Objective To construct and identify the incompetent-replication adenovirus carrying the fusiongene composed of the encoding gene of prepropeptide of mouse nerve growth factor(PN)and human beta-endorphin(?-EP)geue.Methods The gene segments of PN obtained from total RNA of the submandibular glandof a 2-week old Kumning mouse were amplified by RT-PCR and joined with the segment of ?-EP to form the fusiongene which was sequenced.The fusion gene contained in the incompetent-replication adenovirus was formed in theBJ-Ad Easy-1 susceptible cells and identified by PCR so as to choose the positive clone without wild vectors.Thecorrect clone was amplified and purified.The titers of adenovirus were determined using the specific 50% tissueculture infection dosage(TCID 50)method.Three days after the adenovirns was transferred into the cultured A431cells,RT-PCR was performed to showed the transcribed mRNA of this fusion gene and the intracellular ?-EPexpression was quanlitatively detected by inummo-histological method.Finally the concentration of human ?-EP inthe culture medium was determined by quantitative radio-immunoassay on 1st,3rd and 7th day afterinfeetion.Results The sequence of the fusion gene was correct.The titer of recombinant adenovirus Ad-NEP was1.5?10~10 pfu/ml.Three days after infection a 475 bp segment was amplified by RT-PCR and abundant orangegranules were shown in the infected cell.The ?-EP concentration in the culture medium was significantly higher inAd-NEP group than in the control group on 1st,3rd and 7th day(P

Objective To evaluate the reliability and accuracy of three-dimensional digital fusion anatomy in the preoperative evaluation and therapeutic strategy choice of intracranial tumors.Methods MRI scan,including regular MRI,MRA,MRV and DTI,were performed in 87 case.Then tumor themselves as well as tumor-associated structures were reconstructed and fused through iPlan 2.6 software.Based on the reconstructed images,therapeutic strategy were established,preoperative and intraoperative imags were compared.Results The digital reconstruction were successfully finished in all cases.Meanwhile,digital images,originally radiological images and actual images matched well.No approach-associated complication were met in our series.Among tumors in the convexity,the relationship of the reflux veins and the tumors were divided into three types:anterior(12 cases),posterior(19 cases) and overriding(3 cases).All of the relationships were seen in the preoperative fusion image,and the veins were all effectively protected during operation.The tumor-associated arteries could be pushed or wraped by the tumors,and the three-dimensional fusion image could provided their virtually aberrant pathway as well as their relationship with tumor.During microsurgical managment of tumors in the deep brain parenchyma,safe approach were found with the help of comprehensive understanding of the tumors and their adjacent structures.Conclusion Three-dimensional digital fusion anatomy can vividly and accurately display full rang of information about the tumor,and facilitate tumor treatment safely.

OBJECTIVETo investigate the clinical features, surgical transcranial approaches and outcomes of giant pituitary adenomas.

METHODSA series of 112 consecutive cases of giant pituitary adenomas underwent microsurgery through transcranial approaches at People' s Liberation Army General Hospital were retrospectively analyzed. Of the 112 patients, 58 were male and 54 were female, with age ranging from 3 to 72 years(mean age 44. 3 years). There were 91 non-functioning adenomas and 21 hormone-secreting adenomas. The maximum tumor diameter varied from 4. 0 to 7. 2 cm, with mean diameter of 4. 8 cm. Unilateral subfrontal approach was chosen in 16 cases, pterional approach in 41 cases, anterior interhemispheric approach in 34 cases, transcallosal-interforniceal approach in 6 cases, transcortical- transventricular approach in 5 cases, combined approach in 6 cases and other approaches in 4 cases. Postoperative MRI and endocrine function were re-examined routinely to evaluate the therapeutic efficacy. Staged operation through transsphenoidal approach or adjuvant treatments including medical and radiation therapies were administered in patients with hormone-secreting adenomas when hormonal excess persisted after surgery and in patients with non-functioning adenomas who had postoperative MRI evidence of residual tumor.

RESULTSTotal removal of the lesion was achieved in 57 cases (50. 9%) , 26 patients (23. 2%) underwent subtotal resection, and 29 patients (25. 9%) underwent partial removal. Postoperative mortality occurred in 3 patients (2. 7%). Major surgical morbidity occurred in 38 patients (33. 9%). Vision was preserved or improved in 98 patients (87. 5%). The postsurgical follow-up period varied from 3 to 64 months (mean 19. 5 months) . Nineteen of the 21 patients with hormone-secreting adenomas were considered to be in hormonal remission, and 87. 2% of the cases were capable of normal work and life and 8 patients suffered recurrence during the followed-up period.

CONCLUSIONSSelection of appropriate transcranial approaches is the key to successful microsurgery for giant pituitary adenomas according to the morphological characteristics presented in image examinations and clinical symptoms. Staged transsphenoidal operation and/or adjuvant therapies including medical and radiation therapies offer the best chances to control the residual tumors after the maximal surgical removal of giant adenomas through transcranial approaches.

Adenoma ; surgery ; Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; Combined Modality Therapy ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Microsurgery ; methods ; Middle Aged ; Neurosurgical Procedures ; methods ; Pituitary Neoplasms ; surgery ; Postoperative Period ; Retrospective Studies ; Young Adult

Adult ; Aged ; Aged, 80 and over ; China ; epidemiology ; Female ; Helicobacter Infections ; epidemiology ; Helicobacter pylori ; Hospitalization ; Humans ; Inpatients ; statistics & numerical data ; Leprosy ; epidemiology ; microbiology ; Male ; Middle Aged

Objective To investigate the effects of integrin β1 gene expression inhibited by short hairpin RNA (shRNA) on invasion of pancreatic carcinoma PANC1 cells in vitro,and investigate the mechanism.Methods The eukaryotic expression plasmid of shRNA targeting integrin β1 gene ( integrin β1 shRNA) and control eukaryotic expression plasmid shRNA (c-shRNA) was constructed and was transfected into PANC1 cells.The cells without plasmid transfection were used as control.The expressions of integrinβ1,MMP 2,MMP 9 mRNA and protein were detected by real-time PCR and Western blotting.The invasive ability of PANC1 cells was observed with Transwell cell culture chamber.Results Integrinβ1 mRNA expressions in integrinβ1 shRNA group,c-shRNA group and control group were 0.0029 ± 0.0004,0.0131 ± 0.0009,0.0138 ± 0.0005 ; the expressions of integrinβ1 protein were 0.0159 ± 0.0062,0.3215 ± 0.0126,0.3107 ±0.0094; the inhibitory rate of integrinβ1 mRNA and protein expression in integrinβ1 shRNA group was (78.6 ±7.2 ) ％ and (92.9 ± 3.2) ％ ( P ＜ 0.01 ).But there was no difference between the c-shRNA group and control group (P =0.2999).Number of penetrating cells in integrinβ1 shRNA group decreased from 52 ±5 to 21 ±4( P ＜ 0.01 ) ; the expression of MMP 2 and MMP 9 mRNA decreased from 0.592 ± 0.073,0.847 ± 0.069 to 0.102 ± 0.034,0.273 ± 0.071 ; the expression of M MP2 and MMP 9 protein decreased from 0.225 ± 0.046,0.416 ±0.081 to 0.059 ±0.013,0.106 ±0.022(P ＜0.05).Conclusions Recombinant integrinβ1 shRNA expression plasmid can effectively inhibit the expression of integrinβ1 gene and suppress the invasion of PANC1 cells in vitro by down-regulating MMP 2 and MMP 9 gene expression.

OBJECTIVE: To evaluate the applicative value of multimodal navigation combined with intraoperative magnetic resonance imaging system in the treatment of complex lesions of middle skull base. METHOD: Nineteen consecutive patients undergoing complex lesions resection using multimodal microscopic navigation combined with iMRI were included. Preoperative radiological images were imported into navigation planning system, based on which approach and microsurgical window were designed. Transcranial approach and extracranial approach(include trans-oral-nasal-sphenoidal approach and transsphenoidal approach) were performed in our series. After presumptively total resection were finished, intraoperative magnetic resonance (iMRI)were performed, followed by navigation images updating, and continuing resection if necessary. RESULT: iMRI scan were performed from 1 time to 2 times. In 5 cases with residual seen on iMRI scan, continued resection were carried on in 2 of them. There was no iMRI-associated complications. CONCLUSION Microscopic-based neuronavigation, in combination with intraoperative magnetic resonance, could provide objective basis for resection, and improve the safety level of tumor resection.
Adult ; Aged ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Microsurgery ; methods ; Middle Aged ; Neurosurgical Procedures ; Skull Base ; surgery

OBJECTIVETo study the expressions changes of gastric mucosal hypoxia-inducible factor-1alpha (HIF-1alpha) and downstream molecules [such as vascular endothelial growth factor (VEGF) and cyclooxygenase-2 (COX-2)] of verrucous gastritis (VG) patients of different Chinese medicine (CM) syndrome types and their clinical significance.

METHODSTotally 94 VG patients were assigned to Gan-Wei disharmony group (GWDG, 28 cases), the damp-heat in Pi-Wei group (DHPWG, 17 cases), the blood stasis in Wei-collateral group (BSWCG, 20 cases), and the insufficiency of Pi-yang group (IPYG, 29 cases). Another 30 patients with chronic mild non-active superficial gastritis patients accompanied with negative Hp infection were recruited as the control group. The Hp infection was detected using 14C-labeled urea breath test. The expressions of HIF-1alpha, VEGF, and COX-2 in the gastric mucosal tissue were detected using immunohistochemical EnVision two-step test.

RESULTSThe positive Hp infection rate in VG patients was 37.23% (35/94 cases). The positive Hp infection rate in patients of DHPWG (76. 47%) was significantly higher than the other three groups (32.14% in GWDG, 31.03% in IPYG, and 20.00% in BSWCG, P < 0.01). The expressions of HIF-1alpha and COX-2 in VG patients of different syndrome types were obviously higher than those of the control group (P < 0.01, P < 0.05). Of them, the expression of HIF-1alpha was the highest in BSWCG and the expression of COX-2 was the highest in DHPWG. The expression of VEGF was higher in DHPWG and IPYG than in the control group and the GWDG (P < 0.01, P < 0.05).

CONCLUSIONSThe expressions of HIF-1alpha, VEGF, COX-2, and Hp infection showed certain changes in VG patients of different syndrome types. The expression of HIF-1alpha was the strongest in BSWCG. The expressions of VEGF and COX-2 as well as Hp infection were the highest in DHPWG. All showed the specificity of CM syndromes.

Adult ; Aged ; Cyclooxygenase 2 ; metabolism ; Female ; Gastric Mucosa ; metabolism ; pathology ; Gastritis ; diagnosis ; metabolism ; pathology ; Helicobacter Infections ; diagnosis ; metabolism ; pathology ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Medicine, Chinese Traditional ; Middle Aged ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

OBJECTIVETo investigate the feasibility of real-time fluorescent quantitative (qPCR) assay in detecting mycobacterium tuberculosis complex (MTB) in paraffin embedded tissues for diagnostic purpose.

METHODSUsing qPCR assay, 1000 consecutive formalin-fixed and paraffin embedded (FFPE) tissues (from 2011 to 2012) suspected of MTB infection were tested by amplifying the MTB specific insertion sequence 6110 (IS6110). The specificity of the PCR product was confirmed by Sanger sequencing as compared with the MTB genomic DNA of the IS6110 sequence. Tissues with Ziehl-Neelsen acid-fast staining were used as control.

RESULTSIn the 1000 samples, 513 were positive for mycobacterium by Ziehl-Neelsen acid-fast staining (detection rate 51.3%); whereas 546 were MTB positive by qPCR assay (detection rate 54.6%). Concordance rate for both assays was 73.1%. The diagnosis rate increased by 14.4% by combinination of Ziehl-Neelsen acid-fast staining and qPCR results. More interestingly, by analyzing the Ziehl-Neelsen acid-fast staining and qPCR results three cases of M.leprae infection and four cases of non-tuberculous Mycobacterium (NTM) infection were identified.

CONCLUSIONSqPCR detection of MTB in FFPE tissue is more sensitive than Ziehl-Neelsen acid-fast staining assay. Combination of these two assays can increase the detection rate and also identify some rare cases of NTM infection.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Child ; DNA, Bacterial ; genetics ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Paraffin Embedding ; Real-Time Polymerase Chain Reaction ; Sequence Analysis, DNA ; Staining and Labeling ; methods ; Tuberculosis ; diagnosis ; microbiology ; Tuberculosis, Gastrointestinal ; diagnosis ; microbiology ; Tuberculosis, Lymph Node ; diagnosis ; microbiology ; Tuberculosis, Pulmonary ; diagnosis ; microbiology ; Young Adult

OBJECTIVETo investigate the clinical outcomes of anterior corpectomy combined with anterior intervertebral decompression and fusion for multilevel cervical spondylotic myelopathy.

METHODSThe clinical data of 28 patients with multilevel cervical spondylotic myelopathy who underwent surgery from October 2012 to June 2014 were retrospectively analyzed. There were 18 males and 10 females, aged from 45 to 77 years old with an average of (60.11±9.37) years. Three levels were involved in 27 cases, while four levels were involved in 1 case. The preoperative JOA score was 8.89±1.87; the fusion segments angles was (4.87±4.56)°; and the cervical curvature was (11.68±1.25)°. Anterior hybrid decompression and fusion were performed in 28 patients. The fusion segments angles and the cervical curvature were assessed by X-rays at 1, 12 months after operation, respectively. JOA score was used to evaluate the clinical effect.

RESULTSThe operative time was 163 min on average (ranged from 120 to 205 min), and intraoperative bleeding was 198 ml on average(ranged from 100 to 300 ml). Hoarseness occurred in 1 case and got recovery at 3 weeks after operation and choke cough occurred in 1 case, and got improvement at 1 week after operation. All the patients were regularly followed for 12-24 months with an average of(18.46±3.20) months. Graft bone obtained fusion at 12 months after operation and the position of internal fixation was good. The fusion segments angles, the cervical curvature and JOA scores were significantly improved at 1, 12 months after operation(<0.05). The improvement rate of JOA score was(46.46±20.26)% at 12 months after operation, 12 cases got excellent results, 14 good and 2 fair.

CONCLUSIONSAnterior corpectomy combined with anterior intervertebral decompression and fusion is safe and effective and can get satisfactory effects for multilevel cervical spondylotic myelopathy.

OBJECTIVETo explicit whether the expression of the mineral-related proteins is regulated by cbfa1 in human dental papilla cells.

METHODSHuman dental papilla cells were cultured in vitro and transfected with pcDNA3-cbfa1 recombinant plasmids. After selected with G418 sulfate, a cell clone named PC-3, which could stably express the cbfa1 mRNA and protein, was proved by PCR and western blot. Then the amount of ALP and OC and the expression of OPN, BSP, ON, DMP1, DSP and DSPP were detected by immunohistochemistry, Western blot and PCR methods.

RESULTSWe established the human dental papilla cells model PC-3 which could stably express the cbfa1 mRNA and protein. Compared with normal cells, a lot of mineral-related proteins such as ALP, OC, OPN, BSP, ON, DMP1 were upregulated in PC-3 cells.

CONCLUSIONSIn human dental papilla cells, cbfa1 can induce the expression of most mineral-related genes and proteins. It may implicate that cbfa1 must play a key role during tooth development and mineralization.

Alkaline Phosphatase ; biosynthesis ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; physiology ; Dental Papilla ; cytology ; metabolism ; Humans ; Osteocalcin ; biosynthesis