BACKGROUND： Glial fibritlary acidic protein （GFAP） expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could be...BACKGROUND： Glial fibritlary acidic protein （GFAP） expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE： To investigate the inhibitory effects of synthetic small interfering RNAs （siRNAs） on astrocytic GFAP expression in rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment at the cellular and molecular level was performed at the First Hospital of Dalian Medical University between June 2005 and February 2006. MATERIALS： A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was purchased from Ambion, USA, And TransMessengerTM Transfection Reagent from DAKO, Carpinteria, CA. METHODS： Rat astrocytes were isolated and cultured. Three pairs of 21-nucleotide （nt） siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1, 2, 3, and 4 g/L using TransMessengerTM Transfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were regarded as the negative control group, with GAPDH siRNA as the positive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES： GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture. RESULTS： GFAP mRNA expression in the positive control group was significantly less than the negative control group （P 〈 0.01）. GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours （P 〈 0.01 ）, while no differences were detected between the negative control and blank groups （P 〉 0.05）. GFAP protein expression was remarkably less in siRNA-transfected astrocytes compared to the blank control （P 〈 0.01 ）. CONCLUSION： Transfected siRNAs could significantly inhibit GFAP gene expression in astrocytes after 72 hours in culture.展开更多
Cynops orientalis(C.orientalis)has a pronounced ability to regenerate its spinal cord after injury.Thus,exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cor...Cynops orientalis(C.orientalis)has a pronounced ability to regenerate its spinal cord after injury.Thus,exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration.In this study,we established a model of spinal cord thoracic transection injury in C.orientalis,which is an endemic species in China.We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury–during the very acute stage(4 days)and the subacute stage(7 days)–and identified differentially expressed genes;additionally,we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses,at each time point.Transcriptome sequencing showed that 13,059 genes were differentially expressed during C.orientalis spinal cord regeneration compared with uninjured animals,among which 4273 were continuously downregulated and 1564 were continuously up-regulated.Down-regulated genes were most enriched in the Gene Ontology term“multicellular organismal process”and in the ribosome pathway at 10 days following spinal cord injury.We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury,indicating the importance of low metabolic activity during wound healing.Immune response-associated pathways were activated during the early acute phase(4 days),while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen,as well as tight junction proteins,was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury.However,compared with 4 days post-injury,at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated,up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle,and SHH,VIM,and Sox2 were prominently up-regulated.Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury,similar to what is observed in mammalian astrocytes after spinal cord injury,even though axolotls do not form a glial scar during regeneration.We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells,thereby inhibiting reactive gliosis,at early stages after spinal cord injury.Extracellular matrix degradation slows cellular responses,represses the expression of neurogenic genes,and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells.These ependymoglial cells act as neural stem cells:they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons.This provides the regenerative microenvironment that allows axon growth after injury.展开更多
基金the Natural Science Foundation of Liaoning Province, No. 20052165
文摘BACKGROUND： Glial fibritlary acidic protein （GFAP） expression highly correlates with spinal glial scar formation, and is regarded as an important target for scar therapy. Efficient inhibition of expression could benefit recovery from spinal cord injury. OBJECTIVE： To investigate the inhibitory effects of synthetic small interfering RNAs （siRNAs） on astrocytic GFAP expression in rats. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment at the cellular and molecular level was performed at the First Hospital of Dalian Medical University between June 2005 and February 2006. MATERIALS： A total of 100 seven-day-old, Sprague Dawley rats were selected. GAPDH siRNA was purchased from Ambion, USA, And TransMessengerTM Transfection Reagent from DAKO, Carpinteria, CA. METHODS： Rat astrocytes were isolated and cultured. Three pairs of 21-nucleotide （nt） siRNAs specific to rats GFAP mRNA, 401,404 and 854, were synthesized and transfected in primary astrocytes at 1, 2, 3, and 4 g/L using TransMessengerTM Transfection Reagent. Non-transfected astrocytes served as the blank group. Cells transfected with siRNA were regarded as the negative control group, with GAPDH siRNA as the positive control group, and 401 siRNA, 404 siRNA, and 854 siRNA as experimental groups. MAIN OUTCOME MEASURES： GFAP mRNA and protein expression were assessed by RT-PCR and Western blot, respectively, at 24, 48, and 72 hours of culture. RESULTS： GFAP mRNA expression in the positive control group was significantly less than the negative control group （P 〈 0.01）. GFAP mRNA expression in astrocytes from three pairs of siRNA was significantly less than the blank group after 48 hours （P 〈 0.01 ）, while no differences were detected between the negative control and blank groups （P 〉 0.05）. GFAP protein expression was remarkably less in siRNA-transfected astrocytes compared to the blank control （P 〈 0.01 ）. CONCLUSION： Transfected siRNAs could significantly inhibit GFAP gene expression in astrocytes after 72 hours in culture.
基金the National Natural Science Foundation of China,Nos.32270516,31970413the Natural Science Foundation of Anhui Province,No.1908085MC83(to JL)a Start-up grant from Nanjing Agricultural University,No.804090。
文摘Cynops orientalis(C.orientalis)has a pronounced ability to regenerate its spinal cord after injury.Thus,exploring the molecular mechanism of this process could provide new approaches for promoting mammalian spinal cord regeneration.In this study,we established a model of spinal cord thoracic transection injury in C.orientalis,which is an endemic species in China.We performed RNA sequencing of the contused axolotl spinal cord at two early time points after spinal cord injury–during the very acute stage(4 days)and the subacute stage(7 days)–and identified differentially expressed genes;additionally,we performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses,at each time point.Transcriptome sequencing showed that 13,059 genes were differentially expressed during C.orientalis spinal cord regeneration compared with uninjured animals,among which 4273 were continuously downregulated and 1564 were continuously up-regulated.Down-regulated genes were most enriched in the Gene Ontology term“multicellular organismal process”and in the ribosome pathway at 10 days following spinal cord injury.We found that multiple genes associated with energy metabolism were down-regulated and multiple genes associated with the lysosome were up-regulated after spinal cord injury,indicating the importance of low metabolic activity during wound healing.Immune response-associated pathways were activated during the early acute phase(4 days),while the expression of extracellular matrix proteins such as glycosaminoglycan and collagen,as well as tight junction proteins,was lower at 10 days post-spinal cord injury than 4 days post-spinal cord injury.However,compared with 4 days post-injury,at 10 days post-injury neuroactive ligand-receptor interactions were no longer down-regulated,up-regulated differentially expressed genes were enriched in pathways associated with cancer and the cell cycle,and SHH,VIM,and Sox2 were prominently up-regulated.Immunofluorescence staining showed that glial fibrillary acidic protein was up-regulated in axolotl ependymoglial cells after injury,similar to what is observed in mammalian astrocytes after spinal cord injury,even though axolotls do not form a glial scar during regeneration.We suggest that low intracellular energy production could slow the rapid amplification of ependymoglial cells,thereby inhibiting reactive gliosis,at early stages after spinal cord injury.Extracellular matrix degradation slows cellular responses,represses the expression of neurogenic genes,and reactivates a transcriptional program similar to that of embryonic neuroepithelial cells.These ependymoglial cells act as neural stem cells:they migrate and proliferate to repair the lesion and then differentiate to replace lost glial cells and neurons.This provides the regenerative microenvironment that allows axon growth after injury.
