BACKGROUND: Cell division cycle 2 (cdc2) plays an important role in the course of neuronal degeneration in neurodegenerative diseases such as Alzheimer's disease. The silencing of cdc2 gene with adeno-associated virus vector might protect the neurons in neurodegenerative diseases.OBJECTIVE: To pack recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. DESIGN, TIME AND SETTING: Blank control experiment was performed in the laboratory of Department of Neurology at Wuhan Tongji Hospital between October 2007 and August 2008. MATERIALS: Helper Free adeno-associated virus system (pAAV-MCS-EGFP, p-RC, p-Helper) and AAV-293 cells were purchased from Stratagene. METHODS: The pAAV-MCS-U6-cdc2-siRNA-EGFP expressing plasmid was constructed by molecular biological techniques. The cotransfection of this plasmid, together with p-RC and p-Helper into AAV-293 calls was mediated by calcium acid phosphate. The rAAV encoding cdc2-siRNA (rAAV-U6-cdc2-siRNA-EGFP) was packed and harvested. The silencing effect of this virus on cdc2 gene expression in AAV-293 cells was assessed by Western Blot, and its titer was determined by spot hybridization method. MAIN OUTCOME MEASURES: sequencing of pAAV-MCS-U6-cdc2-siRNA-EGFP plasmid inserting U6-cdc2-siRNA; 3 plasmid pAAV-MCS-U6-cdc2-siRNA-EGFP, p-RC, p-Helper cotransfecting AAV-293 cells; cdc2 expression after rAAV-U6-cdc2-siRNA-EGFP infected AAV-293 cells.RESULTS: ①The successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP plasmid was proved by DNA sequencing. ②Green fluorescent protein was expressed in AAV-293 cells, and the contransfection was working well. ③The cdc2 gene expression in AAV-293 cells was down-regulated markedly after infection of rAAV-U6-cdc2-siRNA-EGFP. ④The titer of rAAV-U6-cdc2-siRNA-EGFP was 1×10<'12> v.g/mL.CONCLUSION: The package of rAAV encoding cdc2-siRNA is successful. It can silence cdc2 gene effectively.

Aluminum is a common metal element,and its nervous toxicity probably has an effect on the learning and memory mechanism. Long-term potentiation (LTP) is an available electrophysiology-model,cAMP/protein kinase A (PKA) approach is an important signal transduction pathway of nerve cells,and plays an important role in the process of learning and memory mechanism. Aluminum has some adverse effects on learning and memory through influencing some links,such as neurotransmitter,Ca2 + concentration,protein kinase and so on. According to the recent studies in this field,in electrophysiology and biochemistry,the present paper reviewed the effects of aluminum on the learning and memory mechanism,as well as the role of cAMP/PKA approach.

Objective:To evaluate the effectiveness and treatment feature of maxillary incisor extraction in orthodontics.Methods:9 patients underwent orthodontic treatment with maxillary incisor extraction,5 female and 4 male,with an average age of 1 7.2 years at the start,were included.5 patients were treated by extraction of both upper incisors and lower first premolars,4 by extraction of the abnor-mal incisor and the first premolars in the other three quadrants.Cephalometric and Bolton index analysis were carried out.Results:Sat-isfactory treatment results were observed in all patients.Before treatment the predicted Bolton index of the patients of the anterior ratio and the overall ratio were 80.1 4% and 91 .3%,after orthodontic treatment 78.68% and 90.28%,respectively.Cephalometric analysis showed that U1 -NA(mm),U1 -NA,L1 -NB(mm),L1 -NB,U1 -SN,L1 -MP,UL-E and LL-E were decreased(P <0.05).Conclu-sion:Individual treatment plan based on Bolton index analysis and the corresponding techniques and methods,the patients with abnor-mal upper incisors can be effectively treated with the extraction of maxillary incisors.

BACKGROUND: As a main gene engineering vector, adeno-associated virus (AAV) is characterized by its extensive host cells, lasting and stable expression and less immune response to hosts, and is applied widely. But AAV is a kind of defective virus, and need incasing cells to supply E1 protein. As important and special AAV incasing cells, AAV-293 cells can produce E1 in trans. But AAV-293 cells are delicated and cultivated difficultly, and the biological character is easy to be changed. Therefore, it is necessary to establish a culture method of AAV-293 cells to meet the need of gene engineering.OBJECTIVE: To establish a culture method of AAV-293 cells in vitro.DESIGN: An opening study.SETTING: Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology.MATERIALS: AAV-293 cells line was provided by Stratagene Corporation; high-carbohydrate OMEM (H-DMEM) powder by Gibco Company; there plasmids in AAV Helper-Free by Stratagene Company.METHODS: This experiment was carried out in the neurology laboratory of Tongji Hospital in Wuhan during the period from October 2006 to April 2007. AAV-293 cells were resuscitated and cultivated with H-DMEM growth medium in vitro, and were passaged and stored in liquid nitrogen when the cells monolayer confluence reached 50%. At the same time, their growing state was observed by inverted microscope, and their growth curve was noted. According to whether AAV-293 cells could give out green fluorescence or not (observed by fluorescence inverted microscope) after they were cotransfected with the there AAV system plasmids and infected with AAV supernatant, their biological character of packing AAV was assessed.MAIN OUTCOME MEASURES: ① Morphological observation of AAV-293 cells; ② the growth curve; ③ the package of AAV.RESULTS: ① AAV-293 cells observed by fluorescence inverted microscope were growing adhesively well with irregular polygons, light endochylemas and ambiguous nuclei appearances. ②The growth curve showed that the growing adaptive phase was the first day after AAV-293 cells were passaged, the actively growing phase was from the second day to the fifth day, and the growing platform phase was after the sixth day. ③ AAV-293 cells with green fluorescence observed by fluorescence inverted microscope, and cotransfection of the there AAV system plasmids was successful. AAV-293 cells gave out green fluorescence after infected with AAV supernatant, and AAV package succeeded.CONCLUSION: The culture method established by the authors in the experiment is simple and useful, and the cultured AAV-293 cells remain a good function of AAV package.

Endothelium-derived hyperpolarizing factor (EDHF) is the third factor released by en-dothelial cell other than NO and PGI2. It relaxes smooth muscle accompanied by a hyperpolarization in the membrane potential. EDHF may be epoxye-icosatrienoic acids (EETs) formed from arachidon-ic acid by the action of cytochrome P450. It is synthesized and/or released by endothelial cell as a result of an cytosolic Ca2+ increase, which is stimulated by the action of acetylcholine or bradykinin on endothelial cell. EDHF is shown to activate Ca2+-activated K+ channels and induce a hyperpolarization in the membrane potential in vascular smooth muscle. The hyperpolarization of the membrane inhibits the opening of voltage-dependentcalcium channels, allows calcium sequestration and removal mechanisms to lower intracellular calcium, and leads smooth muscle to relaxation. In large conducing arteries, EDHF may provide a secondary system to NO, which assumes primary importance in endothelium-dependent relaxation and inhibits the release of EDHF. However, in small resistance arteries, EDHF appears to be a major determinant of vascular calibre, and may be of primary importance in the regulation of vascular resistance.

Objective To discuss a method to show the structure of small mammary gland well by computed radiography(CR).Methods By analyzing 150 radiographs of mammary glands,the results showed that the mammary structure showed well whether or not was depended on the size of mammary gland.Using 24 steps aluminum ladder mimiced the small mammary gland and radiography was taken with various parameters and different imaging plate(IP) shading ways,then processed and printed,after that it was measured by density instrument.Results The display of grey scale of 24 steps aluminum ladder was improved if shading 10~15 mm margin of the IP by collimator or shading the central by a square cuprum plate with 22 mm length and 0.2 mm thickness.Radiography of small mammary gland by the same way was performed and the mammary structure was also showed well.Conclusion Shading the exposing areas properly by collimator or cuprum plate during CR can apparently improve the display of small mammary structure.

Objective To evaluate the indications and effectiveness of lower incisor extraction in orthodontics.Methods Twenty-three adult patients with lower incisor extraction were included in the study.The cases consisted of 14 female and 9 male patients with an average age of 23.2 years at the start.One single lower incisor was extracted in 17 patients and the other tow upper premolars were extracted in 6 patients.Cephalometric and casts analyses were carried out.Results Total treatment time was 13-23 months.All extraction space was closed after the treatment.All patients achieved satisfactory treatment results,with normal overbite and overjet and stable occlusal relationship.There were no significant changes before and after treatment on cephalometric analysis.The intercanine width was reduced by from pretreatment to posttreatment [(26.89--2.89) mm vs (23.92-t-1.54) mm (P＜0.05)].Conclusions The intercanine width is reduced after lower incisor extraction.In order to achieve satisfactory clinical effect,we should carefully select cases and pay attention to the corresponding techniques and methods.

Objective To explore the clinical value when treating cervical cancer of supplementing radiotherapy with hyperbaric oxygen treatment. Methods Fifty-eight cervical cancer patients were randomly divided into 2 groups; the HBO group (n=28) received radiotherapy combined with hyperbaric oxygen treatment; a control group (n = 30) received only radiotherapy. Results The complete response rate (CR) of the HBO group was 75.0% , the partial response rate (PR) was 7.1% ,the effective rate(CR + PR) was 92.8% , the total 3 year survival rate was 71.4% , the local recurrence rate was 10.7% , and all of these results were significantly different from the controls (P

Objective To investigate the effects of selenium-and zinc-enriched probiotics on the content of selenium and zinc in blood,antioxidation function and intestinal microflora in canine.Method Eight 18-month native canines,female and male in half,were randomly divided into the control and treatment groups on average.The control group received basal diet,the treatment group received basal diet supplemented with 2.0g selenium-and zinc-enriched probiotics everyday.To determine the experimental indices,the samples were collected on D0,D15 and D30,respectively.Results Compared with the control group,on D15,the content of selenium and zinc in blood,blood GPX,serum SOD,T-AOC and the amount of Lactobacillus in the experimental group were significantly increased,while the amount of Escherichia coli significantly decreased,but the serum MDA and the amount of Bifidobacterium,Staphylococcus and Enterococcus had no significant change.On D30,the content of selenium in blood,serum SOD,T-AOC and the amount of Lactobacillus were very significantly increased,while the content of zinc in blood,blood GPX and the amount of Bifidobacterium significantly increased;but serum MDA and the amount of Escherichia coli,Staphylococcus and Enterococcus very significantly decreased.Conclusion Selenium-and zinc-enriched probiotics could increase content of selenium and zinc in blood,enhance antioxidation function,improve and regulate the intestinal microflora.

In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 (cdk-5) inhibition in mice with Niemann-Pick disease type C (NPC) (npc(-/-)), recombinant adeno-associated virus (rAAV) carrying the small interfering RNA (siRNA) specific for cdk-5 gene was injected into 3-day-old npc(-/-) mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc(-/-) mice and non-surgery age-matched npc(-/-) mice were employed as controls (n=6-10/group). From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc(-/-) mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc(-/-) mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc(-/-) mice, and significantly attenuated the hyperphosphorylation of tau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.