Objective To explore the effect of Latexin (Lxn) gene transfection on proliferation of CD13;MIAPaca-2 pancreatic cancer stem-like cells.Methods CD133+ MIAPaca-2 cells were isolated and sorted by magnetic activated cell sorting from pancreatic cancer MIAPaca-2 celt line.CD133+ MIAPaca-2 cells were cultured in serum-free medium and the capacity for proliferation,and tumorigenicity of CD133+ MIAPaca-2 cells was determined by the floating spheres test and tumor xenograft assays.The CD133+ MIAPaca-2 cells were transfected with Lxn plasmid (1,3,5 μg).After transfection,the protein and mRNA expression of Lxn in CD133+ and CD133+-MIAPaca-2 cells were detected by Western blotting and RT-PCR,respectively.Cell proliferation was assayed by CCK-8.Results CD133+ MIAPaca-2 cells were successfully isolated,and it grew into a ball-suspended way,the tumorigenicity rate in nude mice with subcutaneous injection 1 × 105 cancer cells was 100％.After Lxn plasmid transfection,the expression of Lxn in CD133+ MIAPaca-2 cells was increased in a dose dependent manner,the Lxn protein and mRNA expression of tumor cells transfected with 5 μg plasmid was 20.80 ±0.98,16.80± 2.73,which was significantly higher than that in non-transfected cells (1.02 ± 0.01,1.01 ± 0.01),and the difference between the two groups was statistically significant (P ＜ 0.05).After transfection,cellular proliferation activity also showed a transfection dose and culture time-dependent decrease,the inhibition rate of tumor cells transfected with 0.4 μg plasmid was 36.2％,which was significantly different from that in non-transfected cells (P ＜ 0.05).Conclusions CD133+ MIAPaca-2 pancreatic cancer cells have some characteristics of cancer stem cells.Lxn gene transfection can inhibit the proliferation of CD133+ MIAPaca-2 cells.

Objective To observe the effect of Latexin treatment on the chemoresistance in gemcitabine-resistant pancreatic cancer cell line SW1990, and explore the potential mechanism.Methods Gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was induced and established by increasing gemcitabine dosage intermittently.IC50 of gemcitabine in SW1990 cells and SWl990/GZ cells pre and post Latexin treatment at the dosage of 10, 20 and 40 ng/μl for 48 h was evaluated using CCK-8 assay.The mRNA and protein expression of Latexin gene in SW1990 and SW1990/GZ cells were evaluated using qRT-PCR and Western blot, and the expression of Shh and Gli1 in 40 ng/μl Latexin treated SW1990 and SW1990/GZ cells for 48 h.Results A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was obtained successfully, which can grow stably and passage in the media containing 150 μmol/L gemcitabine.The IC50 values of gemcitabine in SW1990 cells and SWl990/GZ cells were (3.8±0.4)μmol/L and(226.52±13.61)μmol/L, respectively, and the later was greatly higher than the former, which was statistically different (P=0.000).The drug resistance indexes (RI) was 59.6.After treated with different concentrations of Latexin(10,20,40 ng/μl), the IC50 of SW1990 cells was (3.0±0.4)μmol/L, (2.5±0.3)μmol/L and (1.8±0.3)μmol/L, respectively,and the IC50 of SW1990/GZ cells was(113.08±5.01)μmol/L,(70.26±2.31)μmol/L and (42.12±1.31)μmol/L, respectively.Compared with the untreated cells,the IC50 of gemcitabine in 20,40 ng/μl Latexin treated cells was obviously decreased, and the differences were statistically significant (P<0.05).Compared with the SW1990 cells,the expression of Latexin in SW1990/GZ cells was obviously decreased.RI were 37.7, 28.1 and 23.1,respectively.mRNA relative expression of Latexin in SW1990 and SW1990/GZ cells were 0.85±0.08 and 0.31±0.07, and protein relative expression were 0.49±0.09 and 0.13±0.05, and Latexin expression in SW1990/GZ was obviously lower than that in SW1990 cells and the difference was statistically significant (P<0.05).After being treated by 40 ng/μl Latexin, SHH mRNA in SW1990/GZ cells decreased from 0.89±0.09 (control cells) to 0.53±0.06, Gli1 mRNA decreased from 0.58±0.06 to 0.35±0.05, Shh protein decreased from 0.72±0.09 to 0.35±0.06,Gli1 protein level decreased from 0.78±0.08 to 0.28±0.03, and all the differences were statistically significant (P<0.05 or 0.01).Conclusions Latexin can significantly improve the chemosensitivity in gemcitabine-resistant pancreatic cancer cells, and the potential mechanism may be related to the inhibition of sonic hedgehog pathway activation.

Objective To improve detectable rate of gastric schwannoma by endoscopic ultrasonography (EUS). Method Clinical data and endoscopic ultrasonography (EUS) imaging features of 4 cases were retrospectively ana-lyzed which diagnosed as gastric schwannoma pathologically and immunohistochemically while diagnosed as gastric stromal tumor by EUS from May 2008 to June 2015 and reviewed the literature. Results 4 cases of gastric schwan-nomas are female and benign, all 4 lesions are solitary, 3 in gastric body, and 1 in fundus by endoscopic. By EUS, all lesions are originated from muscularis propria, hypoechoic change, even echoes and clear board without calcifica-tion or cystic changes. 2 cases have halo artifacts around the lesion. Literature review found that gastric schwannoma tended to occur in female, halo artifacts could be the feature of gastric schwannoma, calcification or cystic changes were rare in gastric schwannoma which were common in gastric stromal tumors. Conclusion It was difficult to distin-guish gastric schwannoma and gastric stromal tumors that originated from muscularis propria by EUS. For female patients with lesions originated from muscularis propria, originated from muscularis propria and occurred in gastric body, it was necessary to observe lesions whether there was being calcification or cystic and halo artifacts. Integrated all these performance, we should be in consideration of gastric stromal tumors, meanwhile, excluding the possibility of gastric schwannoma.

Objective To investigate the application value of Photoshop in grading invasive risk of gastric stromal tumors (GSTs). Methods EUS image of 97 cases of GSTs confirmed by pathological and immunohistochemical examination were collected. GSTs were divided into four groups (very low risk, low risk, intermediate risk, high risk) by tumor size, mitotic count and rupture of tumor. Mean gray value (intensity of echo) and gray value standard deviation (uniformity of echo) of EUS images of the lesions were determined by Photoshop and then the differences of each group were found by statistical analysis. Results It is difficult to differentiate EUS images of GSTs from each group by visual observation. The mean gray value of EUS image of very low risk group,low risk group, intermediate risk group and high risk group of GSTs respectively were (56.54 ± 6.10), (59.20 ± 7.51), (77.77 ± 10.90) and (83.43 ± 12.47). There was no significant difference between very low risk group and low risk group (P > 0.05). There was no significant difference between intermediate risk group and high risk group (P > 0.05). In addition, the others all had significantly different from that of each group (P < 0.05). The mean gray value standard deviation of EUS image of very low risk group, low risk group, intermediate risk group and high risk group of GSTs respectively were (8.46 ± 2.59), (12.57 ± 5.89), (12.84 ± 4.15) and (16.69 ± 4.69). There was no significant difference between low risk group and intermediate risk group (P > 0.05). In addition, the others all had significantly different from that of each group (P < 0.05). Conclusions The higher risk of GSTs, the higher of echo intensity and the worse of echo uniformity under EUS. Photoshop combined with EUS is helpful for differentiating different risk of GSTs by analyzing mean gray value and gray value standard deviation of the lesions.

Background:Endoscopic ultrasonography( EUS)is the first choice for examining submucosal protuberant lesions in the gastrointestinal tract,however,it is very difficult to differentiate gastric stromal tumor from gastric leiomyoma. Photoshop software now is tried to differentiate some easily misdiagnosed diseases. Aims:To investigate the value of Photoshop software in differentiating EUS image of gastric stromal tumor from gastric leiomyoma. Methods:EUS image of 118 cases of gastric stromal tumor and 42 cases of gastric leiomyoma confirmed by pathological and immunohistochemical examination were collected,mean gray value( intensity of echo)and gray value standard deviation( uniformity of echo)of EUS image of lesion were determined by Photoshop software,and the differentiation value was analyzed. Results:The mean gray value of EUS image of gastric stromal tumor was significantly higher than that of gastric leiomyoma(71. 94 vs. 48. 99,P<0. 01). The gray value standard deviation of EUS image of gastric stromal tumor was significantly higher than that of gastric leiomyoma(16. 63 vs. 9. 80,P<0. 01). Conclusions:Under EUS,gastric stromal tumor exhibits higher intensity of echo and less uniformity of echo than gastric leiomyoma. EUS combined with Photoshop software is helpful for differentiating gastric stromal tumor from gastric leiomyoma by analyzing mean gray value and gray value standard deviation of the lesion.

Objective To study the effects of Lxn on CD133 + PANC-1 pancreatic cancer cells.Methods CD133 + PANC-1 cell were isolated by magnetic activated cell sorting (MACS).The properties of the CD133 + PANC-1 cells and Lxn effects on CD133 + PANC-1 cell proliferation in transplanted tumor in nude mice were determined by floating spheres test and tumor xenograft assays.Cell proliferation was assayed by Cell Counting Kit-8 (CCK-8).The Bcl-2,Bax protein and mRNA expression of CD133 + PANC-1 cells treated by Lxn were analyzed by Western blot and Quantitative real-time PCR (qRT-PCR).Results We successfully isolated the CD133 + PANC-1 cells and cultured in serum free medium,CD133 + PANC-1 cells formed sphere,while CD133-PANC-1 cells grew with adherence slowly and then underwent apoptotic process.CD133 + PANC-1 cells showed high tumorigenic in athymic BALB/c mice.Lxn suppressed the growth of transplanted tumor obviously.Compared with control group [(225.52 ± 34.09) mm3],tumor volume decreased significantly (P ＜ 0.05).Significant reduction in cell proliferation was observed in response to Lxn in PANC-1 CD133 + cells by CCK-8 assay with concentration and time dependent manners (P ＜ 0.05).Treated by Lxn,Bcl-2 expression decreased,Bax expression increased.Conclusions Lxn inhibits the proliferation of CD133 + PANC-1 cells probably through a mechanism down-regualting Bcl-2 and up-regulating Bax.

Dysregulated immune response is crucial for the pathogenesis of inflammatory bowel disease.Recent studies suggested that imbalance among Th1, Th2 and Th17 cells was closely related to the aberrant intestinal immune response.Aims: To investigate the association of imbalance among Th1, Th2 and Th17 cells and Crohn''s disease (CD).Methods: Thirty-six CD patients admitted from Jan.2013 to Dec.2014 at the Second Affiliated Hospital of Wenzhou Medical University were enrolled;40 patients undergoing intestinal polypectomy were collected as controls.Inflamed and normal mucosal tissues were obtained from CD patients and the controls, respectively;expressions of Th1, Th2 and Th17 cells related transcriptional factors (T-bet, GATA-3 and RORγt) and cytokines (IFN-γ, IL-4 and IL-17A) were determined by real-time PCR and immunohistochemistry.Results: In comparison with the controls, mRNA expression of T-bet and RORγt, mRNA and protein expressions of IFN-γ and IL-17A, as well as the ratios for T-bet/GATA-3 and RORγt/GATA-3, which represented balance of Th1/Th2 and Th17/Th2, respectively, were all significantly increased in inflamed mucosal tissue in CD patients (P<0.05).Expression of each of the three transcriptional factors was positively correlated with its corresponding cytokine (P<0.05).IFN-γ+ and IL-17A+ cells mainly located in the intestinal epithelial layer and lamina propria with cytoplasmic immunoreactivities in CD patients.In the stratified analysis, all the above-mentioned parameters were significantly higher in active CD than in inactive CD (P<0.05);furthermore, the ratio for RORγt/T-bet, which represented balance of Th17/Th1, was also increased significantly in active CD (P<0.05).Conclusions: Imbalance among Th1, Th2 and Th17 cells in intestinal mucosal tissue was closely related with CD.Polarization of Th1 and Th17 cells are involved in this process, and Th17 polarization is predominant in active disease.

Objective To design radioactive biliary stents and to evaluate the feasibility and safety of the stents.Methods Plastic stents with inserted iodine-125 seeds were designed and tested in sixteen normal pigs. In the brachytherapy group,the pigs were implanted radioactive stents in the common bile duct (CBD) and then divided into three groups on the basis of radiation dose of the reference point,50 Gy group (n=4),100Gy group (n=4),and 150 Gy group (n=4).In the control group,the same plastic stents with non-radioactive seeds were implanted (n=2),whilst in the blank control group,no stents were implanted (n=2).Blood routine,serum amylase,liver and kidney function were tested before and 1,7,14,30,60 days after the implantation of stents. Animals were sacrificed on the 14,30 and 60 days after stem implantation.Some relevant index such as perito- neal bleeding and inflammation,ascites,injury of adjacent organs,as well as perforation,stricture and dilatation of bile duct were investigated.Bile duct tissues were stained with H-E,and observed under microscopy. Results The radioactive plastic biliary stents were successfully prepared and implanted.There was no effusion, hemorrhage or necrosis in the adjacent organs of radioactive biliary stent implanted group.Perforation of the CBD wall was not observed in the brachytherapy group.By pathological examination in the CBD,obvious hyperplasia of the mucosa and mucosal glands were seen in the control group.Necrosis of mucosal layer existed in brachy- therapy groups.In 50 Gy group,mucosal layer was incomplete and mild hyperplasia of mucosal glands was seen. In 100 Gy group,mucosal layer disappeared and almost no hyperplasia of the mucosal glands could be found.In 150 Gy group,mucosal layer disappeared and mucosal glands obviously decreased.There were no obvious abnormalities noted in blood tests after implantation in each group.Conclusions The design of radioactive biliary stents are feasible and safe.The radioactive stents have obvious radiation effect besides the mechanical effect on the mucosal layer of CBD.

Objective To evaluate the feasibility and safety of radioactive pancreatic duct stents implanted in the pancreatic ducts of pigs by endoscopy. Methods Different doses of 125I radioactive pancreatic duct stents were implanted in the pancreatic ducts of pigs by endoscopy. Blood tests were conducted before and after implantation. 14,30 and 60 days after implantation of the radioactive stents, the pigs were euthanized in batch. All animals underwent post modem examination to exclude intra-abdominal hemorrhage,pancreatic fistula or peritonitis. During autopsy,the liver,bile ducts,head of the pancreas,stomach and duodenum were examined for perforation,stricture or dilation and damage of the surrounding structures.Results Fourteen pigs were implanted with pancreatic duct stents by endoscopic procedures.There was no effusion,hemorrhage or necrosis in the adjacent duodenum,stomach,liver or right kidney.The noral morphological structures of the duct of Wirsung disappeared in all the treated pigs.Histopathological examination revealed that the stents were surrounded by necrotic tissue and outside fibrous tissue. During the follow-up period, the width of outside fibrous tissue gradually increased. There were no serious abnormalities noted in the blood tests after implantation. Conclusion It is indicated that the radioactive stents are safe in all the difierent dose groups. For future clinical application, it is feasible to design a special radioactive stent for each patient according to the size,shape and position of the pancreatic tumor.

Objective:To explore the cleansing effect of Nitric Oxide (NO) sustained-release silica nanoparticles (short for NO sustained-release agent) on endoscopic biofilm and its clinical application.Methods:A total of 160 clinical endoscopes were randomly divided into two groups: the cleansing agent group (80 pieces, disinfected with cleansing agents), NO group (80 pieces, disinfected with NO sustained-release agent). A biofilm model of Pseudomonas aeruginosa was constructed and used as the control for phosphate buffered solution (PBS) treatment. A biofilm model of Pseudomonas aeruginosa on the surface of endoscopic lumen was built first in vitro. Scanning electron microscopy was then used to observe the microstructure of biofilm after treatment with NO sustained-release agent. Viable counting method was used to evaluate the cleansing effect of NO sustained-release agent on biofilm. Finally, at the clinical level, the actual disinfection effect of NO sustained-release agent on clinical endoscopy was evaluated by detecting the protein residues, viable counting and adenosine triphosphate (ATP) biofluorescence detection. Results:The scanning electron microscopy showed that the biofilm was intact in the model group, but scattered bacteria were observed on the biofilm surface in the NO group and the detergent group. Compared with the model group [(4.86±2.67)×10 6(colony-forming units, CFU)/mL], the standard CFUs of the NO group [(1.37±0.61)×10 4CFU/mL] and the detergent group [(1.31±0.21)×10 5CFU/mL] were significantly lower (detergent group VS model group, P=0.009; NO group VS model group, P=0.008), and there was significant difference between the detergent group and the model group ( t=9.53, P=0.000 6). The levels of residual proteins in the endoscopic lumens before and after the treatment were 8.03±1.47 mg/mL and 0.50±0.37 mg/mL in the NO group, 8.01±1.51 mg/mL and 0.91±0.52 mg/mL in the detergent group with significant difference ( P<0.01), and the reduction effect of the NO group was more significant. The disinfection of NO group and cleaning agent group was within the qualifying range, but the ATP bioluminescence value, protein residue and colony count of NO group (78.56±42.59 RLU, 0.50±0.37 mg/mL, 7.55±4.56 CFU) were significantly lower than those of detergent agent group (120.80±54.00 RLU,0.91±0.52 mg/mL,11.50±4.75 CFU, P<0.01). Conclusion:NO sustained-release agent can effectively clear endoscopic biofilm and further improve the disinfection effect on endoscopes, which may be of great significance for improving the effects on treatment and prognosis of patients.