To investigate the effects of ischemia-reperfusion on the levels of nitric oxide and nitric oxide synthase isoforms (nNOS and iNOS), rat organotypic hippocampus slice were cultured in vitro and subjected to ischemia by oxygen-glucose deprivation (OGD) for 30 min and then placed in the normal culture condition. The ischemia-reperfusion produced a time-dependent increase in nitrite levels in the culture medium. Reverse transcriptional-polymerase chain reaction showed augmented levels of mRNA for both nNOS and iNOS when compared with control at 12 h and remained increase at 36 h after OGD (P < 0.05). The protein levels of both nitric oxide synthase isoforms increased significantly as determined by Western Blot. OGD also caused neurotoxicity in this model as revealed by the elevated lactate dehydrogenase (LDH) efflux into the incubation solution. The results suggest that organotypic hippocampus slice is a useful model in studying ischemia-reperfusion brain injury. NO and NOS may play a critical role in the ischemia-reperfusion brain damage in vitro.
Animals, Newborn ; Cell Hypoxia ; Hippocampus/cytology ; Hippocampus/*metabolism ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type I/*metabolism ; Nitric Oxide Synthase Type II/*metabolism ; RNA, Messenger/metabolism ; Rats, Sprague-Dawley ; Reperfusion Injury/*metabolism ; Tissue Culture Techniques

Objective To investigate the possible effect of ginsenoside Rg1 and oligo Aβ1-42 on PKA/CREB pathway.Methods The damage was induced by oligomeric Aβ1-42 in primary cortical neuron.Neurons were incubated with or without glutamate,or incubated in Aβ,or pre-incubated in Rg1 and then co-incubated in Aβ.The proteins of p-CREB,t-CREB,PKA Ⅱ α and BDNF were detected by Western blot.Results After the treatment with Oligo Aβ1-42 for 2 h,the p-CREB/t-CREB level induced by glutamate was obviously lower (P＜ 0.001).However,in neurons pre incubatedwith 2.5,5.0,10.0 μmol/L of ginsenoside Rg1 and then co-incubated with 5μmol/L of oligo Aβ1-42,the p-CREB/t-CREB induced by glutamate was significantly increased as compared with that of Aβ1-42 group (P＜0.05).Upon Aβ1-42 exposure for 2 h,cortical neurons showed a statistically significant increase in PKA Ⅱ α as compared to the control group (P ＜ 0.001).Pre-treatmentwith varying doses of ginsenoside Rg1 (2.5,5,10μmol/L) showed a decrease in PKA Ⅱ α as compared to neurons treated with Aβ1-42 alone for 2 h (P＜0.001).Furthermore,BDNF level significantly increased in Rgl-pretreated cells as compared to cells treated with Aβ1-42 alone for 24h (P＜0.05).Conclusions Ginsenoside Rg1 attenuates the oligo Aβ142 inhibition of PKA/CREB pathway.

Objective Clarify the effect of neutrophil extracellular traps (NETs) on endothelial cell injury, and investigate whether the heparin can exert a protective effect on endothelial cells by reducing the endothelial cell injury induced by NETs.Methods Neutrophils (PMN) were obtained from healthy human peripheral blood by Percoll-Histopaque density gradient centrifugation, and was stimulated with phorbol ester (PMA) to induce NETs. The qualitative and quantitative analysis of NETs was detected by immunofluorescence staining combined with fluorescence detector. The NETs were used to induce human umbilical vein endothelial cells (HUVEC)in vitro. Recombinant DNA hydrolytic enzymes (rhDNase) and heparin intervention were added respectively. The activity of HUVEC was measured by methyl thiazolyl tetrazolium (MTT) method after 6 hours.Results PMA can stimulate PMN to produce NETs. Immunofluorescence staining showed the formation of reticular formation around the PMN. The concentration of cell-free DNA in the supernatant of PMN stimulated by PMA was significant increased compared with the control group through the detection of PicoGreen fluorescent labeling instrument (2 hours: 119.62±14.83 vs. 24.27±0.67, 4 hours: 146.67±21.24 vs. 28.35±2.98, bothP < 0.05). Application of NETs to stimulate the HUVEC, cell damage was dose dependent and inhibition rate increased gradually. The endothelial cell inhibition induced by NETs can be antagonized after adding rhDNase [10μg/L NETs: (8.65±0.51)% vs. (10.99±0.35)%, 20μg/L NETs:(14.85±0.43)% vs. (16.85±0.49)%, 30μg/L NETs: (26.06±3.51)% vs. (27.54±0.62)%, allP < 0.05]. Heparin with different concentrations were added into the experimental group (0.01, 0.1, 1, 10 kU/L). We found that the endothelial cell inhibition rate decreased compared with control group [10μg/L NETs: (8.96±0.70)%, (5.32±1.36)%, (0.70±0.30)%, (0.75±0.20)% vs. (10.99±0.35)%; 20μg/L NETs: (15.57±0.62)%, (13.28±0.65)%, (6.91±0.15)%, (5.86±0.17)% vs. (16.85±0.49)%; 30μg/L NETs: (30.49±0.74)%, (29.41±1.41)%, (23.45±0.75)%, (21.72±1.52)% vs. (27.54±0.62)%, allP < 0.05].Conclusions NETs can induce endothelial cell injury, and the injury degree was increased with the concentration of NETs. Heparin can reduce endothelial cell injury induced by NETs, which may be a potential mechanism for the protective effect of heparin on sepsis.

The aim of this study was to express and purify human histydyl-tRNA synthetase related gene and to prepare its polyantibody. The open reading frame was amplified by PCR, and then recombined into prokaryotic expression vector pQE30 and transformed into E. coli M15 for expression. The expressed products were induced by IPTG after the reconstructed pQE30 was transferred into M15. After purified by Ni affinity chromatography, the product was identified to be a single band by SDS-PAGE. The rabbits were inoculated with purified products. High-titer polyantibody was successfully prepared. Highly-purified expression product and prepared polyantibody may provide a good basis for further study.
Antibodies/*genetics ; Antibodies/immunology ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Histidine-tRNA Ligase/biosynthesis ; Histidine-tRNA Ligase/*genetics ; Histidine-tRNA Ligase/immunology ; Open Reading Frames/genetics ; Prokaryotic Cells/metabolism

Objective To evaluate the application of modified Abbe flap in repairing moderate defects of the upper lip and time point to divide the pedicle.Methods Classic Abbe flap was modified in its design,pedicle cutting and dividing time,which was used to repair moderate defect of the upper lip in 12 cases.Surgery was divided into two phases:one with modified Abbe flap surgery was performed for the combined nasal deformity repair simultaneously,and then the pedicle was divided 9days after surgery.Results 12 patients underwent modified Abbe flap.No vascular complications were found in these flaps.Upper lip shape was well and satisfactory functional recovery,corresponding improvement in nasal appearance.Conclusions The surgery that the modified Abbe flap with the pedicle is divided 9 days after surgery is very simple.On one hand,it greatly improves the patient's appearance and function of the upper lip,improve the overall shape of midface,on the other hand,dividing pedicle time is significantly shorter than in the past,specially reducing the suffering of patients and duration.It is particularly suitable for unilateral and bilateral cleft lip of the upper lip on secondary moderate deformities and combined nasal deformities.

Objective To search for the methods of inducing the high stability rat middle cerebral artery occlusion (MCAO) model and to noninvasively assess the method of model effect. Methods Six kinds of filaments with different diameters were used to induce rat MCAO models and their success rate, incidence of subarachnoid hemorrhage, and infarct volume were compared. The model scores were performed to assess the model effect after the operation and at 24 hours after reperfusion. Results Higher model success rate and lower incidence of subarachnoid hemorrhage were achieved when the 0. 28 mm in diameter and 0.32-0.34 mm in tip diameter filaments were used. 1he sensibility and specificity were higher when the model scores were performed at 24 hours after reperfusion. Conclusions Using the filaments of 0.28 mm in diameter and 0. 32-0. 34 mm in tip diameter for intraluminal thread could improved the stability of the models. The model scores at 24 hours after reperfusion could noninvasively assess the model effect.

Objective To study the clinical efficacy of axillary incision and thoracoscopic surgery for spontaneous pneumothorax.Methods 106 cases of spontaneous pneumothorax in our hospital were given axillary incision surgery(axillary incision group) and thoracoscopic surgery(thoracoscopy group).The intraoperative blood loss,operative time,chest tube drainage time,postoperative hospital stay and surgery costs were compared between the two groups,and the occurrence of complications were observed.Results The armpit small incision group,intraoperative blood loss was (44.5 ± 5.2) ml,the thoracoscopic amount of blood loss was (38.3 ± 6.5) ml (t =6.378,P ＜ 0.01) ;armpit operation time of the small incision group was (68.0 ± 5.3) min,thoracoscopic operative time was (60.8 ±6.0)min; armpit chest tube drainage time of small incision group was (2.8 ± 0.8)d,thoracoscopic group of chest tube drainage time was (2.0 ± 0.5) d; axillary small incision group,length of stay was (4.8 ± 0.7) d,the thoracoscopic group hospitalization time was (4.0 ± 0.6) d,(t =3.552,4.215,3.076,all P ＜ 0.05) ; axillary incision surgery costs was (1 550 ± 348) Yuan,the thoracoscopic group cost of surgery was (4 290 ± 573) Yuan (t =-24.823,P ＜ 0.05).Two groups of patients with no surgical complications,chest X-ray review of lung reexpansion good thoracoscopic group one cases of recurrence of pneumothorax,axillary incision group without recurrence (P ＞ 0.05).Conclusion Axillary small incision and thoracoscopic surgery for spontaneous pneumothorax have the similar efficacy,thoracoscopic surgery is less trauma,faster recovery,shorter hospital stay,but the high cost of surgery,if patients physical condition is acceptable,which can be used axillary incision surgery.

AIM:To construct GSK3?-overexpressed SH-SY5Y cells and to observe the effects of GSK3?-overexpression on tau protein phosphorylation and tubulin acetylation in SH-SY5Y engineered cells. METHODS: The cDNA of GSK3? construct was subcloned into mammalian expression vector pBudCE4.1. The integrity of the GSK3? construct was confirmed by sequence analysis. GSK3? was transiently transfected into SH-SY5Y cells using Lipofectamine2000. Western blotting was used to measured protein levels of GSK3? and phosphorylating GSK3?, as well as, the total tau and phosphorylated tau protein and acetylated tubulin. RESULTS: 36 h after transfection, the levels of GSK3? and phosphorylating GSK3? in SH-SY5Y cells were significantly increased compared with non-transfection group and vector group. After 48 h, the levels of phosphorylated tau protein (Ser199/202, Thr231 and Thr205 residues) but not total tau protein were markedly increased in GSK3?-overexpressed SH-SY5Y cells. In addition, the level of acetylated tubulin was lower than that in non-transfection group and vector group. CONCLUSION: The over-expression of GSK3? in SH-SY5Y cells results in robust increases in tau protein phosphorylation at Ser199/202, Thr231 and Thr205 residues, and decreases in tubulin acetylation.

AIM: To observe the effect of mouse Sim2 (mSim2) eukaryotic expression vector transfection on the cell cycle in PC12 cells in vitro and to explore the role of Sim2 in the pathogenesis of Down syndrome. METHODS: The full open reading frame of mSim2 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into the vector pcDNA3. Then the constructed pcDNA3-Sim2 vectors were transiently transfected into PC12 with Lipofectamine~ TM . The expression of mSim2 was detected by RT-PCR. The effect of mSim2 on the cell cycle was observed by flow cytometry. RESULTS: The eukaryotic expression vector mSim2 was successfully constructed. There was notable expression of mSim2 in the cells transfected with pcDNA3-Sim2. There were more cells in G_0/G_1 phase in the pcDNA3-Sim2 transfected cells than that in the control (P

Aim To explore the possible protective effect of ginsenoside Rg1 on oligomeric Aβ1-42 induced apoptosis and its possible mechanism. Methods The damage was induced by oligomeric Aβ1-42 in primary cortical neurons. Cells were incubated in the absence or presence of Aβ, or co-incubated in sp600125 with Aβ, or pre-incubated in ginsenoside Rg1 then co-incu-bated in Aβ. The p-JNK, JNK, caspase-3 activity and TUNEL-positive cells were detected. Results In Aβ1-42 treated group, the ratio of p-JNK/JNK level was increased more than that in non-treated group for 15 min. However, in neurons preincubated with (2. 5, 5, 10 μmol·L-1 ) ginsenoside Rg1 and then co-incuba-ted with 5 μmol·L-1 oligomeric Aβ1-42 , the p-JNK/JNK ratio, caspase-3 activity and TUNEL positive neu-rons were significantly decreased compared with those of Aβ1-42 treated group. Conclusion Ginsenoside Rg1 can attenuate the oligomeric Aβ1-42-induced apop-tosis by JNK pathway.