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.

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 evaluate the effect of surgical treatment in facial nerve paralysis.Methods Clinical data of 29 cases in facial nerve paralysis were retrospectively analyzed.All of the 29 cases of facial paralysis,18 cases is the suppurative otitis media,9 cases is the temporal bone fracture,2 cases is the neoplasms of the temporal bone.The 29 cases of facial nerve paralysis were surgical treatment.8 cases by vertical segment or horizontal segment of facial nerve decompression,19 cases by the stylomastoid foramen to the geniculate ganglion of facial nerve decompression,2 cases by the itratemporal course of facial nerve decompression.1 case was underwent end-to-end anastomosis,2 cases of the greater auricular or the sural nerve graft for repairing facial nerve defect.All data were analyzed with Rank sum test.Results Makes a follow-up visit for 6～18 months,the facial nerve function(House-Brackman grading system)before the technique Ⅱ 6.9%,Ⅲ17.2%,Ⅳ34.5%,Ⅴ 31.0%,Ⅵ 10.3%,after the technique,restores Ⅰ 6.9%,Ⅱ27.6%,Ⅲ27.6%,Ⅳ 24.1%,Ⅴ 13.8%,statistics analysis facial nerve function restoreS has the significance difference(P＜0.005).Conclusions The facial nerve decompression and the nerve graft are useful method to treat facial paralysis.Surgical treatment of facial paralysis is satisfied in the suppurative otitis media and the temporal bone fracture.

Objective:To explore the expression and biological functions of miR-346 in nasopharyngeal carcinoma.Methods:63 cases nasopharyngeal carcinoma tissue and 34 cases nasopharyngeal non-cancer tissues were collected,the miR-346 expression were detected by Real-time PCR between nasopharyngeal carcinoma tissue and nasopharyngeal non-cancer tissues,6 strains of human nasopharyngeal carcinoma cell lines and 1 strain of normal nasopharyngeal epithelial cell immortalized NP69.Two cell lines with middle expression levels in human nasopharyngeal carcinoma cells lines were selected,and transfected into miR-346 inhibitor,the control group (NC group) were with negative control plasmid transfection,miR-346 expression in two groups were detected by Real-time PCR,the proliferation,apoptosis were detected by Brdu-ELISA and flow cytometry,the migration and invasion were detected by Transwell Chambers experiments.Results:Compared with the nasopharyngeal non-cancer tissues,the miR-346 expression in nasopharyngeal carcinoma tissues was significantly increased (P =0.000);compared with the normal nasopharyngeal epithelial cells NP69.the miR-346 expression in human nasopharyngeal carcinoma cell lines was significantly increased (P＜0.05).The CNE-1 and CNE-2 were chose,after the miR-346 inhibitor transfection,the miR-346 expression were significantly lower compared with NC group,the difference was statistically significant (P＜0.05).The proliferation of two kinds of nasopharyngeal carcinoma cell CNE-1 and CNE-2 were restrained after transfection,the difference showed statistically significant 3 days after transfection (P ＜ 0.05).The apoptosis increased significantly,and the migration cell numbers and invasion cell numbers decreased significantly compared with NC group,the differences were statistically significant (P＜0.05).Conclusion:miR-346 is overexpression in nasopharyngeal carcinoma,down-regu-lation of miR-346 inhibits the proliferation,migration,invasion,promotes the apoptosis,miR-346 may act as a oncogene and play an important role in the pathogenesis of nasopharyngeal carcinoma.

Objective To observe the influence of early postoperative feeding in the healing of intestinal anastomosis in rabbits,and to clarify preliminarily the relationships between early postoperative feeding after gastrointestinal surgery and gastrointestinal anastomotic fistula formation and healing time in rabbits.Methods 48 rabbits were randomly divided into experimental group and control group, then they were treated with gastrointestinal anastomosis.The rabbits in experimental group were fed with liquid diet 24 h after operation,and the rabbits in control group were fed nothing after operation and supplied by total parenteral nutrition.Two rabbits of each group were selected for exploratory laparotomy on the 3rd,5th,7th,10th and 15th day after operation,and the healing rate of anastomosis,the anastomotic bursting pressure,the anastomotic breaking strength,and the hychoxyproline level of anastomosis were observed.Results The healing rate of anastomosis in control group was 91.6%(22/24), and the healing rate of anastomosis in experimental group was 95.8%(23/24),there was no significant difference between two groups(P>0.05).The anastomotic bursting pressures of the rabbits in two groups were decreased remarkably at the 72nd hour after operation,which was the lowest point,and they were increased remarkably on the 5th day after operation,but the anastomic bursting pressure in experimental group was a little lower than that in control group,and it reached the peak on the 7th day after operation in control group. On the 10th day after operation,the anastomic bursting pressure in control group was a little lower than that on the 7th day after operation,but the anastomic bursting pressure in experimental group reached the peak.There were no significant differences of anastomic bursting pressure at different time points between two groups(P>0.05).The anastomotic breaking strength had no significant difference between two groups at the 72nd hour after operation,both of them reached the lowest points,however the anastomtic breaking strengths in two groups were increased remarkably on the 10th day after operation,and reached the peaks.but there were no significant differences of anastomic breaking strength at different time points between two groups (P>0.05 ). The hychoxyproline level of anastomosis:in experimental group was a little lower than that in control group at the 72tnd hour after operation,and both of them reached the peaks on the 7th day after operation;but there were no significant differences of hychoxyproline levels of anastomosis at different time points between two groups(P>0.05).Conclusion Early postoperative feeding can not cause the increase of anasmotic healing time and the incidence rate of gastrointestinal anastomotic fistula.

Objective:To investigate the expression and biological significance of MicroRNA-204 in nasopharyngeal carcinoma (NPC). Methods: qRT-PCR was applied to detect the relative expression of miR-204 in 43 paired nasopharyngeal carcinoma in comparison to the normal nasal mucosa tissues. Pearson chi-square test was used to analyze the relationship between the miR-204 expression and clinical features. The expressions of Bcl-2 and SIRT1 were measured by immunohistochemistry ( IHC ) , Spearman correlation analysis was used to analyze the relationship between miR-204 and Bcl-2,as well as SIRT1. We then transfected the miR-204 mimics into CNE-2 cells,then the Western blot was used to detect the expression of Bcl-2 and SIRT1,which were considered as the potential targets of miR-204. Results:The relative expressions of miR-204 was significantly downregulated in NPC tissues compared to those of the matched normal tumor-adjacent tissues(P<0. 05). Low expression of miR-204 was significantly associated with lymphatic metastasis(P<0. 05) and advanced TNM stage(Ⅲ+Ⅳ,P<0. 05). The expressions of Bcl-2 and Sirt1 in lower miR-204 level group were both higher than in higher miR-204 level group ( P<0. 05 ) . Both the mRNA and protin expression in CNE-2 cells were down-regulated after transfection. Conclusion: Low expression of miR-204 is related to the malignant clinicopathological features in NPC tissues,and miR-204 may through down-regulate Bcl-2 and SIRT1 to suppress NPC genesis and development.

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 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.

Objective To investigate transcription intermediary factor 1β (TIF1β) expression in paracancerous pancreatic tissue and pancreatic tumor tissue by using tissue microarray.The relationship between TIF1β expression and clinicopathological factors in patients with pancreatic cancer was discussed.Method Tissuc microarray and immunohistochemical assay were utilized to detect expression of TIF1β protein in pancreatic cancer tissues and the corresponding non-tumor tissues from 91 cases.Results TIF1β protein were present in pancreatic cancer tissues as well as corresponding non-tumor tissues with varying degrees of expression,and was located in the nucleus.TIF1β expression in pancreatic cancer tissue was significantly higher than that of adjacent tissues (P ＜ 0.05).And it was noted that there was close correlations between TIF1β expression and clinical pathological staging,lymph node metastasis and TNM grading (P ＜ 0.05).Conclusions TIF1β is highly expressed in pancreatic cancer,clinically correlated with pathological staging,lymph node metastasis and TNM grading.TIF1β may play an important role in development of pancreatic cancer.