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 evaluate the application of endoscopic needle- knife precut sphincterotomy (PST) in treating acute suppurative cholangitis. Methods After failure of routine papillary intubation during encoscopic retrograde cholangio-pancreatography (ERCP) in papillary incarcerated stone or inflammatory stenosis cases, needle-knife PST was applied to find the lower opening of the common bile duct. After that, routine papillary sphincterotomy or balloon dilation followed. Then net basket for stone extraction and nasobiliary catheter for drainage were made. Results Eleven of the 12 cases′ stones were extracted successfully, the success rate was 91.7%. In the 11 cases, 5 cases′ incarcerated stones dropped into duodenum automatically after sphincterotomy; 9 cases′ stones were extracted successfully in one treatment while 2 cases′ stones were extracted secondarily after stents were implanted; 1 case′s stone could not be extracted and need surgical treatment after nasobiliary catheter drainage because of stenosis of the lower part of the common bile duct. There was no dead case in all the cases. Conclusions Acute suppurative cholangitis patients, who have papillary incarcerated stones or inflammatory stenosis, can receive more efficacious diagnosis and treatment by applying PST when routine endoscopic papillary intubation fails. PST is an important endoscopic treatment for acute calculous suppurative cholangitis

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 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.

Objective To explore the association between genetic polymorphisms and haplotypes of tumor necrosis factor-related apoptosis inducing ligand (Trail) and ulcerative colitis (UC).Methods A total of 331 patients with UC and 832 age and sex-matched healthy controls were collected.After Trail gene was amplified by PCR,the genetic polymorphisms of three single nucleotides (G1525A/G1588A/C1595T) in 3' non coding regions of Trail gene were examined by direct sequencing.The relation between Trail haplotype and UC was analyzed.Results Compared with control group,the frequencies of variant allele A and genotype GA+ AA in Trail G1525A were significantly lower in UC group (both P＜0.01).The frequencies of variant allele A and T in Trail G1588A and C1595T were also significantly lower in UC group than that of control group,and the difference was statistically significant (both P ＜ 0.01 ).In mild and moderate UC patients,the frequencies of variant allele T and CT+TT in Trail C1595T were 49.15％ and 64.51％,in severe UC patients were 72.37％ and 84.21％,and the differences were significant between the two groups (OR=2.710 and 2.935,95％CI:1.598～4.596 and 1.188～7.249,all P ＜0.05).In severe UC patients,the frequency of variant allele A in Trail G1525A was 48.69％,which was higher than that of mild and moderate patients (35.16％,OR=1.750,95％CI:1.082～2.830,P=0.021).In UC group,the frequency of AAT haplotype was significantly lower than that of controls (43.09％ vs 58.41％,P＜0.01).The frequency of GAT haplotype was significantly higher in UC group (10.15％vs 0.18％,95％ CI:0.005 ～ 0.051,P＜ 0.01).Conclusion The genetic polymorphisms and haplotypes of Trail (G1525A/G1588A/C1595T) gene may be closely correlated with the susceptibility to UC.

Objective To determine the expression of cathepsin D (CD), cathepsin H (CH) and cathepsin L (CL) in human primary hepatocellular carcinoma (HCC), and to investigate their mechanisms. Methods The protein expression of CD, CH and CL in hepatic specimens consisted of control (n = 17), HCC (n = 37) and paracancerous (n = 28) tissues were detected by immunohistochemistry. The relative values of CD, CH and CL protein expression were examined with absorbent density analysis. Data were analyzed using SPSS11. 5 software. The univariate analysis was used to compare the difference among groups. Results The mean absorbance of CD, CH and CL proteins in HCC tissues (1.21± 0.33, 0. 89 ± 0.22 and 1.16± 0. 25, respectively) were significantly higher in comparison with those in control tissues (0. 19 ± 0. 07, 0. 24 ± 0. 12 and 0. 28 ± 0. 14,respectively) and in paracancerous tissues (0.27±0.13,0. 31± 0.14 and 0. 36±0.15)(all P values =0.0001). Whereas there was no difference between control and paracancerous tissues with respect to CD, CH and CL proteins (P ＞0. 05). Three proteins immunohistochemically appeared as a lot of diffused spots and stripes staining in cytoplasma of HCC tissues,but only a few scatted spots staining was found in control and paracancerous tissues. Conclusion The high expression of CD, CH and CL protein in primary HCC may be important markers for carcinogenesis and malignant progress.

Objective To investigate the association between the genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and ulcerative colitis (UC) of Han ethnic population in Zhejiang, China. Methods Two hundred and seventy-four consecutive patients with UC and 726 healthy controls (HC) were studied. The genetic polymorphisms of MTHFR (C677T and A1298C) were genotyped using PCR-RELP methods. Results The frequencies of variant allele and genotype in MTHFR A1298Cgene were higher in UC patients than in the HC (35.77% vs 29. 96%, P =0. 013; 52. 19% vs 44. 90%,P=0.039; respectively). However, there were no significant discrepancies of the allele and genotype frequencies in the MTHFR C677T gene between the UC patients and the HC (P ＞ 0. 05 ). In addition, the MTHFR 677Tr homozygote, T allele and 677CT/1298AC compound genotype were more prevalent in patients with extensive colitis than in those with distal colitis (37. 66% vs 14. 72% ,P = 0. 0002; 49. 35% vs 32.99% ,P =0. 0004; 29. 87% vs 15.23% ,P =0. 006; respectively). Furthermore,the variant allele in the MTHFR A1298C gene (C) in severe UC patients was significantly lower than in mild and moderate UC patients (18.97% vs 33. 88% ,P =0. 022). Conclusion The genetic polymorphisms of MTHFR C677T and A1298C are obviously associated with Han ethnic population with UC in Zhejiang province.

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 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.