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 silence the expression of CTGF by small interfering RNA technology,to observe the influence on fibroblast?like synovial cell apoptosis and several apoptosis?related genes,and to explore the mechanism of action of CTGF in rheumatoid arthritis synovial lesions. Methods Effective CTGF siRNA was screened through real?time PCR. The influence of CTGF siRNA on FLS apoptosis was detected with FITC?PI double staining by flow cytometry. bax,bcl?xl and survivin were detected using real?time PCR when CTGF mRNA has been silenced. Results Compared with other 2 groups of oligo and NC oligo,H1 oligo exhibited the strongest interfering action to CTGF(inhibition ratio>70%),so that it is selected as the effective target gene sequence for the following experiment. Apoptosis of FLS induced by serum deprivation was significantly decreased in the presence of exogenous CTGF. When expression of the CTGFgene was knocked down in FLS,FLS apoptosis was significantly increased,and expres?sion levels of survivin mRNA were decreased significantly(P<0.01). Conclusion FLS survival is positively regulated by CTGF,which may through the sustaining the expression of survivin.

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 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 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.

Objective To study the compliance of examinees,and effectiveness of colorectal distension with partially automated and individualized insufflation of air for dual-energy CT colonography.Methods Forty-six healthy adult volunteers without history of conditions affecting gastrointestinal motor function were enrolled in this study.One day before CT examination,volunteers were asked to orally administered 60 mL 4% diatrizoate meglumine five times for fecal tagging.Air was insufflated by using an inflator in a partially automated and individualized manner.The volunteers were initially asked to assume the right lateral decubitus position,then slowly turn to the supine position.Insufflation rate began at 1.5 L/min,and decreased to 0.5 L/min at later stage.The necessity and volume of air insufflation were decided according to effectiveness of colorectal distension on CT scout images,self-reported sensation of volunteers,and intestinal pressure.Dual-energy CT scanning was performed,and dual-energy blended images were acquired.Compliance of volunteers was statistically analyzed.The effectiveness of colorectal segments distension was statistically analyzed by using Kruskal-Wallis H test.Results No abdominal pain,bloating,nausea or vomiting were noted in the 46 volunteers.All volunteers easily accepted colorectal insufflation of air,with grade 1 compliance.The effectiveness of colorectal distension of grades 1,2,3 and 4 were 0%,2.1%,5.1% and 92.8%,respectively.The difference of effectiveness of colorectal segments distension had no statistical significance(χ2=6.19,P=0.288).The effectiveness of insufflation was poor in 6 colorectal segments,including 2 in sigmoid colon and 2 in rectum.Effectiveness of insufflation was suboptimal in 14 colorectal segments,including 4 in descending colon,4 in sigmoid colon,and 3 in rectum.Conclusion Compliance of examinees with partially automated and individualized insufflation of air for dual-energy CT colonography is excellent,with good effectiveness of colorectal distension.

Objective To activate and amplify γδT cells with low molecular peptide antigen of Mycobacterium tuberculosis low molecular peptide antigen (Mtb-Ag),and to investigate the expression of CD69 molecules on γδT cellular surface.Methods Healthy human peripheral blood mononuclear cells (PBMC) were obtained and separated,then positive cells were isolated by immuno-magnetic beads selection,and the proportion of γδT cells in the PBMCs was detected by fluorescent monoclonal TCR γδT-PE staining and flow cytometry.Expression of CD69 molecules in γδT cells was detected by γδ-PE/CD69 FITC double staining.Results The proportion of γδT cells was (4.9±1.85)％ in freshly obtained PBMC,(69.2±6.57)％ after 10 d of Mtb-Ag activation,and (99.3±8.92)％ after immuno-magnetic beads selection.The expression of CD69 molecules in γδT cells reached the peak (75.2％) at 24 h after initial Mtb-Ag stimulation,and reached the peak (72.0％) at 6 h after second stimulation.Conclusions MtbAg can specifically stimulate the proliferation of γδT cells in the PBMC.Both its initial and the second stimulation can specifically activate γδT cells.

Sulfonamides (SAs), such as sulfaguanidine (SGD), sulfadiazine (SDZ), sulfathiazole (STZ) and sulfamethazine (SMZ), can drastically inhibit the chemiluminescence (CL) intensities generated in both Ag-Luminol and Ni-Luminol systems.Based on these observations, a novel method of high performance liquid chromatography (HPLC) coupled with CL detection was established.Both the Ag-Luminol and Ni-Luminol CL systems were employed as detectors, and the performances of the two detecting systems were compared.After separated by HPLC, four SAs reacted with Ag-Luminol and Ni-Luminol CL system, respectively.Chromatographic conditions were as follows: reversed-phase C18 column (250 mm × 4.6 mm,5 μm), gradient elution, and 0.1% (V/V) formic acid-methanol as mobile phase with flow rate of 1 mL/min.CL conditions were as follows: [Ag]=1.4×10.-4 mol/L (in 0.12 mol/L NaOH);[Ni]=1.5×10.-5 mol/L (in 0.12 mol/L NaOH);[Luminol]=1.2×10.-7 mol/L;and flow rate=1.0 mL/min.Under the optimal conditions, the detection limits of Ag-Luminol CL system were 0.15, 0.96, 1.10, 1.50 μg/mL for SGD, SDZ, STZ, and SMZ, respectively, and the recovery were 81.0%-101.5%.Comparatively, the detection limits of Ni-Luminol CL system were 1.5, 17.2, 16.8 μg/mL for SGD, SDZ and STZ, and the recoveries was 83.9%-110.8%.The result showed that the Ag-Luminol CL system had a much better performance.The method was applied to the determination of the residues of the above four SAs in milk with satisfactory results.

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.