Objective Neurotrophin 3(NTF3)is involved in numerous biological processes;however,its role in hepatocellular carcinoma(HCC)is not well studied.This study investigated NTF3 function in HCC progression and revealed its...Objective Neurotrophin 3(NTF3)is involved in numerous biological processes;however,its role in hepatocellular carcinoma(HCC)is not well studied.This study investigated NTF3 function in HCC progression and revealed its underlying molecular mechanisms.Methods The prognostic relevance of NTF3 was determined through a bioinformatical analysis of publicly available TCGA data.Immunohistochemistry of HCC biopsies was performed to explore the expression of NTF3.Cell growth and proliferation were analyzed using a Cell Counting Kit-8(CCK-8)assay.Cell invasion and migration were analyzed using Boyden Transwell and wound healing assays.Protein expression and mRNA levels were evaluated through immunoblotting and quantitative polymerase chain reaction(qPCR).Cell apoptosis was evaluated with flow cytometry.Results NTF3 expression was significantly lower in HCC tissues than in adjacent non-tumor tissues.Low NTF3 expression was significantly associated with decreased patient survival and specific clinicopathological features.NTF3 overexpression reduced the proliferation,migration,and invasion abilities of HCC cell lines.Conclusion Decreased expression of NTF3 is associated with poor prognosis in HCC patients,likely due to its action in promoting HCC cell proliferation,migration,and invasion.Our findings provide a novel understanding into the pathogenesis of HCC and the role of NTF3 in tumor progression,suggesting that targeting NTF3 has potential therapeutic and diagnostic value for HCC.展开更多
BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and em...BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.展开更多
BACKGROUND： Recent studies have suggested that regeneration of the central nerve fiber following spinal cord injury occurs under specific conditions. OBJECTIVE： To study the effects of Nogo-neutralizing antibody （...BACKGROUND： Recent studies have suggested that regeneration of the central nerve fiber following spinal cord injury occurs under specific conditions. OBJECTIVE： To study the effects of Nogo-neutralizing antibody （IN-1）, in combination with neurotrophin-3 （NT-3）, on axonal regeneration and motor function following spinal cord injury in the rat. DESIGN, TIME AND SETTING： A randomized, controlled, animal study combining immunohistochemistry was performed at the Laboratory of Neuroanatomy of Xiangya Medical College, and Central Laboratory of Xiangya the Third Hospital, Central South University from January 2006 to December 2007. MATERIALS： Eighteen healthy, Sprague Dawley rats were randomly divided into three groups, with six rats per group： control, IN-l, and IN-1/NT-3. Hemisectioned spinal cord injury models were established by cutting the posterior 2/3 of spinal cord, which is equivalent to the Ts level. METHODS： A polyethylene tubing was inserted through into subarachnoid cavity, equivalent to the superior margin at the T8 level. Saline, IN-1, and IN-1/NT-3 were respectively injected into control, IN-1, and IN-1/NT-3 groups, three times/day for seven consecutive days. MAIN OUTCOME MEASURES： At 2 weeks post-surgery, biotin dextran amine （10%） was injected into the right sensorimotor cortex area. At day 28 post-surgery, spinal cord tissue was prepared for frozen sections Positive astrocytic expression was observed with glial fibrillary acidic protein （GFAP） immunohistochemical staining whose proliferation level was represented by gray value, i.e. the higher the gray value was, the less the positive cells were, and growth of positive fibers was observed with a biotin dextran amine histological reaction. Motor function was measured according to BBB scores pre-operatively, as well as at days 1, 7, 14, 21, and 28 post-operatively. RESULTS： Three rats died during experimentation. By random supplement, a total of 18 rats were included. GFAP-positive astrocytes were observed in all the three groups. In the control group, astrocytes were characterized according to active function, hyperplasia, proliferation, hypertrophy, and increasing processes as compared to IN-1 group and IN-1/IN-3 group. Astrocyte hyperplasia represented by gray value in the IN-1 group was less than the control group. Gray value of GFAP-positive products in the IN-1/IN-3 group was higher than other two groups （P 〈 0.05）. Biotin dextran amine tracing demonstrated no corticospinal tract fiber outgrowth following spinal cord injury; the fibers were incapable of passing through the glial scar in the control group. Several fibers were distributed in the proximal scar tissue region in the IN-1 group, and the regenerated fibers were disarranged. Many nerve fibers were distributed throughout the scar tissue, and even several biotin dextran amine-positive fibers were observed at the distal end of the injured segment. Post-operative Basso, Beattie, Bresnahan scores were greater than pre-operative ones, while Basso, Beattie, Bresnahan scores in the IN-1/NT-3 group were significantly greater than the other two groups at days 14, 21, and 28 post-surgery （P 〈 0.05）. CONCLUSION： IN-1, in combination with NT-3, promoted axonal regeneration following spinal cord injury, inhibited the colloidal effect, and enhanced the correlation between proximal and distal processes to recover motor function. The recovery effect of IN-1/NT-3 on motor function was superior that of to IN-1 alone.展开更多
Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remai...Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.展开更多
Schwann ceils and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this stud...Schwann ceils and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this study, we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes. Results showed that neurotrophin-3 was successfully transfected into Schwann cells, where it was expressed effectively and steadily. A composite of Schwann cells transfected with neurotrophin-3 and poly（lactic-co-glycolic acid） biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects. Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination, promote nerve axonal and myelin regeneration, and delay apoptosis of spinal motor neurons. Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.展开更多
In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were ...In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.展开更多
BACKGROUND： Nogo-neutralizing antibody IN-1 accelerates axon growth and enhances recovery of spinal cord function by inhibiting growth inhibitory factors. Neurotrophin-3 （NT-3）contributes to regeneration of nerve f...BACKGROUND： Nogo-neutralizing antibody IN-1 accelerates axon growth and enhances recovery of spinal cord function by inhibiting growth inhibitory factors. Neurotrophin-3 （NT-3）contributes to regeneration of nerve fibers in the spinal cord and motor function recovery. The combination of Nogo-neutralizing antibody IN-1 and NT-3 is hypothesized to produce better outcomes and facilitate axonal regeneration by affecting c-Fos and c-Jun protein expression. OBJECTIVE： To investigate the combined effects of Nogo-neutralizing antibody IN-1 and NT-3 on c-Fos and c-Jun protein levels in the injured spinal cord. DESIGN, TIME AND SETTING： A randomized, controlled study was performed at the Laboratory of Neuroanatomy, Xiangya Medical College, Central South University and the Central Laboratory of Third Xiangya Hospital of China from June 2005 to December 2007. MATERIALS： NT-3 （Peprotech, USA） and Nogo-neutralizing antibody IN-1 （Santa Cruz Biotechnology, USA） were used in this study. METHODS： Hemisectioned spinal cord injury models were established by cutting the posterior 2/3 of rat spinal cord, which is equivalent to the T8 level in the human spine. A total of 120 rats were equally and randomly assigned to three groups： model （0.2 μL saline）, IN-1 （0.2 μL IN-1）, and IN-1/NT-3 （0.2 μL IN-1 ＋ 0.2 μL NT-3）. The compounds were separately infused into transection sites on the side of head. MAIN OUTCOME MEASURES： Western blot analysis was employed to measure c-Fos and c-Jun protein expression in the injured spinal cord at 15, 30 minutes, 1,2, 4, 6, 8, and 12 hours following surgery. RESULTS： Following spinal cord injury, c-Fos and c-Jun protein expression were increased and peaked at 4 6 hours. Following injection of IN-1 or the combination of IN-1 and NT-3, c-Fos protein expression was significantly reduced in the injured spinal cord （P 〈 0.05 or P 〈 0.01） （with the exception of the 15 minute time point）. However, c-Jun protein expression was significantly increased （P〈 0.05 or P〈 0.01） （with the exception of the 15 and 30 minute time points）. Combined application of IN-1 and NT-3 resulted in significantly altered protein expression compared to IN-1 alone. CONCLUSION： IN-1 increases c-Jun protein levels and protects the injured spinal cord by inhibiting c-Fos protein levels. Moreover, the effects of IN-1 combined with NT-3 are more significant than with IN-1 alone.展开更多
Neurotrophin-3 （NT-3） can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with v...Neurotrophin-3 （NT-3） can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein （GFP） to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ＋ 2.78%. Abundant NTo3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NTo3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3.展开更多
BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This eff...BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.展开更多
A spinal cord hemisection injury model was established in rats. Treatment with IN-1 and/or neurotrophin-3 was found to regulate the expression of growth-associated protein 43, nerve growth factor, and basic fibroblast...A spinal cord hemisection injury model was established in rats. Treatment with IN-1 and/or neurotrophin-3 was found to regulate the expression of growth-associated protein 43, nerve growth factor, and basic fibroblast growth factor genes in the injured spinal cord tissues; transcript levels were first increased and then decreased. Expression levels reached a peak at days 7 （growth-associated protein 43） or 14 （nerve growth factor and basic fibroblast growth factor） following spinal cord injury. Combined treatment with neurotrophin-3 and IN-1 achieved the most apparent effect on the expression and recovery of motor function. These findings confirm that combined therapy with neurotrophin-3 and IN-1 can increase expression of growth factors in the injured spinal cord tissues and promote the axonal reaeneration.展开更多
BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and my...BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and myelin sheath repair. However, this effect remains very short-lived. OBJECTIVE： To transfect NT-3 into OECs and to observe the biological activity of OEC-expressing NT-3. DESIGN, TIME AND SETTING： This genetic engineering, in vitro experiment was performed in the Provincial Hospital Affiliated to Shandong University between January 2007 and October 2008. MATERIALS： Trizol Reagent kit was purchased from Gibco, USA; reverse transcription kit, NT-3 Emax ImmunoAssay System reagent was purchased from Promega, USA. METHODS： Neonatal Wistar rat OECs were established as primary cultures and were transfected with pN2A-NT-3 viral vector. The OECs with the highest virus titer and stable cellular growth served as the transfection group; OECs transfected with NT-3-free retrovirus carrier pN2A served as the empty vector group; un-transfected OECs served as the control group. After adherence, the logarithmically cultured PC12-TrkC cells were plated in OECs supernatant from the transfection and empty vector groups, as well as 20 μL PBS, and cultured for 4 days. MAIN OUTCOME MEASURES： NT-3 mRNA expression in OECs, fluorescence of NT-3-positive cells in the transfection group and control group; influence of OECs secreting NT-3 on the differentiation ratio of PC12-TrkC cells. RESULTS： NT-3 mRNA expression was observed 24 hours after transfection and lasted for 28 days which was greater than the control and empty vector groups （P 〈 0.01）. A large number of NT-3-positive cells were observed in the transfection group, and immunofluorescence was greater than the control and empty vector groups. PC12-TrkC cells co-cultured with OECs from the transfection group exhibited a thick and long cell process, increased cell density, and the differentiation ratio was increased （P 〈 0.01）. CONCLUSION： Recombinant double replica retrovirus NT-3 gene was stably and effectively expressed in OECs, and the expressed NT-3 possessed biological activity that promoted neuronal survival.展开更多
In this study, cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3. After 7 days of culture, immunocytochemical staining showed that, 22.4% of cell...In this study, cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3. After 7 days of culture, immunocytochemical staining showed that, 22.4% of cells were positive for nestin, 10.5% were positive for 18-111 tubulin （neuronal marker）, and 60.6% were positive for glial fibrillary acidic protein, but no cells were positive for 04 （oligodendrocytic marker）. At 14 days, there were 5.6% nestin-, 9.6% 13-111 tubulin-, 81.1% glial fibrillary acidic protein-, and 2.2% O4-positive cells. In cells not treated with neurotrophin-3, some were nestin-positive, while the majority showed positive staining for glial fibdllary acidic protein. Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes.展开更多
基金Supported by grants from the National Natural Science Foundation of China(No.ZR2015HM025)Technology Development Program of Shandong Province,China(No.2011YD18003)CSCO-Hengrui Cancer Research Fund(No.Y-HR2018-293 and Y-HR2018-294).
文摘Objective Neurotrophin 3(NTF3)is involved in numerous biological processes;however,its role in hepatocellular carcinoma(HCC)is not well studied.This study investigated NTF3 function in HCC progression and revealed its underlying molecular mechanisms.Methods The prognostic relevance of NTF3 was determined through a bioinformatical analysis of publicly available TCGA data.Immunohistochemistry of HCC biopsies was performed to explore the expression of NTF3.Cell growth and proliferation were analyzed using a Cell Counting Kit-8(CCK-8)assay.Cell invasion and migration were analyzed using Boyden Transwell and wound healing assays.Protein expression and mRNA levels were evaluated through immunoblotting and quantitative polymerase chain reaction(qPCR).Cell apoptosis was evaluated with flow cytometry.Results NTF3 expression was significantly lower in HCC tissues than in adjacent non-tumor tissues.Low NTF3 expression was significantly associated with decreased patient survival and specific clinicopathological features.NTF3 overexpression reduced the proliferation,migration,and invasion abilities of HCC cell lines.Conclusion Decreased expression of NTF3 is associated with poor prognosis in HCC patients,likely due to its action in promoting HCC cell proliferation,migration,and invasion.Our findings provide a novel understanding into the pathogenesis of HCC and the role of NTF3 in tumor progression,suggesting that targeting NTF3 has potential therapeutic and diagnostic value for HCC.
文摘BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.
基金the Foundation of Hunan Public Health Bureau, No.B2005-076
文摘BACKGROUND： Recent studies have suggested that regeneration of the central nerve fiber following spinal cord injury occurs under specific conditions. OBJECTIVE： To study the effects of Nogo-neutralizing antibody （IN-1）, in combination with neurotrophin-3 （NT-3）, on axonal regeneration and motor function following spinal cord injury in the rat. DESIGN, TIME AND SETTING： A randomized, controlled, animal study combining immunohistochemistry was performed at the Laboratory of Neuroanatomy of Xiangya Medical College, and Central Laboratory of Xiangya the Third Hospital, Central South University from January 2006 to December 2007. MATERIALS： Eighteen healthy, Sprague Dawley rats were randomly divided into three groups, with six rats per group： control, IN-l, and IN-1/NT-3. Hemisectioned spinal cord injury models were established by cutting the posterior 2/3 of spinal cord, which is equivalent to the Ts level. METHODS： A polyethylene tubing was inserted through into subarachnoid cavity, equivalent to the superior margin at the T8 level. Saline, IN-1, and IN-1/NT-3 were respectively injected into control, IN-1, and IN-1/NT-3 groups, three times/day for seven consecutive days. MAIN OUTCOME MEASURES： At 2 weeks post-surgery, biotin dextran amine （10%） was injected into the right sensorimotor cortex area. At day 28 post-surgery, spinal cord tissue was prepared for frozen sections Positive astrocytic expression was observed with glial fibrillary acidic protein （GFAP） immunohistochemical staining whose proliferation level was represented by gray value, i.e. the higher the gray value was, the less the positive cells were, and growth of positive fibers was observed with a biotin dextran amine histological reaction. Motor function was measured according to BBB scores pre-operatively, as well as at days 1, 7, 14, 21, and 28 post-operatively. RESULTS： Three rats died during experimentation. By random supplement, a total of 18 rats were included. GFAP-positive astrocytes were observed in all the three groups. In the control group, astrocytes were characterized according to active function, hyperplasia, proliferation, hypertrophy, and increasing processes as compared to IN-1 group and IN-1/IN-3 group. Astrocyte hyperplasia represented by gray value in the IN-1 group was less than the control group. Gray value of GFAP-positive products in the IN-1/IN-3 group was higher than other two groups （P 〈 0.05）. Biotin dextran amine tracing demonstrated no corticospinal tract fiber outgrowth following spinal cord injury; the fibers were incapable of passing through the glial scar in the control group. Several fibers were distributed in the proximal scar tissue region in the IN-1 group, and the regenerated fibers were disarranged. Many nerve fibers were distributed throughout the scar tissue, and even several biotin dextran amine-positive fibers were observed at the distal end of the injured segment. Post-operative Basso, Beattie, Bresnahan scores were greater than pre-operative ones, while Basso, Beattie, Bresnahan scores in the IN-1/NT-3 group were significantly greater than the other two groups at days 14, 21, and 28 post-surgery （P 〈 0.05）. CONCLUSION： IN-1, in combination with NT-3, promoted axonal regeneration following spinal cord injury, inhibited the colloidal effect, and enhanced the correlation between proximal and distal processes to recover motor function. The recovery effect of IN-1/NT-3 on motor function was superior that of to IN-1 alone.
基金supported by Scientific Research Fund of Xinxiang Medical University,No.2013ZD120Science and Technology Innovation Talents in Universities in Ministry of Education of Henan Province in 2010,No.2010HASTIT036
文摘Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.
基金financially supported by the grants for Science and Technology Innovation Talents in Universities in Ministry of Education of Henan Province in 2010,No.2010HASTIT036
文摘Schwann ceils and neurotrophin-3 play an important role in neural regeneration, but the secretion of neurotrophin-3 from Schwann cells is limited, and exogenous neurotrophin-3 is inactived easily in vivo. In this study, we have transfected neurotrophin-3 into Schwann cells cultured in vitro using nanoparticle liposomes. Results showed that neurotrophin-3 was successfully transfected into Schwann cells, where it was expressed effectively and steadily. A composite of Schwann cells transfected with neurotrophin-3 and poly（lactic-co-glycolic acid） biodegradable conduits was transplanted into rats to repair 10-mm sciatic nerve defects. Transplantation of the composite scaffold could restore the myoelectricity and wave amplitude of the sciatic nerve by electrophysiological examination, promote nerve axonal and myelin regeneration, and delay apoptosis of spinal motor neurons. Experimental findings indicate that neurotrophin-3 transfected Schwann cells combined with bridge grafting can promote neural regeneration and functional recovery after nerve injury.
基金supported by grants from the Shanghai Municipal Commission of Health and Family PlanningNo.20114351+3 种基金the Traditional Chinese Medicine Science Funding of Zhejiang Province of ChinaNo.2010ZB091the Natural Science Foundation of Zhejiang Province of ChinaNo.Y2090864
文摘In the present study, we investigated the role of endogenous neurotrophin-3 in nerve terminal sprouting 2 months after spinal cord dorsal root rhizotomy. The left L1-5 and L7-S2 dorsal root ganglia in adult cats were exposed and removed, preserving the L6 dorsal root ganglia. Neurotrophin-3 was mainly expressed in large neurons in the dorsal root ganglia and in some neurons in spinal lamina II. Two months after rhizotomy, the number of neurotrophin-3-positive neurons in the spared dorsal root ganglia and the density of neurite sprouts emerging from these ganglia were increased. Intraperitoneal injection of an antibody against neurotrophin-3 decreased the density of neurite sprouts. These findings suggest that endogenous neurotrophin-3 is involved in spinal cord plasticity and regeneration, and that it promotes axonal sprouting from the dorsal root ganglia after spinal cord dorsal root rhizotomy.
基金a Grant from Department of Health of Hunan Province of China,No.B2005-076
文摘BACKGROUND： Nogo-neutralizing antibody IN-1 accelerates axon growth and enhances recovery of spinal cord function by inhibiting growth inhibitory factors. Neurotrophin-3 （NT-3）contributes to regeneration of nerve fibers in the spinal cord and motor function recovery. The combination of Nogo-neutralizing antibody IN-1 and NT-3 is hypothesized to produce better outcomes and facilitate axonal regeneration by affecting c-Fos and c-Jun protein expression. OBJECTIVE： To investigate the combined effects of Nogo-neutralizing antibody IN-1 and NT-3 on c-Fos and c-Jun protein levels in the injured spinal cord. DESIGN, TIME AND SETTING： A randomized, controlled study was performed at the Laboratory of Neuroanatomy, Xiangya Medical College, Central South University and the Central Laboratory of Third Xiangya Hospital of China from June 2005 to December 2007. MATERIALS： NT-3 （Peprotech, USA） and Nogo-neutralizing antibody IN-1 （Santa Cruz Biotechnology, USA） were used in this study. METHODS： Hemisectioned spinal cord injury models were established by cutting the posterior 2/3 of rat spinal cord, which is equivalent to the T8 level in the human spine. A total of 120 rats were equally and randomly assigned to three groups： model （0.2 μL saline）, IN-1 （0.2 μL IN-1）, and IN-1/NT-3 （0.2 μL IN-1 ＋ 0.2 μL NT-3）. The compounds were separately infused into transection sites on the side of head. MAIN OUTCOME MEASURES： Western blot analysis was employed to measure c-Fos and c-Jun protein expression in the injured spinal cord at 15, 30 minutes, 1,2, 4, 6, 8, and 12 hours following surgery. RESULTS： Following spinal cord injury, c-Fos and c-Jun protein expression were increased and peaked at 4 6 hours. Following injection of IN-1 or the combination of IN-1 and NT-3, c-Fos protein expression was significantly reduced in the injured spinal cord （P 〈 0.05 or P 〈 0.01） （with the exception of the 15 minute time point）. However, c-Jun protein expression was significantly increased （P〈 0.05 or P〈 0.01） （with the exception of the 15 and 30 minute time points）. Combined application of IN-1 and NT-3 resulted in significantly altered protein expression compared to IN-1 alone. CONCLUSION： IN-1 increases c-Jun protein levels and protects the injured spinal cord by inhibiting c-Fos protein levels. Moreover, the effects of IN-1 combined with NT-3 are more significant than with IN-1 alone.
基金supported by the National Natural Science Foundation of China, No. 30973262
文摘Neurotrophin-3 （NT-3） can promote the repair of central nervous system and retinal damage. In previous reports, NT-3 has been expressed by viral vectors. However, plasmid vectors have a safer profile compared with viral vectors in clinical studies. This study recombined amplified human retinal NT-3 with a eukaryotic expression plasmid containing green fluorescent protein （GFP） to construct an NT-3 expression plasmid, pEGFP-N1-NT-3. The transfection efficiency 48 hours after pEGFP-N1-NT-3 transfection to 293T cells was 50.06 ＋ 2.78%. Abundant NTo3-GFP was expressed in 293T cells as observed by fluorescence microscopy, suggesting the construct pEGFP-N1-NT-3 effectively expressed and secreted NT-3-GFP. Secretory vesicles containing NT-3-GFP were observed in a constant location in cells by laser scan confocal microscopy, indicating the expression and secretion processes of NT-3 in eukaryotic cells were in accordance with the physical synthesis processes of secreted proteins. Western blot assay showed that pro-NTo3-GFP had a molecular weight of 56 kDa, further confirming NT-3-GFP expression. At 48 hours after transfection, the concentration of NT-3 in culture medium was 22.3 ng/mL, suggesting NT-3 produced by pEGFP-N1-NT-3 was efficiently secreted. This study constructed a human retinal-derived NT-3 eukaryotic expression plasmid that efficiently expressed and secreted NT-3.
文摘BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.
基金a project by Hunan Provincial Health Department, No. B2005-076
文摘A spinal cord hemisection injury model was established in rats. Treatment with IN-1 and/or neurotrophin-3 was found to regulate the expression of growth-associated protein 43, nerve growth factor, and basic fibroblast growth factor genes in the injured spinal cord tissues; transcript levels were first increased and then decreased. Expression levels reached a peak at days 7 （growth-associated protein 43） or 14 （nerve growth factor and basic fibroblast growth factor） following spinal cord injury. Combined treatment with neurotrophin-3 and IN-1 achieved the most apparent effect on the expression and recovery of motor function. These findings confirm that combined therapy with neurotrophin-3 and IN-1 can increase expression of growth factors in the injured spinal cord tissues and promote the axonal reaeneration.
基金the National Natural Science Foundation of China,No.30770751the Foundation for Youth of Shandong Bureau of Public Health,No.2007QZ002the Doctoral Foundation of Shandong Scientific and Technological Bureau,No. 2008BS03004
文摘BACKGROUND： Previous studies have demonstrated that the combination of olfactory ensheathing cells （OECs） and neurotrophic factor-3 （NT-3） in the rat lateral ventricle can promote nerve axonal regeneration and myelin sheath repair. However, this effect remains very short-lived. OBJECTIVE： To transfect NT-3 into OECs and to observe the biological activity of OEC-expressing NT-3. DESIGN, TIME AND SETTING： This genetic engineering, in vitro experiment was performed in the Provincial Hospital Affiliated to Shandong University between January 2007 and October 2008. MATERIALS： Trizol Reagent kit was purchased from Gibco, USA; reverse transcription kit, NT-3 Emax ImmunoAssay System reagent was purchased from Promega, USA. METHODS： Neonatal Wistar rat OECs were established as primary cultures and were transfected with pN2A-NT-3 viral vector. The OECs with the highest virus titer and stable cellular growth served as the transfection group; OECs transfected with NT-3-free retrovirus carrier pN2A served as the empty vector group; un-transfected OECs served as the control group. After adherence, the logarithmically cultured PC12-TrkC cells were plated in OECs supernatant from the transfection and empty vector groups, as well as 20 μL PBS, and cultured for 4 days. MAIN OUTCOME MEASURES： NT-3 mRNA expression in OECs, fluorescence of NT-3-positive cells in the transfection group and control group; influence of OECs secreting NT-3 on the differentiation ratio of PC12-TrkC cells. RESULTS： NT-3 mRNA expression was observed 24 hours after transfection and lasted for 28 days which was greater than the control and empty vector groups （P 〈 0.01）. A large number of NT-3-positive cells were observed in the transfection group, and immunofluorescence was greater than the control and empty vector groups. PC12-TrkC cells co-cultured with OECs from the transfection group exhibited a thick and long cell process, increased cell density, and the differentiation ratio was increased （P 〈 0.01）. CONCLUSION： Recombinant double replica retrovirus NT-3 gene was stably and effectively expressed in OECs, and the expressed NT-3 possessed biological activity that promoted neuronal survival.
基金The National Natural Science Foundation of China，Medical Scientific Research of Zhejiang Province
文摘In this study, cells from the cerebral cortex of fetal rats at pregnant 16 days were harvested and cultured with 20 μg/L neurotrophin-3. After 7 days of culture, immunocytochemical staining showed that, 22.4% of cells were positive for nestin, 10.5% were positive for 18-111 tubulin （neuronal marker）, and 60.6% were positive for glial fibrillary acidic protein, but no cells were positive for 04 （oligodendrocytic marker）. At 14 days, there were 5.6% nestin-, 9.6% 13-111 tubulin-, 81.1% glial fibrillary acidic protein-, and 2.2% O4-positive cells. In cells not treated with neurotrophin-3, some were nestin-positive, while the majority showed positive staining for glial fibdllary acidic protein. Our experimental findings indicate that neurotrophin-3 is a crucial factor for inducing neural stem cells differentiation into neurons and oligodendrocytes.
