| Background:Spinal cord injury (SCI) is one of critical trauma in central nervous system (CNS), often caused by spinal column dearticulation or bone fracture after car accident and crash. Since the spinal cord is the transduction pathway of the movement, sensation and autonomic nervous system, the main symptom of SCI is the palsy due to the interuption of this transduction pathway. It principally represents as the disorder of movement, sensation and function of autonomic nerve under injury level. The palsy due to spinal cord injury brings heavy burden to the patient himself, his family and society, and also causes severe social problems sometimes. Fortunately, with the advancement of scientific research, the transplantation of neural stem cells (NSCs) which improves regeneration and reparation of spinal cord, gives a new hope for the treatment of SCI.NSCs is a sort of stem cells which could differentiate into nerve cells, they are extensively present in the obfactory bulb, the cerebral cortex, the ventricular zone subventricular zone, the corpora striata and the dentate gyrus of hippocampus. These cells have the potentiality of self-renew and multi-directional differentiation. NSCs can not only facilitate the regeneration of neurons and reparation of brain tissue, but also differentiate into neurons, astrcytes and oligodendrocytes in some conditions. The differentiation of NSCs in vivo and in vitro is affected by various factors, and the outcome of differentiation is closely related with microenvironment. Although the transplantation of NSCs brings a hope of the treatment of CNS diseases, the key point is how to induce NSCs to definitely differentiate into the neurons we want. In this way, the optimal microenvironment for orient differentiation has become hot in this field. Previous studies showed that the traditional Chinese medicine could not only prevent the nerve cells from injury, but also promote the proliferation of NSCs and induce them differentiate into functional neurons to cure the disease of the nerve impairment. Thus it is interesting to study the mechanism of the proliferation and differentiation of the NSCs, and the transplantation of NSCs may be a potential way to treat the disease of the CNS.Recently, traditional medicine monomers (the effective chemical components extracted from Chinese crude herbs) exert a distinct curative effect on the therapy of functional impairment of CNS. Evidence shows that ginkgolide B (GKB) facilitates NSCs to differentiate into neurons. Further researches discovered that GKB shares a similar ability as BDNF in promoting NSCs differentiate into neurons. It was reported that in vitro NSCs were induced to differentiate into neurons when Astragalus mongholicus injection was added in the culture medium. However, the effect of Saponins of Astragalus (SA), the essential component of Astragalus mongholicus on NSCs differentiation remains unclear. It is of great value to elucidate the effect and mechanism of GKB and SA in NSCs differentiation and repair of spinal cord injury. GFP transgenic mouse is a bred of mice with genetical stability to express green fluorescence protein in all cells. The cells isolated from the GFP transgenic mouse expressed the same fluorescence intensity as in vivo, although the cells divided for many times. So GFP transgenic mouse is an ideal model for the research on the biological features of living cells in vivo, and allow new approaches to life science research that general cell culture techniques cannot deliver.Objective:The effects of GKB and SA on NSCs differentiation was investigated in the NSCs derived from Kuming mice fetuses by immunofluorescence method. NSCs derived from GFP transgenic mouse was transplanted into the mice after spinal cord injury, the biological behaviors of the mice and the morphological changes were observed for the effects of the two traditional medicine monomers in this process.Methods1. NSCs, isolated from Kunming mice fetuses, were cultured in the medium pre-supplemented with 40 mg/L GKB or 20, 40 and 60 mg/L SA for 24 h, and 3 and 7 d. The length of the processes of these differentiated cells was observed under and inverted phase contrast microscope. The expressions ofβ-Tubulin, GFAP and CC-1 were detected by immunofluorescence staining, and the percentage of the positive cells was recorded. The effect of GKB and SA on the NSCs differentiation was compared.2. In vivo experiment, there were 3 groups, sham operation group, SCI group and NSCs transplantation group. The SCI animal model was established by opening vertebral canal and entirely cutting off T11 segment of spinal cord. In the NSCs transplantation group, the animals received a microinjection of NSCs derived from GFP transgenic into the lesion immediately after the injury. The growth, integration and differentiation of the transplanted NSCs were observed with immunohistochemical methods for the neural pathological changes. Hind limb BBB functional assessment was performed to evaluate the spinal cord function.Results1. NSCs were successfully isolated from Kunming mice fetuses, and cultured. These NSCs were identified to be Nestin positive.2. The length of processes in the GKB groups (1 and 3 d), were significant longer than that of control groups (P<0.05). The length of processes in the SA groups (20, 40, and 60 mg/L) were significant longer than that control groups (P<0.05). And the length in the three SA groups were significant shorter than that of GKB groups (P<0.05), but there is no significant difference among the three different-dose groups (P<0.05).3. The percentages ofβ-Tubulin+ neuron-like cells and GFAP+ astrocytes in GKB groups were significant larger than control groups (P<0.01), and the percentage of CC-1+ oligodendrocyte-like cells had no marked change compared with control groups. The percentages ofβ-Tubulin+ neurone-like cells in 20 mg/L SA group had no significant difference with control groups, and the percentages of these cells in 40 and 60 mg/L SA groups were significant larger than control groups (P<0.01), and 40 and 60 mg/L SA groups had notable increase in comparison with 20 mg/L SA group (P<0.01), but there was no difference between 40mg/L and 60mg/L SA groups (P>0.05). The percentages of GFAP+ astrocytes in three SA groups had no significant difference with control groups (P>0.05). So did the percentages of oligodendrocytes in SA groups. There was no marked change among these three SA groups (P>0.05).4.In vivo study showed the NSCs which transplanted into the impaired spinal cord could survive more than one month and differentiated into neurons, this result suggested that the transplanted NSCs could at least partly repair the the impaired spinal cord.5 . The data of the hind limb BBB functional assessment indicated that the transplantation of NSCs obviously improved the motor function of the injured hind limb. Conclusion1. Both GKB and SA have the effect to improve the growth of nerve processes in isolated NSCs, but the effect of GKB is more significant. Both of them can induce the NSCs to differentiate into neurons, but GKB also improves the differentiation into astrocytes. There was no significant difference in the CC-1+ oligodendrocyte-orientation differentiation between the two traditional medicine monomers. Our results suggest that combined using these two monomers will be beneficial for the treatment of spinal cord injury. GKB is better for improving the growth of nerve processes, and SA is only to induce the differentiation of NSCs.2. In vivo study indicates that the transplantation of GFP transgenic mice NSCs into impaired spinal cord could promote the reparation of the function of spinal cord. Our research suggests that GKB and SA are able to promote the spinal cord injury reparation, but further study is needed.
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