BACKGROUND: The neural stem cells (NSCs) will be clinically applied extensively in the future, and seeking of more promoting methods of the proliferation and differentiation of NSCs in vitro will be the study direction of NSCs.OBJECTIVE: To introduce an economic and time-saving method for NSCs proliferation.DESIGN: Observation and control experiment.SETTING: Beijing Neurosurgical Institute.MATERIALS: Four Wistar rats pregnant for 14 days with the body mass of (180±20) g (bought from Animal Department of Chinese Academyof Medical Sciences with the batch number of SCXK1100-0006 for experiment animals) were selected and fed at common temperature and humidity.The DMEM/F12 and B27 were bought from Gibco Corporation. The basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) were bought from PeproTech Company. The nidogen monoclonal antibody (MA),glial fibrillary acidic protein (GFAP) polyclonal antibody, galactocerebroside polyclonal antibody and microtubule-associated protein 2 (MAP2) were obtained from Chemicon Company. The fetal bovine serum (FCS) was provided by Hyclone company.METHODS: The experiment was conducted in the Department of Injury and Repair of Beijing Neurosurgical Institute from May to October 2004.Wistar rats were executed by dislocation and sterilized by putting into 75% alcohol. Four rats were used each time, the brain tissues of which were put into Hank's fluid. The cerebral pia mater and vessels were isolated under anatomical microscope, and the brain tissues were sheared off with eye scissors, which were filtered by 200 mesh copper grid and collected into 2 centrifuge tubes, and the supernatant was gotten rid off after that. ①Cells were divided into serum pre-culture group and control group according to whether there were serum pre-culture. The DMEM nutrient fluid of FCS (100 g/L) was added to serum pre-culture group, which was replaced by DMEM/F12 nutrient fluid of EGF, bFGF and B27 at 48 hours after culture. The cells in the control group were added with DMEM/F12 nutrient fluid of EGF, bFGF and B27 to culture in 5% CO2 incubator for one week at 37 ℃. The growth of NSCs at 48 hours after culture was observed in both groups under inverted contrast phase microscope.②On the 5th and 10th day after differentiation induced by 100 g/L FCS, nestin, GFAP, galactocerebroside and MAP2 were stained with immunofluorescence antibody,and the expressions of NSCs in both groups were observed under fluorescent microscope. The PBS buffer solution was used instead of first antibody in the control group, while other procedures were the same as above.MAIN OUTCOME MEASURES: ①Observation of NSCs growth at 48 hours after culture under contrast phase microscope in both groups. ②Detection of NSC specific antigen expressions with immunofluorescence stain in both groups.RESULTS: ①In the cells of the serum pre-culture group at 48 hours after culture, there were large number of regular NSC bulbs, most of which were aggregated by 8-10 cells. The proliferation of cell bulbs was accelerated after the nutrient fluid was replaced to DMEM/F12 nutrient fluid of EGF,bFGF and B27, while irregular lamellar cells could be seen in the control group at 48 hours after culture, and small regular cell bulbs were found at 4-5 days later. ②It could be seen under fluorescence microscope that on the 5th day after induced differentiation, the nestin, GFAP and galactocerebroside were positive, while MAP2 was negative. On the 10th day after induced differentiation, nestin and GFAP were positive, whereas the galactocerebroside and MAP2 were negative (no representation).CONCLUSION: Serum pre-culture can accelerate the bulb-aggregation of NSCs as well as promote the proliferation of NSCs.

Objective To evaluate the methods and results of revision surgery for posterior lumbar cage interbody fusion (cage-PLIF) with postoperative complications, and to analyse the surgical techniques for prevention of these complications. Methods From October 1996 to December 2002, 21 patients with postoperative complications of cage-PLIF underwent reoperations. There were 11 males and 10 females with an average of 43.4 years. The interval between primary and revision surgery ranged from 6 days to 1.5 years with an average of 0.6 year. 16 patients suffering from lumbar disc herniation were treated with the discecto-my and single uninstrumented cage fusion, 5 patients of lumbar spondylolisthesis were treated with cage-PLIF and pedicle screw instrumentation. The complications included cage displacement backward in 20 patients, forward in 1,and cage subsidence in 9 as well. 15 patients complained of low back pain wors-ening or leg radicular pain, of which 4 had intermittent claudication and 10 had leg numbness or weakness during rehabilitation. Revision surgery included re-implantation of the cage filled with iliac crest bone chips in 11 patients, iliac bone autograft after removal of original cages in 7 and decompression of involved nerve root witbout removal of migrated cage because of technical difficulty. Pedicle screw fixations were used in 12 and the intertransverse fusion both with autograft and allograft was added in 7. Results The mean follow-up was 14.2 months (ranged, 7 to 36 months). The cages presented slight retro-displacement in 4 patients shortly after reoperation, without involvement into spinal canal during the subsequent follow-up. Bony fusion occurred in 13 patients, and the pseudarthrosis in 3 patients without further migration of cages. The clinical symptoms relieved in 5 patients, improved in 9, no any change in 6, and worsened in 1. However, low back pain remained in 8 patients, and dysuria in 1 patiant at the last follow-up. Conclusion The results of revi-sion surgery are not satisfactory according to this study, the surgical treatments should be performed as soon as possible if conservative treatments is ineffective. The correct surgical indication and proper technical are the key of prevention of the postoperative complications.

Objective To compare the effects of 0.9% NaCl (normal saline, NS), 10% hydroxyethyl starch (HES, 200/0.5) and hypertonic-hyperoncotic solution (HHS, 7.5% NaCl-10% HES 1:1) on cerebral extracellular excitatory amino acids (EAA) and inhibitory amino acids (IAA) in a rat model of traumatic head injury (THI) combined with acute hemorrhagic shock (AHS). Methods Nineteen healthy adult male SD rats weighing 300-350 g were randomized into 3 groups: NS group (n = 7); HES group ( n = 6) and HHS group ( n = 6). THI was produced by modified Feeney method (a 20g weight drops from a height of 40 cm on the parietal region) and AHS was induced by modified Wiggers method (Blood was removed from femoral artery and MAP was maintained at 40 mm Hg for 1 hour). Fluid resuscitation was started at 1 hour of AHS with 0.9% NS (3 times the volume of the removed whole blood) or 10% HES (in a one to one ratio) or HHS 4 ml?kg-1 administered over 15 min. The extracellular fluid of injured brain tissue was collected using intracerebral microdialysis before head injury (baseline) during THI + AHS (1h) and resuscitatin (2h) for determination of the levels of EAA (glutamate, aspartate) and IAA (glycine, GABA, taurine) by HPLC.Results The 5 amino acids were significantly increased during THI + AHS as compared with their baseline values. Glutamate level was further increased during resuscitation with NS. GABA and taurine concentrations were maintained at high level during resuscitation with HES or HHS. The increase in glutamate was inhibited by resuscitation with HES and the increase in glutamate, aspartate and glycine were inhibited by HHS. Conclusions In a rat model of THI combined with AHS, resuscitation with NS can increase cerebral extracellular EAA. Both HES and HHS resuscitation can inhibit the increase in cerebral extracellulaar EAA and HHS is more effective.

Objective To analyze the diagnostic values of ultrasonography and computed tomography (CT)for gallbladder adenomyomatosis. Methods The ultrasound and CT findings of 28 cases of pathologically confirmed gallbladder adenomyomatosis in our hospital were retrospectively analyzed.The diagnostic values of the two imaging tools for gallbladder adenomyomatosis were analyzed with the pathological diagnosis as the gold standard.Comparison of rates was made by chi-square test;multiple comparisons of rates were made by partition of chi-square.Results Before operation,among the 28 patients,15 were diagnosed with gallbladder adenomyomatosis by ultrasonography,and 9 were diagnosed by CT;the diag-nostic rate of CT was 32.14%,and the diagnostic rate of ultrasonography was 53.57%.The chi-square test showed no difference between the di-agnostic rates of ultrasonography and CT for gallbladder adenomyomatosis (χ2 =2.63,P=0.10>0.05).In addition,the statistical results showed no differences between the diagnostic rates of ultrasonography and CT for various types of gallbladder adenomyomatosis (segmental type:χ2 =0,P=0.11>0.0125;diffuse type:χ2 =2.57,P=1.00>0.0125;focal type:χ2 =1.42,P=0.23>0.0125).Conclusion CT and ultrasonography are two important imaging methods for the diagnosis of gallbladder adenomyomatosis.The detection rate of gallbladder adenomyomatosis can be in-creased through the combination of convex array probe and linear array probe in ultrasonography.

[Objeetive] To develop the recombinant human bone morphogenetic protein-2(rhBMP-2)loaded amorphous calcium phosphate(ACP)delayed release nano-sized material,investigate its cytotoxicity of cell,and provide a reference for the experiment of composite material in vivo.[Method]The rhBMP-2/ACP delayed release nano-sized material were prepared by chemical wet method and cultured on rabbit bone marrow-derived mesenchymal stem cells(BMSCs)in vitro.Then the adhesion,proliferation,growth and functional expression of BMSCs were measured.[Result]Cytotoxicity test demonstrated that rhBMP-2/ACP delayed release nano-sized material had not affect the percentage of cell's proliferation with material-extracted liquid cultured with BMSCs and the cytotoxicity was graded zero.The adhesion,proliferation,configuration of the cells on the surface of this material were identical to the control group.[Conclusion]It was suggested that rhBMP-2/ACP delayed release nano-sized material might have good cellular biocompatibility,no cytotoxicity and not effected the normal functional expression of BMSCs in vitro.

[Objective]To study the biocompatibility and security of the recombinant human bone morphogenetic protein-2(rhBMP-2) loaded amorphous calcium phosphate(ACP) delayed release nano-sized material and investigate the feasibility of the clinical use as a kind of bone substitute in bone engineering.[Method]The in vitro hemolyzation,cytotoxicity,microkernel test in marrow smear,acute systemic toxicity,pyrogenicity,subcuticular stimulation reaction and short-term intramuscular implantation,as well as endosseous implantation were performed on the rhBMP-2/ACP delayed release nano-sized material.[Result]The material-extracted liquid induced no hemolyzation,no toxic effects of genetic,no pyrogenic reaction in rabbits,no cytotoxicity cultured in rabbit bone marrow-derived mesenchymal stem cells(BMSCs) in vitro and no acute toxicity in mice.The intramuscular implantation and endosseous implantation in rabbits induced no inflammatory reaction,tissue necrosis and the material was degraded gradually,fused organically with tissues.[Conclusion]The biocompatibility and security of the rhBMP-2/ACP delayed release nano-sized material could meet the requirements given in biological standards for implanted biomaterials ISO10993 and GB/T16886,suggesting that it could be a good bone substitution for the clinical trial.

Objectives:To study the effects of gene therapy with tissue type plasminogen activator(t PA)cDNA on the formation of thrombo embolism in vascular anastomotic sites. Methods:①The cDNA encoding t PA was amplified by RT PCR using the isolated total RNA as the template from the Bowes melanoma cells.②Recombinant plasmid pAdCMV t PA was cotransfected into 293 cells with pJMa 17 ,and the infectious but replication deficient AdCMV t PA was generated.③The rats were randomly divided into the control and treatment groups.11 0 nylone medical suture was applied to perform rat carotid artery end to end anastomoses.In the treatment group,AdCMV t PA solution was injected into the vascular anastomotic site while AdCMV (no containing t PA DNA) solution was injected into the control group. By means of RT PCR and chromogenic plasmin substrates,the following results were obtained. Results:①The t PA cDNA was successfully cloned and its eukaryotic expressing vector was constructed.②When the isolated RNA was performed with RT PCR,1.69 kb band appeared in the treatment group while the band could not be found in the control group.The t PA activity could be detected postoperatively on the 1st,2 nd,3 rd,4 th,5 th,6 th,7 th,10 th and 13 th day of the treatment,but could not be detected in the control group. Conclusions:The t PA gene can produce t PA having biological activity at anastomotic sites, possibly prevent the formation of thrombus embolism effectively and develop the anastomotic patency.

Third-class medical metrology station is being established in hospital for implementing national and military medical metrology law and regulations. The goal, significance and main functions are elaborated. It is suggested that the leader should take the initiative to grasp not only the overall design and the supplementary equipment construction, but the civil engineering technological design and the layout of metrology standard equipment. The general steps of establishing third-class medical metrology station are discussed.

BACKGROUND:Cerebrovascular drag, occlusion and other mechanical stimulations inevitably occur during some craniocerebral operations, which cause acute mechanical cerebrovascular vasospasm. At present, the mechanism underlying the patho-physiology as well as the pathological prognosis of this acute mechanical vasospasm remains unclear.OBJECTIVE: To observe changes in the vascular diameter of the middle cerebral artery, cerebral blood flow (CBF), and ultrastructure of vascular wall and vascular endothelial cells, during the early stage (2 hours) of mechanical cerebral vasospasm in cats.DESIGN: Open experiment.SETTING: Department of Neurology, Beijing Tiantan Hospital, Capital Medical University; Beijing Institute of Neurosurgery.MATERIALS: Six healthy adult hybrid cats, of either gender, weighing from 2.5 to 3.5 kg, were provided by the China Medical Science Institute of Experimental Animals. Laser Doppler flowmetry (Periflux 5010, Sweden Perimed Company)was used.METHODS: This study was carried out in the Beijing Institute of Neurosurgery between August 2005 and March 2006. For all experimental surgical procedures, the cats were anesthetized by intraperitoneal injection of 200 g/L chloral hydrate, at 2 mL/kg, and then placed in a prone position. A median incision was made in the scalp and a square bone window, 8×10 mm, was opened at 1.5 cm posterior and 1.5 lateral to the anterior fontanel, after which the dura mater was pricked out. The fine detecting head of the Laser Doppler flowmetry was fixed to a region of the cerebral surfacewith no vessels or with only a few vessels. Subsequently, the cats were placed in lateral position. Under the surgical microscope, the right middle cerebral artery was exposed through a suborbital approach. Blunt apparatus was used to stimulate middle the middle cerebral artery repeatedly, at a frequency of 100 time/min within 30 minutes.The diameter of the middle cerebral artery was measured and a perfusion index of cortical brain tissue was monitored, separately, before and then at 0, 0.5, 1.0, 1.5, 2.0 hours after stimulation. Ultrastructural changes in the vascular wall and the vascular endothelial cells were observed during the early stage (2 hours) of mechanical cerebral vasospasm in cats.MAIN OUTCOME MEASURES: Diameter of the middle cerebral artery and the perfusion index of cortical brain tissue before and then at 0, 0.5, 1.0, 1.5, 2.0 hours after stimulation, as well as any ultrastructural changes in the vascular wall and endothelial cells 2 hours after stimulation.RESULTS: The results from six cats were were analyzed. ①The diameter of the middle cerebral artery was (0.617±0.129), (0.723±0.082), (0.840±0.084) mm 0, 0.5 and 1.0 hours after stimulation, respectively, which was significantly smaller than that before stimulation [(0.897±0.066) mm,t =4.74, 4.017, 1.299,P ＜ 0.01]. ② The perfusion index of cortical brain tissue was 67.8±18.5, 82.5±17.5, 89.8±24.0, 94.0±22.2 and 98.5±21.0 at 0, 0.5, 1.0,1.5 and 2.0 hours after stimulation, which was significantly lower than that before stimulation (159.2±23.5, t =4.716-7.469, P ＜ 0.01 ). ③ At the early stage of acute mechanical stimulation (2 hours) to middle cerebral artery, endothelial cell chromatin aggregated at the edge of the cells and achromocyte formed, but mitochondrial crista was unclear.CONCLUSION: Mechanical stimulation to the middle cerebral artery in cats can lead to cerebral vasospasm. Apoptosis of endothelial cells appears at the early stage of stimulation (2 hours). These results indicate that, in order to prevent against cerebral vasospasm, cerebrovascular mechanical stimulation should be as minimal as possible and that as few as possible craniocerebral operations should be performed.

Objective To evaluate the accuracy of dual-source CT (DSCT) in diagnosing the degree of coronary artery stenosis,and compare it with coronary artery angiography (CAG).Methods One hundred patients with suspected coronary heart disease,clinical diagnosis of angina pectoris,myocardial infarction and patients after percutaneous coronary intervention,were given DSCT and CAG examination.The CAG results were considered as the reference standard,and the results of the two examinations were compared.The sensitivity,specificity,positive predictive value and negative predictive value of DSCT in diagnosing coronary artery stenosis (mild ＜ 50％,middle 50％-75％,severe ≥75％) were computed.Results CAG displayed coronary artery 1320 segments,DSCT displayed coronary artery 1287 segments,the appreciable rate was 97.5％.The sensitivity of DSCT in diagnosing mild,middle and severe coronary artery stenosis were 64.4％ (105/163),88.7％ (235/265) and 97.4％ (187/192),specificity all were 99.7％ (617/619),positive predictive value were 98.1％ (105/107),99.2％ (235/237) and 98.9％ (187/189),negative predictive valne were 91.4％ (617/675),95.4％ (617/647) and 99.2％ (617/622).There was significant correlation between DSCT and CAG in diagnosing coronary artery stenosis by Spearman test (P＜ 0.01).Conclusions The results of DSCT have high accordance with CAG in diagnosing coronary artery stenosis.It is a kind of non-invasive examination with low cost,and could reduce the pain of invasive examination.So DSCT is a good choice in diagnosing coronary artery disease.