Objective To establish in vitro model for perineural invasion (PNI) of pancreatic cancer,and observe the process of PNI.Methods Mouse dorsal root ganglia (DRG) and pancreatic cancer Capan-2 cell line were co-cultured in Matrigel matrix.Two models were constructed:model 1 for observing areas of cell colonies and model 2 for observing distance of cell migration.Neurite outgrowth and cell colony growth were observed by invert microscope,images were analyzed by using Image pro plus software.Results Neurite outgrowth was not affected by co-culture.DRG neurite appeared radial growth under either co-culture or single culture.It was observed that Capan-2 cell could migrate to DRG,and grows around the nerve fiber as spindleshaped,then continue to migrate to DRG.At the 5th day,in the co-culture group of model 1,the colony area was (309.28 ±19.11) μm2,which was significantly bigger than that in single culture model [(208.57±7.94) μm2,P ＜0.01].At the 5 th day,in the co-culture group of model 2,Capan-2 migrated (284.1 ±12.9) μm to DRG,while in the side of blank Matrigel,cell migrated less than 150 μm,the difference was statistically significant (P ＜0.01 ).Conclusions The in vitro models for perineural invasion in pancreatic cancer were constructed successfully,which lay a good foundation for future studies.

Objective To investigate the role of mucosal mast cells infiltration and degranulation with nerve growth factor (NGF)in development of visceral hypersensitivity in Sprague-Dawley (SD)rats. Methods The model of visceral hypersensitivity of irritable bowel syndrome (IBS)was established in 19 neonate SD rats with intestinal stimulation (rectalballon distention)on 8th,10th and 12th postnatal days. The other 19 neonate SD rats without colonic distention were assigned to the control group.After rats grew up (six to eight weeks old),the visceral sensitivity was tested by abdominal withdrawal reflex (AWR)in 10 rats of each group.Mast cell infiltration and degranulation were observed with toluidine blue staining in colon tissue slides.The NGF level of intestinal tissues was detected by enzyme-linked immunosorbent assay (ELISA)methods in the left nine rats of each group.The culture system of dorsal root ganglias (DRG)from the neonatal rats was set up.The changes of electrophysilogical characters of DRG stimulated with NGF (100 ng/mL)for four days were recorded with patch-clamp.Paired t test was performed for comparison between groups.Results The results of AWR indicated that neonatal colonic stimulation could significantly increase visceral sensitivity after growing up.Under 20,40 and 60 mmHg (1 mmHg=0.133 kPa)distention pressure,visceral sensitivity scores of visceral hypersensitivity rats and rats of control group were 1 .00±0.50 vs 1 .67 ±0.50,1 .89 ±0.31 vs 2.89 ±0.34 and 2.89 ±0.33 vs 3.89±0.33,the differences were statistically significant (t=-2.83,-6.00 and -6.00,all P <0.05 ). The results of master cells staining in tissue slides showed colonic master cells infiltration was obvious in rats with visceral hypersensitivity,and part of mast cells were degranulation.The result of ELISA demonstrated that NGF level of visceral hypersensitivity rats was significantly higher than that of control group ((11.07±3.06)pg/mg vs (2.38 ±1.88)pg/mg,t =-6.93,P <0.05).The results of electrophysilogical tests of primary cultured DRG indicated that compared with blank control growp,the action potential threshold of neuron in NGF 100 ng/mL group significantly decreased ((-18.0±2.1 )mV vs (-29.0 ± 2.5 )mV,t = 12.26,P <0.05)and discharge frequency increased ((5 .0± 1 .4 )/800 ms vs (12.0 ± 3.2)/800 ms,t=-8.40,P <0.05 ).Meanwhile,neuron voltage-gated K+ current density remarkably decreased,most were sustained delayed rectifier K+ current (I K )decreasing ((279.0 ±48.0)pA/pF vs (203.0±39.0)pA/pF,t=6.18,P <0.05).Conclusion Colonic stimulation in neonatal rats could cause intestinal master cells infiltration and degranulation,which induced changes of neuron electrophysilogical characters and resulted in visceral hypersensitivity after growing up.

Objective To detect and analyze the value of adipokines in the diagnosis and prognosis of patients with pancreatic cancer complicated with diabetes.Methods From July 2012 to June 2013,24 pancreatic ductal adenocarcinoma (PDAC) complicated with diabetes mellitus (DM) (study group),30 PDAC (control group A) and 31 DM (control group B) cases were collected.Serum levels of adiponectin,resistin,leptin and visfatin were tested and the differences were compared among different groups.Receiver operator characteristic (ROC) curves were used to analyze the diagnostic value of the above cytokines.The patients were followed up for two to 58 months.Kaplan-Meier survival curve was performed to analyze the prediction value of above cytokines in recurrence-free survivals (RFS) in patients with PDAC complicated with DM.Chi square test,t test and Mann-Whitney U test were used for statistical analysis.Results The median levels of serum adiponectin and resistin of study group were both higher than those of control group B (36.92 mg/L,15.80 mg/L to 101.57 mg/L vs 12.84 mg/L,5.64 mg/L to 21.39 mg/L;9.07 μg/L,6.39 μg/L to 12.19 μg/L vs 5.93 μg/L,4.22 μg/L to 7.68 μg/L,respectively),and the differences were statistically significant (Z=-3.462 and-2.868,P=0.001 and 0.004,respectively).However the serum levels of leptin and visfatin of study group were both lower than those of control group B (1.02 μg/L,0.11 μg/L to 2.06 μg/L vs 1.92 μg/L,0.96 μg/L to 2.72 μg/L;2.43 μg/L,0.48 μg/L to 4.28 μg/L vs 4.18 μg/L,2.43 μg/L to 7.28 μg/L),and the differences were statistically significant (Z=-1.986 and-2.336,both P＜0.05).The results of ROC curve analysis of adipokines in differential diagnosis of pancreatic cancer from diabetes indicated that the areas under the curve (95 ％ confidence interval) of serum adiponectin,resistin,leptin and visfatin in the diagnosis of PDAC were 0.774 (0.645,0.903),0.727 (0.593,0.861),0.657 (0.504,0.810) and 0.685 (0.543,0.826),respectively,with statistical significance (P=0.001,0.004,0.047 and 0.020,respectively).The sensitivity of combination of CA19-9 and adiponeetin in the diagnosis of PDAC complicated with DM was 0.917 and the negative predictive value was 0.940.The leptin level in advanced PDAC patients was lower than that in non advanced PDAC patients (0.61 μg/L,0.11 μg/L to 2.28 μg/L vs 1.86 μg/L,0.79 μg/L to 4.14 μg /L);and the difference was statistically significant (Z=-2.210,P=0.027).Higher serum adiponectin level was correlated with early recurrence after operation in PDAC patients complicated with DM (P=0.035).Conclusions Serum adipokines may be valuable in screening PDAC in patients with DM.And adipokines may be valuable in the prediction of recurrence after operation in PDAC patients complicated with DM.