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
Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibi...Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.展开更多
目的:研究子宫内膜异位症(内异症)患者在位内膜神经生长因子(NGF)及其受体trkA、p75NTR的表达,探讨上述因子与内异症疼痛的关系。方法:选择就诊于北京协和医院并进行了腹腔镜手术的28例子宫内膜异位症患者作为研究组,非子宫内膜异位症患...目的:研究子宫内膜异位症(内异症)患者在位内膜神经生长因子(NGF)及其受体trkA、p75NTR的表达,探讨上述因子与内异症疼痛的关系。方法:选择就诊于北京协和医院并进行了腹腔镜手术的28例子宫内膜异位症患者作为研究组,非子宫内膜异位症患者10例作为对照组(B组),收集上述患者分泌期在位子宫内膜。根据患者有无痛经,分为内异症疼痛组(A1组,18例)及内异症非疼痛组(A2组,10例)。采用免疫组化比较各组病灶中NGF及其受体trkA、p75NTR的表达,并分析其与痛经的关系。结果:各组在位内膜腺上皮中NGF的表达显著高于间质(P<0.05);内异症疼痛组腺上皮NGF表达较非疼痛组明显升高(359.9±18.7 vs 201.3±34.3,P<0.05)。p75NTR主要在子宫内膜间质细胞中表达;内异症组明显高于非内异症组(58.8±21.1、22.5±16.1 vs 0,P<0.05);内异症疼痛组较非疼痛组p75NTR表达明显升高(58.8±21.1 vs 22.5±16.1,P<0.05)。trkA在非内异症组子宫内膜间质中表达明显高于腺上皮(P<0.05);在内异症组腺上皮中,trkA的表达较非内异症组明显增高(P<0.05);trkA的表达量与内异症患者疼痛无关。结论:p75NTR可能参与内异症的发病,NGF及其受体p75NTR在在位内膜中的表达与患者疼痛相关,表明其可能参与了内异症疼痛发病机制。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a Ph D fellowship by FCT-Fundacao para a Ciência Tecnologia (SFRH/BD/135868/2018)(to SSC)。
文摘Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.
文摘目的:研究子宫内膜异位症(内异症)患者在位内膜神经生长因子(NGF)及其受体trkA、p75NTR的表达,探讨上述因子与内异症疼痛的关系。方法:选择就诊于北京协和医院并进行了腹腔镜手术的28例子宫内膜异位症患者作为研究组,非子宫内膜异位症患者10例作为对照组(B组),收集上述患者分泌期在位子宫内膜。根据患者有无痛经,分为内异症疼痛组(A1组,18例)及内异症非疼痛组(A2组,10例)。采用免疫组化比较各组病灶中NGF及其受体trkA、p75NTR的表达,并分析其与痛经的关系。结果:各组在位内膜腺上皮中NGF的表达显著高于间质(P<0.05);内异症疼痛组腺上皮NGF表达较非疼痛组明显升高(359.9±18.7 vs 201.3±34.3,P<0.05)。p75NTR主要在子宫内膜间质细胞中表达;内异症组明显高于非内异症组(58.8±21.1、22.5±16.1 vs 0,P<0.05);内异症疼痛组较非疼痛组p75NTR表达明显升高(58.8±21.1 vs 22.5±16.1,P<0.05)。trkA在非内异症组子宫内膜间质中表达明显高于腺上皮(P<0.05);在内异症组腺上皮中,trkA的表达较非内异症组明显增高(P<0.05);trkA的表达量与内异症患者疼痛无关。结论:p75NTR可能参与内异症的发病,NGF及其受体p75NTR在在位内膜中的表达与患者疼痛相关,表明其可能参与了内异症疼痛发病机制。
