ObjectiveTo study the change of autophagy of human pancreatic cancer cell MiaPaCa-2 before and after Ad5-KAI1 tranfection,and to investigate the possible mechanism.MethodsThe MiaPaCa-2 cells without KAI1 expression were infected with Ad5-KAI1 with KAI1 target gene,and Ad5-null was used as negative control,and parental cell was used as blank control.The formation of autophagosomes was observed by electromicroscopy.The green fluorescent protein-labeled light chain 3 (LC3) associations with autophagosome membranes was detected by confocal microscopy.PD98059,LY294002 were applied to pre-treat the cells.The expression levels of beclin 1,AKT,ERK,the phosphorylation of AKT and ERK protein and the ratio of LC3-Ⅱ to LC3- Ⅰ were detected by Western blotting.ResultsAfter 100 MOI Ad5-KAI1 infections for 24 h,the rate of cell expressing KAI1 protein reached (84.97 ±8.56)％,number of LC3 increased from 4 to 20; and swelling,degeneration of mitochondria was observed,and bilayer-like structure in cytoplasm was found.The expression of beclinl increased (1.4 ±0.3 ) folds,and the expression of LC3-Ⅱ/LC3- Ⅰ increased (8.00 ±2.78) folds.PI3K blockade LY294002 pretreatment significantly suppressed the phosphorylation of AKT of MiaPaCa-2 (2.756 vs 1.516),but it did not inhibit the increase of ratio of LC3-Ⅱ to LC3- Ⅰ(0.770 vs 1.403).ERK blockade PD98059 pretreatment not only significantly suppressed the phosphorylation of ERK of MiaPaCa-2 ( 1.637 vs 0.403 ),but also inhibit the up-regulation of beclin 1 protein expression ( 2.377 vs 1.150) and increase of ratio of LC3- Ⅱ to LC3- Ⅰ (2.225 vs 0.680).ConclusionsKAI1 can significantly induce autophagy of human pancreatic cell line MiaPaCa-2 through phosphorylation of ERK rather than AKT.

Objective To explore the pathway of KAI1 induced autophagy regulating apoptosis in human pancreatic cancer cell line MiaPaCa-2.Methods There were three groups in the experiment,which were extracellular regulated protein kinases (ERK) phosphorylation inhibitor PD98059 pretreated group,Caspase-3 activation inhibitor VAD-FMK pretreated group and no PD98059 or VAD-FMK pretreated groups.And each group was divided into three sub groups with different treatment,which were adenovirus AD5-null vector infected control group,the human KAI1 gene recombinant adenovirus vector AD5 KAI1 infected group and autophagy inhibitor 3-MA pretreated and AD5-KAI1 infected group.The cell apoptosis was observed by AnnexinV-FITC/PI double staining.Caspase-3 activation level was evaluated by flow cytometry.ERK phosphorylation and poly(ADPribose) polymerase (PARP) cleavage were determined by Western blot.Results After the cancer cells infected with AD5 KAI1,KAI1 protein was expressed and GFP-LC3 green particles increased.Caspase-3 activation,PARP cleavage,ERK phosphorylation and apoptosis increased obviously.After autophagy inhibitor 3-MA pretreated,the percentage of apoptosis increased from (63.0 ± 7.9)％ to (88.0±4.5) ％ and Caspase-3 activation increased from (34.0±2.8) ％ to (44.2±4.0) ％ and PARP cleavage more.The apoptosis induced by 3-MA could be totally inhibited by Caspase-3 activation inhibitor VAD-FMK pretreated but could not be inhibited by ERK phosphorylation inhibitor PD98059.Conclusion KAI1- induced autophagy inhibits apoptosis through the downregulation of Caspase-3activation and PARP cleavage instead of ERK phosphorylation.

[Objective] To investigate the effectivity and reliability of individualized method to localize the entrance point of the lumbar pedicle screws by vertebral plate borderline.[Methods]Four vertebra specimen were taken the X-ray film of anterior-posterior views as the center of L2.On the X-ray film,the distance between the centre-point of the pedicle to the lateral and superior margins of the vertebral plate were measured,after applied barium at the centre-point of the pedicle,the specimen were taken the X-ray film once more,the relation of the barium to the centre-point of the pedicle was measured;the distance between the centre-point of the pedicle to the lateral and superior margins of the vertebral plate were measured on the normal lumbar X-ray film in 107 cases,the data in different sex and different projection 's center were compared;the method were applied to 40 patients of lumbar pedicle internal fixation,the information in hospital were reviewed to 30 patients of the lumbar pedicle screw localization by transverse process and transverse process.[Results]All(except L5)of lumbar plate borderline were displayed clear in the experiment,accurate rate of setpoint to be 92.5%.The principal X-ray affected scarely to the relation of the pedicle with the plate borderline of nearby two segment,but affected obviously far from three segment on the normal lumbar X-ray film;the accurate rate of one time 's setpoint in clinical application was 96%,average operation 's time being 96 minute,average lose blood being 212 ml,transverse articular process 's group showed significant difference.[Conclusion]Individualized localization of the lumbar pedicle screw entrance points by vertebral plate borderline is one of feasible method,displayed less trauma and accurate rate highly in the clinical application.

[Objective]To evaluate the radiographic and clinical results of locking compression plate and interlocking intramedullary nail in the treatment of distal tibial shaft fracture(4 to 11cm proximal to tibiotalar joint).[Method]A total of 65 cases of distal tibia shaft fractures were retrospectively reviewed from 2003 to 2007.Thirty-seven cases were treated with locking compression plate as group A,and 28 cases were treated with interlocking intramedullary nail as group B.According to AO classification system,32 cases were type A,12 were type B,21 were type C.[Result]All cases were followed up for 12-24 months(mean,16.5 months).Five cases were found delayed or nonunion,with 1 case in group A and 4 cases in group B.One case in group A and 2 cases in group B were found developed deep infection.Two cases in group A and 8 cases in group B received second operation.The good-to-excellent rates of ankle joint function were 91.9% in group A and 71.4% in group B.The good-to-excellent rates of knee joint function were 97.3% in group A and 78.6% in group B.[Conclusion]Distal tibial shaft fracture could be treated successfully with either percutaneous plates or intramedullary nails.Percutaneous locking plate is better than intramedullary nail in aspect of prevention of delayed union,malunion and second operation.

Objective To evaluate urinary vascular endothelial growth factor (VEGF) in the diagnosis of bladder cancer. Methods VEGF in urine was measured by enzyme linked immunosorbent assay in 33 cases of transitional cell bladder carcinoma,10 benign urological conditions and in 10 normal individuals. Results The mean urinary VEGF level in 33 patients with bladder cancer was ( 309.8 ? 86.6 )ng/g Cr, significantly higher than in the 20 controls which was (107.6?35.4)ng/g Cr ( P

Objective:To produce and identify antiCK20 monoclonal antibody.Methods:Lymphocytes from the spleen of mice being immunonized by CK20 antigen were fused with the myeloma cell line(SP2/0) using PEG4000.Hybridodma cells were established by selective growth of the fusion cells in the HT medium,and the presence of antiCK20 antibody was screened by inderect ELISA,and the clonality was achieved by limiting dilution.We have incubated cloning cells into mouse abdominal cavity to produce ascitic fluid contained monoclonal antibody.Chromatography with SPA-Sepharose CL-4B affinity column were emploied to isolate the monoclonal antibody from ascitic fluid.Finally,the antibody were tested the activities and sentivities,isoforms and titer through Western blot,two directions immuning diffusion of agar and ELISA.Results:Only one hybridoma cell line,secreating McAb against CK20,had been established.The modal number of chromosome is 101(99-103).The results of identifications showed that the antibodies kept high activitis and sensitivitis in detecting sample.The titer of ascitic fluid and the McAb purified are 1∶10~6 equally.The immunoglobulin of the McAb is classified as IgG1.Conclusion:AntiCK20 monoclonal antibody have been produced succesfully with high sensitive and active and was named L20031030.

Objective To discuss the application values of image reconstructive technique in 16-slice helical CT scanning. Methods 32 cervical spinal cord lesion images which have processed in MPR, MIP and VR were reviewed to discover the best image reconstructive form. Results By grading, reconstruction score of MPR, MIP and VR was 4.75, 1.75 and 4.5, MPR and VR in top. Conclusion 16-slice CT scanning with MPR and VR image reconstructive of cervical spinal cord can be satisfied the clinical demands.

Objective To study the effect of PcG member NSPc1 on proliferation of HeLa cells.Methods Using bioinfomatic analysis to design the siRNA sequence to knockdown NSPc1.Detecting the expression level of NSPc1 in HeLa cell line using semi-quantitative RT-PCR,Real-time PCR and Western blot after transfection of the designed siRNA.Transient transfecting pSUPER-NSPc1 into Hela cells and performing BrdU incorporation assay.Establishing NSPc1 stably knockdown cell line,comparing proliferation abilities with the control cells.Results(1)The designed siRNA did efficiently knockdown the expression of NSPc1;(2)Transient knockdown of NSPc1 could repress BrdU incorporation;(3) The established NSPc1-knockdown cell lines had a significantly lower proliferation rate than that of control cells.Conclusion The expression of NSPc1 is necessary for the normal proliferation of HeLa cells.The NSPc1 stably knockdown cell pool is a useful model for further study of pathway related to NSPc1.

OBJECTIVE: To investigate the clinical outcomes of artificial bone (hydroxyapatite) and bone cement implantation in treating young patients with avascular necrosis of the femoral head caused by different reasons. METHODS: A total of 18 patients (23 hips) with Ficat stage Ⅲ and Ⅱ avascular necrosis of the femoral head were treated with dead bone debridement, artificial bone and cement implantation in Department of Orthopedics, Third Affiliated Hospital of Guangxi Traditional Chinese Medical College from October 2003 to July 2008. The patients were followed up for 24.6 months (ranging 3 months to 5 years). The affected hip function was evaluated by modified Merled'Aubigne Scores and X-ray. RESULTS: Mean hip scores improved significantly from 11.65 to 15.09 after surgery. Postoperative radiographs demonstrated the improved collapse and restoration of femoral head sphericity. The femoral heads of most patients remained the appearance after surgery, and the necrosis range did not enlarge. The patients were satisfactory to the treatment results. CONCLUSION: Artificial bone and cement implantation could restore head sphericity and prevent further collapse. The method can be used as an alternative for treatment of avascular necrosis of femoral head at Ficat stage Ⅱ and Ⅲ, especially for young patients.

ObjectiveTo investigate the effects of heterogeneous phosphatase and tensinhomologue deleted on chromosome ten (PTEN) on cell cycles,proliferation,invasion,tumorigenicity,metastasis and the expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR)proteins in human pancreas cancer cell line ( ASPC-1 ).MethodsASPC-1 cells was transfected with plasmid pE-PTEN containing PTEN,and empty plasmid pE-PTEN transfection was used as control,then ASPC-1-pE-PTEN (A-pE-P) cell and ASPC-1-pE (A-pE) cell was obtained.The expression of PTEN mRNA was determined by RT-PCR. PTEN,VEGF and EGFR proteins were measured by cell immunohistochemical method.Clone formation assay was used to observe the numbers of clone.Transwell was used to test the invasion ability of cells.The growths of tumor were detected by nude mice subcutaneous injection of cancer cells in vivo.Results Compared with ASPC-1,the expressions of PTEN mRNA of A-pE-P increased by 179.3％,and the expressions of PTEN protein were also significantly increased.The expressions of VEGF protein were significantly decreased.The expressions of EGFR protein were not significantly changed.Number of G2/M phase cells was significantly increased from (26.81 ± 1.03)％ to (31.5 ± 1.76)％ (P ＜0.05).The numbers of clone was decreased by 28％ (P ＜0.05).The number of penetrating cells was decreased[(46.3 ±6.6) vs (63.8 ±7.5) per high power field,P ＜0.05].The tumor volumes were significantly reduced [(142.4 ±30.9) vs (202.7 ±43.6) mm3,P ＜0.05].The tumor inhibitory rate was 42.4％.The distant metastases were significantly reduced [(2.0 ±0.7) vs (5.0 ± 1.3),P ＜0.01 ].Conclusions Heterogeneous PTEN can not only inhibit the proliferation,invasion and metantasis of ASPC-1 cells,arrest the cell growth at G2/M phase,but also decrease the expressions of VEGF.