 were much stronger than the Dulbecco 's modified Eagle medium group. The grey level showed that the distinction between the two group has statistics signification ( P <0.05).Conclusion1. The hippocampus derived neural stem cells can differentiate to neuron with the cooperation of NGF and IGF - 1.2. During the differentiation of neural stem cells into neuron, GAP - 43 has close correlation with the differentiation of neural stem cells. GAP -43 maybe plays an important role on switching and guiding the growth of process.3. The neural stem cells can survive, integrate and immigrate in the spinal cord after transplantation.4. The neural stem cell transplantation can improve the resume of neuraxon plasm transporation.5. The neural stem cells transplantation can improve the expression of GAP -43 and GDNF, this may be the mechanism of repair the injuried spinal cord.
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