Neurotrophin-3、LncRNA H19与小儿癫痫持续状态严重程度的关系及对预后预测效能研究

目的探讨神经营养因子-3(Neurotrophin-3)、长链非编码核糖核酸H19(LncRNA H19)与小儿癫痫持续状态(SE)严重程度的关系及对预后预测效能。方法选取2020年1月—2023年12月山西医科大学附属运城市中心医院儿内科收治的SE患儿152例为SE组,根据儿童癫痫持续状态严重程度评分(STEPSS)分为轻度亚组67例、中度亚组52例、重度亚组33例;根据院内结局分为良好预后亚组91例和不良预后亚组61例。另选取同期医院健康体检儿童60例为健康对照组。采用酶联免疫吸附法检测血清Neurotrophin-3,实时荧光定量聚合酶链反应检测LncRNA H19水平;Spearman法分析血清Neurotrophin-3、LncRNA H19水平与SE患儿STEPSS评分的相关性;多因素Logistic回归分析SE患儿不良预后的影响因素;受试者工作特征(ROC)曲线分析血清Neurotrophin-3、LncRNA H19水平对小儿SE不良预后的预测价值。结果SE组血清Neurotrophin-3水平低于健康对照组,LncRNA H19水平高于健康对照组(t/P=11.877/<0.001、20.966/<0.001);随着病情加重,轻度亚组、中度亚组、重度亚组血清Neurotrophin-3水平依次降低,LncRNA H19水平依次升高(F/P=184.107/<0.001、114.394/<0.001);152例SE患儿不良预后发生率为41.13%(61/152)。不良预后亚组STEPSS评分、SE发作时间≥1 h、全面性发作、气管插管比例、血清LncRNA H19水平高于良好预后亚组,血清Neurotrophin-3水平低于良好预后亚组(χ^(2)/t/P=8.090/<0.001、11.931/0.001、11.566/0.001、8.752/0.003、6.467/<0.001、7.846/<0.001);SE患儿STEPSS评分与血清Neurotrophin-3水平呈负相关,与LncRNA H19水平呈正相关(rs/P=-0.764/<0.001,0.748/<0.001);多因素Logistic回归分析显示,SE发作时间≥1 h、STEPSS评分升高、全面性发作、LncRNA H19升高为小儿SE不良预后的独立危险因素[OR(95%CI)=3.216(1.406~7.354)、2.001(1.366~2.931)、3.970(1.229~11.691)、1.592(1.245~2.034)],Neurotrophin-3升高为独立保护因素[OR(95%CI)=0.943(0.919~0.967)];血清Neurotrophin-3、LncRNA H19水平及二者联合预测SE患儿不良预后的AUC分别为0.808、0.780、0.891,二者联合的AUC大于单独预测(Z/P=3.194/0.001、3.521/<0.001)。结论血清Neurotrophin-3水平降低和LncRNA H19水平升高与SE患儿病情加重和不良预后有关,血清Neurotrophin-3、LncRNA H19水平联合对SE患儿不良预后有较高的预测效能。

Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury

Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

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.

Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury

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.

Effects of Nogo-neutralizing antibody and neurotrophin-3 on axonal regeneration following spinal cord injury in rats

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.

Nanoparticles carrying neurotrophin-3-modified Schwann cells promote repair of sciatic nerve defects

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.

Endogenous neurotrophin-3 promotes neuronal sprouting from dorsal root ganglia

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.

Effects of combined application of Nogo-neutralizing antibody IN-1 and neurotrophin-3 on c-Fos and c-Jun expression in a rat model of hemisection spinal cord injury

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.

Effect of neurotrophin-3 on SOD and MDA in rats after acute spinal cord injury

Objective:To investigate the effect of neurotrophin-3 on the expressions of SOD and MDA in the injured spinal cord of rats. Methods: Totally 105 SD rats were randomly divided into 3 groups (n = 35): sham group, control group and experimental group. Animal model of acute spinal cord was inflicted with Allen's method by a thin plastic tube situated in subarachnoid space below the injury level for perfusion. Rats in experimental group received 20μl NT-3 (200 ng) from the tube at 0, 4. 8. 12. 24 h and 3. 7 d after injury, and those in control group got the equal volume of normal saline at the same time points. The animals in sham group only received opening vertebral plate and putting tube in subarachnoid space. The rats were sacrificed at 4, 8. 12. 24 h, and 3. 7, 14 d postinjury (n = 5). And the levels of superoxide dismutase (SOD) and malondialdehyde (MDA) in blood were observed with colorimetric method. Results: The serum level of SOD reduced obviously and the level of MDA raised obviously in rats after the injury, and the activity of SOD reached the lowest on day 3 and the concentration of MDA reached peak at the 7 d. In the experimental group, the SOD level was obviously higher (P<0. 01). and MDA level was lower than the control (P<0. 01). Conclusion:NT-3 can mitigate secondary injury of spinal cord in vivo. One of mechanisms is that inhibits abnormal expression of MDA and elevates the activity of SOD. thus the injury of free radical and lipid peroxidation is attenuated.

Construction of a eukaryotic expression plasmid for human retina-derived neurotrophin-3

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.

IN-1 combined with neurotrophin-3 for axonal growth-related gene expression after spinal cord injury

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.

Location and expression of neurotrophin-3 and its receptor in the brain of human embryos during early development

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.

Effects of neurotrophin-3 on the differentiation of neural stem cells into neurons and oligodendrocytes

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.

Expression and biological activity of double replica retrovirus carrier-mediated neurotrophin-3 in olfactory ensheathing cells

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.

Delayed peripheral treatment with neurotrophin-3 improves sensorimotor recovery after central nervous system injury

Neurotrophin-3 (NT3) is a growth factor found in many body tissues including the heart, intestines, skin, nervous system and in skeletal muscles including muscle spindles (Murase et al., 1994). NT3 is required for the survival, correct connectivity and function of sensory (“proprioceptive”) afferents that innervate muscle spindles;these neurons express receptors for NT3 including tropomyocin receptor kinase C. These proprioceptive afferents are important for normal movement (Boyce and Mendell, 2014) and signals from muscle spindles are important for recovery of limb movement (e.g., after spinal cord lateral hemisection)(Takeoka et al., 2014). The level of NT3 declines in most tissues during postnatal development;its level is low in adult and elderly humans and other mammals (Murase et al., 1994). Elevation of NT3 has been shown to improve outcome in various animal models of neurological disease and injury. For example, many groups have shown that delivery of NT3 directly into the central nervous system promotes recovery after spinal cord injury but this often involved invasive routes or gene therapy (Boyce and Mendell, 2014;Petrosyan et al., 2015;Wang et al., 2018).

Influence of neurotrophin-3 on Bcl-2 and Bax expressions in spinal cord injury of rats

Objective： To study the protective mechanisms of neurotrophin-3 （NT-3） on the spinal cord injury. Methods：Totally 105 SD rats were randomly divided into 3 groups： control group, experimental group and sham operation group. Rats from the former 2 groups were inflicted to animal model of acute spinal cord injury according to Allen＇s （WD） by situating a thin plastic tube in the subarachnoid space below the injury level for perfusion. Rats in experimental group received 20 ul NT-3 （200 ng） from the tube at 0, 4, 8, 12, 24 h and 3, 7 d after injury, and those in control group got an equal volume of normal saline at the same time. The animals in sham operation group only received opening vertebral plate and tube was put in subarachnoid space. The rats were sacrificed at 4, 8, 12, 24 h and 3, 7, 14 d post injury （n=5）. The expression levels of Bcl-2 and Bax proteins in spinal cord of rats were detected by immunohistochemistry assay. Results： The level of Bax protein in control group significantly increased as compared with those in sham operation group, and the peak reached at 8 h after spinal cord injury. The Bcl-2 proteins were always weakly positive. The Bax proteins in NT-3 group significantly decreased but the Bcl-2 proteins obviously increased as compared with those in control group. Conclusion： NT-3 can protect spinal cord from injury in vivo. One of the mechanisms is that NT-3 can inhibit abnormal expression of Pax protein, and increase the expression of Bcl-2 protein, then inhibit apoptosis after spinal cord injury.

Effects of neurotrophin-3 intervention on on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis

Objective:To study the effects of neurotrophin-3 (NT-3) intervention on bone marrow mesenchymal stem cell osteoblast differentiation as well as cell proliferation and apoptosis. Methods: Bone marrow mesenchymal stem cells were cultured and divided into control group, 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group, they were treated with different doses of NT-3 for 24 h, and then osteoblast marker gene, cell proliferation gene and apoptosis gene expression were determined.Results: RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression in 25 ng/mL NT-3 group, 50 ng/mL NT-3 group and 100 ng/mL NT-3 group were significantly higher than those in control group whereas Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression were significantly lower than those in control group, and the larger the dose of NT-3, the higher the RUNX2, Osterix, ALP, OCN, BMP-2, Bcl-2, Nrf2, ERK1/2 and PCNA mRNA expression whereas the lower the Bim, Bax, Caspase-3, CHOP and Beclin1 mRNA expression.Conclusion: NT-3 intervention in bone marrow mesenchymal stem cells can promote osteoblast differentiation and cell proliferation and inhibit apoptosis.

THE CLONING OF HUMAN NEUROTROPHIN-3 GENE

In the present study, we have cloned the gene of human neurotrophin3 (hNT3) from the genomic DNA of white blood cells (WBC) by polymerase chain reaction (PCR). The amplification products were cloned into pUC19 and sequenced. Genomic sequence comparison of the cloned fragment and the reported hNT3 (GenBank M61180) reveals 7 base differences: 1 in the signal peptide, 3 in the prepro peptide, and 3 in the mature hNT3. Except the 2 varied bases (16th, T to G; 285th, A to C) in the signal peptide and prosequence resulted in the change of their encoded aminoacids (TyrAsp; GlnHis), the other varied bases have no influence on their respective encoded aminoacids, and all the changes have no influence on the open reading frame (ORF) of the hNT3.

沈阳市O_(3)与PM_(2.5)关系及污染主控因素分析

PM_(2.5)与O_(3)的协同控制是空气质量持续改善的关键所在,厘清PM_(2.5)与O_(3)的关系,识别O_(3)主控因素以及量化气象和人为排放贡献是实施二者协同控制的基础.本研究基于沈阳市大气复合立体超级站2019−2022年地面观测数据,分析PM_(2.5)和O_(3)协同关系及成因;利用逐步回归模型得到影响O_(3)变化的主控因素,并估算各气象因素对O_(3)的贡献.结果表明:①沈阳市2019−2022年夏季PM_(2.5)浓度与O_(3)浓度呈正相关,有明显的协同增长效应,其余三季均呈明显负相关.究其原因,主要是由于夏季高温和高太阳辐射条件利于大气光化学反应,促进了O_(3)、PM_(2.5)中二次无机成分〔主要是硫酸盐(SO_(4)^(2−))、硝酸盐(NO_(3)−)和铵盐(NH_(4)^(+)),简称“SNA”〕共同增长所致;而冬季高排放和高大气稳定度等气象条件利于SNA和二次有机碳(SOC)非均相生成,但弱太阳辐射和低温等条件不利于O_(3)光化学生成,加之高NO的滴定效应,使SNA和SOC浓度均与O_(3)浓度呈负相关.②在观测的相关污染物和气象因子中,过氧乙酰硝酸酯(PAN)与O_(3)浓度的关系最为密切,尤其在夏季.③气象因素中,O_(3)浓度与气温高度相关,与风速也呈正相关,而与相对湿度则在各季节均呈负相关.冬、春、秋三季PM_(2.5)均对O_(3)起抑制作用,冬季尤为突出.在高浓度O_(3)污染(O_(3)浓度>160μg/m^(3))过程中,主控因素中气温和风速的抬升促进O_(3)浓度升高,而高NO2和相对湿度(RH)则有利于降低O_(3)浓度.在2019−2022年高浓度O_(3)污染过程中,气象因素对沈阳市O_(3)浓度变化的贡献高于O_(3)前体物排放的贡献,总贡献为57μg/m^(3),对污染形成起着主导作用.

CdS/In_(2)O_(3)/g-C_(3)N_(4)复合材料的制备及光催化性能研究

采用溶剂热法成功合成了一种新型的Z型CdS/In_(2)O_(3)/g-C_(3)N_(4)三元复合光催化材料。通过XRD、SEM、TEM、XPS和紫外-可见漫反射光谱仪对光催化材料的相结构、形貌、原子价态和光响应性能等进行表征,通过可见光降解苯酚评价其光催化活性。结果表明,具有零维结构的CdS、一维结构的In_(2)O_(3)和三维结构的g-C_(3)N_(4)形成了0D/1D/3D三元复合材料,该材料在180 min可有效降解90%的苯酚,降解速率是CdS的2.9倍、g-C_(3)N_(4)的6倍,且具有较高的稳定性。复合材料光催化能力的增强主要归因于三维多孔g-C_(3)N_(4)与CdS和In_(2)O_(3)形成的三维空间电场。三维多孔结构不仅有利于污染物的高效吸附,而且为光催化反应提供活性位点,三维空间和网络互连结构有利于光生电荷的定向迁移,增加载流子寿命。