BACKGROUND: The glial scar formation after spinal cord injury in mammals is the physical and chemical barriers for neural regeneration, and relieving or delaying glial scar formation can provide benefit conditions for the regeneration of injured spinal cord.OBJECTIVE: To design and screen short hairpin RNA (shRNA) interfere molecular targeting the gene coding region of glial fibrillary acidic protein (GFAP) in rat, and reconstruct the eukaryotic vector of shRNA.DESIGN: An observational animal experiment.SETTING: Department of Spine Surgery, Shenzhen Hospital affiliated to Southern Medical University.MATERIALS: Twenty-five Wistar rats of clean degree, either male or female, weighing 20-25 g, were used. DMEM/F12,lipofectamine2000, Trizol RNA separating kits); fetal bovine serum (Hyclone); BamH Ⅰ, HindⅢ, Pstl, Sail and T4 ligases;Plasmid mini preparation kit and DNA gel extraction kit.METHODS: The experiments were carried out in the Shenzhen Hospital affiliated to Southern Medical University from October 2005 to June 2006. Three pairs of shRNA template which composed of 19 bp reverse repeated motif of GFAP target sequence with 9 bp spacer were designed and synthesized, then they were inserted directionally into plasmid Psilencer 2.1 respectively to generate small interfering RNA (siRNA) eukaryotic expression vector. ShRNA molecules were transfected by liposome via ex vivo expression repressive model of GFAP of rat spinal astrocytes. The effects of expressive suppression were detected with real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, and then the optimal shRNA eukaryotic vector of repressive expression of GFAP was screened.MAIN OUTCOME MEASURES: ① Interfering sequence specific shRNA template synthesis; ② Constructing specific recombinant plasmid eukaryotic expression vector. ③ Culturing rat spinal astrocytes in vitro; ④ Effects of expressive suppression on GFAP in primary astrocytes after RNA interference detected with real-time fluorescence quantitative RT-PCR and Western blot.RESULTS: Sequence analysis showed that GFAP-shRNA recombinant plasmid eukaryotic expression vector was successfully constructed, and optimal GFAP-shRNA eukaryotic vector was screened using real-time fluorescence quantitative RT-PCR and Western blot. The GFAP expressions were reduced by 81%, 63% and 56% at the levels of mRNA and protein respectively.CONCLUSION: GFAP-shRNA eukaryotic expression vectors were successfully constructed and screened. The gene expression GFAP of primitive rat astrocyte can be suppressed significantly by the GFAP-shRNA eukaryotic expression recombinant optimized via ex vivo cellular expression suppression model, which should pave the way for the following multiple targets of RNAi genetic manipulation in the treatment of suppression of glial scar formation after spinal cord injury.

Objective To investigate the effects of bortezomib on cellular migration and invasion ability in cuhured human prostatic cancer cells DU145 and its mechanism.Methods Transwell method was used to detect cell migration.Invasion was assayed by Matrigel-coated invasion chambers.Expressions of FAK and Tyr397 proteins were analyzed by Western blot.Results After treated by bortezomib at the concentration of 10,20 nmol/L for 24 h,the invasion index of DU145 cells were (69.05±10.56) and (52.55±6.98),they were gradually reduced compared with untreated group (81.55±10.56) (P ＜ 0.05).The migration index were (39.35±6.45),(32.05±4.22),they were also reduced compared with untreated group (58.75±5.41) (P ＜ 0.05).The group of treated by bortezomib showed Tyr397 protein expression had been suppressed.However FAK protein had not marked change.Conclusions FAK is involved in the regulation of cellular migration and invasion function.Bortezomib might inhibit cells migration and invasion function by down regulation of Tyr397 expression.

Objective To study the signal transduction pathway of norepinephrine (NE)-induced expressions of oncogenes c-fos and c-myc in the rat heart. Methods mRNA levels of c-fos and c-myc in the left ventricle of the Langendorff-perfused rat heart was quantitated by reverse transcription and polymerase chain reaction (RT-PCR). Results NE increased the expressions of c-fos and c-myc while the effects of NE on the expressions of c-fos and c-myc were blocked by ? 1-adrenergic blocker prazosin (5?10 -6 mol/L) completely. Neomycin (5 mmol/L) and ruthenium red (10 ?mol/L) decreased the NE-induced c-fos and c-myc mRNA levels. Conclusion NE can induce the expressions of oncogenes c-fos and c-myc in the ventricle mediated by ? 1-adrenergic receptor. The signal transduction pathway of NE-induced c-fos and c-myc expressions may be associated with inositol phosphates-Ca 2+ system and diacylglycerol-protein kinase C system.

Objective To analyse the relationship between the personality and occupation tension of anesthesiologists.Methods One hundred anesthesiologists working at different hospitals in Shenzhen volunteered to complete the questionnaire.Ninety-one valid questionaires were recovered.The questionnaire survey included the Eysenck Personality Questionnaire and Job Content Questionnaire and anesthesia events.The standard scores of internal and external factor,stubborn factor,emotional stability factor,social support,sense of personal achievement and work demand/control ratio (D/C) were calculated.Results Internal and external factor (E) and stubborn factor (P) were negatively correlated with demand/control ratio (D/C).The correlation coefficients were-0.7805and-0.2375.There was no correlation between emotional stability factor (N) and D/C.Social support and sense of personal achievement were negatively correlated with D/C.The correlation coefficients were-0.225 and -0.264.More than 50％ of the study subjects suffered from mental tension during op.eration and fatique after night shift,overtime work and emergency operation.Conclusion Occupation tension is lower in extroverted and higher in introverted anesthesiologists and is also relatively low in those with stubborn personality.Strong social support and deep sense of personal achievement can lower occupation tension.

BACKGROUND: It is demonstrated that bone marrow stem cells (BMSCs) can generate neurosphere structures, which is similar to cloning sphere of neuron-specific enolase (NSE), in a specially induced system in vitro; therefore, BMSCs draw more and more attention as seed cells to repair central nerve injury.OBJECTIVE: To investigate the differentiation from BMSCs into neuron-like calls induced by fetal spinal cord tissue.DESIGN: Observational study.SETTING: Department of Spine Surgery, Second People's Hospital of Shenzhen.MATERIALS: SD rats (16 pregnant days old and 2 months old) were provided by Animal Center, Tongji Medical College,Huazhong University of Science and Technology. Single antibody of NSE, multi-antibody of glial fibrillary acidic protein (GFAP) and single antibody of neurofilament (NF200) were provided by Wuhan Boster Company.METHODS: The experiment was carried out in Immune Opening Laboratory and Central Laboratory, Basic Medical Department, Tongji Medical College, Huazhong University of Science and Technology in September 2006. Bones of lower extremities of rats were collected to centrifuge BMSCs. Fetal spinal cord tissue homogenate was extracted from 16 pregnant days old rats to make inducing solution and induce differentiation of BMSCs. Otherwise, embryo muscle tissue was used to make muscle tissue homogenate as the same way so as to regard as the controls.MAIN OUTCOME MEASURES: BMSCs underwent morphological observation after induction; in addition, anti-NSE, NF200 and anti-GFAP were used to label neurons and astrocytes, respectively. Ten non-overlapping sights were randomly selected from positive reactive-induced cells after immunohistochemical staining under optic microscope to calculate ratio of positive cells of NSE and NF200 counting for total numbers of cells.RESULTS: ① Morphological changes of BMSCs after induction: During early induction, optic microscope indicated that soma of partial cells rebounded; whose apophysis was long and thin; apophysis of differentiated cells grew gradually and intercrossed each other. It was similar to nerve cells and some branches were similar to dendrite branches. However,morphological changes of cells in the control group were not obvious. ② Expression of relevant antigens differentiated from BMSCs into neuron-like cells at one week after induction: Most cells in spinal cord homogenate group expressed as positive NSE and NF200; a few of cells expressed as GFAP. While, positive staining of nerve cell antibody was not observed in the control group; meanwhile, positive reaction of nerve cell antigen was not observed in the control group,too. Immunohistochemistry examination demonstrated that positive rates of NSE and NF200 expressions were (68±1.7)%and (76.2±2.9)%, respectively.CONCLUSION: Fetal spinal cord tissue homogenate can induce differentiation from BMSCs into neuron-like cells.

Objective:To investigate the clinical significance of Her2 pathological expression in the breast and gastric cancer patients for providing a reference for cancer prevention. Methods: Selected surgical resection specimens of 60 diagnosed in advanced gastric cancer and 60 diagnosed with advanced breast cancer from March 2009 to May 2014 in our hospital,all specimens were given the Her2 pathological expression of immunohistochemical analysis and investigation the clinical and pathological data. Results: The Her2 positive expression rates in the breast cancer and gastric cancer specimens were 40. 0% and 36. 7%,respectively that compared to no significant difference (P>0. 05). The Her2 expression was not related to the cancer patient’s age,histological type,and there were sig-nificantly correlated to the TNM stage and lymph node metastasis ( P<0. 05 ) . Spearman correlation analysis showed that Her2 expression in the breast cancer and gastric cancer were significant positive correlation to the Survivin, Bcl-2 expression ( P<0. 05 ) . Conclusion:Breast cancer and gastric cancer patients have shown high pathological expression of Her2,and are related to the TNM stage and lymph node metastasis, it may be through inhibited the apoptosis regulating proteins involved in the pathogenesis and metastasis of tumors.

OBJECTIVEOptical coherence tomography images may be distorted by motion artifacts in dynamic imaging, so it may lead to misdiagnosis in clinical diagnosis. Motion artifacts correction has become an urgent issue in optical coherence tomography imaging.

METHODSFirstly, using the improved complex nonlinear diffusion preprocessing filtering reduced the noise of the sequence images, then using the image mass center aligned the distortion data in the Y direction, finally, using the method of deviation average corrected motion artifacts along the Y direction.

RESULTSAfter correction, the motion artifacts in the longitudinal 2D images and the 3D image disappeared, the surface of the 2D and the 3D image became more smooth, the structure between layers of the images got clear and distinct, retinal en-face single slice image was sharp, and the fundus tissue structure could be observed.

CONCLUSIONThe algorithm of correction makes the physical structure of the retinal display truly after motion artifacts correction.

Objective To investigate whether there has any difference of gastric and small bowel transit time and completion rates between two capsule endoscopes with different size and weight. Methods Clinical data of patients who had undergone OMOM or MiroCam (smaller and lighter than OMOM) capsule endoscopy were retrospectively studied. Comparison of gastric and small bowel transit time and completion rates were made between the two kinds of capsule endoscopy. Results 1, 448 patients (628 in OMOM group and 820 in MiroCam group) were finally includ-ed. In patients with Crohn's disease or suspected Crohn's disease, gastric transit time of OMOM was significantly longer than that of MiroCam [(53.4 ± 52.6) minutes vs (41.1 ± 47.9) minutes, = 0.022]. In patients with gastroin-testinal bleeding, gastric transit time in OMOM was significantly shorter than that in MiroCam [(42.1 ± 44.8) minutes vs (62.0 ± 78.6) minutes, = 0.016). No significant difference in small bowel transit time or completion rate was found. Conclusions We conclude that the differences of gastric transit time, small bowel transit time and completion rates between the two kinds of capsule endoscopy with different size and weight are not significantly. Whereas, in patients with Crohn's disease or suspected Crohn's disease, gastric transit time of smaller and lighter capsule en-doscopy is shorter in patients with gastrointestinal bleeding, but longer of gastric transit time in smaller and lighter capsule endoscopy.

Objective To study the signal transduction pathway for myocyte contraction induced by Substance K (SK) in rats. Methods The fluorescent Ca 2+ indicator Fluo 3AM was used to quantitate the calcium signal directly in the primary cultured myocardial cells. Changes of free Ca 2+ concentration in cardiac myocytes were detected by flow cytometry. The phospholipase C (PLC) inhibitor, neomycin and IP3 receptor antagonist, heparin, were used to block signal system of phosphates Ca 2+ in order to investigate whether they took part in the SK induced changes of [Ca 2+ ]i in myocytes. Results SK(1.78?10 -5 mol/L) elevated [Ca 2+ ]i in myocytes. The effects could be blocked by neomycin and heparin. Conclusion SK can elevate [Ca 2+ ]i in myocyte, which may be mediated by a signal system of phosphates Ca 2+ .

Objectives:Using IE 1 gene promoter of Autograph californica nuclear polyhedrosis virus(AcNPV),a transfer vector with an immediately early gene promoter was constructed.　Methods:Transfer vector pAcPIneo which contains neomycin resistance gene(neo)coding sequence downstream of IE 1-promoter was constructed and cotransfected with the wild type of AcNPV DNA into Sf9 insect cells.Recombinant virus was selected by G418 resistance since the neo gene can be expressed in Sf9 cells.　Results:Northern blot hybridzation with 32　P labeled neo gene fragement as probe showed that the neogene was integrated in plogene of AcNPV genome.　Conclusions:Transfer vector with an immediately early gene promoter of baculovirus was constructed successfully,the neo gene was transcribed from the immediately early phase to the very late phase in infected cells.