Abstract:

Objective:To investigate the possibility of survivin inhibition by lentiviral vector-mediated RNA interference and the influence on cell apoptosis in pancreatic cancer cell line.
Methods:The lentiviral vector of SiRNA targeted against survivin(LV-shRNA-survivin-1, LV-shRNA-survivin-2, LV-shRNA-survivin-3) was constructed and transfected into the packaging cells 293T, and then the supernatant with virus was collected to transfect SW1990 cells. Quantitative real-time fluorescent PCR and Western-blot were used to detect the expression of survivin. DAPI staining and detection of enzymatic activity of caspase 3/7 were employed to examine cell apoptosis.
Results:Three lentiviral vector-survivin-shRNA were constructed successfully. In the LV-shRNA-survivin-1 group,the survivin mRNA and protein expression inhibitory rate was 73.50% and 87.64% respectively; when compared to control group, the activity of caspase-3/7 increased significantly,which showed a 14.5-fold increase, and apoptosis increased 11.95%.
Conclusions:Lentiviral vector-mediad RNA interference targeted against survivin can effectively inhibit survivin expression and increase cell apoptosis significantly.

Abstract:

Objective:To investigate the effects of histone deacetylase inhibitor trichostatin A (TSA), on the cell growth of human pancreatic cancer cell line PANC-1 in vitro and its mechanism.
Methods:The proliferating activity of PANC-1 cells was observed by MTT assay. The cell apoptosis and cell cycle distribution were detected by flow cytometry. Western blot and real-time PCR were used to assess the expression levels of inhibitor of apoptosis gene bcl-2 and cell cycle regulation gene p21 WAF1/CIP1.
Results:A dose dependent inhibition was remarkably confirmed in PANC-1 cells treated by TSA. As compared with control group, apoptosis rate of PANC-1 cells in TSA group was increased obviously at 72h (P＜0.05). The percentage of G 0/G 1 phase increased markedly, while the percentage of S phase decreased significantly (P＜0.05). Furthermore, the expression level of bcl-2 mRNA and protein in TSA group was significantly lower than that in control group (P＜0.05). The expression level of p21 WAF1/CIP1 mRNA and protein in TSA group was significantly higher than that in control group (P＜0.05).
Conclusions:Our data demonstrate that TSA-induced growth arrest of pancreatic cancer cell line is induced by a block in the G 0/G 1 phase and apoptosis, which may occur through down-regulated expression of apoptosis gene bcl-2 and up-regulated expression of cell cycle regulation gene p21 WAF1/CIP1.

Abstract:

Objective:To investigate the expression of TLR2 and TLR4 in pancreatic cancer and the clinicopathological significance.
Methods:The mRNA of TLR2 and TLR4 in 30 cases of pancreatic cancer and its adjacent tissues were detected with real-time PCR, and the protein of TLR2 and TLR4 in 65 cases of pancreatic cancer and 38 cases of adjacent tissues were detected with immunohistochemieal methods, and the relationship between TLR2 or TLR4 and clinico pathological features of pancreatic cancer were analyzed．Kaplan-Meier method was used to assess the impact of TLR2 or TLR4 expression on survival.
Results:The relative quantification of TLR2 mRNA and TLR4 mRNA in pancreatic cancer tissues was 0.84±0.17 and 0.81±0.10 respectively, which was significantly higher than that in adjacent tissues（0.70±0.13 and 0.70±0.16 respectively，P＜0.05), and protein expression of TLR2 and TLR4 in pancreatic cancer tissues was 63.10% and 69.2% respectively,which was significantly higher than that in adjacent tissues(34.2% and 39.5% respectively，P＜0.05).There was no significant correlation between the expression of TLR2 or TLR4 mRNA or protein with the age，gender，tumor location or the degree of differentiation in patients with pancreatic cancer(P>0．05)．However，there was significant correlation between the expression of TLR2 or TLR4 mRNA or protein with tumor size, lymph node metastasis,venous invasion and clinical staging （P＜0.05）. The mean survival of patients with negative TLR2 or TLR4 expression in tumor tissue was 18.4 and 19.7 months respectively,which was significantly longer than those with TLR2 or TLR4 positive expression（12.4 months，P＜0.05）.
Conclusions:TLR2 and TLR4 overexpressed in pancreatic cancer, and TLRs signaling pathway may promote development of pancreatic cancer．

Abstract:

Objective:To investigate low molecular weight heparin (LMWH) therapy for pancreatic encephalopathy in severe acute pancreatitis (SAP).
Methods: SD rats were randomly divided into 3 groups: (1) Sham operation (S) group，(2) SAP group and (3) LMWH treatment (LT) group：LMWH was administrated at 4 hours after construction of SAP model. The levels of serum amylase, myelin basic protein (MBP), tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), brain water contents and pathological changes of pancreas and brain were measured at 24 h after models were set up in SAP and in S group，and 24 h after LMWH was administrated in LT group.
Results:(1) The levels of serum amylase, TNF-α, IL-6 in SAP group were significantly higher than those in S group and LT group (all P＜0.001); the levels of serum MBP in SAP group were significantly higher than those in S group and LT group(P＜0.01,<0.05 respectively), and the level of serum amylase and IL-6 in LT group were significantly  higher than those in S group (P＜0.05,<0.001 respectively); but the levels of TNF-α and MBP showed no significant difference between LT group and S group (all P＞0.05). (2) The brain water content in SAP group was significantly higher than that in S group and LT group (P＜0.01, <0.05 respectively). (3) Patch necrosis of acinar cells and massive hemorrhagic changes of pancreas could be observed in SAP group, but the necrosis of acinar cells and hemorrhagic changes in LT group were markedly less severe than those in SAP group. (4) In encephalon tissue of SAP group, demyelination, neuronal apoptosis, and mitochondrial vacuolation in neuronal cells were observed; but in LT group, there were significantly less demyelination and mitochondrial edema of neuronal cells than those in SAP group, and neuronal apoptosis was not found in LT group.
Conclusions: LMWH treatment can significantly reduce the release of pancreatic enzymes, reduce neuronal apoptosis and inhibit the production of inflammatory mediators,and thus reduce the occurrence of pancreatic encephalopathy in SAP.

Abstract:

Objective:To study  the expressive levels of EZH 2 and PTEN in pancreatic cancer and non-cancerous tissue, and the effects on carcinogenesis of rat pancreas.
Methods:dimethylbenzathracene (DMBA) was directly implanted into the parenchyma of rat pancreas (group A,group B). The rats of group B were treated with 1 mL trichostatin A (TSA) solution (1μg/mL) intravenously per weer 1 week after the model were set up. The rats of group A, B were killed within 3-5 months
 and the rats in the control (C group) were executed at 5 months to observe the develope of pancreatic cancer by macrograph and microscopy, The EnVision TM immunohistochemistry was used to assay the expression of EZH 2 and PTEN in above pancreatic specimens.
Results:(1) The incidence of pancreatic cancer within 3-5 months in group A was 48.7% (18/37), including 17 cases of ductal adenocarcinoma and 1 case of fibrosarcoma. The incidence of pancreatic cancer in group B was 33.3% (12/36), including 11 cases of ductal adenocarcinoma and 1 cases of fibrosarcoma. The mean maximal diameter of mass was significantly higher in  group A than that in group B (P＜0.05). No pathological changes were found in pancreas of group C or other main organs of group A and group B. (2) the positive rxpression rates of EZH 2  were significantly higher in ductal adenocarcinoma of group A + group B than those in non-cancerous pancreatic tissues (P＜0.01). The positive expression rate of PTEN was significantly lower in ductal adenocarcinoma in group A + group B than that in non-cancerous pancreatic tissues (P＜0.05). But no stastical difference was found among the positive rate of PTEN in ductal adenocarcinoma of group A or group B and in non-cancerous pancreatic tissues of group A or group B (P＞0.05). The non-cancerous pancreatic tissues with positive expression of EZH 2 and/or negative expression of PTEN showed mild to severe atypical hyperplasia of ductal epithelium. An inconsistency was found between the expression of EZH 2 and PTEN in ductal adenocarcinoma（P=0.045）. Pancreas of group C showed negative expression of EZH 2 and positive expression of PTEN. Two cases of fibrosarcoma showed negative expression of EZH 2 and PTEN.
Conclusions:By use of higher dose of DMBA  directly implanted into the parenchyma of pancreas,  high incidence  of pancreatic cancer can be obtained. TSA could have an inhibitive effect on carcinogenesis and growth of pancratic cancer. The activation of EZH 2 gene and inactivation of  PTEN gene might have key effects on  pancreas carcinogenesis induced by DMBA in rat.

Abstract:

Objective:To study the effect of Qing Yi Tang (QYT) on bacterial and endotoxin translocation from intestine in acute hemorrhagic necrotizing pancreatitis (AHNP) in rats.
Methods:SD rats were  randomly divided into 3 groups: AHNP group, AHNP +QYT treatment group and sham operation(SO) group. AHNP models were set up in the former two group, while the pancreas was only mobilized in SO group. After setting up the model, QYT was gavaged in the QYT group, while normal saline was gavaged in the SO group and AHNP group. The concentration of  endotoxin(ET) and the DNA of E. coli expressoin in portal vein blood in three groups of rats were measured 24h later.
Results:The concentration of ET and amount of E.coli DNA in portal vein of QYT group were lower than those of  AHNP group.
Conclusions:QYT can reduce significantly the concentration of ET and E.coli DNA in portal vein blood and decrease bacterial and endotoxin translocation from intestine in the AHNP rats.

Abstract:

Objective:To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human SIRT1 gene,  assay the expression of SIRT1 in pancreatic carcinoma PANC-1 cells after transfecting with recombinant plasmids, and detect the RNAi effect of shRNA.
Methods:Three plasmid expression vectors coding for shRNA targeting SIRT1 gene sequence and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in E.coli. DH5α was identified by restriction digestion, PCR and sequencing. The vectors were transfected into PANC-1 cells. SIRT1 expression was assayed with real-time quantitative PCR and Western blot.
Results:The successful construction of recombinant plasmids was confirmed by DNA sequencing of the inserted segments. Transfection of shRNA plasmids significantly down-regulated SIRT1 expression in PANC-1cells. Recombinant plasmid 1 had the strongest effect, with an inhibition ratio of 79.43% at the mRNA level and 83.27% at the protein level, which showed a significant difference from plasmids 2 and 3 (P＜0.05).
Conclusions:Plasmid vector expressing shRNA against SIRT1 has been successfully constructed and it can down-regulate SIRT1 expression after transfected into PANC-1 cells. This finding could facilitate further studies on SIRT1 function and its application in tumour gene therapy．

Abstract:

Objective:To detect the inhibitory effect of HSP27-siRNA on hepatocellular carcinoma (HCC) cells.
Methods:HCC cell QGY lines were cultured with HSP27-siRNA in a differtent range of concentration  for various time periods.Cell activity was studied by MTT.The changes of cell cycle and apoptosis were analyzed by FCM. RT-PCR was used to detect the effect of HSP27-siRNA on QGY cell expression of HSP27 mRNA. Western blot was used to detect the inhibition efficiency of HSP27-siRNA on HSP27protein.
Results:The proliferation of QGY cell was inhibited by HSP27-siRNA,and HSP27-siRNA decreased the expression of HSP27 protein.HSP27-siRNA inhibited the proliferation of QGY cell line and induced apoptosis in vitro, and its effect was both dose-and time-dependent.
Conclusions:HSP27-siRNA can inhibit proliferation and induce apoptosis of HCC cancer cell lines. Thus, it may become a method for effective treatment of HCC cancer.

Abstract:

Objective:To investigate the effect of IGF-I on the apoptosis and the expression of survivin through the signaling pathway of PI-3K/Akt in colon cancer cell line SW480. 
Methods:When colon cancer cell line SW480 was culfured, Western blot was used to detect the expression of survivin protein in SW480 cells after they were stimulated by different concentrations of IGF-I for different lengths of time. The phosphorylation of Akt in SW480 cells was determined by Western blot after SW480 cells were stimulated by 100 ng/mL  IGF-I with in 2 h.Before and after the specific inhibitor LY294002 was treated in SW480 cells to block the pathway of PI-3K/Akt,  the phosphorylation level of Akt protein was detected by western blot, survivin protein by western blot and cell immunofluorescence and the cell apoptosis rates of different treated-groups were measured by flow cytometry.
Results:IGF-I up-regulated the expression of survivin in a dose-dependent and time-dependent manner in colon cancer line SW480（when treated with IGF-I for 12h, the expression of Survivin reached  the peak and when treated with 100ng/ml IGF-I, the Survivin reached the highest expression level）; IGF-I actived the signaling pathway of PI-3K/Akt of SW480 cells quickly（the expression of p-Akt proteins in SW480 reached the maximum level when cultured with IGF-I for 15 and 30min, but after that they quickly decreased）; IGF-I induced survivin expression through the PI-3K/Akt in colon cancer line SW480; IGF-I could inhibited the apoptosis of SW480 cells through the PI-3K/Akt［use of flow cytometry for detection in the blank, stimulation and block groups showed the SW480 cell apoptosis rate was(5.18±0.415)%, (0.85±0.052)%,(3.15±0.411)% respectirely, with significant differences between the groups(P＜0.05）］.
Conclusions: IGF-I could induce survivin expression through the PI-3K/Akt in colon cancer line SW480, then lead to the inhibition of its apoptosis.

Abstract:

Objective:To study the antitumor effectivity of special cytotoxic T lymphocytes(CTLs) induced by B lymphocytes in mice.
Methods:B lymphocytes were collected, isolated and purfied. Cells were initially activated by CD40L and rmIL-4, then cocultivated with T lymphocytes. T lymphocyte proliferation was examined. Total RNA, which was extracted from Hepal-6(a hepatocelluar carcinoma cell line), were transfected into B lymphocytes, as experimental group; while transfected with RNA of mice liver cells,liposimes and 1640 were as control groups, The expression of antigen presenting cell(APC) markers (CD40,CD80 and CD86) and major histocomability complex(MHC) on B cell surface after transfection were deteced. CTL were obtained by stimulating T lymphocytes with transfected B lymphocytes. Hepal-6 was cell-targeted and examined as index of CTL killing activity. The IFN-r secretion of stimulated CTL was quantified.
Results: T cell proliferation in experimental group had a higher degree than that in RNA control group (P＜0.05), liposome control group and blank control group(P＜0.01). and the MHC and co-stimulating molecules expression of experimental group were significantly higher than other groups. Compared to control groups, the  killing activity and IFN-γ secretion of in experimental group was significantly increased (P＜0.05).
Conclusions:HCC RNA-transfected B lymphocyte has capability of inducing anti-tumor effects through CTL response, which may present a potential pathway for future HCC treatment.

Abstract:

Objective:To study the efficiency enhancement and toxicity reduction effects of scutellaria barbata polysaccharides (SBPS) on mice treated with cytoxan (CTX).
Methods:Transplanted H 22 hepatoma mice were randomly divided into negative control group, CTX group and combined SBPS and CTX group. After 10 d of consecutive drug administration, the tumor inhibitory rate, blood  white cell count, phagocytic index and activity of IL-2 were measured.
Results:SBPS increased the tumor inhibitory rate and reduced the toxicity of cytoxan, and enhanced the function of mononuclear phagocytes and the activity of IL-2 and TNF-α.
Conclusions:SBPS has efficiency enhancement and toxicity reduction effects on cytoxan, and the mechanism may be by increasing the immunologic function of the organism.

Abstract:

Objective:To discuss the risk factors of postoperative bleeding after pancreaticoduodenectomy (PD).
Methods:The clinical data of 305 patients receiving PD in our hospital from December 2004 to January 2008 were analyzed retrospectively.
Results:The incidence of postoperative bleeding was 9.18%.Univariate analysis showed that hypertension, preoperative blood serum bilirubin level, skeletization procedure, internal drainage, operative blood loss, intraoperative blood transfusion,post-operative hemoglobin levels and intra-abdominal infection were significantly associated with postoperative bleeding after PD. Multivariate analysis, using logistic regression, identified 4 variables as independent factors associated with the occurrence of bleeding, i.e., hypertension, preoperative blood serum bilirubin level, operative blood loss,and intra-abdominal infection. The predictive equation was P=1/{1+exp［-(-4.8785+1.2892gxy+0.0082dhs+0.0004sxl+1.5721fqgr)］}.
Conclusions:The independent risk factors for postoperative bleeding after PD are hypertension,preoperative blood serum bilirubin level more than or equal to 171 μmol/L, operative blood loss more than or equal to 700 mL, and intra-abdominal infection.Thus,appropriate perioperative management and degree of surgical skill of the operator can effectively decrease postoperative bleeding after PD.

Abstract:

Objective:To study the effect of early enteral nutrition(EN) and total parenteral nutrition (TPN) in patients with severe acute pancreatitis by a mata analysis.
Methods:Randomized controlled trials of TPN and early EN in patients with severe acute pancreatitis were searched in Medline,Pubmed and Embase from Jan 1970 to June 2009,and Cochrane Library (Issue 2, 2009). Seven studies were enrolled into the analysis. The detail about the trial design,characters of the subjects and results of the studies were reviewed by two independent authors and analysed by using Revman 5.0.18 software.
Results:Compared with TPN,early EN was associated with a significantly lower incidence of pancreatic infections (OR=0.38,95% CI:0.18～0.82, P=0.01),organ failure (OR=0.43,95% CI:0.23～0.79, P=0.007), fewer surgical interventions (OR=0.34,95% CI:0.18～0.63, P=0.0006) and mortaily (OR=0.41, 95%  CI:0.19～0.88, P=0.02). However, there was no significant difference in total infections ［OR=0.43，95% CI（0.17，1.10）,P=0.08］between patients with TPN and early EN.
Conclusions:Early EN could be the preferred nutrition feeding method for patients with severe acute pancreatitis.

Abstract:

Objective:To study  the operative method for severe duodenal trauma.
Methods:The clinical data of 38 cases of severe duodenal trauma complicated with pancreatic injury who underwent different operations between 1992—2006 year were reviewed.
Results:In 8 cases duodenal diverticulization was performed, of which 5 cases were cured, 2 cases developed intestinal fistula, and one  died.Of 9 cases who underwent panceaticoduodenectomy(PD), 3 were cured but 6 had pancreatic leakage, and 3 of them died.Of 16 cases who had primary repair of ruptured duodenum with simple suture or patch suture, 13 cases cured, 2 cases developed intestinal fistula and one patient died.Five patients underwent duodenal repair and excision of head of pancreas plus pancreaticojejunostomy (PD with preserved duodenum), 4 cases recovered and 1 had pancreatic leakage, but 3 with wrap-type pancreaticojejunostomy had no complications.
Conclusions:The repair of ruptured duodenum with simple suture plus duodenostomy and jejunostomy is preferential option for majority of cases of duodenal trauma. This procedure plus removal of head of pancreas and  pancreaticojejunostomy (PD with preserved duodenum) can be used for severe pancreatic injury with excellent result, but duodenal diverticulization or  panceaticoduodennectomy must not be undertaken lightly.

Abstract:

Objective:To investigate clinical features and clinical experience of diagnosis and treatment of solid-pseudopapillary tumor of the pancreas (SPTP).
Methods:The clinical records of  757 cases of SPTP were analyzed retrospectively, which included 748 cases concerning SPTP published in Chinese medical journals and 9 cases of SPTP admitted to our hospital.
Results:Among the patients, 66 were males, 691 were females, and the mean age was 26.6 years. Major clinical manifestations were abdominal pain or discomfort (45.3%) and palpable abdominal masses (35.5%),but some cases (22.0%) were completely asymptomatic and the lesions were detected by routine examinations. In 95.9% of cases,  B ultrasonic examinations could detect the tumors，but the accuracy for judging the location of tumors by CT(87.5%) and MRI(90%) was better than that by B ultrasonic exam (71.2%). The tumors were often located in the head (39.1%) or the body and tail (54.1%) of the pancreas. The main treatment for SPTP was surgical procedure including enucleation (34.1%), pancreatoduodenectomy (23.7%) and distal pancreatectomy with splenectomy (22.5%).It was found that 60.3% of the tumors were solid and cystic, had complete capsule, and had a mean diameter of 8.0 cm.Among the 458 cases that were followed up, 450 cases were alive, including 3 cases with recurrences, 3 cases treated with tumor reductive surgery and 1 case with hepatic metastases after surgery. Of the 450 cases 8 died.
Conclusions:SPTP occurs predominantly in young females. Surgical procedure is the main effective treatment, and the prognosis is good, even for the cases with metastases or recurrences, the aggressive surgical therapy is good.

Abstract:

Objective:To investigate the methods and effects  of applying choledochoscope in  drainage of retroperitoneal abscess.
Methods:Eighteen cases of retroperitoneal abscess, who underwent open surgical debridement and drainage or PCD but the drainage was ineffective,  were selected for use of choledochoscope to irrigate and debride the abscess repeatedly.
Results:All of the 18 patients had effective choledoscopic treatment; in 17 patients the abscess healed and drainage catheter was successfully removed; but in one patient, who developed pancreatic fistula, was cured by internal drainage operation performed 6 months later.
Conclusions:Use of choledochoscope as an adjunctive treatment after drainage operation of retroperitoneal abscess is simple, safe, and effective.