1.Growth differentiation factor 11 promotes proliferation of mouse neural stem cells and activates both transforming growth factor-β/Smads and Wnt/β-Catenin signal pathways
Haoju ZHANG ; Fobao HUANG ; Hao QIN ; Yiwu DAI ; Ruxiang XU
Chinese Journal of Neuromedicine 2017;16(5):433-438
Objective To investigate the effects of growth differentiation factor 11 (GDF11) on proliferation of mouse neural stem cells (NSCs) and expression levels of transforming growth factor (TGF)-β/Smads and Wnt/β-Catenin signal key proteins.Methods NSCs,derived from the subventricular zone of E14 d CD1 mice,were cultured and induced differentiation;specific proteins nestin and SOX2 were confirmed by immunofluorescence assay.Neuron marker nucleus antigen (NeuN)and astrocyte marker glial fibrillary acidic protein (GFAP) were identified by immunofluorescent staining.The cells of third generation in their exponential phase were chosen and randomly divided into experimental group (adding GDF11 to make the final concentration as 40 ng/mL) and control group (adding equal amount of culture fluid).The proliferation of the cells in the two groups was detected by 5-ethynyl-2'-deoxyuridine (EdU) kits and protein expressions of Smad2/3,phosphorylated (p)-Smad2/3,Smad4 and β-Catenin were measured by Western blotting one and 6 h after treatment.Results Round and bright cells suspended in culture medium were observed through optical microscope.Immunofluorescence assay showed that over 90% cells expressed both nestin and SOX2,and some of them expressed NeuN or GFAP.EdU proliferation test showed that the percentage of EdU positive cells in the experimental group (0.34±0.08) was significantly higher than that in the control group (0.24±0.03,P<0.05).Western blotting showed that the expression levels ofp-Smad2/3,Smad4 and β-Catenin were significantly increased one and 6 h after treatment as compared with those in the control group (P<0.05).Conclusion GDF11 can promote the proliferation of NSCs in vitro and probably is on account of activating TGF-β/Smads and Wnt/β-Catenin signal pathways.
2.Ultrastructure of neurovascular unit in chronic compressive cervical myelopathy rat model
Jinghui XU ; Houqing LONG ; Wenli CHEN ; Xing CHENG ; Yangliang HUANG ; Fobao LI
Chinese Journal of Microsurgery 2016;39(1):52-57
Objective To observe the ultrastructure of the neurovascular Unit (NVU) in chronic compressive cervical myelopathy rat model at different stages.Methods From March, 2014 to March, 2015, 32 rats were divided into two groups: sham control group (n =8) and compressive spinal cord injury group (n =24).The model was established by inserting the compression sheet made of polyurethane at the level of C6.BBB and somatosensory evoked potentials (SEP) were used to evaluate the spinal cord function status of model rat.Transmission Electron Microscopy (TEM) examination of compressive cervical spinal cords was performed separately at the 14th, 21st, 28th and 42nd day after modeling.Results At the 14th, 21st, 28th and 42nd, the BBB score were 17.571 ± 0.870, 15.952 ± 0.870, 15.476 ± 0.602 and 16.190 ± 0.632 were significantly lower than those in the control group (the BBB score of 4 points were 19.600 ± 0.516, 19.500 ± 0.527, 19.600 ± 0.699 and 19.800 ± 0.6232 respectively) (P < 0.05).Latency prolongation and amplitude reduction of somatosensory evoked potentials (SEP) were presented in the compressive spinal cord injury group.At the 14th day, edema around the capillaries was observed, the morphological structure of endothelial cells and basement membrane was normal, the tight junction between endothelial cell was intact, the mitochondria in the axons, oligodendrocytes and astrocyte foot processes were edematous.At the 21st day, extensive edema, even partial necrosis around the capillaries were found, the surrounding structure were arranged loosely, partial loss of endothelial cells and basement membrane, cavitation occurrence in endothelial cells, basal membrane density was significantly lower, mitochondria vacuoles and shrinkage in the cytoplasm, axon myelin loose or broken;at the 28th day, the edematous range surrounding capillaries narrowed, low density of basement membrane and endothelial cells, vacuoles in endothelial cells, loose axon myelin, while some mitochondrias backed to normal.At the 42nd day, capillary integrity, no abnormalities were found in endothelial cells, basement membrane, tight junction and mitochondria, double layers of endothelial cells and basement membrane could be seen, local broken and loose structure were presented in part of the axons;The TEM of the sham control group showed normal ultrastructure of NVU.Conclusion The ultrastructure of NVU in chronic cervical spinal cord compression presented various in the different periods, NVU disruption were found in the early stages (14th-28th days), and compensatory and repair process were developed incompletely later.
3.Effect of miR-184 on proliferation of neural stem cells and its underlying mechanism
Fobao HUANG ; Haoju ZHANG ; Yugang LIU ; Dan LUO ; Ruxiang XU ; Yiwu DAI
Chinese Journal of Neuromedicine 2017;16(3):262-268
Objective To investigate the effect ofmiR-184 on proliferation of neural stem cells (NSCs) and its mechanisms in mice.Methods The pHBLV-U6-GFP-miR-184 over-expression plasmid and pHBLV-U6-GFP-miR-184 inhibitor plasmid were used to construct recombinant lentivirus.And the NSCs derived fiom subventricular zone of E14d CD1 mouse were confirmed by immunofluorescence assay.There were four groups that contain a miR-184 overexpression group,a miR-184 inhibitor group and two control groups.The NSCs which infected with lentiviral vectors were selected for puromycin resistance for 5-7 days,and then surviving cells were cultivated to three generations.The expression level ofmiR-184 was detected by real time-quantitative PCR (RT-qPCR).And the target genes ofmiR-184 were predicted through TargetScan,IRTarBase and MiRanda,and were confirmed by Western blotting and RT-qPCR.The cells in the four groups were culttared under proliferating conditions incorporated bromodeoxyuridine (BrdU) in cell proliferation analyses.The protein expressions of Hesl and Hes5,the target proteins of Notch signaling pathways,and their mRNA expressions were detected by Western blotting and RT-qPCR.Results There were 90% of cells in each group expressing both Nestin and Sox2.The miR-184 level in the miR-184 overexpression group was 67.63±7.53 times of that of the control group,with significant difference (P<0.05).The percent of BrdU+/DAPI+ of the miR-184 overexpression group was 1.47±0.05 times of that in the control group,with significant difference (P<0.05);and the percent of BrdU+/DAPI+ of the miR-184 inhibitor group was 0.84±0.03 times of that in the inhibitor control group,with significant difference (P<0.05).Numbl was a target gene ofmiR-184 indicated by IRTarBase and MiRanda.The miR-184 could inhibit Numbl protein expression;the Numbl protein expression level in the miR-184 overexpression group was 0.73±0.07times of that in the control group,and the Numbl protein expression level in the miR-184 inhibitor group was 1.30±0.05 times of that in the control group,with significant difference (P<0.05);but miR-184 did not change the Numbl mRNA level.The miR-184 could activate Notch signaling pathway through inhibiting the Numbl protein expression,and increase the Hes1 and Hes5 protein and mRNA expression levels (P<0.05).Conclusion The miR-184 promotes the NSCs proliferation through inhibiting the Numbl protein translation and further activating the Notch signaling pathway.
4.Galangin induces apoptosis of glioma cells through Wnt/β-Catenin signal pathway
Jianglei HOU ; Yuyang LIU ; Miaochun BAI ; Guangyun ZHENG ; Chen LIU ; Haoju ZHANG ; Fobao HUANG ; Ruifeng WANG ; Yiwu DAI ; Ruxiang XU
Chinese Journal of Neuromedicine 2017;16(7):657-664
Objective To investigate the effect of galangin on proliferation and apoptosis of glioma cells in vitro.Methods (1) The glioma cells U87 and U25 1were divided into blank control group,DMSO group,100,200,300 and 400 μmol/L galangin treatment groups.MTT was used to study the effects of drugs on the proliferation of U251 and U87 cells.(2) Hoechest staining was used to observe cell apoptosis in the presence of different concentrations of galangin (0,100 and 200 μmol/L).(3) Flow cytometry was employed to detect the apoptosis of U251 and U87 cells in the presence of different concentrations of galangin (1 00 and 200 μmol/L).(4) Western blotting was employed to detect the expressions of apoptosis-related protein 3-Catenin,B-cell lymphoma-2 (Bcl-2),Bcl-2 related protein gene (Bax),cleaved-caspase-3,cleaved-caspase-9 and poly (ADP-ribose) polymerase (PARP) in the presence of different concentrations of galangin.Results (1) The proliferation of U251 and U87 cells was obviously inhibited atter 100,200,300 and 400 μmol/L galangin treatments,and dose-effect relation was noted.The concentrations of galangin at half rate of inhibition (IC50) were 281,321,276 and 229 μmol/L in U251 cells,and 289.4,261.1,247.4 and 225.3 μ mol/L in the U87 cells after 100,200,300 and 400μmol/L galangin treatments for 24 h.(2) Under the action of galangin,corresponding increase in apoptosis rates of U251 and U87 cells was noted following the increase of galangin concentrations (0,100 and 200 μmol/L),with significant differences (P<0.05).(3) The detection of cell apoptosis by flow cytometry found similar changes.(4) Western blotting results indicated that galangin at the concentration of 0,100 and 200 μmol/L could significantly decrease the expressions of apoptosis-related protein 3-Catenin and Bcl-2,and increase the Bax,cleaved-caspase-3 and cleaved-caspase-9,and cleaved-PARP expressions;significant differences were noted between each two concentrations (P<0.05).Conclusion Galangin can inhibit proliferation of glioma cells U251 and U87,and induce mitochondrial pathway of apoptosis via Wnt/β-Catenin signaling.
5.Extra-long subcutaneous tunnel in external ventricular drains
Yehai LI ; Jianhao LIN ; Jing YE ; Guilu HE ; Ruirui YUN ; Fobao HUANG ; Qiao LI ; Liang ZHANG
Chinese Journal of Neuromedicine 2020;19(7):700-705
Objective:To explore the clinical value of extra-long subcutaneous tunnel ventricular drainage in patients with hydrocephalus.Methods:From March 2016 to March 2020, 33 patients who were not suitable for ventriculoperitoneal shunt, who would have expected time of external ventricular drainage longer than 7 d, who had external ventricular drainage reaching for 7 d and still could not expect for drainage tube drawing for the next 7 d, or who had hydrocephalus after external ventricular drainage were chosen in our study. These patients accepted extra-long subcutaneous tunnel ventricular drainage. The curative effects in the patients were analyzed retrospectively.Results:The drainage tube was kept for a maximum of 24 months and the shortest time was 13 d, with average of 69.3 d; 32 patients (97%) had drainage time longer than 14 d. There was no secondary infection after operation.Conclusion:Extra-long subcutaneous tunnel extraventricular drainage tube has a long duration of catheter placement, could avoid multiple drainage and secondary intracranial infection, so it is a safe and effective new technology for hydrocephalus.