ObjectiveTo explore the effect of bFGF-chitosan carriers on inducing bone marrow-derived mesenchymal stem cells (MSCs) to differentiate into nerve cells.MethodsMSCs were detected by immunohistochemistry and Western blot after they were induced by bFGF-chitosan carriers to differentiate into neurons. The MTT chromometry assay was carried out to determine cell viability.ResultsThe proportion of express neural stem cells marker Nestin, and neuronal markers class Ⅲ β-tubulin and MAP-2 was 83.54% after MSCs induced by bFGF-chitosan carriers.ConclusionbFGF-chitosan carriers can induce MSCs to differentiate into nerve cells with a high percentage.

BACKGROUND:Neural stem cells (NSCs) hold self-renewal and multi-directional differentiation potential. NSCs differentiation into neurons in high proportion under induction conditions exhibits broad application prospect. OBJECTIVE:To explore the effect of basic fibroblast growth factor (bFGF)-chitosan carrier on the NSCs differentiation into neurons in vitro, and whether the differentiated neurons could form synaptic-like connection with myocytes. METHODS:After purification, the NSCs were co-cultured with chitosan, soluble bFGF or bFGF-chitosan carrier. After 7-day induction, the NSCs differentiation into neurons was observed by immunofluorescence staining of beta tubulin Ⅲ. The NSCs differentiation into cholinergic neurons was observed through double immunofluorescence staining of ChaT and beta tubulin Ⅲ. The synaptic-like connection between the neurons and myocytes was observed by triple staining of beta tubulin Ⅲ and MHC. RESULTS AND CONCLUSION:The percentage of differentiated neurons in the bFGF-chitosan carrier group was 74%, which was significantly higher than that in the other two groups. Additionally, the synaptic-like connection formed between the differentiated neurons and myocytes. To conclude, the bFGF-chitosan carrier promotes the NSCs differentiation into neurons to form synaptic-like connection with the co-cultured myocytes.

Objective To explore the effect of inhibition of miR-214 expression on the proliferation of hepatocellular carcinoma cells via regulation of E2F3 expression.Methods The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was examined by RT-PCR.Hepatocellular carcinoma cells were transfected with miR-214 NC and miR-214 mimics with liposomes.The expression of miR-214 was detected by RT-PCR.The cell viability was detected by MTT assay.Cell apoptosis was detected by Hoechst staining.Cell cycle was detected by flow cytometry.Western blot, RT-PCR and dual luciferase reporter gene assay were used to detect whether E2F3 was a downstream target gene of miR-214.Results The expression of miR-214 in SMMC-7721, HepG2, SK-Hep-1 and Huh 7 cells was 0.83±0.08, 0.32±0.03, 0.33±0.03, and 0.08±0.01, respectively.The expression of miR-214 in the HepG2 cells was the lowest, so HepG2 cells were selected as the subsequent experimental cell line.Compared with the miR-214 NC group, the expression of miR-214 (0.65±0.06 vs.0.14±0.01) was up-regulated, the cell viability (0.35±0.03 vs.0.69±0.06) was decreased, cell apoptosis rate [(36.37±3.43)% vs.(3.74±0.34)%] was increased, the G1 phase of the cell cycle (57.79±5.78 vs.45.319±4.53) was prolonged, the expression of E2F3 protein (0.23±0.02 vs.0.98±0.09) and mRNA (0.24±0.02 vs.0.99±0.10) was significantly down-regulated in the miR-214 mimics group (P<0.01).Conclusion miR-214 mimics suppress the HepG2 cell proliferation via targeted down-regulation of E2F3 expression.

Objective To explore the effect of basic fibroblast growth factor (bFGF)-chitosan carriers on neural differentiation of neural stem cells (NSCs). Methods NSCs were isolated from spinal cord of a neonatal Wistar rat and cultured. Purity of cultured NSCs was identified with Nestin immunofluorescent staining. The 10 mg/ml chitosan carriers, 20 ng/ml bFGF or 10 mg/ml bFGF-chitosan carriers were added into medium of P3~P4 NSCs respectively. NSCs were observed with immunofluorescent staining: 3 days after incubation with Nestin and β-tubulin III; 7 days after incubation with microtubule-associated protein-2 (MAP2), glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP); and 14 days after incubation with synapsin-1 and MAP2. The electrophysiological activity of cells was detected with MED64. Results 3 days after incubation, all the NSCs differentiated into Nestin+/β-tubulin III+, and the length of neurofilament was the highest in those co-cultured with bFGF-chitosan carriers. 7 days after incubation, NSCs differentiated into MAP2+, GFAP+ and MBP+, and more NSCs differentiated into MAP2+ with bFGF-chitosan carriers. 14 days after incubation, NSCs differentiated with bFGF-chitosan carriers express synapsin-1+/MAP2+ and showed electrophysiological activity. Conclusion bFGF-chitosan carriers can induce NSCs to differentiate into neuron with high percentage and the differentiated neurons can form synapses with electrophysiology activity.

Objective To explore the induction of basic fibroblast growth factor (bFGF)-chitosan carrier transforming the adult rat bone marrow mesenchymal stem cells (rMSCs) into nerve cells. Methods rMSCs were detected qualitatively and counted quantitatively by immunohistochemistry after they were induced into nerve cells, such as neural stem cells neurons and astrocytes. The methyl thiazolyl tetrazolium (MTT) chromometry assay was carried out to determine the cell viability. Results rMSCs induced by bFGF-chitosan carrier expressed neural stem cell marker nestin, neuron marker β-tubulin Ⅲ and astrocytes marker glial fibrillary acidic protein (GFAP). Nestin expressed more in the bFGF-chitosan group, and reached its maximum (49.40%) at the 9th day. Conclusion bFGF-chitosan carrier can induce the adult rMSCs differentiate into neural stem cells in a high proportion.

Objective To observe the effects of neurotrophin 3 (NT3)-chitosan on motor function, and proliferation and differentiation of the neural stem cells (NSCs) in the injury area and subventricular zone (SVZ) in rats with motor cortex injury. Methods Sixty-five Wistar rats were divided into control group (n=7), injury group (n=29) and NT3-chitosan group (n=29). The motor cortex was aspirated and re-moved as cerebral injury model. NT3-chitosan was immediately implanted into the injured area after operation, and the control group re-ceived no intervention. Pellet reaching test was performed to detect the recovery of the forelimb function, HE staining was used to observe the lesion cavity size, and immunofluorescence staining was used to observe the proliferation and differentiation of NSCs 3 days, 7 days, 14 days, 1 month, 2 months and 3 months after operation. Results The grasp success rate was higher (F>6.00, P≤0.05), and the lesion cavity size was significantly smaller (F>629.5, P<0.001) in the NT3-chitosan group than in the injury group. In the NSCs differentiation experi-ment, the number of BrdU cells at all the time points was significantly higher in the NT3-chitosan group than in the injury group (F>171.43, P<0.001). In the NSCs proliferation experiment, the number of BrdU positive cells was still significantly higher in the NT3-chitosan group than in the control group and in the injury group (F>155.06, P<0.001), the number of Dcx positive cells was significantly higher in the NT3-chitosan group than in the injury group (F=62.367, P<0.001), and the number of BrdU/Dcx positive cells was significantly higher in the NT3-chitosan group than in the control group (F=33.527, P<0.001). Conclusion NT3-chitosan could activate NSCs in the SVZ, and pro-mote endogenous neurogenesis and forelimb function recovery in rats after motor cortex injury.

Objective To analyze the effect of different treatment conditions on cells synchronization in G0/G1 phase to get the best con-dition, and to explore its effect on neural differentiation of bone marrow mesenchymal stem cells (BMSCs) induced by basic fibroblast growth factor (bFGF). Methods BMSCs were isolated and cultured in 5%, 1%, 0.5%, 0.1%, 0 fetal bovine serum (FBS) respectively, for 24 hours and 48 hours. After PI staining, cell cycle proportions of each phase were detected by flow cytometry, and were compared with the normal group (10%FBS). After the optimal treatment condition was got, 20 ng/ml bFGF was added into synchronization group and unsyn-chronization group 3 days and 7 days, respectively. The expression of Nestin and Tuj-1 were detected with immunofluorescence. Results Adult rat BMSCs were isolated from bone marrow and cultured, after passage, the cells were with long spindle shape. Compared with the normal group, the cell proportion of G1/G0 phase increased under different treatments, peaked with (94.274 ± 0.468)%under 1%FBS, 48 hours (F=39.91, P<0.001). After bFGF induction for 3 days, the Nestin+cell number was higher in the synchronization group than in the un-synchronization group [(80.3 ± 2.4)%vs. (12.1 ± 1.5)%] (F=28.25, P<0.001). After bFGF induction for 7 days, the Tuj-1+cell number was higher in the synchronization group than in the unsynchronization group [(74.8±3.2%)%vs. (19.3±2.5)%] (F=17.95, P<0.001). Conclusion 1%FBS, 48 hours is the optimal condition to BMSCs synchronization in G0/G1 phase, which can promote the neural differentiation of BM-SCs.

Objective To compare the application of CUBIC and iDISCO clearing methods in observing 3D imaging of spinal cord with immunofluorescent staining. Methods 1 mm thick spinal cord coronal sections were processed with CUBIC and iDISCO, respectively. The neurofilament (NF) protein was detected by immunofluorescent staining and then was observed by a laser confocal microscope. Results Compared with CUBIC, iDISCO had the advantages of shorter time, higher transparency (F=6.64, P<0.01), and deeper penetration (F=5117.55, P<0.01). Conclusion Immunofluorescent staining combined with iDISCO could completely observe the spinal axons with shorter time and better stain effect.

Objective To investigate the potential mechanism of basic fibroblast growth factor (bFGF)-chitosan carrier to induce neural stem cells to differentiate into neurons. Methods After purification, the neural stem cells were cocultured with chitosan, soluble bFGF and bFGF-chitosan carrier. Three hours, twenty-four hours, three days and seven days after induction, immunofluorescence staining of Nestin, beta tubulin III, microtubule-associated protein-2 (MAP2), and fibroblast growth factor receptor 1 (FGFR1) were used to observe the expres-sion of FGFR1;real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) and Western blotting were used to detect RNA and protein level changes after induction. Results Three hours after induction, there was no significant difference in the expression of FGFR1 among three groups. Twenty-four hours after induction, the expression level of FGFR1 was significantly higher in the bFGF-chitosan carrier group than in the chitosan group and the soluble bFGF group (P<0.001);three days and seven days after induction, the expression of FGFR1 decreased significantly in the chitosan group and soluble bFGF group (P<0.001), however, it was still higher in the bFGF-chitosan carrier group;moreover, the expression of genes associated with the pathway of extracellular regulated protein kinases/mitogen activated protein ki-nase (Erk/MAPK) was significantly higher in the bFGF-chitosan carrier group than in the chitosan group and soluble bFGF group (P<0.001). Conclusion bFGF-chitosan carrier might upregulate the expression of FGFR1, then activate Erk/MAPK signal pathways, and finally promote the differentiation of neural stem cells into neurons.

Objective To explore the physiological characteristics of synaptic transmission of anterior horn early development in thorac-ic spinal cord mediated byα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in rats. Methods 36 Wistar rats were divided into embryonic 17 days group (E17, n=12), embryonic 20 days group (E20, n=12) and postnatal 7 days group (P7, n=12). Immuno-fluorescent staining of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) was used to test the distribution of AMPA receptors. Multi-elec-trode array technique (MED-64 system) was used to test the changes of field excitatory post-synaptic potential (fEPSP) of synaptic transmis-sion mediated by AMPA receptor. Results There was small amount of CaMKⅡ-positive neurons existing in gray matter of spinal cord at E17, CaMKⅡ-positive neurons migrated to the center, and the number of neurons became more and more in E20 and P7 rats. The number of evoked fEPSP gradually increased in rats from E17 to P7, and could be blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The range of synaptic connection in spinal cord gray matter significantly reduced (P<0.001). Conclusion AMPA receptors participate in the early development of spinal cord in rats and act as the main excitatory receptor of functional synaptic connection in neural network of ventricornu.