Isolation, Differentiation And Influencing Factors Of Mouse Spinal Cord Neural Stem Cells

Objective:Neurodegenerative diseases are caused by neurons loss or myelin loss, which worsen gradually and lead to dysfunction eventually.The pathological mechanism of neurodegenerative diseases is largely unknown due to the complexity of their pathogenesis and the poorly understanding of the pathogenesis of these diseases. There was no positive clinical treatments and effective measures yet. However, neural stem/progenitor cell therapy bring hopes for the treatment of such diseases as neural stem/progenitor cells have great potential values for clinical diseases. The regulated mechanisms for the proliferation and differentiation of stem cells needed to be clarified before the clinical therapy of stem cells, so we can modified the strategies and methods to directed differentiation of stem cells for clinical uses. Rho/Rho(Ras homologue) kinase signal transduction pathway is one of the important signaling pathway in the regulation of stem cell proliferation and differentiationand the regulated effect is not clear in the spinal cord neural stem cells. Here, we uesthe Rho kinase inhibitor, Y-27632, to study the regulated effect of proliferation and differentiation in the neural stem cell from mouse spinal cord.Method:Select 13-15 day-old (embryonic stage)C57 mice and isolated the spinal cord neural stem cells through trypsin digestion. Neural stem cells were cultured in suspended system and identified with neural stem cell specific marker Nestin and Sox2 by immunofluorescence staining, The Nestin and Sox2 positive neural stem cells were divided into control group and experimental group with addition of Y-27632 (10μmol/L) in order to decipher the effect of Y-27632 on the proliferation and differentiation of neural stem cells. To add continuly Y-27632 for 5-7 times to the culture system for neural stem cells; To detect the expression level of neural stem cell special marker, differentiated cell markers and Brdu/Nestin double positive cells through immunofluorescence staining,real-time-RT-PCR and Western Blot methods; To analyze the effect of Y-27632 on the differentiation of neural stem cells by adding Y-27632 to the differentiated system containg 0.5% FBS, through immunofluorescence staining,real-time-RT-PCR and Western Blot methods.Results:Spinal cord neural stem cells can be successfully obtained from 13-15-day old embryonic mice by trypsin digestion and the percentage of neural stem cells with Nestin/Sox2 double marked by immunofluorescence staining is above 90%. From cell quantity to RNA expression level and to protein expression level, Y-27632 decrease the proliferation capacity of spinal cord neural stem cells cultured in suspension and facilitate the differentiation compared to the control by immunofluorescence staining assay, Western blot and RT-PCR analysis. To further decipher the effect of Y-27632 on the differentiation of neural stem cells, Y-27632 is added to the spontaneous differentiation system and Y-27632 benefit the spinal cord neural stem cells differentiation into the GFAP positive cells compared to the control.Conclusion:Y-27632 (10μmol/L)decrease the proliferation capacity of spinal cord neural stem cells in mice And promote the differentiation of spinal neural stem cells into GFAP positive astrocytes.Objective:Neurogenesis in the mammalian central nervous system is believed to end after birth. So brain and spinal cord injuries typically lead to permanent functional loss due to lack of neurogenesis. However, neural stem cells were isolated and cultured in vitro successfully from the striatum of the adult mouse brain and from the adult mouse spinal cord in 1992 and in 1996, respectively, which indicated neurogenesis exists continuously along the life period. So stem cell therapy will be potentially used to treat the neurodegenerative diseases. Research on neural stem cells experienced from embryonic stem cells, embryonic cultured neural stem cells from an adult animal to obtain neural tissue directly on neural stem cells. Study on the top of the two, due to the inevitable rejection, the use of immunosuppressant to high dose. The neural stem cells obtained from adult animals have no problems in this aspect. But the culture of spinal neural stem cells is more difficult due to the length of spinal cord, impurities, and inhomogeneity of different segments in spinal cord.Here, the experiments were performed to find which segments in mice spinal cord have enriched spinal cord neural stem cells and to discover the characters of neural stem cells from different segments of the spinal cord and to modify the culture system for spinal cord neural stem cells in the adult mice.Method:Choose between 8 and 12 weeks of adult C57 mice, isolated spinal cord tissue, in accordance with the cervical, thoracic, lumbar and sacral tail points into four sections, each after enzymatic digestion and cultured obtain neural stem cells. Immunofluorescence staining was identified with neural stem cell specific marker nestin, Sox2 cells and were counted and compared. After passage, respectively, doubling the number of. Each segment of the neural stem cells to the differentiation medium of natural differentiation for 7 days with 0.5%FBS, were observed by immunofluorescence staining. The mouse spinal cord segments were extracted RNA and protein, The content of neural stem cells was detected by blot western and RT-PCR. For each segment of the spinal cord after frozen section for immunofluorescence staining after neural stem cells compared to the situation.Results:From 8 to 12 weeks of the adult C57 mice isolated spinal cord tissue by enzymatic digestion and culture can be succeed neural stem cells, by immunofluorescence staining assay with neural stem cell specific marker nestin, Sox2 cells, from cell counts on the thoracic< cervical< lumbar< sacral. The number of cells from multiplying:thoracic< cervical< lumbar< sacral. The content of RNA in stem cell and protein level of neural:cervical< thoracic< lumbar< sacral. Cells in the neural stem cell count after staining sections of cervical< thoracic< lumbar< sacral.Conclusion:Contents of stem cells in the spinal cord of adult C57 mice:cervical < thoracic< lumbar< sacral. From the effectiveness of cell culture, at least the caudal segment of the foreign body, the highest levels of neural stem cells, in vitro culture of neural stem cells are the most.