| Objective(1)To investigate the neural stem cells cultured in vitro by Bone marrow-derived mesenchymal stem cells-conditioned medium and bone morphogenetic protein,the influence of differentiation and its possible mechanism;(2)to explore the effect of BMSCs-CM on the recovery of spinal cord injury model in SD rats and its possible mechanism.Methods(1)Cell experiment: 24 hours of brain tissue of newborn SD rats were isolated and cultured into NSCs,and cultured to the second generation.NSCs with good growth status were randomly divided into control group,BMP4 group,BMP4+BMSCs-CM group.Differentiated and cultured,respectively,until the seventh day of culture,using cell immunofluorescence technique to detect microtubule-associated protein 2(MAP-2)and Glial fibrillary acidic protein(GFAP).The expression of Smad1/5/8 and p-Smad1/5/8 protein in GFAP and MAP-2 and BMP signaling pathways was detected by Western Blot(WB).(2)Animal experiment: 96 healthy female SD rats were selected and divided into control group(single spinal cord injury group)and experimental group(BMSCs-CM micro-osmotic pump injection group after spinal cord injury test)by simple randomization..The spinal cord tissues of the two groups were taken out on the 1st,3rd,5th,and 7th day after modeling,and the expression of BMP4 and p-Smad1/5/8 were detected by WB technique.The two groups of rats were taken out in the fourth week.Spinal cord tissue was observed by HE staining.The expression of MAP-2 and GFAP in the spinal cord tissue was detected by tissue immunofluorescence technique and WB technique.The first week to the sixth week after spinal cord modeling BBB exercise scores(Basso,Beattie &Bresnahan locomotor rating scale,BBB test)were performed on two groups of SD rats per week.Results(1)Cell experiment: Through WB detection and cell immunofluorescence staining,we found that compared with the control group,BMP4 group promoted the differentiation of NSCs into astrocytes(P<0.05);decreased its differentiation into neuronal cells(P<0.05).At the same time,the expression of p-Smad1/5/8 protein was also increased(P<0.05).Compared with BMP4 group,BMP4+BMSCs-CM group increased the differentiation of NSCs into neurons(P<0.05),and decreased the differentiation of NSCs into astrocytes(P<0.05).Meanwhile,p-Smad1/ The expression of the 5/8 protein was also significantly reduced(P < 0.05).(2)Animal experiments: By WB detection and immunofluorescence staining of tissues,we found that BMP4 expression reached the highest level on the first day after SCI,compared with the SCI group,BMP4 expression level in the injured lesion at the early time point after BMSC-CM treatment.Significantly decreased(P < 0.05).At the same time,the expression of p-Smad 1/5/8 protein increased on day 1 and reached the highest level on day 3 after SCI,compared with the SCI group,at the early time point of damage after BMSC-CM treatment.The expression level of p-Smad 1/5/8 was also significantly decreased(P < 0.05).Moreover,in the BMSCs-CM group compared with the SCI group,the expression of MAP-2 around the injured spinal cord tissue was significantly increased(P<0.05),while the expression of GFAP was significantly decreased(P<0.05);the BBB score was also significantly improved(P <0.05).Conclusion(1)Cellular experiments: BMP4 promotes the differentiation of NSCs into astrocytes and prevents them from differentiating into neuronal cells,a phenomenon associated with increased expression of p-Smad1/5/8 protein.BMSCs-CM can reverse the effect of BMP4 on the expression of these proteins.(2)Animal experiments: BMSC-CM promotes the differentiation of neural stem cells into neurons by down-regulating the expression of BMP4 in the injured lesions and inhibiting the activation of BMP/Smad signaling pathway during the recovery of SCI,while reducing the neural stem cells to the star glue.Differentiation of cytoplasmic cells,thereby improving neurological function after spinal cord injury. |
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