This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices.Organotypic spinal cord slices,normally cultured for 6 days,were divided into control ...This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices.Organotypic spinal cord slices,normally cultured for 6 days,were divided into control group(Ctrl)and transplanted group(T).The choroidal plexus epithelial cells were dissociated and primary cultured(C group).The choroidal plexus epithelial cells cultured for 6–7 days were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanineperchlorate(CM-Dil),and were identified by transthyretin(TTR)in immunocytochemistry.They were adjusted to the density of 0.5–1×107/ml,then 2μl cells suspension were transplanted to the spinal cord slices in the T group.The same amount of basal medium was dripped on the spinal cord slices in the Ctrl group.After 14 days of transplantation,the differentiations into neurons and astrocytes,and the synapses were identified by immunofluorescence histochemistry.At the same time,the ratios of cell differentiations and synapses in new system,and the changes of MAPK signaling pathway were tested by western blotting.The choroid plexus epithelial cells were well labeled by CM-Dil and were immune-stained by TTR in immunocytochemistry.The choroid plexus epithelial cells bodies were small when transplanted on the spinal cord slices,but big when transplanted on the polyester membrane inserts.The transplanted cells could differentiate into astrocytes,and possibly differentiate into neurons,and there were a large number of synaptophysin positive vesicles between transplanted cells and organotypic spinal cord slices in immunofluorescence histochemistry.The levels of GFAP,TUB-III and synaptophysin in the T group were higher than which in the Ctrl and C groups in western blotting(P<0.05).And the ratios of p-JNK/JNK and p-P38/P38 in the T group were significantly lower than which in the Ctrl and C groups(P<0.05).But the ratio of p-ERK/ERK in the three groups was of no significant difference.The transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system.展开更多
Objective:Using neuromyelitis optica immunoglobulin G(NMO-IgG)to induced ex vivo mice spinal cord slice model.Methods:Vibratome-cut transverse spinal cord slices from 7-day-old C57BL/6Jmouse pups were cultured on tran...Objective:Using neuromyelitis optica immunoglobulin G(NMO-IgG)to induced ex vivo mice spinal cord slice model.Methods:Vibratome-cut transverse spinal cord slices from 7-day-old C57BL/6Jmouse pups were cultured on transwell porous supports for 7days,then randomly divided into the control group and NMO model group.Slices of the control group were further cultured with human serum complement,while slices from NMO model group were exposed to complement and NMO-IgG.After 24-hour incubation,slices of both groups were measured for aquaporin-4(AQP4),glial fibrillary acidic protein(GFAP),myelin basic protein(MBP)and neurofilament light chain(NFL)by immunofluorescence.Results:Slices exposed to NMO-IgG showed astrocyte swelling,and a significant loss of AQP4and GFAP staining.Ratios of the loss of AQP4and GFAP staining were 77.74%±6.75%and 75.62%±5.76%respectively in the model group,and NMO-like injury score was 3.11±0.60.But there were no obvious losses of AQP4and GFAP staining in the control group,and NMO-like lesion score was 0.00.There were significant differences between the two groups with regards to the above indexes(P<0.01).Ratios of the loss of MBP and NFL staining in the model group were 37.60%±4.88%and46.29%±4.98%respectively,while the corresponding figures in the control group were 9.10%±1.63%and 5.80%±0.81%,and the differences between the two groups were statistically significant(P<0.01).Conclusion:These results suggested that NMO-IgG-induced ex vivo spinal cord slice model possesses typical features of NMO,and this model might be useful for relevant fundamental studies.展开更多
Background Treatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated. Using an alternative methods, in vitro organotypic slice culture m...Background Treatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated. Using an alternative methods, in vitro organotypic slice culture method, would be useful to transplant cells and assessing the effects. This study was to determine the possibility of differentiating human bone marrow precursor cells into cells of the neuronal lineage by transplanting into canine spinal cord organotypic slice cultures. Methods Bone marrow aspirates were obtained from posterior superior iliac spine （PSIS） of patients that had undergone spinal fusion due to a degenerative spinal disorder. For cell imaging, mesenchymal precursor cells （MPCs） were pre-stained with PKH-26 just before transplantation to canine spinal cord slices. Canine spinal cord tissues were obtained from three adult beagle dogs. Spinal cords were cut into transverse slices of 1 mm using tissue chopper. Two slices were transferred into 6-well plate containing 3 ml DMEM with antibiotics. Prepared MPCs （lx104） were transplanted into spinal cord slices. On days 0, 3, 7, 14, MPCs were observed for morphological changes and expression of neuronal markers through immunofluorescence and reverse transcription-polymerase chain reaction （RT-PCR）. Results The morphological study showed： spherical cells in the control and experiment groups on day 0; and on day 3, cells in the control group had one or two thick, short processes and ones in the experiment group had three or four thin, long processes. On day 7, these variously-sized processes contacted each other in the experiment group, but showed typical spindle-shaped cells in the control group. Immunofluorescence showed that PKH-26（＋） MPCs stained positive for NeuN （＋） and GFAP（＋） in experimental group only. Also RT-PCR showed weak expression of β-tubulin III and GFAP. Conclusions Human bone marrow mesenchymal precursor cells （hMPCs） have the potential to differentiate into the neuronal like cells in this canine spinal cord organotypic slice culture model. Furthermore, these findings suggested the possibility that these cells can be utilized to treat patients with spinal cord injuries.展开更多
目的探讨细胞外高C a2+对脊髓灰质c-fos基因表达的影响。方法用原位杂交方法观察离体培养脊髓组织在高钙离子环境下30 m in^12 h不同时间点c-fos基因的表达变化。结果高钙损伤后30 m in^6 h脊髓组织灰质中c-fos表达明显较对照组强,4~6 ...目的探讨细胞外高C a2+对脊髓灰质c-fos基因表达的影响。方法用原位杂交方法观察离体培养脊髓组织在高钙离子环境下30 m in^12 h不同时间点c-fos基因的表达变化。结果高钙损伤后30 m in^6 h脊髓组织灰质中c-fos表达明显较对照组强,4~6 h达到高峰,8 h即恢复至对照水平。结论高钙离子能诱导离体脊髓组织灰质c-fos基因的表达,提示钙离子在脊髓继发性损伤的作用可能是通过介导c-fos等即早基因的表达实现的。展开更多
基金This study was supported by National Natural Science Foundation of China(81471247).
文摘This study aimed to evaluate the integration of transplanted choroidal plexus epithelial cells with organotypic spinal cord slices.Organotypic spinal cord slices,normally cultured for 6 days,were divided into control group(Ctrl)and transplanted group(T).The choroidal plexus epithelial cells were dissociated and primary cultured(C group).The choroidal plexus epithelial cells cultured for 6–7 days were labeled by 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanineperchlorate(CM-Dil),and were identified by transthyretin(TTR)in immunocytochemistry.They were adjusted to the density of 0.5–1×107/ml,then 2μl cells suspension were transplanted to the spinal cord slices in the T group.The same amount of basal medium was dripped on the spinal cord slices in the Ctrl group.After 14 days of transplantation,the differentiations into neurons and astrocytes,and the synapses were identified by immunofluorescence histochemistry.At the same time,the ratios of cell differentiations and synapses in new system,and the changes of MAPK signaling pathway were tested by western blotting.The choroid plexus epithelial cells were well labeled by CM-Dil and were immune-stained by TTR in immunocytochemistry.The choroid plexus epithelial cells bodies were small when transplanted on the spinal cord slices,but big when transplanted on the polyester membrane inserts.The transplanted cells could differentiate into astrocytes,and possibly differentiate into neurons,and there were a large number of synaptophysin positive vesicles between transplanted cells and organotypic spinal cord slices in immunofluorescence histochemistry.The levels of GFAP,TUB-III and synaptophysin in the T group were higher than which in the Ctrl and C groups in western blotting(P<0.05).And the ratios of p-JNK/JNK and p-P38/P38 in the T group were significantly lower than which in the Ctrl and C groups(P<0.05).But the ratio of p-ERK/ERK in the three groups was of no significant difference.The transplanted choroidal plexus epithelial cells can integrate with organotypic spinal cord slices into a new system.
基金supported by the National Natural Science Foundation of China (No.81460194 No.81260188)
文摘Objective:Using neuromyelitis optica immunoglobulin G(NMO-IgG)to induced ex vivo mice spinal cord slice model.Methods:Vibratome-cut transverse spinal cord slices from 7-day-old C57BL/6Jmouse pups were cultured on transwell porous supports for 7days,then randomly divided into the control group and NMO model group.Slices of the control group were further cultured with human serum complement,while slices from NMO model group were exposed to complement and NMO-IgG.After 24-hour incubation,slices of both groups were measured for aquaporin-4(AQP4),glial fibrillary acidic protein(GFAP),myelin basic protein(MBP)and neurofilament light chain(NFL)by immunofluorescence.Results:Slices exposed to NMO-IgG showed astrocyte swelling,and a significant loss of AQP4and GFAP staining.Ratios of the loss of AQP4and GFAP staining were 77.74%±6.75%and 75.62%±5.76%respectively in the model group,and NMO-like injury score was 3.11±0.60.But there were no obvious losses of AQP4and GFAP staining in the control group,and NMO-like lesion score was 0.00.There were significant differences between the two groups with regards to the above indexes(P<0.01).Ratios of the loss of MBP and NFL staining in the model group were 37.60%±4.88%and46.29%±4.98%respectively,while the corresponding figures in the control group were 9.10%±1.63%and 5.80%±0.81%,and the differences between the two groups were statistically significant(P<0.01).Conclusion:These results suggested that NMO-IgG-induced ex vivo spinal cord slice model possesses typical features of NMO,and this model might be useful for relevant fundamental studies.
文摘Background Treatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated. Using an alternative methods, in vitro organotypic slice culture method, would be useful to transplant cells and assessing the effects. This study was to determine the possibility of differentiating human bone marrow precursor cells into cells of the neuronal lineage by transplanting into canine spinal cord organotypic slice cultures. Methods Bone marrow aspirates were obtained from posterior superior iliac spine （PSIS） of patients that had undergone spinal fusion due to a degenerative spinal disorder. For cell imaging, mesenchymal precursor cells （MPCs） were pre-stained with PKH-26 just before transplantation to canine spinal cord slices. Canine spinal cord tissues were obtained from three adult beagle dogs. Spinal cords were cut into transverse slices of 1 mm using tissue chopper. Two slices were transferred into 6-well plate containing 3 ml DMEM with antibiotics. Prepared MPCs （lx104） were transplanted into spinal cord slices. On days 0, 3, 7, 14, MPCs were observed for morphological changes and expression of neuronal markers through immunofluorescence and reverse transcription-polymerase chain reaction （RT-PCR）. Results The morphological study showed： spherical cells in the control and experiment groups on day 0; and on day 3, cells in the control group had one or two thick, short processes and ones in the experiment group had three or four thin, long processes. On day 7, these variously-sized processes contacted each other in the experiment group, but showed typical spindle-shaped cells in the control group. Immunofluorescence showed that PKH-26（＋） MPCs stained positive for NeuN （＋） and GFAP（＋） in experimental group only. Also RT-PCR showed weak expression of β-tubulin III and GFAP. Conclusions Human bone marrow mesenchymal precursor cells （hMPCs） have the potential to differentiate into the neuronal like cells in this canine spinal cord organotypic slice culture model. Furthermore, these findings suggested the possibility that these cells can be utilized to treat patients with spinal cord injuries.
文摘目的探讨细胞外高C a2+对脊髓灰质c-fos基因表达的影响。方法用原位杂交方法观察离体培养脊髓组织在高钙离子环境下30 m in^12 h不同时间点c-fos基因的表达变化。结果高钙损伤后30 m in^6 h脊髓组织灰质中c-fos表达明显较对照组强,4~6 h达到高峰,8 h即恢复至对照水平。结论高钙离子能诱导离体脊髓组织灰质c-fos基因的表达,提示钙离子在脊髓继发性损伤的作用可能是通过介导c-fos等即早基因的表达实现的。
