Objective:To construct the highly efficient expression recombinant adenovirus that expresses elafin protein for further study.Methods:The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV. The linearized shuttle plasmid was homogenously recombined with pAdEasy-1 in BJ5183. The candidate clone was further analyzed by restriction endonuclease digestion, PCR, and sequence scanned. Then the recombined adenovirus plasmid was digested with Pac Ⅰ and transfected into 293 cells for packaging and amplifying. Infection titer and rate were monitored by green fluorescent protein(GFP) expression. The expression of elafin protein was detected by Western blot and ELISA to appraise the function of elafin protein for oppressing the activity of elastic protease.Results:Digestion with restriction endonuclease, PCR, Western blot and ELISA confirmed that elafin gene was cloned into the adenovirus vector successfully.Conclusion:The recombined adenovirus Ad-elafin is constructed successfully, which will be benefit for future study.

Objective To construct the recombinant adenovirus for nature antibiotic elafin protein and observe its expression in primary airway epithelia.Methods The elafin gene was amplified by RT-PCR from lung tissues,then the fragment was inserted into the multiple clone site of pAdTrack-CMV.The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183.The candidate clone was further analyzed by restriction endonuclease digestion,PCR,and sequencing.Then the recombinant adenovirus plasmid was digested with PacⅠ and transfected into 293 cells for packaging and amplification.Infection titer and rate were monitored by green fluorescent protein(GFP)expression.Finally the primary airway epithelium was infected with Ad-elafin,and the GFP expression was observed by fluorescence microscope in epithelial cells 24 h after transfection.The elafin protein was detected by ELISA in the supernatant.Results Restriction endonuclease and PCR confirmed that elafin gene was cloned into the adenovirus vector successfully.Twenty-four hours after transfection,the GFP expression was seen by fluorescence microscopy.The concentration of elafin protein was(2.2?0.4)ng/ml in supernatant of transfected group,while that of control group was(1.9?0.3)ng/ml,P

BACKGROUND: Multiple applications of opium medicines can induce the accommodative changes of morphology and function in some intracerebral nerve positions. These accommodative changes are important neurobiological bases inducing drug-desire and re-addiction after detoxification. However, the actual molecular mechanism is unclear at present.OBJECTIVE: To investigate the impacts of the generation of heroin-dependence and detoxification on brain-derived neurotrophic factor (BDNF) in rat to provide a laboratorial gist for the participation of BDNS in heroin-dependence and detoxification.DESIGN: A randomized controlled study by employing experimental animals as subjectsSETTING: Mental health center of a medical university affiliated hospital MATERIALS: The study was conducted in the Laboratory of Pharmacology,Faculty of Pharmacology, Chongqing Medical University between March 2004and July 2004. Totally 30 inbreeding clean male SD rats with a bodymass between 200 g and 250 g were obtained from the Experimental Animal Center of the Third Military Medical University of Chinese PLA. Rats were randomly divided into blank control group(control group), heroin-dependent group (heroin group), and naloxone detoxification group(naloxone group) with 10rats each.METHODS: Morphine was subcutaneously injected into the rat with dose-increasing method to establish heroin-dependence rat model. Rats of naloxone group received subcutaneously injection of 2 mg/kg of naloxone to excite abstinent symptoms. The same dose of normal saline (NS) was injected in rats of control group. Model rats of each group were observed biologically and behaviorally. BDNF expression at different brain zone of rats in three different groups was tested with immunohistochemistry and digoxin-labeled oligonucleoide probe in situ hybridization technique.Comparison of the evaluation of abstinent symptoms in rats of each group.RESULTS: In the heroin group, the relative content of BDNF protein was higher in frontal lobe cortex, locus caeruleus and hippocampus than that of the control group( P ＜ 0.05); BDNFmRNA relative content was higher in frontal lobe cortex than that of the control group( P ＜ 0. 05) . In naloxone group, BDNF and its mRNA relative contents in frontal lobe cortex, locus caeruleus and hippocampus were higher than that of heroin group and control group ( P ＜ 0.05 ).CONCLUSION: Chronic administration of heroin could affect BDNF protein and its mRNA expressions in the corresponding brain areas of the rats, which suggests that the change of BDNF expression participates in heroin-dependence and detoxification.

BACKGROUND: Recent studies have proven the existence of the regeneration of central nervous tissue. But abjective results,such as lacked of neurogenesis after injury,also have been found in many experiments. The greatest difficulty in conventional brain transplantation or brain tissue transplantation has been the survival and development of the graft. Additionally,the stability of therapeutic effects and the rehabilitation of brain functions also need confirmation.OBJECTIVE: To investigate an approach to intrathecal injection of neural stem cells(NSCs) in stroke therapy,and observe therapeutic effects and side effects as well,so as to make the evaluation of the safety and feasibility.DESIGN: A confirmative before-after study based on stroke patients.SETTING: A neurology department in a municipal hospital and a microbiology and immunology department affiliated to a university hospital.PARTICIPANTS: From November 2002 to September 2003,26 stroke inpatients in the Neurology Department of Anyang Municipal People' s Hospital were selected. Of all the c ases,3 were diagnosed as acute cerebral hemorrhage,and the other 23 had been suffering strokes for durations ranging from 3months to 30 years,an average of (4.2 ± 6. 6) years. They were 20 male and 6female between the ages from 36 -72 years old,an average of(56.3 ± 12.7)years old. Fifteen of them were ischemic and 11 were hemorrhagic. Nineteen were associated with hypertension,2 with coronary heart disease,4 with diabetes and 4 associated with hyperlipodemia.INTERVENTIONS: On each of the 3 patients with acute cerebral hemorrhage (hematoma volume,35 - 40 mL),a microinvasive intracerebral hematoma puncture was performed,and then a suspension of NSCs were conducted to the stroke by a drainage tube. For the rest of the patients,suspensions were intrathecally administered into the subarachnoid and then flowed to the cerebral surface through cerebrospinal fluid(CSF) circulation. Afterwards,physical therapy(PT),occupational therapy (OT) and speech therapy(ST) were jointly applied to facilitate the rehabilitation of the stroke patients. Therapeutic effects was calculated according to the European stroke scale(ESS) and the Barthel Index(BI) . If ESS index went beyond or equal to 1 score,the case would be defined as effective; otherwise,it would be defined as not effective. Additionally,CT,MRI,EEG,chest x-ray,and blood biochemical variables were also measured.MAIN OUTCOMEMEASURES: Therapeutic effects and side effects were taken as main outcomemeasurements.RESULTS: Of the 23 patients who ntrathecal administration,19had positive therapeutic effect and 4 did not. Post-transplantation ESS was higher than that of pre-transplantation(54. 1 ±21.2 vs 51.4 ±21.1,t = 5.8,P = 0. 000 007 6),while post-transplantation BI also increased significantly as compared with that of pre-transplantation(41.1 ± 31.3 vs 36. 1 ± 32. 1,vasive intracerebral hematoma puncture had successful rehabilitation and regained self-care ability. Of all the patients,4 got a transitory fever and 2felt slight post-operation headache.CONCLUSION: Conclusion can be drawn from the study that stroke patients are ameliorated to various extents after neural stem cell transplantation which has no toxicity or side effects. It shows that neural stem cell transplantation is viable and feasible in improving the motor function and self-care ability in stroke patients.

Objective To evaluate the feasibility and safety of the modified panceaticojejunostomy called Kissing panceaticojejunostomy by animal experiment,and to explore the pathological process of primary healing between the pancreatic duct and the jejunal mucous.Methods The tolerance pressure at the anastomotic intestine was measured during surgery to ensure close contact suture of Kissing panceaticojejunostomy.Exploratory laparotomy,pancreatic ductography and pathology was performed at 2,4 weeks after panceaticojejunostomy.Results Kissing panceaticojejunostomy was performed successfully on 6 Beagle dogs.Tolerance pressure in all dogs was 90 cm H2O and above.No bleeding、fistula or contrast extravasation was found,and healing anastomosis was identified by laparotomy and pancreatic ductography.Cicatricial healing was found between pancreatic duct and jejunal mucosa 4 weeks after anastomosis.Conclusions Kissing panceaticojejunostomy is easy to perform,ensuring close-up cicatricial healing without hydrops or abscess between pancreatic duct and jejunal mucosa.

Objective To observe the application of endoscopy combined with 125I seed implantation in the pancreatic cancer palliative surgery in elderly patients.Methods A total of 78 elderly patients with pancreatic cancer who can not be treated with radical operation were divided into observation group and control group (n=39 each group).All patients were treated with stent intervention.Patients in the ation group were treated with 125I seed implantation guided by CT or ultrasonangraphy additionally.The clinical symptoms were compared between groups.The survival time and complications of the patients were recorded during the follow-up.Results The clinical symptoms of patients in both two groups were greatly improved (x2 =0.06,P＞0.05).Compared with the control group,the pain remission was better and the incidence of pain caused by pancreatic cancer was lower in the observation group (46.2％ vs.76.9％,x2 =7.80,P＜0.05).There was a significant difference in the average of survival time between the observation group and control group [(13.7±3.4) months vs.(8.4±3.8) months,t=3.73,P＜0.01],but no statistical difference was found in the long-term survival rate between groups.There was no significant difference in the incidence of complications between the two groups (10.3％ vs.7.7％,x2=0.15,P＞0.05).Conclusions Endoscopy combined with 125Ⅰ seed implantation is an effective treatment for elderly patients with advanced pancreatic cancer,which can effectively relieve the clinical symptoms,proiong the survival time,reduce the pain and complications.

Objective:To investigate if in vitro chemotherapy can induce the EMT progress in gastric cancer (GC) cells. Method:The GC cell line, SGC7901, was treated using 5-Fu at a concentration of 30 μg/mL. The residual cells after four cycles of 5-Fu therapy were named as SGC7901/Fu. The morphological changes and malignant biological features, including the invasiveness and clone formation ability and the characteristics of cancer stem cell and biomarkers of EMT between SGC7901 and SGC7901/Fu, were compared. Results:The SGC7901/Fu cells displayed a mesenchymal appearance, decreased the expression of epithelial markers, and increased the expression of mesenchymal markers. The 50% inhibitory concentrations in the SGC7901/Fu and SGC7901 cells were (43.8 ± 7.2) and (64.6 ± 5.5)μg/mL, respectively. The number of cells that migrated through the basement-membrane of the Transwell chamber was 51.4 ± 8.7 and 93.2 ± 9.5, respectively. The rate of clone formation was 5.2%± 1.0%and 13.2%± 2.2%, respectively. The portions of the CD44+/CD24-cells were 4.13%±0.81%and 7.97%±0.50%, respectively. All differences were statistically significant (P<0.05). Conclusion:The residual GC cells underwent EMT progress after 5-Fu treatment, with increased chemoresistance and ability of invasiveness and acquired the property of cancer stem cells.

Objective:To establish a highly sensitive method by immuno PCR for measurement of GH in Urine.Methods:Sandwich ELISA was operated in PCR tubes which treated with glutarakdehyde,and PCR was tested in the same PCR tube.The PCR production were electrophoresis in Agarose gel and Scan for gel.Results:The results show that ELISA could be correctly reacted in PCR tubes which treated with glutarakdehyde,more than 0.1 pg/ml GH could be detected.Results of Scan showed that the GH values in urine overnight have significantly deference between 30 normal children and 10 GHD patients which were diagnosed by GH provocative test.Conclusion:The sensitivity of Immuno PCR is 10 3～10 4 times higher than that of ELISA,Immuno PCR could be used in detection of GH in urine of children.

Objective To evaluate the technique of transpancreatic septum precut for cannulation of inaccessible common bile duct in endoscopic retrograde cholangiopancreatography (ERCP). Methods Data of 109 patients with difficult biliary cannulation in ERCP, of whom 56 underwent transpancreatic septum precut and 53 had needle-knife sphincterotomy from January 2006 to July 2008, were analyzed retrospectively, and the success rate of cannulation and the occurrence of complications were compared between the two methods. Results Of 109 patients accepted precut papillotomy, common bile duct cannulation was successfully achieved in 97. The success rates of transpancreatic septum precut group and needle-knife sphincterotomy group were 96.4% (54/56) and 81.1% (43/53) respectively, which was significantly different (P<0.05). Complications occurred in 11 cases, including bleeding(n =4), acute pancreatitis(n=5), cholangitis(n=2). The tolal frequency of complications of the transpancreatic septum pre-cut papillotomy group was lower than that of needle-knife sphincterotomy group(3.6% vs. 17.0%, P<0.05). Conclusion In patients with inaccessible bile ducts, transpancreatic septum precut is a safe and effective procedure in cannulation, exhibiting a higher success rate and lower occurrence of complication when compared with needle-knife sphincterotomy.

Objective To explore the manipulation,feasibility and safety of laparoscopy assisted natural orifice translumenal endoscopic surgery(NOTES).Methods The technique was performed in 4 female porcine models.Diagnostic laparoscopy was performed first,and followed by transgastric access through the anterior gastric wall under the monitor of laparoscopy.The stoma was extended with the dilation ballon and the endoscope was sent to the abodominal cavity.Such procedures as endoscopic peritoneoseopy,fallopian tube ligation,oophorectomy,eholeeysteetomy and closure of gastrostomy were performed.The drainage tube and antibiotics of 3 days were used after operation according to the bleeding and potential risk of fistula and peritonitis.The animals were feeded on a semiliquid diet.Two weeks later,routine gastroseopy was pedbrmed to examine the healing of luminal incision.Subsequently,all models were sacrificed for the evaluation of the validity of NOTES.Results With the assistance of the laparoscope,endoscopic peritoneoseopy and,fallopian tube ligation,oophorectomy and cholecystectomy,twice for each,were successfully performed in all models.Abdominal drainage were used in two animals'after eholecystectomy.All porcines survived for 2 weeks postoperatively with no weight loss.Autopsic examination showed good healing of transgastric incisions,with little adhesion,no massive heinan'hage,abcesses,or injury to adjacent organs.Conclusion Combined laparoscopy and endoscopy for NOTES is feasible with relative safety.Laparoscopic assistance could facilitate the NOTES procedures at the present stage.