Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant p...Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.展开更多
The peripheral nervous system has the potential to regenerate after nerve injury owing to the intrinsic regrowth ability of neurons and the permissive microenvironment.The regenerative process involves numerous gene e...The peripheral nervous system has the potential to regenerate after nerve injury owing to the intrinsic regrowth ability of neurons and the permissive microenvironment.The regenerative process involves numerous gene expression changes,in which transcription factors play a critical role.Previously,we profiled dysregulated genes in dorsal root ganglion neurons at different time points（0,3 and 9 hours,and 1,4 and 7 days） after sciatic nerve injury in rats by RNA sequencing.In the present study,we investigated differentially expressed transcription factors following nerve injury,and we identified enriched molecular and cellular functions of these transcription factors by Ingenuity Pathway Analysis.This analysis revealed the dynamic changes in the expression of transcription factors involved in cell death at different time points following sciatic nerve injury.In addition,we constructed regulatory networks of the differentially expressed transcription factors in cell death and identified some key transcription factors（such as STAT1,JUN,MYC and IRF7）.We confirmed the changes in expression of some key transcription factors（STAT1 and IRF7） by quantitative reverse transcription-polymerase chain reaction.Collectively,our analyses provide a global overview of transcription factor changes in dorsal root ganglia after sciatic nerve injury and offer insight into the regulatory transcription factor networks involved in cell death.展开更多
Peripheral nerve injuries（PNI） are caused by a range of etiologies and result in a broad spectrum of disability. While nerve autografts are the current gold standard for the reconstruction of extensive nerve damage,...Peripheral nerve injuries（PNI） are caused by a range of etiologies and result in a broad spectrum of disability. While nerve autografts are the current gold standard for the reconstruction of extensive nerve damage, the limited supply of autologous nerve and complications associated with harvesting nerve from a second surgical site has driven groups from multiple disciplines, including biomedical engineering, neurosurgery, plastic surgery, and orthopedic surgery, to develop a suitable or superior alternative to autografting. Over the last couple of decades, various types of scaffolds, such as acellular nerve grafts（ANGs）, nerve guidance conduits, and non-nervous tissues, have been filled with Schwann cells, stem cells, and/or neurotrophic factors to develop tissue engineered nerve grafts（TENGs）. Although these have shown promising effects on peripheral nerve regeneration in experimental models, the autograft has remained the gold standard for large nerve gaps. This review provides a discussion of recent advances in the development of TENGs and their efficacy in experimental models. Specifically, TENGs have been enhanced via incorporation of genetically engineered cells, methods to improve stem cell survival and differentiation, optimized delivery of neurotrophic factors via drug delivery systems（DDS）, co-administration of platelet-rich plasma（PRP）, and pretreatment with chondroitinase ABC（Ch-ABC）. Other notable advancements include conduits that have been bioengineered to mimic native nerve structure via cell-derived extracellular matrix（ECM） deposition, and the development of transplantable living nervous tissue constructs from rat and human dorsal root ganglia（DRG） neurons. Grafts composed of non-nervous tissues, such as vein, artery, and muscle, will be briefly discussed.展开更多
背景:研究表明,背根神经节中主要的胶质细胞类型——卫星胶质细胞,可以通过多种途径与背根神经元胞体相互作用和信息交流,参与慢性疼痛的发生与调节。以往研究重点集中在卫星胶质细胞敏化神经元胞体,对于两者间抑制性调节关注较少,且迄...背景:研究表明,背根神经节中主要的胶质细胞类型——卫星胶质细胞,可以通过多种途径与背根神经元胞体相互作用和信息交流,参与慢性疼痛的发生与调节。以往研究重点集中在卫星胶质细胞敏化神经元胞体,对于两者间抑制性调节关注较少,且迄今为止两者间交流尚未见有研究进行汇总,并讨论其内在联系。目的:从卫星胶质细胞与神经元的结构形态、离子通道、蛋白受体等方面,以及相邻卫星胶质细胞间相互作用的角度,对近年来两者间可能存在的相互作用、信息交流等机制的研究进行综述。方法:以"pain,satellite glial cell,DRG"和"疼痛,卫星胶质细胞,背根神经节,缝隙连接"为检索词,检索了在PubMed、Web of Science、SinoMed及CNKI数据库收录的2008年1月至2019年2月的相关文献,并追踪所得文献中所引用的相关性较高的参考文献,最终筛选出77篇相关度较高的文献进行分析。结果与结论:在背根神经节中,卫星胶质细胞包绕单个神经元形成的卫星胶质细胞鞘,通过"神经元-卫星胶质细胞"这一功能单位,实现了相邻神经元之间的相互调控。大多数的文献支持了卫星胶质细胞在慢性疼痛疾病发生过程中起着发生、维持的重要作用;但是,也有部分文献发现卫星胶质细胞与神经元之间存在抑制性调节,激活P2Y1受体、谷氨酸转运体都可以抑制疼痛的发生,在背根神经节中调节P2Y1受体、谷氨酸转运体的活性可以作为治疗疼痛的新靶点。展开更多
基金Supported by the National Natural Science Foundation of China (30600665)the Natural Science Foundation Project of CQ CSTC (CSTC, 2008BB5107)+1 种基金the Youth Scientific Research Foundation of Third Military Medical University (06XG048)the Open Project Program of the State Key Laboratory of Trauma, Burns and Combined Injury (2006A-3)
文摘Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.
基金supported by the National Natural Science Foundation of China,No.31500823the Natural Science Foundation of Jiangsu Province of China,No.BK20150403the Natural Science Fund for Colleges and Universities in Jiangsu Province of China,No.16KJB180024
文摘The peripheral nervous system has the potential to regenerate after nerve injury owing to the intrinsic regrowth ability of neurons and the permissive microenvironment.The regenerative process involves numerous gene expression changes,in which transcription factors play a critical role.Previously,we profiled dysregulated genes in dorsal root ganglion neurons at different time points（0,3 and 9 hours,and 1,4 and 7 days） after sciatic nerve injury in rats by RNA sequencing.In the present study,we investigated differentially expressed transcription factors following nerve injury,and we identified enriched molecular and cellular functions of these transcription factors by Ingenuity Pathway Analysis.This analysis revealed the dynamic changes in the expression of transcription factors involved in cell death at different time points following sciatic nerve injury.In addition,we constructed regulatory networks of the differentially expressed transcription factors in cell death and identified some key transcription factors（such as STAT1,JUN,MYC and IRF7）.We confirmed the changes in expression of some key transcription factors（STAT1 and IRF7） by quantitative reverse transcription-polymerase chain reaction.Collectively,our analyses provide a global overview of transcription factor changes in dorsal root ganglia after sciatic nerve injury and offer insight into the regulatory transcription factor networks involved in cell death.
基金supported,in part,by a research grant from Baylor Scott&White Health Central Texas Foundation and NIH grant R01-NS067435(JHH)
文摘Peripheral nerve injuries（PNI） are caused by a range of etiologies and result in a broad spectrum of disability. While nerve autografts are the current gold standard for the reconstruction of extensive nerve damage, the limited supply of autologous nerve and complications associated with harvesting nerve from a second surgical site has driven groups from multiple disciplines, including biomedical engineering, neurosurgery, plastic surgery, and orthopedic surgery, to develop a suitable or superior alternative to autografting. Over the last couple of decades, various types of scaffolds, such as acellular nerve grafts（ANGs）, nerve guidance conduits, and non-nervous tissues, have been filled with Schwann cells, stem cells, and/or neurotrophic factors to develop tissue engineered nerve grafts（TENGs）. Although these have shown promising effects on peripheral nerve regeneration in experimental models, the autograft has remained the gold standard for large nerve gaps. This review provides a discussion of recent advances in the development of TENGs and their efficacy in experimental models. Specifically, TENGs have been enhanced via incorporation of genetically engineered cells, methods to improve stem cell survival and differentiation, optimized delivery of neurotrophic factors via drug delivery systems（DDS）, co-administration of platelet-rich plasma（PRP）, and pretreatment with chondroitinase ABC（Ch-ABC）. Other notable advancements include conduits that have been bioengineered to mimic native nerve structure via cell-derived extracellular matrix（ECM） deposition, and the development of transplantable living nervous tissue constructs from rat and human dorsal root ganglia（DRG） neurons. Grafts composed of non-nervous tissues, such as vein, artery, and muscle, will be briefly discussed.
文摘背景:研究表明,背根神经节中主要的胶质细胞类型——卫星胶质细胞,可以通过多种途径与背根神经元胞体相互作用和信息交流,参与慢性疼痛的发生与调节。以往研究重点集中在卫星胶质细胞敏化神经元胞体,对于两者间抑制性调节关注较少,且迄今为止两者间交流尚未见有研究进行汇总,并讨论其内在联系。目的:从卫星胶质细胞与神经元的结构形态、离子通道、蛋白受体等方面,以及相邻卫星胶质细胞间相互作用的角度,对近年来两者间可能存在的相互作用、信息交流等机制的研究进行综述。方法:以"pain,satellite glial cell,DRG"和"疼痛,卫星胶质细胞,背根神经节,缝隙连接"为检索词,检索了在PubMed、Web of Science、SinoMed及CNKI数据库收录的2008年1月至2019年2月的相关文献,并追踪所得文献中所引用的相关性较高的参考文献,最终筛选出77篇相关度较高的文献进行分析。结果与结论:在背根神经节中,卫星胶质细胞包绕单个神经元形成的卫星胶质细胞鞘,通过"神经元-卫星胶质细胞"这一功能单位,实现了相邻神经元之间的相互调控。大多数的文献支持了卫星胶质细胞在慢性疼痛疾病发生过程中起着发生、维持的重要作用;但是,也有部分文献发现卫星胶质细胞与神经元之间存在抑制性调节,激活P2Y1受体、谷氨酸转运体都可以抑制疼痛的发生,在背根神经节中调节P2Y1受体、谷氨酸转运体的活性可以作为治疗疼痛的新靶点。
