Single-Cell Sequencing Technology and Its Application in the Study of Central Nervous System Diseases.

The mammalian central nervous system consists of a large number of cells, which contain not only different types of neurons, but also a large number of glial cells, such as astrocytes, oligodendrocytes, and microglia. These cells are capable of performing highly refined electrophysiological activities and providing the brain with functions such as nutritional support, information transmission and pathogen defense. The diversity of cell types and individual differences between cells have brought inspiration to the study of the mechanism of central nervous system diseases. In order to explore the role of different cells, a new technology, single-cell sequencing technology has emerged to perform specific analysis of high-throughput cell populations, and has been continuously developed. Single-cell sequencing technology can accurately analyze single-cell expression in mixed-cell populations and collect cells from different spatial locations, time stages and types. By using single-cell sequencing technology to compare gene expression profiles of normal and diseased cells, it is possible to discover cell subsets associated with specific diseases and their associated genes. Therefore, scientists can understand the development process, related functions and disease state of the nervous system from an unprecedented depth. In conclusion, single-cell sequencing technology provides a powerful technology for the discovery of novel therapeutic targets for central nervous system diseases.

Effects and mechanisms of salidroside on the behavior of SPS-induced PTSD rats.

Post-traumatic stress disorder (PTSD) is a complex mental disorder, closely associated with stress and traumatic events. Salidroside (Sal) has been reported to possess neuroprotective effects. However, the behavioral effects and mechanisms of Sal on PTSD remain unknown. In this study, we utilized a rat model of PTSD induced by single prolonged stress (SPS) and administered Sal intraperitoneally (25, 50, 75 mg/kg/d) for 14 days. We then examined the behavioral effects and underlying mechanisms of Sal on SPS-induced PTSD rats. Our findings demonstrated that Sal alleviated anxiety-like behavior and spatial learning and memory impairment in SPS-induced PTSD rats. Furthermore, Sal treatment preserved the histomorphology of the hippocampal region. It was observed that Sal protected against hippocampal neuronal apoptosis in PTSD rats by reducing the number of TUNEL-positive cells and modulating apoptosis-related proteins (Bcl-2 and Bax). Additionally, Sal inhibited the activation of the NF-κB/iNOS/COX-2 signaling pathway in the hippocampus of PTSD rats, thereby suppressing the release of inflammatory factors (TNF-α and IL-1ß) and the activation of microglia. Notably, Sal increased the expression of synapse-associated proteins PSD95 and Synapsin I in the hippocampus, while also enhancing dendritic density in the region. In conclusion, our results demonstrated that Sal could attenuate SPS-induced PTSD-like behaviors by inhibiting hippocampal neuronal apoptosis, enhancing hippocampal synaptic plasticity, and reducing neuroinflammatory responses. These findings may provide a foundation for the potential clinical application of Sal in the treatment of PTSD.

Effects and mechanisms of tanshinone IIA on PTSD-like symptoms.

BACKGROUND: In recent years, Salvia miltiorrhiza and its active substances have remarkably progressed in treating central neurological disorders. Tanshinone IIA (TSA) is an active ingredient derived from the rhizome of Salvia miltiorrhiza that has been found to alleviate the symptoms of several psychiatric illnesses. Post-traumatic stress disorder (PTSD) is a mental disorder that results after experiencing a serious physical or psychological injury. The currently used drugs are not satisfactory for the treatment of PTSD. However, it has been reported that TSA can improve PTSD-like symptoms like learning and memory, cognitive disorder, and depression through multi-target regulation. PURPOSE: This paper discusses the ameliorative effects of TSA on PTSD-like symptoms and the possible mechanisms of action in terms of inhibition of neuronal apoptosis, anti-neuroinflammation, and anti-oxidative stress. Based on the pathological changes and clinical observations of PTSD, we hope to provide some reference for the clinical transformation of Chinese medicine in treating PTSD. METHODS: A large number of literatures on tanshinone in the treatment of neurological diseases and PTSD were retrieved from online electronic PubMed and Web of Science databases. CONCLUSION: TSA is a widely studied natural active ingredient against mental illness. This review will contribute to the future development of TSA as a new clinical candidate drug for improving PTSD-like symptoms.

The role of RNA splicing factor PTBP1 in neuronal development.

Alternative pre-mRNA splicing, which produces various mRNA isoforms with distinct structures and functions from a single gene, is regulated by specific RNA-binding proteins and is an essential method for regulating gene expression in mammals. Recent studies have shown that abnormal change during neuronal development triggered by splicing mis-regulation is an important feature of various neurological diseases. Polypyrimidine tract binding protein 1 (PTBP1) is a kind of RNA-binding proteins with extensive biological functions. As a well-known splicing regulator, it affects the neuronal development process through its involvement in axon formation, synaptogenesis, and neuronal apoptosis, according to the most recent studies. Here, we summarized the mechanism of alternative splicing, structure and function of PTBP1, and the latest research progress on the role of alternative splicing events regulated by PTBP1 in axon formation, synaptogenesis and neuronal apoptosis, to reveal the mechanism of PTBP1-regulated changes in neuronal development process.

Syntaphilin mediates axonal growth and synaptic changes through regulation of mitochondrial transport: a potential pharmacological target for neurodegenerative diseases.

Mitochondria are a crucial energy source for maintaining neuronal growth and synaptic function. Neurons possess unique morphological characteristics, which make the proper regulation of mitochondrial transport essential for meeting their energy demands. Syntaphilin (SNPH) is capable of specifically targeting the outer membrane of axonal mitochondria, anchoring them to microtubules, and thereby preventing their transport. SNPH also interacts with other mitochondrial proteins to regulate mitochondrial transport. The regulation of mitochondrial transport and anchoring mediated by SNPH is indispensable for axonal growth during neuronal development, maintenance of ATP levels during neuronal synaptic activity, and regeneration of mature neurons following damage. Precise blocking of SNPH may be an effective therapeutic strategy for neurodegenerative diseases and related mental disorders.

The latest role of nerve-specific splicing factor PTBP1 in the transdifferentiation of glial cells into neurons.

Central nervous system injury diseases can cause the loss of many neurons, and it is difficult to regenerate. The field of regenerative medicine believes that supplementing the missing neurons may be an ideal method for nerve injury repair. Recent studies have found that down-regulation of polypyrimidine tract binding protein 1 (PTBP1) expression can make glial cells transdifferentiate into different types of neurons, which is expected to be an alternative therapy to restore neuronal function. This article summarized the research progress on the structure and biological function of the PTBP family, the mutual regulation of PTBP1 and PTBP2, their role in neurogenesis, and the latest research progress in targeting PTBP1 to mediate the transdifferentiation of glial cells into neurons, which may provide some new strategies and new ideas for the future treatment of central nervous system injury and neurodegenerative diseases. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing.

The ameliorative effects and mechanisms of abscisic acid on learning and memory.

Abscisic acid (ABA), a conserved hormone existing in plants and animals, not only regulates blood glucose and inflammation but also has good therapeutic effects on obesity, diabetes, atherosclerosis and inflammatory diseases in animals. Studies have shown that exogenous ABA can pass the blood-brain barrier and inhibit neuroinflammation, promote neurogenesis, enhance synaptic plasticity, improve learning, memory and cognitive ability in the central nervous system. At the same time, ABA plays a crucial role in significant improvement of Alzheimer's disease, depression, and anxiety. Here we review the previous research progress of ABA on the physiological effects and clinical application in the related diseases. By summarizing the biological functions of ABA, we aim to reveal the possible mechanisms of ameliorative function of ABA on learning and memory, to provide a theoretical basis that ABA as a novel and safe drug improves learning memory and cognitive impairment in central system diseases such as aging, neurodegenerative diseases and traumatic brain injury.

The Effects of the Olig Family on the Regulation of Spinal Cord Development and Regeneration.

Neurons and glial cells in the central nervous system (CNS) are generated from neuroepithelial cells in the ventricular zone that surrounds the embryonic neural tube. The proliferation and distinct differentiation of neural precursors occurs at certain stages and are regulated by a series of transcription factors leading to the generation of neuronal and glial cell subtypes. In this manuscript, we review the effects of the Olig family, namely, members Olig1, Olig2 and Olig3, on the distinct differentiation of glial and neuronal cells in the developing spinal cord and injured neural tissue.

Transplantation of NSCs Promotes the Recovery of Cognitive Functions by Regulating Neurotransmitters in Rats with Traumatic Brain Injury.

Transplantation of neural stem cells (NSCs) may be a potential strategy for traumatic brain injury treatment (TBI) due to their intrinsic advantages, such as cell replacement, secretion of neurotrophins and formation of functional synapses with host. However the underlying effects of transplanted NSCs on host micro-environment still need to be further elucidated. In this manuscript the effects of NSCs on release of neurotransmitter, survival of hippocampal neurons, reactivity of astrocytes and recovery of cognitive function after TBI were observed. The NSCs were isolated from cortex of neonatal Sprague-Dawley rat and then transplanted into injured brain regions caused by free-weight drop. The proliferation of astrocytes around injured sites were examined by GFAP immunofluorescent staining on 3, 7, 14 days after injury. The survival of neurons at CA1 regions of hippocampus toward contused regions was observed by HE staining on 3 and 14 days post-injury. The content of glutamic acid (Glu) and GABA in hippocampal tissues was examined on 1, 3, 7, 14, 28 days after injury by ELISA. On third day post-injury, hippocampal-dependent spatial memory was measured for 5 days without intermittent. NSCs in culture have the ability to proliferate and differentiate into different phenotypes of neural cells. After transplantation of NSCs, the proliferation of astrocytes around injured site was significantly inhibited compared to the injured group. At the same time the survival of neurons in hippocampal CA1 region were much more than those in injured group on 14 days post-injury. Meanwhile, the cognitive functions in NSC transplanted group was remarkably improved compared with injured group (p < 0.05). Furthermore, NSCs transplantation dramatically inhibited the release of Glu and maintained the content of GABA in injured hippocampal tissues on 1, 3, 7, 14, 28 days post-injury, which was of difference in statistics (p < 0.05). NSCs transplantation can effectively alleviate the formation of glial scar, enhance the survival of hippocampal neurons and improve cognitive function defects in rats with TBI. The underlying mechanism may be related to their effects on inhibiting the release of Glu and maintaining the content of GABA, so as to down-regulate excitotoxicity of neurotransmitter and improve the micro-environment in injured sites.

[Effect of electroacupuncture on degradation of myosin heavy chain of gastrocnemius muscle in diabetes rats].

OBJECTIVE: To explore the effect of electroacupuncture（EA）on the expression of muscle-specific ring finger protein 1（MuRF1/Trim63），F-box only protein 32（Fbxo32），myosin heavy chain-IIa（Myh2），myosin heavy chain-IIb（Myh4）and myosin heavy chain-I（Myh7）in diabetes rats. METHODS: Thirty-six male Wistar rats were equally randomized into control, model and EA groups. The diabetes model was established by intraperitoneal injection of 0.1% Streptozocin (STZ) solution (50 mg/kg). After that, EA (2 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36), "Yinlingquan" (SP9) and "Shenshu" (BL23) for 10 min, once a day, 6 times a week for 2 weeks. The fasting blood glucose (FBG) and fasting serum insulin (FINS) contents were assayed by using ELISA, and the homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. The body weight, and wet weight of bilateral gastrocnemius muscles were measured. The cross-sectional area (CSA) of the gastrocnemius muscle was measured after H.E. stai-ning. The expression of MuRF1, Fbxo32, Myh2, Myh4 and Myh7 mRNAs in the gastrocnemius tissue was tested using quantitative real time-PCR. RESULTS: Compared with the control group, the FBG and HOMA-IR were significantly higher (P<0.05), and the FINS, body weight were significantly lower (P<0.05) after intravenous injection of STZ for 1, 2, 3, 4, 5 weeks respectively. Following EA treatment and compared with the model group, the FBG and HOMA-IR were significantly down-regulated (P<0.05), and the FINS and body weight were considerably increased (P<0.05). Following modeling and compared with the control group, the wet weight of gastrocnemius muscle, CSA, and expression levels of Myh2, Myh4 and Myh7 mRNAs were obviously decreased, and the expression of MuRF1 and Fbxo32 mRNA was obviously increased in the model group (P<0.05). After EA treatment, the gastrocnemius muscle wet weight, CSA, expression levels of Myh2, Myh4 and Myh7 mRNA were significantly up-regulated (P<0.05), and the expression levels of MuRF1 and Fbxo32 mRNA were markedly down-regulated in comparison with those of the model group (P<0.05). CONCLUSION: EA treatment can delay the atrophy of gastrocnemius muscle (skeletal muscle) in diabetes rats possibly by improving the degradation of myosin heavy chain via regulating the expression of muscular MuRF1, Fbxo32, Myh2, Myh4 and Myh7 mRNAs.

Motor neuron degeneration following glycine-mediated excitotoxicity induces spastic paralysis after spinal cord ischemia/reperfusion injury in rabbit.

Spinal cord ischemia and reperfusion (SCIR) injury is the major cause of a wide range of complications, including neural degeneration and devastating paraplegia. Decrease of inhibitory neurotransmitters and increase of excitory neurotransmitters are the major cause for the excitotoxicity of neurons. However, no study has reported the temporal loss of motor neuron in the ventral horn of spinal cord area following SCIR-induced spastic paralysis, not even the mechanism under it. In the present study, we found that the rabbits were mainly spastic paralyzed after spinal cord ischemia-reperfusion injury. And the ischemia 60 min group is the optimal treating condition, because of the higher rate of spastic paralysis and lower mortality. Motor neurons in the ventral horn of spinal cord were significant degeneration at 3 h following spastic paralysis and only 12.5% motor neurons were observed at 72 h post-operation, compared with control group. ELISA results indicated that Glycine and GABA were both downregulated following spastic paralysis. But Glycine immediately decreased at 10 min post-operation and lasted for the whole process (at least 72 h). Meanwhile GABA only significantly decreased at 72 h. Furthermore, Glutamic expression was significant upregulation at 3 hours post-operation, and the upregulation back to the base level at 72 h post-operation. Glutamic receptor-(NR1) and Glycine α1 receptor upregulated accordingly, whereas GABBR2 didn't upregulate significantly until at 72 h post-operation. Abundant extracellular Ca2+ influxed into cytoplasm in neurons following spastic paralysis. The type of paraplegia is mainly spastic paraplegia after SCIR (ischemia 60 min treatment). Following spastic paraplegia, motor neuron in the ventral horn of spinal cord area was significant degeneration at early stage and last for the whole process. It may contribute to the decrease of Glycine at early stage and followed exitotoxicity, which caused intracellular calcium overload to make neurons dead. It would lay the foundation for better understanding the motor neuron degeneration and mechanism following spastic paralysis. And it would supply a novel and effective target for spastic paralysis prevention and therapy.

Local injection of Lenti-Olig2 at lesion site promotes functional recovery of spinal cord injury in rats.

AIMS: Olig2 is one of the most critical factors during CNS development, which belongs to b-HLH transcription factor family. Previous reports have shown that Olig2 regulates the remyelination processes in CNS demyelination diseases models. However, the role of Olig2 in contusion spinal cord injury (SCI) and the possible therapeutic effects remain obscure. This study aims to investigate the effects of overexpression Olig2 by lentivirus on adult spinal cord injury rats. METHODS: Lenti-Olig2 expression and control Lenti-eGFP vectors were prepared, and virus in a total of 5 µL (108 TU/mL) was locally injected into the injured spinal cord 1.5 mm rostral and caudal near the epicenter. Immunostaining, Western blot, electron microscopy, and CatWalk analyzes were employed to investigate the effects of Olig2 on spinal cord tissue repair and functional recovery. RESULTS: Injection of Lenti-Olig2 significantly increased the number of oligodendrocytes lineage cells and enhanced myelination after SCI. More importantly, the introduction of Olig2 greatly improved hindlimb locomotor performances. Other oligodendrocyte-related transcription factors, which were downregulated or upregulated after injury, were reversed by Olig2 induction. CONCLUSIONS: Our findings provided the evidence that overexpression Olig2 promotes myelination and locomotor recovery of contusion SCI, which gives us more understanding of Olig2 on spinal cord injury treatment.

Retinoic acid induced the differentiation of neural stem cells from embryonic spinal cord into functional neurons in vitro.

Retinoic acid is an important molecular taking part in the development and homeostasis of nervous system. Neural stem cells (NSCs) are pluripotent cells that can differentiate into three main neural cells including neuron, astrocyte and oligodendrocyte. However, whether retinoic acid can induce NSCs derived from embryonic spinal cord differentiating into functional neurons and its efficiency are not clear. In this experiment, NSCs were isolated from embryonic 14 d spinal cord of rats. The growth and neuronal differentiation of NSCs induced by 500 nM RA was examined in vitro. It was indicated that compared with the control group, there were more differentiated cells with longer cytodendrites in the medium treated with RA at different time. And more, there were more neuronal marker positive cells in 500 nM RA group than the control group seven days after differentiation. At the same time, the expression of ß-tublin III protein in RA group was higher than those in control group, which was contrary to the expression of astrocyte marker GFAP protein at seven days after differentiation. However the differentiated neurons, whether treated with RA or not both exhibited biological electrical reactivity after stimulated by glutamine. Therefore, these findings indicated that RA could promote growth of cellular dendrites and neuronal differentiation of NSCs, which also induce functional maturation of differentiated neurons finally.

Wnt signaling pathway participates in valproic acid-induced neuronal differentiation of neural stem cells.

Neural stem cells (NSCs) are multipotent cells that have the capacity for differentiation into the major cell types of the nervous system, i.e. neurons, astrocytes and oligodendrocytes. Valproic acid (VPA) is a widely prescribed drug for seizures and bipolar disorder in clinic. Previously, a number of researches have been shown that VPA has differential effects on growth, proliferation and differentiation in many types of cells. However, whether VPA can induce NSCs from embryonic cerebral cortex differentiate into neurons and its possible molecular mechanism is also not clear. Wnt signaling is implicated in the control of cell growth and differentiation during CNS development in animal model, but its action at the cellular level has been poorly understood. In this experiment, we examined neuronal differentiation of NSCs induced by VPA culture media using vitro immunochemistry assay. The neuronal differentiation of NSCs was examined after treated with 0.75 mM VPA for three, seven and ten days. RT-PCR assay was employed to examine the level of Wnt-3α and ß-catenin. The results indicated that there were more ß-tublin III positive cells in NSCs treated with VPA medium compared to the control group. The expression of Wnt-3α and ß-catenin in NSCs treated with VPA medium was significantly greater compared to that of control media. In conclusion, these findings indicated that VPA could induce neuronal differentiation of NSCs by activating Wnt signal pathway.

Ameliorative Effects of p75NTR-ED-Fc on Axonal Regeneration and Functional Recovery in Spinal Cord-Injured Rats.

As a co-receptor of Nogo-66 receptor (NgR) and a critical receptor for paired immunoglobulin-like receptor (PirB), p75 neurotrophin receptor (p75NTR) mediates the inhibitory effects of myelin-associated inhibitors on axonal regeneration after spinal cord injury. Therefore, the p75NTR antagonist, such as recombinant p75NTR protein or its homogenates may block the inhibitory effects of myelin and promote the axonal regeneration and functional recovery. The purposes of this study are to subclone and express the extracellular domain gene of human p75NTR with IgG-Fc (hp75NTR-ED-Fc) in prokaryotic expression system and investigate the effects of the recombinant protein on axonal regeneration and functional recovery in spinal cord-injured rats. The hp75NTR-ED-Fc coding sequence was amplified from pcDNA-hp75NTR-ED-Fc by polymerase chain reaction (PCR) and subcloned into vector pET32a (+), then the effects of the purified recombinant protein on neurite outgrowth of dorsal root ganglion (DRG) neurons cultured with myelin-associated glycoprotein (MAG) were determined, and the effects of the fusion protein on axonal regeneration, functional recovery, and its possible mechanisms in spinal cord-injured rats were further investigated. The results indicated that the purified infusion protein could promote neurite outgrowth of DRG neurons, promote axonal regeneration and functional recovery, and decrease RhoA activation in spinal cord-injured rats. Taken together, the findings revealed that p75NTR still may be a potential and novel target for therapeutic intervention for spinal cord injury and that the hp75NTR-ED-Fc fusion protein treatment enhances functional recovery by limiting tissue loss and stimulating axonal growth in spinal cord-injured rats, which may result from decreasing the activation of RhoA.

Inhibiting PTEN protects hippocampal neurons against stretch injury by decreasing membrane translocation of AMPA receptor GluR2 subunit.

The AMPA type of glutamate receptors (AMPARs)-mediated excitotoxicity is involved in the secondary neuronal death following traumatic brain injury (TBI). But the underlying cellular and molecular mechanisms remain unclear. In this study, the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GluR2-lacking AMPARs mediated neuronal death was investigated through an in vitro stretch injury model of neurons. It was indicated that both the mRNA and protein levels of PTEN were increased in cultured hippocampal neurons after stretch injury, which was associated with the decreasing expression of GluR2 subunits on the surface of neuronal membrane. Inhibition of PTEN activity by its inhibitor can promote the survival of neurons through preventing reduction of GluR2 on membrane. Moreover, the effect of inhibiting GluR2-lacking AMPARs was similar to PTEN suppression-mediated neuroprotective effect in stretch injury-induced neuronal death. Further evidence identified that the total GluR2 protein of neurons was not changed in all groups. So inhibition of PTEN or blockage of GluR2-lacking AMPARs may attenuate the death of hippocampal neurons post injury through decreasing the translocation of GluR2 subunit on the membrane effectively.

The effects of different phenotype astrocytes on neural stem cells differentiation in co-culture.

Astrocytes were reported to show neuroprotective effects on neurons, but there was no direct evidence for a functional relationship between astrocytes and neural stem cells (NSCs). In this experiments, we examined neuronal differentiation of NSCs induced by protoplasmic and fibrous astrocytes in a co-culture model respectively. Two types of astrocytes and NSCs were isolated from E13 to 15 cortex of rats. The neuronal differentiation of NSCs was examined after co-culture with two kinds of astrocytes. There were more neuronal marker ß-tublin III positive cells from NSCs co-cultured with protoplasmic astrocytes. However the differentiated neurons, whether co-cultured with protoplasmic astrocytes or fibrous astrocytes, both expressed glutamate AMPA receptor subunit GluR2 protein and exhibited biological electrical reactivity after stimulated by glutamine. Therefore, these findings indicated that two types of astrocytes could induce the differentiation of NSCs and also possibly induce functional maturation of differentiated neurons, among which protoplasmic astrocytes have the ability to promote neuronal differentiation of NSCs compared with fibrous astrocytes.

High-dose glucocorticoids induce decreases calcium in hypothalamus neurons via plasma membrane Ca(2+) pumps.

In our previous studies, we occasionally found that high-dose glucocorticoids (GC) induced decrease in [Ca(2+)](i) in hypothalamus neurons. In previous articles, modulation of Ca(2+) channels by GC has been shown to contribute to the elementary regulation of several neuronal functions. However, little is known about the regulation of the Ca efflux pathways that counterbalance the Ca(2+) influx in neurons caused by high-dose GC. In this study, we demonstrate that a high-dose of GC (10 M dexamethasone) caused a 20% decrease in [Ca(2+)](i) within 2 s in cultured hypothalamic neurons; furthermore, we show that an antagonist of the GC receptor blocks this action. To ascertain the temporal sequence of relevant calcium transport mechanisms we selectively blocked the main calcium transporters, including sodium/calcium exchanger (NCX), plasma membrane calcium pumps (PMCA), and P-type Ca(2+)-ATPases of the sarcoplasmic reticulum (SERCA). The GC-induced [Ca(2+)](i) decrease disappeared completely when PMCA was blocked, but not when NCX and SERCA were blocked. These results suggest that high-dose GC (10(-6) M) rapidly decreases [Ca(2+)](i) by activating PMCA but not NCX or SERCA.

The use of a gold nanoparticle-based adjuvant to improve the therapeutic efficacy of hNgR-Fc protein immunization in spinal cord-injured rats.

As a common receptor for three myelin associated inhibitors, Nogo-66 receptor (NgR) mediates their inhibitory activities on neurite outgrowth in the adult mammalian central nervous system (CNS). Therapeutic vaccination protocol targeting NgR emulsified with Freund's adjuvant (FA) has been used in spinal cord injury (SCI) models. However, the vaccine emulsified with FA may induce some side effects, which are not suitable for further clinical application. As an adjuvant, gold nanoparticles (GNPs) could stimulate a stronger immune response without producing detectable toxicity and physiological damage than FA. There is, however, uncertainty regarding the efficacy of axon regeneration and neuroprotection in vaccines with GNPs as an adjuvant. In this investigation, a recombinant protein vaccine targeting NgR, human NgR-Fc (hNgR-Fc) fusion protein conjugated with 15 nm GNPs was prepared and its effects on axonal regeneration and functional recovery in spinal cord-injured rats were investigated. The results showed that adult rats immunized with the protein vaccine produced higher titers of anti-NgR antibody than that with FA, and the antisera promoted neurite outgrowth in presence of MAG in vitro. In a spinal cord dorsal hemisection model, vaccine immunized with GNPs promoted axonal regeneration more effectively than FA, resulted in significant protection from neuronal loss, and improved functional recovery. Thus, as an adjuvant, 15 nm GNPs can effectively boost the immunogenicity of hNgR-Fc protein vaccine, and promote the repair of spinal cord-injured rats. The utilization of GNPs, for clinical considerations, may be a more beneficial supplement than FA to the promising therapeutic vaccination strategy for promoting SCI repair.

[Detecting protein expression of EphrinB2 ligand and its receptor EphB4 in astrocytoma using confocal laser scanning microscopy].

BACKGROUND & OBJECTIVE: EphrinB2 is a novel angiogenic factor. EphrinB2 and its receptor EphB4 express in several kinds of tumor cells,and correlate with tumorigenesis and neoangiogenesis. This study was designed to explore the characteristics of EphB4 and EphrinB2 protein expression in astrocytomas. METHODS: Double labelling immunofluorescence was used to detect co-expression of EphB4/EphrinB2 with glial fibrilary acid protein (GFAP)or CD34 protein in 35 fresh glioma specimens, and 2 kinds of human glioma cell lines (CHG-5,and SHG-44). RESULTS: EphB4/EphrinB2 and CD34 proteins co-expressed in some tumor stromal microvessels, and mainly localizing in endothelial cells. Co-expression of EphB4/EphrinB2 and GFAP proteins was also noticed in tumor cells,and 2 glioma cell lines. In poorly-differentiated SHG-44 cells, the average fluorescence intensity of EphB4 was 72.48+/-33.78,and that of EphrinB2 was 96.80+/-36.98, both higher than those in well-differentiated CHG-5 cells (56.7+/-21.7, and 53.6+/-18.8). But the green fluorescence intensity of GFAP in SHG-44 cells was 22.3+/-15.3, while in CHG-5 cells was 47.5+/-16.7. CONCLUSION: Expressions of EphB4 and EphrinB2 proteins may be related to differentiation degree of tumor cells.