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Objective:To clarify the neuroprotective effects of neural cell adhesion molecule (NCAM) derived peptide P2 on in vitro cultured neuron and ischemic stroke rat. Methods:Primary cortical neurons were extracted and cultured, and CCK-8 method was used to observe the protective effect of different concentrations of P2 on cortical neurons under oxygen-glucose deprivation (OGD) conditions.The levels of apoptosis-related proteins and extracellular signal regulated kinase 1/2 (Erk1/2) were observed by Western blot. Clean grade male SD rats were selected for animal experiments. The middle cerebral artery occlusion (MCAO) method was used to establish the rat model of cerebral ischemia/reperfusion injury. The rats with successful model were divided into sham operation group, MCAO group and MCAO+ P2 group according to the random number table, with 12 rats in each group. After operation, rats in MCAO+ P2 group were subcutaneously injected with 1 mg/kg P2 once a day until 14 days after operation, and rats in the other two groups were subcutaneously injected with 0.9% sodium chloride solution of the same volume.Beam-walking test was used to evaluate the motor function of rats.Immunofluorescence staining and Western blot were used to detect the in-situ apoptosis of neuronal cells and the expression of Erk1/2 in ischemic penumbra of rat brains, respectively. All statistical analyses were performed using SPSS 22.0.Repeated measurement ANOVA was used to evaluate the beam-walking experimental data, and one-way ANOVA were used to analyze other experimental data among multiple groups.Results:Compared with OGD group, 0.5, 1.0 and 2.0 μmol/L P2 improved the activity of neurons under OGD conditions, of which 1 μmol/L P2 had the best effect ((2.436±0.284), (1.551±0.410), P<0.05). Western blot showed that the protein levels of bax ((76.120±3.232)%, (88.965±5.208)%, P<0.05), cleaved caspase-3 ((76.736±4.306)%, (97.781±8.111)%, P<0.05) and cleaved caspase-9 ((88.833±6.581)%, (104.962±4.788)%, P<0.05) in 1 μmol/L P2 treated group were all lower than those in OGD group, while the protein levels of bcl-2 ((56.146±3.882)%, (43.170±6.945)%, P<0.05) and phosphorylated Erk1/2 ((73.583±8.557)%, (55. 219±4.615)%, P<0.05) in 1 μmol/L P2 treated group were both higher than those in OGD group. Compared with MCAO group, on the 14th day after P2 intervention, the slip ratio of hindlimb of the paralyzed hind limbs of rats was lower ((23.438±11.540)%, (41.733±13.631)%, P<0.05), the apoptosis rate of neurons around the focus was lower ((13.144±6.485)%, (26. 699±6. 402)%, P<0.05), and the level of phosphorylated Erk1/2 protein in the brain tissues around the infarct focus was higher ((74.062±7.458)%, (53.327±7.093)%, P<0.05). Conclusion:Low doses of neural cell adhesion molecule derived peptide P2 exert neuroprotective effects on OGD neurons and ischemic stroke rats. The underlying mechanism may be related to the activation of Erk.
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Objective To observe the effect of epigallocatechin gallate (EGCG) on the spatial learning memory deficit in amyloid procursor protein (APP) / presenilin-1 (PS1) double transgenic mice, synaptic ultrastructure and expression of neural cell adhesion molecule in hippocampal CA1 region. Methods Eight weeks old male APP / PS1 double transgenic mice were selected as Alzheimer’s disease (AD) model and divided into the model group, the EGCG group and the donepezil hydrochloride group, 12 in each group.Besides,normal mice of the same brood (with no transgene) were recruited as a normal group (n = 12). Related indices were detected after 6 months continuous gastrogavage. The spatial learning-memory deficit of APP / PS1 double transgenic mice was detected by Morris water maze test. The synaptic ultrastructure of hippocampal CA1 region was observed by transmission electron microscopy. The expression levels of neural cell adhesion molecule (NCAM) and polysialyltranseferase α2,8-polysialic acid (ST8Sia Ⅱ) protein in hippocampal CA1 region of APP / PS1 transgenic mice were detected by immunofluorescence and Western blotting. Results Compared with the normal group, the mean value of escape latency in the model group was extended, and compared with the model group, the mean value of escape latency in the EGCG group and donepezil hydrochloride group were increased (P < 0. 05) . The result of electron microscope showed that the changes of synaptic interface curvature of EGCG group and donepezil hydrochloride group were not obvious. Compared with the model group, the width of the synaptic gap becomes narrower and the thickness of the post-synaptic compact were increases (P < 0. 05) . Immunofluorescence showed that the expression of NCAM and ST8Sia Ⅱ proteins in the hippocampus CA1 region was expressed in the cytoplasm of neurons, the expressions of NCAM and ST8Sia Ⅱ in hippocampal CA1 region were significantly increased in EGCG group and donepezil hydrochloride group (P< 0. 05) . Their contents also showed higher levels of expression in Western blotting (P < 0. 05) . Conclusion EGCG shows improvement on the spatial learning-memory deficit in APP / PS1 double transgenic mice,which may be associated with affecting the synaptic structure of hippocampus and improving the expressions of neural cell adhesion molecule.
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Objective To understand the distribution of nerve fibers and the types of neural cells in Aspidogaster conchiola. Methods Whole worms were subjected to silver staining, histochemical staining and hematoxylin-eosin (HE) staining, and the nervous systems of the worms were observed. Results There were 3 types of neural cells in the worm head near the cerebral ganglion, including unipolar, bipolar and multipolar neurons, which were divided into 7 types according to the morphology. There was a nerve network on the surface of pharynx and intestinal tract, as well as the reproductive organ, including testis, ovary, lower uterus and penis sac. The nerve network was consisted of circular and longitudinal nerve fibers, and the structure of the nerve network around the mouth was similar to central nerve. Conclusions The structure of the A. conchiola central nervous system is very complicated, and the neural networks may be associated with the physiologic activity of the worm. Different neural cells may have diverse functions.
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Pituitary adenomas account for 10-15% of primary intracranial tumors. Growth hormone (GH)-secreting adenomas account for 13% of all pituitary adenomas and cause acromegaly. These tumors can be aggressive, invade surrounding structures and are highly recurrent. The objective of this study was to evaluate E-cadherin, Slug and neural cell adhesion molecule (NCAM) expression in GH-secreting pituitary adenomas and its relationship to tumor invasiveness. A cross-sectional study of patients who underwent hypophysectomy due to GH-secreting pituitary adenoma from April 2007 to December 2014 was carried out. The medical records were reviewed to collect clinical data. Immediately after surgery, tumor samples were frozen in liquid nitrogen and stored in a biofreezer at -80°C for assessment of E-cadherin 1 (CDH1), SLUG (SNAI2), and NCAM (NCAM1) by real-time PCR. The samples were fixed in formalin and embedded in paraffin for immunohistochemical analysis of E-cadherin and NCAM. Thirty-five patients with acromegaly were included in the study. Of these, 65.7% had invasive tumors. Immunohistochemically, E-cadherin was expressed in 96.7% of patients, and NCAM in 80% of patients. There was no statistically significant relationship between tumor grade or invasiveness and immunohistochemical expression of these markers. Regarding gene expression, 50% of cases expressed CDH1, none expressed SNAI2, and 53.3% expressed NCAM1. There was no statistically significant relationship between tumor grade or invasiveness and gene expression of CDH1, SNAI2, and NCAM1. The absence of Slug overexpression and of E-cadherin and NCAM suppression suggests that expression of these markers is not associated with tumor invasiveness in GH-secreting pituitary adenomas.
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Humans , Male , Female , Adolescent , Adult , Middle Aged , Aged , Young Adult , Acromegaly/pathology , Adenoma/pathology , Cadherins/analysis , Neural Cell Adhesion Molecules/analysis , Snail Family Transcription Factors/analysis , Acromegaly/genetics , Acromegaly/metabolism , Immunohistochemistry , Biomarkers, Tumor/analysis , Adenoma/genetics , Adenoma/chemistry , Gene Expression , Cross-Sectional Studies , Neoplasm GradingABSTRACT
Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.
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Objective To investigate the role of cell adhesion molecule L1 like (CALL) in the genesis and development of esophageal squamous cell carcinoma (ESCC).Methods From July 2007 to December 2010,a total of 100 patients with ESCC who received radical resection of esophageal cancer were enrolled.The ESCC tissues and corresponding tumor-adjacent normal tissues were obtained.The expression of CALl was determined by tissue microarray technology and immunohistochemical staining.The CALL over-expressed esophageal cancer cell line was established.The effects of CALL on cell migration and invasion were detected by wound-healing assay and Transwell assay,respectively.The effects of CALL on actin microfilament was analyzed by filamentous actin (F-actin) staining.Chi square test,Fisher's exact test,multivariate analysis and t test were performed for statistical analysis.Results The positive expression rate of CALL in ESCC tissues was 56 ％ (56/100),which was lower than that of tumor-adjacent normal tissues (95％,95/100),and the difference was statistically significant (x2=41.114,P＜0.01).There were statistically significant differences in CALL expression at protein level among patients with ESCC of different differentiation degree,different pathological T stage,lymph node metastasis and different TNM stage (x2=13.702,5.317,21.453,Fisher's exact test;all P＜ 0.05).The five year disease related survival rate of ESCC patients with down-regulated expression of CALL was 0(0/49),which was lower than those with normal CALL expression (25.5％,13/51),and the difference was statistically significant (x2 =43.338,P＜0.01).The median survival time of CALL expression down-regulated group was 17 months,and that of normal expressed group was 38 months.CALL expression was an independent risk factor of disease special survival rate (hazard ratio (HR) 0.353,95％ confidence interval (CI) 0.188 to 0.666,P=0.001).The results of wound-healing assay showed that the migration ability of CALL overexpressed CALL-k30 cells was lower than that of Vec-k30 cells in control group on 24 hours after wound.The results of Transwell invasion test showed the number of migrating cells penetrating CALL k30 cells attached to the inferior surface of the membrane was 44.000±13.748,which was less than that of the Vec k30 cells (154.333±25.007),and the difference was statistically significant (t=5.136,P=0.036).The results of F-actin staining demonstrated that actin filaments of CALL-k30 cells was 234.667 ± 65.118,which was lower than that of Vec-k30 cells (597.000± 119.929),and the difference was statistically significant (t=4.707,P=0.042).Conclusions CALL lowers the migration and invasion abilities of esophageal cancer cells by inhibiting F-actin microfilaments.Its abnormal expression may play an important role in the genesis,development and prognosis of ESCC.
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Men with diabetic erectile dysfunction (ED) respond poorly to the currently available oral phosphodiesterase-5 inhibitors. Therefore, functional therapies for diabetic ED are needed. Stromal vascular fraction (SVF) and the adenovirus-mediated cartilage oligomeric matrix angiopoietin-1 (Ad-COMP-Ang1) gene are known to play critical roles in penile erection. We previously reported that SVF and Ad-COMP-Ang1 have only a short-term effect in restoring erectile function. Further improvements to ED therapy are needed for long-lasting effects. In the present study, we aimed to test if the combination of SVF and Ad-COMP-Ang1 could extend the erection effect in diabetic ED. We found that the combination therapy showed a long-term effect in restoring erectile function through enhanced penile endothelial and neural cell regeneration. Combination therapy with SVF and Ad-COMP-Ang1 notably restored cavernous endothelial cell numbers, pericyte numbers, endothelial cell-cell junctions, decreased cavernous endothelial cell permeability, and promoted neural regeneration for at least 4 weeks in diabetic mice. In summary, this is an initial description of the long-term effect of combination therapy with SVF and Ad-COMP-Ang1 in restoring erectile function through a dual effect on endothelial and neural cell regeneration. Such combination therapy may have therapeutic potential for the treatment of diabetic ED.
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Animals , Male , Mice , Angiopoietin-1/genetics , Diabetes Mellitus, Experimental/metabolism , Endothelium, Vascular/metabolism , Erectile Dysfunction/therapy , Genetic Therapy/methods , Intercellular Junctions/metabolism , Mesenchymal Stem Cell Transplantation , Penile Erection/physiology , PermeabilityABSTRACT
OBJECTIVE: Repair of sensorial nerve defect is an important issue on peripheric nerve surgery. The aim of the present study was to determine the effects of sensory-motor nerve bridging on the denervated dermatomal area, in rats with sensory nerve defects, using a neural cell adhesion molecule (NCAM). METHODS: We compared the efficacy of end-to-side (ETS) coaptation of the tibial nerve for sural nerve defect repair, in 32 Sprague-Dawley rats. Rats were assigned to 1 of 4 groups: group A was the sham operated group, group B rats had sural nerves sectioned and buried in neighboring muscles, group C experienced nerve sectioning and end-to-end (ETE) anastomosis, and group D had sural nerves sectioned and ETS anastomosis was performed using atibial nerve bridge. Neurological evaluation included the skin pinch test and histological evaluation was performed by assessing NCAM expression in nerve terminals. RESULTS: Rats in the denervated group yielded negative results for the skin pinch tests, while animals in the surgical intervention groups (group C and D) demonstrated positive results. As predicted, there were no positively stained skin specimens in the denervated group (group B); however, the surgery groups demonstrated significant staining. NCAM expression was also significantly higher in the surgery groups. However, the mean NCAM values were not significantly different between group C and group D. CONCLUSION: Previous research indicates that ETE nerve repair is the gold standard for peripheral nerve defect repair. However, ETS repair is an effective alternative method in cases of sensorial nerve defect when ETE repair is not possible.
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Animals , Rats , Methods , Muscles , Neural Cell Adhesion Molecules , Peripheral Nerves , Rats, Sprague-Dawley , Skin , Sural Nerve , Tibial NerveABSTRACT
Objective To explore the effect of neural cell adhesion molecule (NCAM) on adhesion,migration and morphology of mouse bone marrow-derived mesenchymal stem cells (BMSCs).Methods We isolated and cultured BMSCs from wild-type and NCAM gene knockout mice.The expression of NCAM was detected by Western blot and immunofluorescence.Wound healing and adhesion assays were used to detect cell migration and adhesion ability respectively.The morphological changes were observed and the expressings of protein β1 integrin,E-cadherin,β-catenin and N-cadherin were analysed by Western blot.Results The migration and adhesion of BMSCs were significantly reduced after NCAM gene knockout.Meanwhile,the expression of β1 integrin was lower than those in wild-type BMSCs (P<0.01).The morphology of NCAM gene knockout BMSCs changed from irregular to flattened,and expressed epithelial identification marker E-cadherin and β-catenin (P<0.05).However,the expression level of mesenchymal identification marker N-cadherin was decreased (P<0.01).Conclusions NCAM is involved in adhesion and migration of BMSCs via regulating the expression of β1 integrin.Furthermore,NCAMmay negatively regulate the mesenchymal-epithelial transitions of BMSCs.
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Background:Dysregulation of microRNAs is associated with intestinal mucosal barrier injury,intestinal inflammation and intestinal dysfunction. Abnormal expression of microRNAs occurs in patients with inflammatory bowel disease(IBD). Aims:To investigate the expression and significance of microRNA-595( miR-595)in IBD. Methods:A total of 100 patients with IBD at Nanjing General Hospital of Nanjing Military Command of PLA from July 2012 to July 2014 were enrolled,in which 63 cases were ulcerative colitis(UC)and 37 cases were Crohn’s disease(CD). According to disease activity,patients were divided into active UC(aUC)group,remissive UC(rUC)group,active CD(aCD)group and remissive CD(rCD)group. A total of 42 healthy subjects were served as normal control(NC)group. Specimens of serum and intestinal tissue were collected. Expression of miR-595 in serum and intestinal tissue was determined by fluorescence quantitative PCR. Luciferase report gene plasmid containing the 3’UTR of neural cell adhesion molecule 1(NCAM1)or fibroblast growth factor receptor 2(FGFR2)and plasmid containing miR-595 were co-transfected into human colon cancer cell line HCT116 to detect the effect of miR-595 on transcriptional activities of NCAM1 and FGFR2. Results:Expression of miR-595 in serum and intestinal tissue in UC and CD groups was significantly higher than that in NC group(P < 0. 05), and that in aUC and aCD groups was significantly higher than that in rUC and rCD groups,respectively(P < 0. 05). MiR-595 could down-regulate the transcriptional activities of NCAM1 and FGFR2 through directly binding to the 3’UTR of NCAM1 and FGFR2. Conclusions:Expression of miR-595 in serum and intestinal tissue is increased in patients with IBD and correlates with disease activity. MiR-595 inhibits the expressions of tight junction protein NCAM1 and FGFR2,thereby inducing injury of intestinal mucosal barrier and promoting intestinal inflammation. MiR-595 can serve as a serum biomarker for diagnosis of IBD and disease activity evaluation.
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Objective:To study the spinal cord injury, spinal cord transection and persistent placeholder damage on the influence of secondary neural cell apoptosis in rats.Methods: Select 60 healthy male Wistar rats, numbered after using the random number table method is divided into A (18,spinal cord contusion),B (18,spinal cord transection),C (18,continuous placeholder),D (6,control),E (6,the control group only) groups of five,were observed at the 1,4,7 D after 5 group of rats nerve cell apoptosis index, spinal cord tissue Bcl-2,the expression of Bax,caspase 3 protein.Results:A,B,C three groups of rats after building 1 d are gray and white matter positive markers, and the gray matter and white matter of three groups of rats nerve cell apoptosis index differences statistically significant ( P<0.05);4 d,7 d after building gray matter and white matter of three groups of rats tend to place increased ap-optotic cells in the spinal cord index ( P<0.05);in building 1,4,7 d group C after rat spinal cord grey matter and white matter of apoptotic cell index was significantly higher than that of group A and group B, group B were significantly higher in group A and the differences were statistically significant (P<0.05).1,4,7 d after building A,B,C,D,E five group rats the Bcl-2,Bax,caspase-3 protein expression differences were statistically significant (P<0.05),1,4,7 d after building A,B,C the Bcl-2 of three groups of rats, Bax,caspase-3 protein expression was significantly higher than that of group D and group E ( P<0.05).Conclusion: Secondary rats after spinal cord injury of nerve cells apoptosis,apoptosis time,severity,and damage type and severity.
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Objective To investigate the clinical significance and prognostic value of PKD3 expression in human hepatocellu-lar carcinoma (HCC)after hepatectomy.Methods We analyzed mRNA expression of L1CAM in 1 10 HCCs by quantitative real-time PCR (qRT-PCR)and western blot,and the relationship among the overall survival of HCCs.Results The relative protein and mRNA expression level of L1CAM was up-regulated in HCCs comparing with adjacent non tumor liver tissues (P <0.01).L1CAM expression in the well-differentiated group was higher than that in the poor-differentiated group (P < 0.01 ).The expression of L1CAM mRNA was significantly correlated with tumor differentiation and TNM stage (P <0.05).The prognosis of patients with high expression L1CAM was poor (P <0.01).Conclusion L1CAM expression is related to occurrence and development of HCCs and may predict the prognosis of HCCs after hepatectomy.
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Objective To evaluate the influence of sevoflurane anesthesia on the expression of growth-associated protein 43 (GAP-43) and neural cell adhesion molecule (NCAM) in hippocampal neurons of neonatal rats.Methods Thirty-six pathogen-free Sprague-Dawley rats,aged 7 weeks,weighing 15-20 g,were randomly divided into 4 groups (n =9 each) using a random number table:control group (C group),1.5％ sevoflurane 6 h group (L group),3％ sevoflurane 2 h group (H1 group) and 3％ sevoflurane 6 h group (H2 group).Group L inhaled 1.5％ sevoflurane in oxygen for 6 h.H1 and H2 groups inhaled 3％ sevoflurane in oxygen for 2 and 6 h,respectively.Group C inhaled 30％ oxygcn only.When the neonatal rats were 14 days old,the rats underwent Morris water maze test for 7 consecutive days.Place navigation and spatial probe tests were carried out.After the end of Morris water maze test,the rats were sacrificed,and the hippocampus was obtained for determination of the expression of GAP-43 and NCAM in hippocampal neurons.Results Compared with group C,the escape latency was significantly prolonged,the time of staying at the original platform quadrant was shortened,and the expression of GAP-43 was down-regulated in L,H1 and H2 groups,and the frequency of crossing the original platform was decreased in L and H2 groups.There was no significant difference in NCAM expression among the four groups.Conclusion The mechanism by which sevoflurane anesthesia decreases the cognitive function may be related to down-regulated expression of GAP-43,but not related to NCAM expression in hippocampal neurons of neonatal rats.
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This study was performed to assess the neurotoxic effects of methylmercury, arsanilic acid and danofloxacin by quantification of neural-specific proteins in vitro. Quantitation of the protein markers during 14 days of differentiation indicated that the mouse ESCs were completely differentiated into neural cells by Day 8. The cells were treated with non-cytotoxic concentrations of three chemicals during differentiation. Low levels of exposure to methylmercury decreased the expression of GABAA-R and Nestin during the differentiating stage, and Nestin during the differentiated stage. In contrast, GFAP, Tuj1, and MAP2 expression was affected only by relatively high doses during both stages. Arsanilic acid affected the levels of GABA(A)-R and GFAP during the differentiated stage while the changes of Nestin and Tuj1 were greater during the differentiating stage. For the neural markers (except Nestin) expressed during both stages, danofloxacin affected protein levels at lower concentrations in the differentiated stage than the differentiating stage. Acetylcholinesterase activity was inhibited by relatively low concentrations of methylmercury and arsanilic acid during the differentiating stage while this activity was inhibited only by more than 40 microM of danofloxacin in the differentiated stage. Our results provide useful information about the different toxicities of chemicals and the impact on neural development.
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Animals , Mice , Acetylcholinesterase/metabolism , Arsanilic Acid/toxicity , Cell Differentiation/drug effects , Embryonic Stem Cells/cytology , Environmental Pollutants/toxicity , Fluorescent Antibody Technique , Fluoroquinolones/toxicity , Gene Expression Regulation/drug effects , Methylmercury Compounds/toxicity , Nerve Tissue Proteins/metabolism , Neurons/cytology , Tetrazolium Salts/metabolism , Thiazoles/metabolismABSTRACT
Objective: To investigate the effects of miR(microRNA)-200a on migration and invasion abilities of glioma cells, and to explore its possible mechanism. Methods: Differential expression levels of miR-200a in glioma U87 cells were achieved by transfecting with hsa-miR-200a mimic, hsa-miR-200a inhibitor or hsa-miR-negative control by Lipofectamine™ 2000. The migration and invasion abilities of U87 cells were detected by wound-healing assay and Transwell invasion assay, respectively. Bioinformatics software was used to predict downstream target genes of miR-200a and their binding sites. The potential target genes were verified by Luciferase Reporter Assay and Western blotting. Results: Exogenous overexpression of miR-200a could promote migration and invasion abilities of U87 cells (P < 0.05), while miR-200a inhibitors could generate the opposite results (P < 0.05). Luciferase Reporter Assay and Western blotting revealed that hsa-miR-200a negatively regulated the protein expression of NCAM1 (neural cell adhesion molecule 1) gene which was regarded as the target gene. Conclusion: The miR-200a can promote the migration and invasion abilities of glioma U87 cells, in which NCAM1 may be one of the target genes. Copyright © 2013 by TUMOR.
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Objective To identify the features of the NCAM+ c-Kit+ subset of hepatic progenitor cells in the intrahepatic cholangiocarcinoma (ICC) cell line RBE.Method Magnetic activated cell sorting was used to isolate NCAM+ c-Kit+/NCAM-c-Kit-subset cells,which were tested for hepatic progenitor cell properties and proliferation,colony formation,and invasive abilities in nude mice.Resuits The cell proliferation ability of NCAM+c-Kit+ subset cells was stronger than that of NCAMc-Kit-subset cells (P＜0.01).In serum-free condition,the number of colonies formed by NCAM+c-Kit+ subset cells was more than that of NCAM-c-Kit-cells (P＜0.01).1 × 104 NCAM+c-Kit+ cells were enough to form tumors in nude mice after subcutaneous inoculation for two weeks,while 1 × 106 NCAM-c-Kit-cells were necessary to form tumors for three weeks.The tumor formation rate of NCAM+c-Kit+ cells was higher than that of NCAM-c-Kit-cells (P=0.04).Conclusions It is possible that NCAM+c-Kit+ subset cells in RBE have the properties of hepatic progenitor cells,and NCAM combined with c-Kit might be a valuable marker for isolating and purifying ICC stem/progenitor cells.
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Objective To investigate the impact of polyclonal neural cell adhesion molecule antibody (P-NCAM-Ab) on the potency of botulinum toxin A (BTX-A).Methods Ninety male Sprague-Dawley rats were randomly divided into 3 equal groups:a normal control group,a BTX-A group and a P-NCAM-Ab group.The rats in the normal control group were injected with 100 μl of saline solution in their right gastrocnemius,while those in the BTX-A and P-NCAM-Ab groups were injected with 100 μl of BTX-A (0.5 U).In addition,the rats in the P-NCAM-Ab group were also injected with 100 μl of P-NCAM-Ab (the dosage was 20 U) at the same site on the 3rd day after the BTX-A injection.The rats' gastrocnemius muscle strength was evaluated with a self-made system for evaluating neuromuscular function before and after the toxin injection,on the 3rd day,as well as 1,2,4,6,8,10 and 12 weeks after the BTX-A injection.Any wet weight changes in the muscles were observed,and immunochemistry methods were employed to observe any structural changes in the motor endplates and nerve fibers at the different time points.Results After the saline injection,the average gastrocnemius muscle strength of the control group increased with time,while strength in the BTX-A and P-NCAM-Ab groups demonstrated a decrease in strength followed by a gradual increase.The average gastrocnemius muscle strength of the rats in the BTX-A and P-NCAM-Ab groups was significantly lower than that of the control group at all time points.Compared with the BTX-A group,the muscle strength of the P-NCAM-Ab group rats decreased further.Strength recovery in the BTX-A and P-NCAM-Ab groups was significantly slower than in the control group.The wet weight percentage in the BTX-A and P-NCAM-Ab groups at first decreased and then recovered with time.After the BTX-A injection,the average wet weight percentage of the P-NCAM-Ab group rats was significantly lower than that of the BTX-A group after 3 days,and 1,2 and 4 weeks.Karnovsky-Roots AchE staining showed that the motor endplates' color in the BTX-A and P-NCAM-Ab groups deepened gradually,though the color of the P-NCAM-Ab group was lighter than that of the BTX-A group at each time point.The mean optical density of the motor endplates' positive reaction area increased with time in both groups,but the P-NCAM-Ab group was lower than that of the BTX-A group at 1,2,4,8 and 12 weeks.Counting the nerve fibers dyed by gold chloride showed similar trends with both experimental groups significantly different from the control group.Conclusion P-NCAM-Ab can increase the potency of BTX-A and prolong its action.
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Background Optic neuropathy is one of the diabetic eye complications.Rosiglitazone,a peroxisome proliferator activated receptor γ(PPARγ) agonist,plays a very important role in arresting the pathogenesis and development of diabetes.However,the role of PPARγ in diabetic optic neuropathy is unclear.Objective This study was to investigate the protective effect of rosiglitazone against diabetic optic neuropathy and its mechanism.Methods Male Sprague-Dawley rats were randomly divided into the control group,diabetic group and rosiglitazone group,with 10 rats for each group.Diabetic models were induced by injecting 50 mg/kg of streptozotocin via the caudal vein,and rosiglitazone(5 ng/[kg· d])was used in the rats of the rosiglitazone group by intragastric administration every day for four weeks.At the end of the experiment,the fasting blood sugar(FBS) was tested in all the animals.The level of vascular endothelial growth factor(VEGF) in the blood plasma was detected by ELISA.Optical neural tissues were obtained from the rats of each group,and Lauck fast Blue myelin stain was used to examine the morphology of the optical myelin.The expression of neural cell adhesion molecule (NCAM) mRNA and protein in the optic nerve was detected by real time PCR and Western blot,respectively.Results The levels of FBS,blood plasma VEGF,NCAM mRNA and protein in the optic nerve were significantly different among the control group,diabetic model group and the rosiglitazone group after the administration of 5 nmg/(kg · d) rosiglitazone for 4 weeks (F =6.12,P＜0.01 ; F =5.14,P＜0.05 ; F =4.75,P＜0.05 ; F =4.87,P＜0.05).Compared with the control group,the level of FBS significantly increased in the diabetic model group(t =2.26,P＜O.05),and that in the rosiglitazone group significantly declined in comparison with the diabetic model group(t=2.08,P＜0.05).The optic nerve exhibited a normal morphology in the control group as revealed by the Lauck fast Blue myelin staining;however,severe demyelination of the optic nerve and proliferation of glial cells were found in the diabetic model group,and mild demyelination of the optic nerve and proliferation of glial cells were seen in the rosiglitazone group.Blood plasma VEGF was(28.76±4.21)ng/L in the control group and(134.28±11.36)ng/L in the diabetic model group,showing a significant difference between them (t=2.36,P ＜ 0.05).Compared with the model group,the blood plasma VEGF was significantly lower in the rosiglitazone group ([42.67 ± 5.83] ng/L) than that in the diabetic model group (t =2.17,P＜ 0.05).Expression of NCAM mRNA and protein in the optic nerve significantly decreased in the diabetic model group compared with the control group(t =2.21,t =2.58,both at P＜0.05);while those in the rosiglitazone group were significantly elevated in comparison with the diabetic model group(t =2.19,t =2.67,both at P＜O.05).Conclusions Rosiglitazone can protect optic nerve from damage in diabetic rats mainly by downregulating blood plasma VEGF level and upregulating NCAM expression.
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Background Retinitis pigmentosa (RP)is a common hereditary blinding eye disease in ophthalmology.Current researches documented that RP may have the common pathophysiologic basis to Alzheimer disease and chronic neurodegenerative disease.Understanding this mechanism will offer a new therapeutic target for RP.Objective The purpose of the present study was to investigate the roles of cyclin-dependent kinase 5 (Cdk5)/P25 activation in the apoptosis of retinal neural cells of RCS rats.Methods Eighteen SPF RCS rats and 18 RCS-rdy+ rats were randomized into 17-,25-and 35-day groups respectively and 6 rats for each.The rats were sacrificed at corresponding time points and retinal hemogenete was prepared.Expressions of CdkS,P35,P25 and tau phosphorylation in the retinas were detected by Western blot,and the kinase activity of Cdk5/P25 was analyzed by quantitative colorimetric assay.Results The expressing level of P35 protein(A340) in the retinas of 17-day-old RCS rats was near that of 17-day-old RCS-rdy+ rats(t =0.52,P＞0.05).In 25-and 35-day-old RCS rats,the expressing levels of P35 protein were 2.20±0.48 and 1.23±0.14,which were higher than those of RCS-rdy+ rats(1.43±0.13 and 0.93±0.10),showing significant differences between them(t =3.78,4.28,P＜0.05).The expression of P25 was undetectable at postnatal 17 days in RCS rats and RCS-rdy+ rats,but it showed significantly higher in RCS rats(0.300±0.003 and 0.230±0.004) than that in RCS-rdy+ rats(0.040±0.004 and 0.070±0.004) at postnatal 25 days and 35 days(t=121.81,77.51,P＜0.01).No significant difference was found in the expression of Cdk5 in RCS rats and RCS-rdy+ rats at different ages (t =-0.60,0.19,1.62,P＞ 0.05).The kinase activity of Cdk5/P25 did not show significantly different between RCS and RCS-rdy+ rats at postnatal 17 days(t =0.19,P＞0.05),but significantly higher kinase activity of Cdk5/P25 was seen in RCS rats (0.0058 ±0.0005 and 0.0056±0.0004) than that in RCS-rdy+ rats(0.0038±0.0003 and 0.0032 ±0.0007) at postnatal 25 days and 35 days (t =8.07,5.97,P＜ 0.01).No expression of tau phosphorylation was detected in RCS rats at postnatal 17 days,but significantly higher tau phosphorylation level was seen in RCS rats at postnatal 25 days and 35 days(1.80±0.22 and 1.23±0.17),which were significant different in comparison with RCS-rdy+ rats at postnatal 25 days and 35 days(1.60 ±0.20 and 1.04 ±0.12)(t=4.71,3.17,P＜0.05).Conclusions The Cdk5/P25 kinase activity shows a consistent trend with theexpressions of P25 and tau phosphorylation in the RCS rats,indicating that the upregulation of P25 induces the enhance of enzyme activity of Cdk5,which phosphorylate its substrates to result in more apoptosis of retinal neural cells.
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PURPOSE: Cell transplantation of myelin-producing exogenous cells is being extensively explored as a means of remyelinating axons in X-linked adrenoleukodystrophy. We determined whether 3,3',5-Triiodo-L-thyronine (T3) overexpresses the ABCD2 gene in the polysialylated (PSA) form of neural cell adhesion molecule (NCAM)-positive cells and promotes cell proliferation and favors oligodendrocyte lineage differentiation. MATERIALS AND METHODS: PSA-NCAM+ cells from newborn Sprague-Dawley rats were grown for five days on uncoated dishes in defined medium with or without supplementation of basic fibroblast growth factor (bFGF) and/or T3. Then, PSA-NCAM+ spheres were prepared in single cells and transferred to polyornithine/fibronectin-coated glass coverslips for five days to determine the fate of the cells according to the supplementation of these molecules. T3 responsiveness of ABCD2 was analyzed using real-time quantitative polymerase chain reaction, the growth and fate of cells were determined using 5-bromo-2-deoxyuridine incorporation and immunocytochemistry, respectively. RESULTS: Results demonstrated that T3 induces overexpression of the ABCD2 gene in PSA-NCAM+ cells, and can enhance PSA-NCAM+ cell growth in the presence of bFGF, favoring an oligodendrocyte fate. CONCLUSION: These results may provide new insights into investigation of PSA-NCAM+ cells for therapeutic application to X-linked adrenoleukodystrophy.