Objective To explore the impact of insulin secretory function of adult islet cells from barium-alginate microencapsulation in vitro. Methods After weighting the pancreas,human pancreatic islets were isolated with type V collagenase and purified by Ficoll's discontinuous density gradient centrifugation.The islet cell yield and purity were evaluated with microscope by DTZ staining.Human islets were coated by barium-alginate microencapsulation,and the viability was assessed by insulin release assay in vitro.Results After isolation,the average number of islet was about 3600 ±447 per gram pancreas.It was about 2140±207 after purification with more than 70％ purity.At day 2,4 and 6 after islet cell culture in vitro,basal insulin concentrations from the culture medium was measured,and the mean insulin concentration (mU/L) in media of the microencapsulated islet group at 2nd,4th and 6th day were 3.302±1.63、3.504±1.10 and 2.921±1.13 respectively,and those non-microencapsulated islet group were 3.814 ± 1.49、4.175 ±1.60、3.617± 1.34.There were no significant differences between these two groups (P＞0.05).Conclusions The bioartificial pancreas has effective insulin secretory function in vitro without being affected by barium-alginate microencapsulation.

Pancreatic cancer is a common malignancy of gastrointestinal system, with features of early metastasis, easy invasion to adjacent tissues and organs and neural metastasis. Therapies include surgery, chemotherapy, radiotherapy, physi-cal and biological therapy and so on. Surgical management, including radical resection and palliative operation, is a major approach. Radiotherapy and chemotherapy could improve the resectional rate and decrease the tumor dissemination. Physical and biological therapies are widely recommended, and there is a rapid progress in zoopery. However, the efficacy of all the thera-pies is far from satisfactory. Recently, the therapy consisting of surgical resection, radiotherapy, chemotherapy, physical and biological therapy in increasing the long-term survival rate and improving the life quality of patients, along with combined thera-py has attracted the attention of surgeons.

Objective To investigate the methods and feasibility of islets isolation and purification,and to get more islets with high purity and good function for clinical transplantation.Methods After being weighted,human pancreatic islets were isolated with type Ⅴ collagenase,and purified by Ficoll's discontinuous density gradient centrifugation.The yield and purity of islets were evaluated by dithizone(DTZ) staining under microscope,and the viability was assessed by insulin release assay in vitro.Results The average number of islets was about 3 600 ± 447 per gram pancreas after isolation and it was about 2140 ±207 after purification with more than 70％ purity.2,4,6 days after islet cell culture,the basal insulin concentration of the culture medium was measured,and it was 3.302 ± 1.63,3.504 ±1.10,and 2.921 ±1.13 (mIU/L/100 islets) respectively.Conclusion Collagenase digest and Ficoll's discontinuous density gradient centrifugation are effective methods for isolation and purification of human pancreatic islets.

Objective To investigate the clinical efficacy of two types of duodenum-preserving pancreatic head resection (Beger procedure and Berne procedure) for chronic pancreatitis with mass in the head of the pancreas.Methods The clinical data of 46 patients with chronic pancreatitis and mass in the head of the pancreas who were admitted to the Affiliated Hospital of Guiyang Medical College from September 2008 to April 2012 were retrospectively analyzed.There were 24 patients received Beger procedure (Beger group),and 22 received Beme procedure (Berne group).The complications,life quality and pain after the operation were evaluated.Patients were followed up via phone call and out-patient examination till April 2013.The measurement data were analyzed using the Mann-Whitney U test,and the constituent ratios were compared using the chi-square test.Results The operation time and volume of blood loss were (377 ± 21) minutes and (746 ± 129) mL in the Beger group,and (323 ± 17) minutes and (577 ± 111)mL in the Berne group,with significant difference between the 2 groups (U=14.0,88.0,P ＜0.05).Four patients in the Beger group and 1 in the Berne group were complicated with pancreatic leakage,with no significant difference between the 2 groups (x2=0.714,P ＞ 0.05).The scores of life quality evaluation (physical condition,work capacity,cognitive ability,emotion,social competence and overall life quality) were 82 ± 14,74±24,90 ± 18,78±20,83 ± 18,73 ± 18 in the Beger group,and 79 ± 16,71 ±20,92 ±21,76 ± 18,80 ±21,70 ± 16 in the Berne group,with no significant difference between the 2 groups (U =177.5,183.5,187.5,178.0,189.5,192.0,P ＞ 0.05).The scores of symptom evaluation (fatigue,nausea and vomitting,pain,anorexia,dyspnea,sleep disorders,obstipation,diarrhea,financial worries) were 28 ± 16,24 ± 10,20±12,23 ± 14,4 ± 1,32 ± 12,6 ±2,18 ± 14,36± 18 in the Beger group,and 26 ± 18,26 ±20,22 ± 16,26 ± 16,3 ± 1,30 ± 10,5 ± 1,16 ± 12,38 ± 20 in the Berne group,with no significant difference between the 2 groups (U=194.5,215.5,182.5,180.5,213.0,199.0,195.0,184.5,181.5,P＞0.05).In the Beget group,19 patients did not have acute onset of pain,and 5 patients had acute onset of pain once a year; 6 patients were administered antalgesic occasionally.In the Berne group,20 patients did not have acute onset of pain,and 2 patients had acute onset of pain once a year; 4 patients were administered antalgesic occasionally,with no significant difference between the 2 groups (x2=0.485,0.041,P ＞ 0.05).All the patients were followed up,and the median time of follow-up was 36.3 months.No perforation of duodenum and steatorrhea was observed.No patient died perioperatively.Conclusion The clinical efficacy of the Berne procedure is similar to that of the Beger procedure,while the Berne procedure has advantages of easy manipulation and less operation time.

Hormone ingredient in the egg oil, egg and Oviductus Ranae of Rana temporaria chensinensl is David were studied as well as the effect of egg-oil on platelet aggregation and blood lipid.Results proved that the estradiol content in the egg-oil is very high.The egg-oil can obviously inhibit blood platelet aggregation and showed an hypolipemia activity. It is expeetedthat the egg-oil of R. temporaria chensinensis can be developed as an efficient medicine for the prevention and cure of atherosclerosis and hyperlipemia in elderly.

Objective To assess the clinical significance of QT interval dispersion (QTd) in the diagnosis and prognosis of early heart damage in patients with severe acute pancreatitis.Methods All patients received complete ECG,There were 58 patients with SAP (the SAP group) and 189 patients with mild acute pancreatitis (the control group).These patients were analyzed retrospectively and 60 normal people were used as the healthy control group.The QT interval were measured respectively in serial 12-lead electrocardiogram and QTd,QTcd were calculated.Result QTd and QTcd were significantly longer in the SAP group than in the MAP group and in the healthy control group (P＜0.01).QTd,QTcd were not remarkably extended in the MAP group than in the healthy control group (P＞0.05).Conclusions QTd and QTcd have clinical values to diagnose and to predict early heart damage in patients with SAP.They might be useful in evaluating the condition of cardiac function in patients with SAP.

Objective To investigate whether KAP-1 could induce CSC-self renewal in pancreatic cancer cell lines.Methods KAP-1 expression was examined in 14 cases of pancreatic cancer with immunohistochemistry.KAP-1 shRNA amplified by PCR was inserted into pGC-LV in vector,and then identified by restriction endonuclease digestion and nucleotide sequencing.The lentiviral vector pGC-shRNA-KAP-1 was co-transfected with pHelper 1.0 and pHelper 2.0 packaging plasmids into HEK 293T cells,and the lentivirus was collected and virus titer was measured.The expressions of KAP-1 and vimentin were detected by Western blot and RT-qPCR when human pancreatic cancer cell line Panc-1 was infected by the lentivirus.Sphere forming assay was conducted to assess the capacity of CSC or CSC-like cell self-renewal in this study.Results The KAP-1 expression level in cancerous tissues on immunohistochemistry was significantly higher than in the corresponding normal tissues (P=0.002).After infected by lentivirus,the expressions of KAP-1 and vimentin were knocked down,which could be detected by Wesren blot and RT-qPCR.Compared to Panc-1-GFP (NC) cells,the outcomes suggested that knocking down the expression of KAP-1 could decrease the formation of pancreatospheres,which further suggested the capacity of CSC-self-renewal in primary and secondary pancreatospheres of Panc-1-shRNA-KAP-1 and NC cells.Conclusions KAP-1 expression in pancreatic cancer tissues has been identified.The lentiviral vector for shRNAs targeting KAP-1 was constructed successfully.The formation of pancreatospheres decreased by knocking down the KAP 1 gene.KAP-1 is involved in the regulation of CSC phenotype.The mechanism is probably related to the upregulated expression of the EMT marker vimentin.

Objective To investigate the effects of KAP-1 in promoting the epithelial-mesenchymal transition (EMT) of pancreatic cancer cell line Capan-2.Methods The lentiviral vector of LV-plenti-GFP-KAP-1 was constructed.Capan-2 cells were divided into the experimental group (cells transfected by lentiviral vector of LVplenti-GFP-KAP-1),negative control group (cells transfected by empty vector) and blank control group 1 (cells cultured in 1640 medium plus 10％ fetal calf serum).Capan-2 cells in the experimental group were subdivided into the miR-100-5p inhibitor transfection group (cells transfected with miR-100-5p inhibitor),empty vector control group (cells transfected with microRNAs inhibitor),blank control group 2 (cells cultured in 1640 medium plus 10％ fetal calf serum).The lentivirus was identified by double endonuclease restriction and sequencing,and the virus titer was detected.The morphological changes of the cells were observed after transfecting lentiviral vector of LV-plenti-GFP-KAP-1 to the Capan-2 cells.The expressions of KAP-1,genes of EMT proteins and mRNA of miR-100-5p were detected by real-time quantitative polymerase chain reaction.The protein expressions of KAP-1,EMT proteins in all the groups were detected by Western blot.The measurement data were presented by mean ± standard deviation,and were analyzed using the analysis of variance.Results The lentiviral vector of LV-plentiGFP-KAP-1 was successfully constructed,and the virus titer was 2 × 108 TU/ml.Compared with the control group,the mesenchymal transition of the Capan-2 cells was detected in the experimental group after transfecting the Capan-2 cells with lentiviral vector of LV-plenti-GFP-KAP-1 for 48 hours.The relative mRNA expressions of KAP-1,N-cadherin,vimentin,E-cadherin,miR-100-5p were 1.77 ± 0.83,2.62 ± 0.71,2.50 ± 0.21,7.20 ± 1.17 and 1.81 ±0.40 in the experimental group,5.03 ±0.29,5.07 ±1.53,3.83 ±0.57,7.83 ±0.78,7.01 ± 0.96 in the negative control group,5.13 ± 1.14,5.81 ± 1.49,4.92 ± 0.90,3.07 ± 0.36,6.87 ± 0.35 in the blank control group 1,with significant difference among the 3 groups (F =5.99,7.62,7.88,6.62,4.64,P ＜0.05).The relative mRNA expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 1.56 ± 0.42,4.89 ± 0.61,5.20 ± 0.38,with significant difference among the 3 groups (F =5.14,P ＜ 0.05).The relative mRNA expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.10 ± 1.37,3.44 ± 0.94,3.08 ±1.16,with no significant difference among the 3 groups (F =0.49,P ＞ 0.05).The results of western blot showed that the relative protein expressions of KAP-1,N-cadherin,vimentin,E-cadherin were 2.77 ± 1.99,1.31 ±0.38,4.25 ± 0.63,0.62 ± 0.06 in the experimental group,0.83 ± 0.46,0.41 ± 0.37,1.03 ± 0.33,1.17 ± 0.45 in the negative control group,0.71 ± 0.26,0.08 ± 0.04,1.37 ± 0.92,3.04 ± 0.65 in the blank control group 1,with significant difference among the 3 groups (F =5.54,4.68,3.19,8.18,P ＜ 0.05).The relative protein expression of KAP-1 in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 2.27 ±0.71,0.56 ±0.43,0.61 ±0.39,with significant difference among the 3 groups (F =4.81,P ＜0.05).The relative protein expressions of vimentin in the miR-100-5p inhibitor transfection group,empty vector control group,blank control group 2 were 3.19 ± 0.55,3.93 ± 0.06,3.61 ± 0.73,with no significant difference among the 3 groups (F =0.04,P ＞ 0.05).Conclusion KAP-1 promotes the EMT of Capan-2 cells by specifically down-regulating the miR-100-5p expression.

Objective To investigate the possibility of transfection of adipose-derived mesenchymal stem cells (ADMSCs) into the insulin-secreting cells in vitro,and assay the secretion of insulin of ADMSCs in high and low glucose environment.Methods The ADMSCs that untransfected were in the control group,the ADMSCs that contained PcDNA3.1 were in the vacant vector group,and the ADMSCs that contained PcDNA3.1-hINS were in the recombinant vector group.After transfection,the recombinant vector group were sub-divided into the 1,6,12,18 days groups.According to the concentrations of glucose,the recombinant vector 18 days group were divided into the high glucose group and low glucose group.Human insulin gene was amplified by RT-PCR,and the eukaryotic expression recombinant vector PcDNA3.1-hINS that contained the human insulin gene was constructed.The ADMSCs were obtained by digestion and centrifugation,and then underwent flow cytometry for identification.The transcription of insulin DNA was assayed by RT-PCR,and the levels of insulin were assayed by ELISA.Glucose test was done in the recombinant vector 18 days group.The measurement data was shown in the format of (x) ± s,the measurement data in multiple groups were compared by randomized analysis of variance,and the comparison among groups was performed by the t test.ResuIts The expressions of CD44,CD90,CD106 were positive,and the expressions of CD34,CD45 and CD11b were negative.No insulin DNA transcription was detected in the control group and vacant vector group.The levels of insulin secreted were (4.7 ± 0.8) mIU/L,(8.8 ± 0.5) mIU/L,(8.9 ± 0.8)mIU/L,(8.6 ± 0.6)mIU/L in the recombinant vector 1,6,12,18 days group,which were significantly higher than (1.3 ± 0.6) mIU/L in the control group and (1.7 ± 0.8) mIU/L in the vacant vector group (t =10.09,32.64,22.20,55.53 ; 9.23,27.56,19.43,51.25,P ＜ 0.05).There were significant differences in the levels of insulin secreted between the recombinant vector 1 day group and the recombinant vector 6,12,18 days groups (t =12.77,12.26,13.93,P ＜0.05).There were no significant difference in the levels of insulin secreted between the recombinant vector 6,12,18 days groups (F =45.67,P ＞ 0.05).There was a significant difference in the level of insulin secreted between the high glucose group and the low glucose group (t =2.03,P ＜ 0.05).The result of the glucose stimulation test was negative.Conclusion The ADMSCs are transfected into insulinsecreting cells in vitro successfully,and the secretion of insulin is stable.Although the secretion of insulin can't change in line with the concentration of glucose,it is a new seed cell for the treatment of diabetes with stem cells.

Objective To explore the overexpression of the 14-3-3 sigma (14-3-3σ) gene effects proliferation in pancreatic cancer cells.Methods The 14-3-3σ protein level in 46 pancreatic cancers and 9 normal pancreases specimens were analyzed by immunohistochemistry.The mRNA and protein expression of 14-3-3σ in pancreatic carcinoma cell lines (BxPC3,CAPAN-1,PANC-1,AsPC-1,SW1990,MiaPaCa-2 and CFPAC-1) were detected by RT-qPCR and Western blot respectively.A recombinant plasmid of pEGFP-14-3-3σ was constructed and transfected into the pancreatic cancer cell PANC-1 by liposome,and the expression of 14-3-3σ was detected by Western blot and real time fluorescence quantitative PCR.Cell proliferation activity was determined by MTS assay.Results The 14-3-3σ protein level was higher in pancreatic cancer tissue than in normal pancreatic tissue.mRNA and protein expression of 14-3-3σ was highest in BxPC3,high in AsPC-1,SW1990 and CFPAC-1,low in PANC-1 and Capan-1,and lowest in MiaPaCa-2.The successfully constructed pEGFP-14-3-3σ was confirmed by RT-qPCR and Western blot.MTS assay showed cell proliferation activity was significantly enhanced by overexpression of the 14-3-3σ gene compared to negative and blank control cells.Conclusion The expression of 14-3-3σ was higher in pancreatic cancer compared with normal pancre atic tissue,and the 14-3-3σ gene enhanced the cell proliferation activity of PANC-1.Therefore,14-3-3σ may play an important role in pancreatic cancer development.