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Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.
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Humanos , Hipóxia Celular , Linhagem Celular Tumoral , Glioblastoma/patologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Microambiente Tumoral , Neoplasias Encefálicas/patologiaRESUMO
Objective To explore the changes of brain structure network in patients with Parkinson's disease with mild cognitive impairment(PD-MCI),and to provide novel markers for the early recognition of PD-MCI. Methods Total 47 patients with primary PD were continuously enrolled at the De-partment of Neurology,Affiliated Hospital of Xuzhou Medical University from May 2017 to May 2018. Twenty-four healthy volunteers were selected as the healthy control (HC) group. General demographic data were col-lected from all subjects. The overall cognitive function and the five cognitive domains (attention and working memory,executive function,language,memory and visual spatial function) were comprehensively evaluated, then the patients with PD were further divided into PD-MCI group(n=22) and PD-NC group(n=25) based on their cognitive evaluation results. All subjects completed the diffusion tensor imaging( DTI) scan and the individual structural brain connection was obtained by deterministic diffusion-tensor tractography. By graph theory analysis technology,the network properties (global properties and node properties) and edge-wise dis- tributions were compared to assess structural connectivity differences among the three groups. Results Com-pared with the PD-NC and HC groups,the PD-MCI group had a larger characteristic path length(PD-NC:(8. 33±0. 95),HC:(8. 18±1. 35),PD-MCI:(9. 20±1. 52),F=4. 14,P<0. 05) and reduced global efficien-cy (PD-NC:(0. 13±0. 05),HC:(0. 13±0. 04),PD-MCI:(0. 10±0. 04),F=3. 73,P<0. 05) in addition to a lower nodal efficiency in frontoparietal areas,thalamus,cingulate gyrus,and insular(P=0 . 003-0. 040,FDR correction). Compared with the HC group,the PD-MCI group had a large frontotemporoparietal areas,basal ganglia,and insular network with decreased connection strength (P<0. 05,FDR correction). Compared with the PD-NC group,the PD-MCI group had a lower network connection strength in the frontoparietal areas (P<0. 05,FDR correction). Conclusion A disruption of structural connections that make up the brain network can lead to changes in information integration and delivery,leading to cognitive dysfunction in patients with PD. The distribution pattern of brain network structure connection changes is expected to become a new marker for identifying PD-MCI.
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Objective To investigate the correlation between mild cognitive impairment (PD-MCI) and serum homocysteine (Hcy) in patients with Parkinson's disease.Methods 85 patients with PD (43 with PD-MCI,42 with PDN) as the observation group and 43 healthy elderly patients as the control group.The demographic information,clinical and neuropsychological assessments were conducted and the Hcy of fasting venous blood were detected in all subjects.The I-Icy level and its associated factors were also analyzed in the PD group.Results Serum levels of Hcy in the PD patients((13.53±2.29) μmol/L) were higher than those in the HC group((11.77±1.56) μmol/L),the difference was statistically significant (t=4.57,P ＜0.05).There was significant difference in serum Hcy levels among HC group,PDN group ((12.85 ± 1.88) μmoL/L) and PD-MCI group((14.22±2.47) ±mol/L) (F=16.13,P＜0.01).Hcy level of the PDN group was higher than that of the HC group (P＜0.05),and PD-MCI group was higher than PDN group (P＜0.05).Serum levels of Hcy in patients with PD-MCI were significantly associated with UPDRS-Ⅲ (r=0.305),H-Y (r=0.313),LEDD (r=0.424),MMSE (r =-0.470),MoCA (r=-0.503),duration of connection test B (r =0.617) and semantic fluency tests (r=0.557) (all P＜0.05).Conclusion Elevated serum Hcy levels are expected to be biomarkers for predicting PD-MCI.
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Objective To investigate expression of CXCR4 and CXCR7 protein and mRNA, which are the receptors of stromal cell derived factor-1α(SDF-1α), in the bone marrow mesenchymal stem cells (BMSCs);to explore the role of SDF-1α/CXCR4/CXCR7 axis in migration of BMSCs in vitro and the possible mechanism .Method BMSCs were isolated from rats and cultured in vitro.CD29, CD44 and CD34 of the cells were identified by flow cytometry .CXCR4-selective antagonist AMD 3100 and CXCR7-specific neutralizing antibody were applied to block CXCR 4 and CXCR7 respectively.The expressions of CXCR4 and CXCR7 mRNA and protein on BMSCs were detected with RT-PCR and Western blotting .Transwells chamber test was used to observe the migration of BMSCs .The BMSCs were divided into the BMSCs group ( A ) , the AMD3100 pretreated BMSCs group ( B ) , the CXCR7-specific neutralizing antibody pretreated BMSCs group(C), the AMD3100 +CXCR7-specific neutralizing antibody pretreated BMSCs group ( D).Result Flow cytometry showed that the expressions of CD 44 and CD29 were positive, while the expression of CD34 was negative in the third passage of BMSCs (P3-BMSCs).CXCR4 and CXCR7 protein and mRNA were both expressed in P3-BMSCs. Compared with the A group, the expression of CXCR4 and CXCR7 protein declined significantly in the B group and the D group;the protein expression of CXCR7 in the C group was lower compared with the A group (P<0.05).However, the expression of CXCR4 mRNA and CXCR7 mRNA had no significant difference between groups .SDF-1αfactor promoted migration of BMSCs ( P <0.05 ).Compared with the 0μg/L group, the numbers of migrated cells were increased significantly in both of the 10μg/L group and the 100μg/L group ( P<0.01 ) .The number of migration of BMSCs was significantly higher in the 100μg/L group than that of the 10μg/L group ( P <0.01 ) .AMD3100 and CXCR7-specific neutralizing antibody both inhibited significantly the migration of BMSCs ( P<0.05 ) , and the attenuate effect was more significant when they worked together ( P<0.05 ) .Conclusion CXCR4 and CXCR7 receptors are co-expressed in P3-BMSCs;the SDF-1αfactor can promote the migration of BMSCs in the concentration dependent manner ;SDF-1α/CXCR4/CXCR7 axis is involved in the migration of BMSCs , and both of the CXCR4 and CXCR7 receptors have a synergistic promoting effect to the BMSCs migration .
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In order to investigate the chemical and morphornetrical properties of the neuronal precursor cells derived from thesubventricular zone(SVZ) of the postnatal rat forebrain in vitro. The cell-type specific antibodies were used for the immunocy-tochemical staining ,and the morphometric parameters which were the mean soma diameter and the ellipticity index (i. e. , thesmallest soma diameter divided by the largest soma diameter) of every SVZ-derived cell were measured for identifying the pheno-types of the SVZ cells in vitro. The experiment animals were SD rats (weights: 100～ 150 g), the SVZ cells derived from thepostnatal rats were cultured on poly-D-lysine-coated 24-well glass chamber slides in the Neurobasal Medium supplemented withB27 in 5% CO2 at 37 C. The following results were obtained.. At 1 day in vitro, almost all SVZ cells (〉90%) from the postna-tal rat forebrain expressed Tujl, an antibody that recognizes neuron-specific tubulin. Likewise, the preponderance of the SVZcells expressed the polysialylated neural cell adhesion molecule (PSA-N-CAM) ; The majority of the SVZ Tujl-positive cells cul-tured were the cells that had oval-shaped bodies with two short, unbranched processes protruded from every two poles, theirmean soma diameter were 8.42±1.03μm and their ellipticity index were 0.57±0.12. Meanwhile, there were approximately20% of the SVZ cells in culture that were sphere-shaped cells with mean soma diameter 7.20±l.04 μm , and it might be observed that these cells connected with one another. As the time in culture went on, these sphere-shaped SVZ-derived cells alsotransformed to oval-shaped ones as described above, but it could be observed that the cells were still connected in the processesof them. By 3 and 5 days in culture, the SVZ cells had larger cell somas (average diameter 9. 07±1.07 μm), and often consider-ably longer processes but still with few branches. Immunocytochemical staining revealed that the majority of the SVZ cells in cul-ture remained Tujl-positive, PSA-N-CAM-positive. By 7 days in culture, the Tujl-positive cells in culture showed remarkablemorphological changes, and possessed typical neuronal phenotypes, which had more larger cell somas (average diameter 12.8 ±1.13 μm), and had more longer, branched processes. Our results showed that the SVZ in the postnatal SD rats contained theneuronal precursor cells which were PSA-N-CAM-positive and could differentiate into new neurons in vitro.
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Objective To explore the effect of glial cell line-derived neurotrophic factor(GDNF) on the expressing of calbindin D28K(CB) in substantia nigra(SN) of rat model of Parkinson's disease(PD) and the possible role of neural cell adhesion molecule(NCAM) in this course.Methods 36 rat models of PD were made,and divided into GDNF group,NCAM blocked group and control group(each group had 12 rats).Immunohistochemistry and Western blot method were used to detect the number of CB-positive cells and the expressed level of CB protein in SN of rats.Results The number of CB-positive cells(46.50?6.28) and the expressed level of CB protein(33770.60?6929.76) in SN of GDNF group were significantly higher than control group [(27.00?8.60),(18281.00?5266.78) respectively](all P0.05).Conclusions GDNF may protect the injured dopaminergic neurons through up regulating the expressing of CB,but NCAM is not involved in this mechanism.
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Objective To observe the proliferation and differentiation of the neural progenitors in the midbrain substantia nigra(SN) of adult rat when dopaminergic neurons(DA neurons) were destroyed. Methods The Parkinson disease model(PD model) was induced in adult SD rats by steriotaxic injection of 6-OHDA into the right striatum,and then assessed by behavioral analysis to screen the qualified models.After varied survival period,the brain of model rats were perfused and fixed by 4% paraformaldehyde,moved the segment of SN,embedded with paraffin and coronally sectioned continuously.The microsections were processed by immunohistochemistry labeling separately the neural progenitors with anti-nestin monoclonal antibody,the dividing cells with anti-PCNA(proliferation cell nuclear antigen),the neuronal precursors with anti-Tuj1(?-tubulin isotype Ⅲ),and DA neurons with anti-TH(tyrosine hydroxylase).The labeled cells were counted under microscope and analyzed statistically.(Results It) was found in the right SN of PD model rats that: 1.Nestin positive(Nestin~+) cells appeared 10d after 6-OHDA injection,became abundant on 14d,declined in number on 17d,and disappeared on 21d.2.Weakly positive PCNA(PCNA~+) cells appeared on 7d.PCNA~+ cells were abundant on 14d,decreased in number from 21d,with only a few positive cells noticed on 28d.3.Tuj1 positive cells appeared in small number on 10d,became abundant on 14d,decreased in number from 17d and dropped nearly to zero on 21d.4.The number of TH positive neurons was significantly less than the normal control(by 24%) on 7d,and became even less as time elapsed.Conclusion When 6-OHDA is injected into the striatum of adult rats to cause degeneration and death of the DA neurons there,there would be a certain period of time in which a number of neural progenitors will be induced to proliferate actively and differentiate toward neuronal cells(except DA neurons).
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Objective To explore the possible roles of PI3K pathway and MAPK pathway in mediating the survival and differentiation-promoting effect of glial cell line-derived neurotrophic factor(GDNF) on midbrain dopaminergic neurons.(Methods Midbrain) slices of early postnatal rats were cultured,the slices were divided into four groups according to the different substances added into the culture medium: i.e.blank control group,GDNF group,Wortmanin plus GDNF group,and PD98059 plus GDNF group.On the 6th day,some slices were fixed,embedded and sectioned.The sections were processed for tyrosine hydroxylase(TH) immunohistochemistry,and then examined under microscope to determine the morphologic index for statistic analysis(n=6).Meanwhile,some slices were taken for Western blot to examine the expression of TH protein in midbrain slices(n=4). Results GDNF group slices showed the morphologic maturity,the density and the diameter of TH positive neurons,as well as the level of TH expression,were all significantly higher than that of the control groups.The survival effect of GDNF on DA neurons was almost abolished when the PI3K pathway was blocked with Wortmannin;while the MAP kinase and ERK kinase(MEK) inhibitor PD 98059 was added to block the MAPK pathway,the diameter of TH positive neurons was decreased significantly.Conclusion The PI3K pathway might mediate the survival effect of GDNF,while the MAPK pathway seems to be involved in the differentiation process.