Objective To clone and express the alkaline protease AprA , one important virulence factor secreted by Pseudomonas aeruginosa(PAE)in Escherichia coli, to clone and express the inhibitor of AprA (AprI) and its substrate flagellin , and to detect the function of AprA and the inhibitory function of AprI .Methods The genes encoding AprA ,AprI and flagellin gene were amplified respectively by PCR using PAE PAO 1 genome DNA as the template .The expression vec-tors (pET-28a-AprA, pET-28a-AprI and pET-28a-Flagellin) were constructed and transformed into E.coli BL21(DE3) respectively.The recombinant AprA protein was expressed by IPTG induction and purified via denaturing and renaturation. The recombinant AprI and flagellin were expressed and purified by Ni 2+affinity chromatography .The cleavage activities of AprA on flagellin were detected by SDS-PAGE.Results Recombinant AprA , AprI and flagellin protein were expressed and purified .It was demonstrated that AprA cleaved flagellin , which was blocked by AprI .Conclusion Recombinant AprA could cleave its substrates as an alkaline protease , and its inhibitor AprI inhibits the activities of AprA .This study will contribute to further investigations on the role of AprA in the pathogenesis of PAE .

Objective To investigate the effects of cryopreservation on the growth and osteogenesis capa- bility of human bone marrow stromal cells(BMSCs)on demineralized bene matrix(DBM).Methods Bone marrow aspirates were obtained from the lilac crests of three donors.The BMSCs were isolated from the bone marrow by density gradient centrifugation.Cells of passage 3 were cryopreserved in liquid nitrogen for 24 hours,and then re- covered.The non-cryopreserved BMSCs were used as the control,The cryopreserved and control BMSCs were cul- tured in osteogenic media,collected and labeled with Dil to be seeded onto the DBM when cells were confluent.The percentage of BMSCs adhered to the DMB was detected.The cell morphology and matrices secreted by BMSCs on the DBM were observed by the inverted phase-contrasted microscope,fluorescence microscope and scanning electron microscope(SEM).The growth and viability of BMSCs on the DBM were determined using the modified MTT ashy. The osteogenesis ability of BMSCs on the DBM was determined by assessment of the alkaline phosphatase(ALP) activity and osteocalcin(OCN)content.Results The percentages of the cryopreserved and control cells adhered to DBM were(97.25?1.17)% and(97.00?1.09)% respectively.The cells adhered well to the DBM and grew rapidly.Large amounts of matrices on the DBM were observed by the light microscope and SEM.The cells embedded in the matrices could be observed by fluorescence microscope.There were no significant differences in the assay values of MTT,ALP and OCN between the cryopreserved and control BMSCs on the DBM.Conclusion Since cryopreservation does not affect the growth and osteogenesis capability of BMSCs on DBM,the cryopreserved BMSCs can be used as a cell source in bone tissue engineering.

Objective To investigate the clinical application of a new thermosensitive embolic agent in interventional arterial embolization treatment, and to evaluate its effectiveness and safety in treating inoperable advanced hepatocellular carcinoma (HCC). Methods Prospective, open and single center clinical study of a new thermosensitive embolic agent was conducted. A total of 10 patients with clinically confirmed inoperable advanced HCC were treated with percutaneous arterial embolization using thermosensitive embolic agent; a maximum of 4 therapeutic cycles were performed until the disease progressed. Objective effect was evaluated at (30 ±7) days after the treatment according to mRECIST standard; the progression free survival (PFS) time and the survival period (OS) were recorded. The adverse reactions and adverse events were determined by NCI-CTC 3.0 version standard. Results Ten patients (23 target lesions in total) were enrolled in this study. After the treatment, complete remission (CR) was obtained in one patient, partial remission (PR) in 3 patients, stable disease (SD) in 5 patients, and progress disease (PD) in one patient. The objective response rate (CR+PR) was 40%, and the disease control rate (CR+PR+SD) was 90%. The PFS of hepatic lesions was 18.4 weeks (95%CI:14.15-22.65), the cumulated median survival period was 46.2 weeks (95%CI:38.18-54.22). The most common adverse reactions included pain, fever, hepatic function damage, small amount of ascites, diarrhea, etc. Conclusion The new thermosensitive embolic agent is not sticky to vessel and is visible under X-ray; it has reliable embolization effect, and the therapeutic results can be easily evaluated. This embolic agent is not perfect, and it has some limitations in clinical use. The adverse reactions are mild, which can be easily tolerated by the patients. It is worth trying to carry out large sample and randomized controlled studies in order to open up more areas for arterial chemoembolization therapy of tumors.

Objective To repair segmental mandibular defects with autogenous bone marrow stromal cells(BMSCs)and?-triealcium phosphate.Methods Isolated BMSCs were in vitro expand- ed.A 3 cm-long segmental mandibular defect was created at right mandible in 12 canines,of which de- fects in six canines were repaired with BMSCs and?-tricalcium phosphate(?-TCP)and that in other six cases repaired with?-TCP,which was used as control.The engineered bone was evaluated by X-ray, CT,DXA,gross and histological examination,immunohistochemistry and biomechanical test 4,12,26,32 weeks after operation respectively.Results In induced BMSCs,histochemistry showed AKP activity. Oral X-ray showed obvious callus formation 4-26 weeks after operation in experimental group but minimal bone formation in control group.At 32 weeks after operation,gross observation,X-ray and CT demonstra- ted well bony-union in experimental group but bony-nonunion in control group.DXA indicated that the bone density of experimental group was significantly higher than that of control group.Biomechanical test revealed no statistical difference upon mechanical strength of mandibula between experimental group and normal group.Conclusions Canine segmental mandibular defects can be well repaired with the tissue- engineered bone generated by autogenous osteogenic BMSCs and?-TCP scaffold.

OBJECTIVETo investigate the application of tissue-engineering bone with ADSCs (adipose-derived stem cells) and coral scaffold for repairing of cranial bone defect in canine.

METHODSAutologous ADSCs isolated from canine subcutaneous fat were expanded, osteogenically induced, and seeded on coral scaffolds. Bilateral full-thickness defects (20 mm x 20 mm) of parietal bone were created (n = 7). The defects were either repaired with ADSC-coral constructs (experimental group) or with coral alone (control group). Radiological, gross, biomechanical and histological observations were done to evaluate the bone regeneration.

RESULTSThree-dimensional CT scan showed that new bones were formed in the experimental group at 12 weeks after implantation, while coral scaffolds were partially degraded in the control group. By radiographic analysis at 24 weeks post-transplantation, it showed that an average repair percentage of each defect was (84.19 +/- 6.45)% in experimental group, and (25.04 +/- 18.82)% in control group (P < 0.01). The maximum compression loading was (73.45 +/- 17.26) N in experimental group, and (104.27 +/- 22.71) N in control group (P <0.01). Histological examination revealed that the defect was repaired by typical bone tissue in experimental group, while only minimal bone formation with fibrous connection in the control group.

CONCLUSIONSThe tissue-engineering bone with autologous osteogenic ADSCs and scaffold could successfully repair the cranial defects in canine models.

Adipocytes ; cytology ; transplantation ; Animals ; Anthozoa ; Bone Regeneration ; Bone Substitutes ; Bone and Bones ; Cell Culture Techniques ; Cells, Cultured ; Dogs ; Female ; Male ; Skull ; surgery ; Stem Cell Transplantation ; Tissue Engineering ; methods ; Tissue Scaffolds ; Transplantation, Autologous

Adult ; Blood Coagulation ; Emergency Treatment ; Female ; Humans ; Intraoperative Period ; Liver Transplantation ; Male ; Middle Aged

OBJECTIVETo analyze the incidence and survival rates of primary liver cancer (PLC) among residents in Yangpu district of Shanghai.

METHODSA total of 9 730 736 permanent residents (male 4 996 390 and female 4 734 346) of Yangpu district in Shanghai were recruited in the study between year 2002 and 2010; among whom 2726 PLC cases were diagnosed and 2427 death cases were certified. The incidence and mortality of PLC were calculated under the analysis of the new PLC cases and death cases between year 2002 and 2010. The rates were standardized by the demographic composition developed in the Fifth Nationwide Census in year 2000, in order to analyze the survival condition of PLC patients and explore the prognosis of surgical excision treatment.

RESULTSAmong the new PLC cases between year 2002 and 2010, 1966 cases were male, whose average age of onset was 59.81; while 760 cases were female, whose average age of onset was 68.93. The crude incidence rate was 39.35/100 000 in male, which was higher than it in female, as 16.05/100 000. The difference showed statistical significance (U = 7.32, P < 0.01). The standardized incidence rates were 21.98/100 000 and 6.96/100 000 in male and female, respectively. The difference showed statistical significance (U = 221.76, P < 0.01). There were 2427 PLC death cases in total, including 1734 male death cases and 693 female death cases. The crude mortality rate was 34.71/100 000 in male and 14.64/100 000 in female, whose difference were statistically significant (U = 6.68, P < 0.01). The standardized mortality rate was 19.16/100 000 in male and 6.06/100 000 in female, whose difference were statistically significant (U = 207.18, P < 0.01). The incidence and mortality rates both increased apparently since males aging over 35 and females over 45. The 1 - 5 year survival rates of PLC patients were 33.95%, 23.11%, 17.04%, 14.42% and 12.29%, respectively. In the surgical excision treatment group (321 cases), the 1 - 5 year survival rates were separately 66.78%, 52.87%, 41.88%, 33.57% and 32.64%; while in the non-surgical treatment group (2405 cases), the 1 - 5 year survival rates were separately 29.04%, 18.58%, 13.30%, 11.16% and 9.01%. The differences in 1 - 5 year survival rates between surgical and non-surgical groups all showed statistical significance (U = 12.78, 10.52, 8.28, 5.56 and 5.12, respectively, P < 0.01).

CONCLUSIONThe incidence of PLC was close to its mortality in Yangpu district of Shanghai. The male incidence and mortality rates were significantly higher than female corresponding rates. The surgical excision treatment could improve the prognosis of PLC.

Aged ; China ; epidemiology ; Female ; Humans ; Incidence ; Liver Neoplasms ; epidemiology ; mortality ; surgery ; Male ; Middle Aged ; Prognosis ; Survival Rate

OBJECTIVETo analyze the incidence and mortality of lung cancer patients among residents in Yangpu District of Shanghai from 2002 to 2010.

METHODSThe data of lung cancer cases in permanent residents of Yangpu district were collected from the database of the registration and management system in Shanghai city. Temporal trend in the incidence of lung cancer was analyzed by using annual percent change (APC) mode1. The survivals were calculated using SPSS 16.0 and Joinpoint Regression Program 3.5.1 software.

RESULTSA total of 5726 cases of lung cancer were diagnosed from January 2002 to December 2010. Of those, 3865 were males with an average age of onset 70.08 years, and 1861 were females with an average age of onset 70.88 years. The crude incidence rate was 77.36/10(5) in men, significantly higher than 39.31/10(5) in women (U = 24.84, P < 0.01). The standardized incidence rate was 25.23/10(5) in men, significantly higher than 13.47/10(5) in women (U = 13.24, P < 0.01). A total of 5248 cases died of the disease in this period. Of those, 3586 were men. The crude mortality was 71.77/10(5) in men, significantly higher than 35.11/10(5) in women (U = 24.67, P < 0.01). The standardized mortality was 21.72/10(5) in men, significantly higher than 11.08/10(5) in women (U = 13.01, P < 0.01). Both the incidence and mortality rates increased apparently in those older than 45 years. The 1- to 5-year survival rates in all cases were 37.42%, 22.71%, 15.73%, 12.32%, and 10.53%, respectively. The 1- to 5-year survival rates in male cases were significantly lower than those in female cases (P < 0.05 for each comparison). The 1- to 5-year survival rates of the patients whose tumor had been surgically removed were significantly higher than those in the patients without surgery (P < 0.05 for each comparison).

CONCLUSIONSThe incidence of lung cancer is close to the mortality in the residents of Yangpu District of Shanghai city. Surgical resection treatment improves the prognosis of lung cancer.

Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; China ; epidemiology ; Female ; Follow-Up Studies ; Humans ; Incidence ; Lung Neoplasms ; epidemiology ; mortality ; surgery ; Male ; Middle Aged ; Sex Distribution ; Survival Rate ; Young Adult

OBJECTIVETo investigate the value of Diffusion-Weighted Imaging (DWI) in the diagnosis of early stage liver diffuse lesions.

METHODSDiethylnitrosamine (DEN) was used to induce liver lesions in rats. Sequential DWI studies were performed on the livers from 1 to 14 weeks after DEN was administered through drinking water. Comparing studies with a blank control group was set and pathohistological examinations of the livers were performed.

RESULTSNo obvious routine MRI morphological change was found in either group during this period, but DWI demonstrated heterogeneous changes in the test group at the cirrhosis stage. There was no significant alteration of the apparent diffusion coefficient (ADC) value in the control group during this period (P > 0.05). The ADC values of the test group began to decline from the fifth week. Until the tenth week, the ADC value of the test group decreased drastically and when b = 300 s/mm2 statistic, the results showed an obvious difference between the two groups. There were also differences between the ADC values at the 10th, the 9th and the 1st weeks of the test group (P < 0.05). When b = 600 s/mm2 and 1000 s/mm2, significant differences were found after the sixth week between the two groups (P < 0.01). The main pathohistological liver change in the test group during the 1 to 4 week period after DEN was administered was swelling of hepatocytes; during the 5 to 8 week period it was fibrous tissues hyperplasia, and in the 9 to 14 week period it was cirrhotic nodule formation.

CONCLUSIONMR functional DWI could detect liver diffuse lesions earlier than conventional MR imaging. Measurement of ADC value may be of use in early diagnosis of liver diffuse diseases and for monitoring the changes of the lesions.

Animals ; Chemical and Drug Induced Liver Injury ; Diethylnitrosamine ; Diffusion Magnetic Resonance Imaging ; methods ; Liver Diseases ; diagnosis ; pathology ; Male ; Rats ; Rats, Wistar

Adenocarcinoma/surgery* ; Esophageal Neoplasms/surgery* ; Esophagogastric Junction/surgery* ; Gastrectomy ; Humans ; Margins of Excision ; Neoadjuvant Therapy ; Retrospective Studies ; Stomach Neoplasms/surgery*