ObjectiveTo investigate the distribution and expression of y-secretase subunit (APH-1)in the central nervous system (CNS) of APP/PS1 double transgenic Alzheimer's disease (AD) adult mouse model,and to detect the expression difference of APH-1 in developmental brain between AD model mouse and wild-type littermates in order to further clarify the relationship between APH-1 and AD. MethodsOffspring bred by APP/PS1 double transgenic AD mice were genotyped.Immunohistochemical staining was used to detect APH-1 distribution and expression in the CNS of adult APP/PS1 double transgenic AD mouse model,in the brain of AD model mouse and its wild-type littermates on postnatal day 1,7,21 and 120.Results APH-1 was widely expressed in almost all regions of the CNS,especially in the cerebral cortex,hippocampus,olfactory bulb,hypothalamus,ventral striatum,caudate putamen,raphe magnus nucleus,cerebellum,brainstem and spinal cord of the adult APP/PS1 double transgenic mice.APH-1 expression was higher in the cortex of both AD and wild type mouse on postnatal day 1 than on postnatal day 7 and 21 with increased level of APH-1 protein in adult mouse brain.APH-1 expression in the brain of AD mice was higher than in its wild type littermates at any stage(P＜0.05).Conclusions Distribution of APH-1 is ubiquitous and region-dependent in the CNS.The different distribution and expression between APP/PS1 double transgenic mouse model and its wild type littermate indicate that APH-1 may be related to AD.

AIM: To explore the possibility that proteasome is involved in nicastrin(NCT) degradation and NCT is ubiquitinated before degradation.METHODS: Following the generation of NCT stable cell lines,the methods of Western blotting,pulse-chase metabolic labeling technique,double immunofluorescent staining,combined with proteasomal inhibition were used to investigate the NCT expression in NCT stable cell line.RESULTS: Treatment of the cells with proteasomal inhibitors significantly increased both endogenous NCT(produced by the cell itself) and exogenous NCT(produced by the gene transfection) in SH-SY5Y cells.The effect of specific proteasomal inhibitor lactacystin on NCT expression was in time-and dose-dependent manners.Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radio-labeled nicastrin protein was blocked by lactacystin.The results of double immunofluorescent staining showed that NCT and ubiquitin were co-located in the cells.CONCLUSION: The proteasome is involved in the degradation of NCT in neuronal cells,and NCT is ubiquitinated before degradation.

Objective To investigate whether valproic acid (VPA) affect spatial learning memory and senile plaques in the APP/PS1 double transgenic AD mouse model of different gender. MethodsTwenty 3-month old APP/PS1 double transgenic AD mice,male and female mouse evenly,were randomly divided into VPA-treated and saline-treated groups ( 10 for each group). 30 mg· kg-1 · d-1 of VPA and the same amount of saline were peritoneally injected into mice for 4 weeks. Morris water maze was conducted to check the effect of VPA on the capability of spatial learning and memory of AD mouse model. Immunohistochemical staining was used to examine the effect of VPA on the morphological changes in the brains of mice. ResultsVisible platform test showed that VPA-treated and saline-treated mice had similar escape latency (P＞0.05) and path length (P＞0.05) ,the swimming speed between male and female mice had no difference (P＞0.05). Hidden platform test showed that VPA treated mice had a significantly shorter latency (P＜0.01) and path length (P＜0.01) to reach the platform compared with saline-treated mice. Meanwhile, both in VPA-treated and control groups, the male mice had a shorter correlation escape latency and path-length than female mice had(P＜0.05 ). Immuohistochemical staining showed that the number (11.23±3.78) of senile plaques (SP) in the cerebral cortex and hippocampus of VPA-treated male mice were notably decreased than that(28.17 ±3.46) in the control group ( t= 14.67, P＜0.01 ),furthermore,the number of SP in the cerebral cortex and hippocampus of VPA-treated male mice was significantly reduced,as compared with which (20.36 ±4.21)in the VPA-treated female mice(P＜0.05). ConclusionVPA can significantly lower formation of SP, and remarkably improve the capability of spatial learning and memory of APP/PS1 transgenic mice,which have gender difference.

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.

Objective To examine the changes of Aβ expression and its related metabolic enzymes in the brains of AD and T2DM mice, so as to explore the possible mechanism of type 2 diabetes mellitus combined with AD.Methods Five-month-old APP/PS1 double transgenic mice, ob/ob mice and the wild-type control mice were employed in this study.Immunohistochemical (IHC) staining, Elisa and Western blot were used to detect SP, Aβ and its related metabolic enzymes.Results A certain number of SPs were observed in the cerebral cortex and hip-pocampus of APP/PS1 mice;SPs were occasionally observed in the cortex of ob/ob mice, while no SP appeared in wild-type mice.Aβ40 and Aβ42 levels were significantly increased in APP/PS1 and ob/ob mice brains as compared with controls (P<0.05), thought both Aβ40 and Aβ42 levels in AD mice were significantly higher than those of ob/ob mice (P<0.05).APP expression level was highest in APP/PS1 mice among 3 groups, and its expressed higher in ob/ob mice than that of control mice (P<0.05).BACE1 expression was notably increased in APP/PS1 and ob/ob mice as compared with control(P<0.05), however, it expressed higher in APP/PS1 mice than ob/ob mice (P<0.05).The expression of Aβ degradation enzyme IDE was reduced in APP/PS1 and ob/ob mice(P<0.05), while lowest in ob/ob mice.Conclusions Overexpression of Aβ may be one of main reasons for T2DM combined AD.

Objective To investigate the changes of dopamine-?-hydroxylase(DBH) and activator protein 2-?(AP-2?) expression in spinal cord under the condition of stress or pain stimulation,so as to explore the mechanism for changes of noradrenergic(NA) neurons in the spinal cord of rat pain model.Methods Immunohistochemical staining,double immunofluorescent staining,Western blotting and computing-image analysis system were used to detect the changes of DBH/AP-2? expression in the spinal cord of formalin-induced rat pain model.Results A small number of DBH-positive neurons were sparsely distributed in the ventral horn of the normal spinal cord,while in the formalin-treated group,much more darkly-stained DBH-positive neurons appeared primarily in the ventral horn,intermediate zone,and the dorsal horn,which reached the highest level on day 3 after formalin-injection.The grey value and number of DBH-positive neurons on day 7 after injection began to decrease,but still higher than that in the control group.Compared with control group,the number of noradrenergic neurons in spinal cord of formalin-treated rat was increased significantly,which was also confirmed by Western blotting.Double immunofluorescent staining showed that DBH and AP-2? co-existed in the cells of the spinal cord.The changes of AP-2? expression were similarly to that of DBH in the spinal cord of rat pain model.Conclusion Our results indicated that some non-noradrenergic neurons with different chemical properties might convert into noradrenergic neurons under pain stimulation;noradrenaline may be involved in the formalin-induced pain and behavior regulation;As one of transcription factors,AP-2? may promote the DBH synthesis.

Objectives To investigate whether degradation of anterior pharynx decfective-1(Aph-1) goes through proteasomal pathway or lysosomal pathway.Methods Various methods such as cell culture,Western blotting,pulse-chase metabolic labeling technique,double immunofluoresecnt staining,combined with proteasomal and lysosomal inhibition were used to check Aph-1 expression level in stable Aph-1-transfected or non-transfected neuronal(SH-SY5Y)cell line.Results Using Western blotting,treating the neuronal cells with proteasome specific inhibitors significantly increased the expression of both endogenous and exogenous Aph-1.The effect of the proteasome inhibitors on Aph-1 expression was dose-and time-dependent Lysosomal pathway was not involved in Aph-1 degradation. Pulse-chase metabolic labeling experiment showed that the turnover of newly-synthesized radiolabeled Aph-1 protein was blocked by Lactacystin.Double immunofluorescent staining revealed colocalization of Aph-1 and ubiquitin in the same cells.Conclusion Degradation of Aph-1 protein is mediated by proteasomal pathway in neuronal cells,and is not related to lysosomal pathway.Aph-1 protein is ubiquitinated before degradation.

Objective To explore the role of transcription factor activator protein 2?(AP-2?) on the expression of tyrosine hydroxylase(TH) in noradrenergic neurons of locus ceruleus(LC) after traumatic brain injury.Methods Oinety rats were!randomly divided into control group and injury group that comprised 5 subgroups.There were 15 rats in each group.The rat model of traumatic brain injury was established with Feeney's methods.Brain stems were dissected from decapitated heads 1,3,6,24 and 72 h after traumatic injury and freeze-mounted for cryo-sectioning.TH and AP-2? expressions in noradrenergic neurons of LC were analyzed with double immunofluorescence.Results The number and fluorescence intensity of TH and AP-2?-positive neurons in LC at various intervals after injury significantly increased as compared with control group(P

Objective: To study the chemical constituents in the roots of Aconitum iochanicum Ulbr.Methods: The air-dried roots of A.iochanicum were powdered and extracted by methanol with a percolation method.After removing the solvent under reduced pressure, the crude extract was dissolved in1.5% HCl solution, and then extracted by ecetic ether.The acidic solution was basified to pH 9.0 by NaOH (5%) and extracted with ethyl acetate to obtain crude alkaloidal extract after the removal of ethyl acetate.The compounds were isolated and purified by column chromatography and identified based on spectral analysis (1H-NMR, 13C-NMR and MS).Results: Totally 18 compounds were isolated from A.iochanicum and characterized as 14-O-acetylsachaconitine (1), franchetine (2), crassicaudine (3), indaconoitine (4), 14-benzoyl chasmanine (5), 14-O-acetyltalatisamine (6), talatisamine (7), chasmannine (8), crassicauline A (9), bikhaconine (10), 13,15-dideoxyaconitine (11), crassicautine (12), kongboensine (13), liljestrandisine (14), ludaconitine (15), 8-deacetyl-yunaconitine (16), yunaconitine (17) and ouvrardiantine (18).Conclusion: It''s the first time to study the chemical constituents of A.iochanicum, and 18 diterpenoid alkaloids are isolated.

This paper proposed the idea of building the applied anatomical experiment and research platform for surgical postgraduates with professional degree,establishing double-tutorial system and applying applied anatomical teaching in basis course learning,clinical skill training and research capacity cultivating after analyzing the reasons of poor applied anatomical background of surgical postgraduates with professional degree.These ideas were intended to improve the cultivation quality.