胰体尾导管腺癌术后胰瘘危险因素分析

目的探讨胰体尾导管腺癌术后胰瘘危险因素。方法单中心、回顾性分析华中科技大学同济医学院附属协和医院2016年1月至2018年12月间因胰体尾导管腺癌行胰体尾切除术的所有病例。收集病例术前、术中、术后资料,并用SPSS v22.0对数据行单因素、多因素分析。胰瘘的定义及分组按照2016年国际胰瘘研究组制定标准执行,所有病例均随访至少3个月。结果共91个病例被纳入研究,总体胰瘘率为25.27%(23/91),术后90 d内无死亡病例发生,共3个胰瘘危险因素被鉴定出:胰腺质地(软)[比值比=8.965,95%置信区间(2.400,33.490),P=0.001],合并心血管病[比值比=9.148,95%置信区间(1.936,43.225),P=0.005],术后第1天白蛋白<26.50 g/L[比值比=6.100,95%置信区间(1.846,20.157),P=0.003]。结论胰腺质地软、合并心血管病、术后第1天较低白蛋白水平是胰体尾导管腺癌术后胰瘘发生的独立危险因素。由于研究的局限性,结果尚待进一步验证。

胰体尾癌行机器人扩大根治术的临床疗效

目的:比较达芬奇机器人手术系统行扩大的胰体尾导管腺癌淋巴结清扫术与标准的淋巴结清扫术的临床疗效。方法:回顾性分析我院外科自2010年4月至2015年9月收治54例胰体尾导管腺癌病人的临床资料,其中37例行标准的淋巴结清扫术,17例行扩大的淋巴结清扫术。比较两组病人一般资料、手术时间、术中出血量、术后住院天数、淋巴结清扫数、术后并发症发生率。结果:扩大清扫组与标准清扫组病人一般情况基本一致。扩大清扫组手术时间长于标准清扫组,有统计学差异(231.5 min比141.4 min,P<0.001)。扩大清扫组术后住院天数长于标准清扫组,有统计学差异(22.8 d比20.4 d,P<0.05)。扩大清扫组淋巴结清扫个数多于标准清扫组,有统计学差异(11.2枚比6.1枚,P<0.001)。两组术中出血量、胰漏、胃排空障碍、术后出血、术后感染发生率差异均无统计学意义(P>0.05)。结论:机器人手术系统行胰体尾导管腺癌的淋巴结扩大清扫是安全的,与标准清扫无统计学差异,不增加病人的并发症发生率。

经屈氏韧带路径的腹腔镜根治性前入路顺行模块化胰体尾癌切除术的临床应用

目的评价经屈氏韧带路径的腹腔镜根治性前入路顺行模块化胰体尾癌切除术(L-RAMPS)的可行性和安全性。方法回顾性分析笔者所在科室于2017年11月收治的1例行L-RAMPS的胰腺癌患者的临床资料。结果该例患者的手术历时255 min,术中出血量为200 mL,未输血,术后无胰瘘、消化道出血、胃排空延迟等并发症发生。术后已获访12个月,无肿瘤复发或转移,继续随访。结论 L-RAMPS在严格选择的早期胰体尾癌患者中是安全可行的。

Persistence of side population cells with high drug efflux capacity in pancreatic cancer

AIM: To investigate the persistence of side population (SP) cells in pancreatic cancer and their role and mechanism in the drug resistance. METHODS: The presentation of side population cells in pancreatic cancer cell line PANC-1 and its proportion change when cultured with Gemcitabine, was detected by Hoechst 33342 staining and FACS analysis. The expression of ABCB1 and ABCG2 was detected by real- time PCR in either SP cells or non-SP cells. RESULTS: SP cells do exist in PANC-1, with a median of 3.3% and a range of 2.1-8.7%. After cultured with Gemcitabine for 3 d, the proportion of SP cells increased significantly (3.8% ± 1.9%, 10.7% ± 3.7%, t = 4.616, P = 0.001 < 0.05). ABCB1 and ABCG2 expressed at higher concentrations in SP as compared with non-SP cells (ABCB1: 1.15 ± 0.72, 5.82 ± 1.16, t = 10.839, P = 0.000 < 0.05; ABCG2: 1.16 ± 0.75, 5.48 ± 0.94, t = 11.305, P = 0.000 < 0.05), which may contribute to the efflux of fluorescent staining and drug resistance. CONCLUSION: SP cells with inherently high resistance to chemotherapeutic agents do exist in pancreatic cancers, which may be candidate cancer stem cells contributing to the relapse of the tumor.

Lentivirus-mediated shRNA interference targeting STAT3 inhibits human pancreatic cancer cell invasion

AIM： To investigate RNA interference targeting signal transducer and activator of transcription-3 （STAT3） on invasion of human pancreatic cancer cells.METHODS： We constructed three plasmids of RNA interference targeting the STAT3 gene. After LV （lentivirus）-STAT3siRNA （STAT3 small interfering RNA） the vector was transfected into the human pancreatic cell line, SW1990 and cell proliferation was measured by the MTT assay. Flow cytometry was used to assess cell cycle. Vascular endothelial growth favor （VEGF） and matrix metalloproteinase-2 （MMP-2） mRNA and protein expression were examined by quantitative PCR and western blotting, respectively. The invasion ability of SW1990 cells was determined by cell invasion assay.RESULTS： We successfully constructed the LVSTAT3siRNA lentivirus vector and proved that it can suppress expression of STAT3 gene in SW1990 cells. RNA interference of STAT3 by the LV-STAT3siRNA construct significantly inhibited the growth of SW1990 cells, in addition to significantly decreasing both VEGF and MMP-2 mRNA and protein expression. Moreover, suppression of STAT3 by LV-STAT3siRNA decreased the invasion ability of SW1990 cells.CONCLUSION： The STAT3 signaling pathway may provide a novel therapeutic target for the treatment of pancreatic cancer since it inhibits the invasion ability of pancreatic cancer cells.

Helicobacter species ribosomal DNA in the pancreas, stomach and duodenum of pancreatic cancer patients

AIM： To determine whether gastric and enteric Helicobacter species are associated with pancreatic cancer. METHODS： Patients with exocrine pancreatic cancer （n = 40）, neuroendocrine cancer （n = 14）, multiple endocrine neoplasia type 1 （n = 8）, and chronic pan- creatitis （n = 5） were studied. Other benign pancreatic diseases （n = 10） and specimens of normal pancreas （n = 7） were included as controls. Pancreatic tissue specimens were analyzed by Helicobacter-specific PCR-assay and products were characterized by denaturing gradient electrophoresis and DNA-sequencing. From a subset of the pancreatic cancer patients, gastric and/or duodenal tissue as well as gallbladder and ductus choledochus tissue were analyzed. Gallbladder and choledochus samples were included as controls. Stomach and duodenum samples were investigated to analyze whether a gastric helicobacter might disseminate to the pancreas in pancreatic cancer patients. Pancreatic specimens were analyzed by Bacteroidesspecific PCR for detecting the translocation of indigenous gut microbes to the diseased pancreas. RESULTS： He/icobacter DNA was detected in pancreas （tumor and/or surrounding tissue） of 75% of patients with exocrine cancer, 57% of patients with neuroendocrine cancer, 38% of patients with multiple endocrine neoplasia, and 60% of patients with chronic pancreatitis. All samples from other benign pancreatic diseases and normal pancreas were negative. Thirtythree percent of the patients were helicobacterpositive in gastroduodenal specimens. Surprisingly, H. bilis was identified in 60% of the positive gastroduodenal samples. All gallbladder and ductus choledochus specimens were negative for helicobacter. Bacteroides PCR-assay was negative for all pancreatic samples. CONCLUSION： Helicobacter DNA commonly detected in pancreatic cancer suggests a possible role of the emerging pathogens in the development of chronic pancreatitis and pancreatic cancer.

Suppression of pancreatic carcinoma growth by activating peroxisome proliferator-activated receptor γ involves angiogenesis inhibition

AIM： TO Study the possible actions and mechanisms or peroxisome proliferator-activated receptor γ （PPARγ）, a ligand-activated transcription factor, in pancreatic car- cinogenesis, especially in angiogenesis. METHODS： Expressions of PPARy and retinoid acid receptor （RXRα） were examined by reverse-transcription polymerase chain reaction （RT-PCR） with immunocyto- chemical staining. Pancreatic carcinoma cells, PANC-1, were treated either with 9-cis-RA, a ligand of RXRα, or with 15-deoxy-△12,14 prostaglandin J2 （15d-PGJ2）, a ligand of PPART, or both. Antiproliferative effect was evaluated by cell viability using methyltetrazolium （MTT） assay. A pancreatic carcinoma xenograft tumor model of nude mice was established by inoculating PANC-1 cells subcutaneously. Rosiglitazone, a specific ligand of PPARy, was administered via water drinking in experimental group of nude mice. After 75 d, all mice were sacrificed. Expression of proliferating cell nuclear antigen （PCNA） in tumor tissue was examined with immunohistochemical staining. Expression of vascular endothelial growth factor （VEGF） mRNA in PANC-1 cells, which were treated with 15d-PGJ2 or 9-cis-RA at various concentrations or different duration, was detected by semi-quantitative RT-PCR. Effects of Rosi- glitazone on changes of microvascular density （MVD） and VEGF expression were investigated in xenograft tumor tissue. Neovasculature was detected with immu- nohistochemistry staining labeled with anti-Ⅳ collagen antibody, and indicated by MVD. RESULTS： RT-PCR and immunocytochemical stain- ing showed that PPARγ and RXRα were expressed in PANC-1 cells at both transcription level and translation level. MTT assay demonstrated that 15d-PGJ2, 9-cis-RA and their combination inhibited the growth of PANC-1 cells in a dose-dependent manner. 9-cis-RA had a com- bined inhibiting action with 15d-PGJ2 on the growth of pancreatic carcinoma. In vivo studies revealed that Rosiglitazone significantly suppressed the growth of pancreatic carcinoma as compared to control group （0.48 ± 0.23 cm^3 vs 2.488 ± 0.59 cm^3, P 〈 0.05）, and the growth inhibition rate was 80.7%. Immuno- histochemistry study showed that PCNA was down regulated in Rosiglitazone-treated group compared to the control group. 15d-PGJ2, 9-cis-RA and their com- bination inhibited the expression of VEGF mRNA in PANC-1 cells in a dose- and time-dependent manner. MVD was decreased more significantly in Rosiglitazone- treated mice （10.67±3.07） than in the control group （31.44±6.06） （P 〈 0.01）. VEGF expression in xeno- graft tumor tissue was also markedly down-regulated in Rosiglitazone-treated mice. CONCLUSION： Activation of PPARγ, inhibits the growth of pancreatic carcinoma both in vitro and in vivo. Sup- pression of tumor angiogenesis by down-regulating the expression of VEGF may be one of the mechanisms by which PPARγ, activation inhibits the growth of pancre- atic carcinoma.

Body mass index does not affect the survival of pancreatic cancer patients

AIM to evaluate the association of body mass index(b MI) with the overall survival of pancreatic ductal adenocarcinoma(PDAC) patients.METHODS A retrospective analysis of PDAC patients diagnosed in the National Cancer Center of China between January 1999 and December 2014 was performed. these patients were categorized into four b MI groups(< 18.5, 18.5-22.9, 23-27.4 and ≥ 27.5 kg/m2). χ2 tests for comparison of the proportions of categorical variables, and Student's t-test or Mann-Whitney test for continuous variables were employed. Survival analysis was performed with the Kaplan-Meyer method. their HRs of mortality and 95%CIs were estimated using the Cox proportional hazards model.RESULTS With a median age of 59.6 years(range: 22.5-84.6 years), in total 1783 PDAC patients were enrolled in this study. their mean usual b MI was 24.19 ± 3.53 for the whole cohort. More than half of the patients(59.3%) experienced weight loss during the disease onset and progression. Compared with healthy-weight individuals, newly diagnosed patients who were overweight or obese had more severe weight loss during their disease onset and progression(P < 0.001). Individuals who were overweight or obese were associated with positive smoking history(P < 0.001). A significant difference in comorbidity of diabetes(P = 0.044) and coronary artery disease(P < 0.001) was identified between high b MI and normal-weight patients. After a median follow-up of 8 mo, the survival analysis showed no association between b MI and the overall survival(P = 0.90, n = 1783). When we stratified the whole cohort by pancreatic cancer stage, no statistically significant association between b MI and overall survival was found for resectable(P = 0.99, n = 217), unresectable locally advanced(P = 0.90, n = 316) and metastatic patients(P = 0.88, n = 1250), respectively. the results did not change when we used the b MI at diagnosis.CONCLUSION Our results showed no significance of b MI for the overall survival of PDAC patients.

Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices

AIM： To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS： Human pancreatic tissue samples （malignant and normal） were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% 02. Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection.RESULTS： With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION： The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo

AIM:To generate an adenoviral vector specifically targeting the EphA2 receptor(EphA2R) highly expressed on pancreatic cancer cells in vivo.METHODS:YSA,a small peptide ligand that binds the EphA2R with high affinity,was inserted into the HI loop of the adenovirus serotype 5 fiber knob.To further increase the specificity of this vector,binding sites for native adenoviral receptors,the coxsackie and adenovirus receptor(CAR) and integrin,were ablated from the viral capsid.The ablated retargeted adenoviral vector was produced on 293T cells.Specifi c targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines.Upon demonstrating specifi c in vitro targeting,in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector.RESULTS:Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold.Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor.YSA-mediated transduction was inhibited by addition of synthetic YSA peptide.The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro.In a subsequent in vivo study in a nude(nu/nu) mouse model however,no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein,nor upon injection into the peritoneum.CONCLUSION:Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.

Effects of α-adrenoreceptor antagonists on apoptosis and proliferation of pancreatic cancer cells in vitro

AIM: To discuss the expression of α-adrenoreceptors in pancreatic cancer cell lines PC-2 and PC-3 and the effects of α1- and α2-adrenoreceptor antagonists, yohimbine and urapidil hydrochloride, on the cell lines in vitro. METHODS: We cultured the human ductal pancreatic adenocarcinoma cell lines PC-2 and PC-3 and analyzed the mRNA expression of α1- and α2-adrenergic receptors by reverse transcription polymerase chain reaction (RT-PCR). The effects of yohimbine and urapidil hydrochloride on cell proliferation were assessed by 3-(4,5-dimethylthiasol-2-yl)- 2,4,-diphenyltetrazolium bromide (MTT) assay. Apoptosis was detected using the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay and flow cytometry (FCM). RESULTS: PC-2 expressed mRNA in α1- and α2- adrenoreceptors. MTT assays showed that urapidil hydrochloride had no effect on PC-3 cell lines. However, exposure to urapidil hydrochloride increased DNA synthesis in PC-2 cell lines as compared to the control group. PC-2 cell lines were sensitive to both drugs. The proliferation of the 2 cell lines was inhibited by yohimbine. Cell proliferation was inhibited by yohimbine via apoptosis induction. CONCLUSION: The expression of α1- and α2- adrenoreceptors is different in PC-2 and PC-3 cell lines, which might be indicative of their different functions. The α2-adrenoceptor antagonist, yohimbine, can inhibit theproliferation of both cell lines and induce their apoptosis, suggesting that yohimbine can be used as an anticancer drug for apoptosis of PC-2 and PC-3 cells.

Extracorporeal shock wave lithotripsy for pancreatic and large common bile duct stones

Extraction of large pancreatic and common bile duct(CBD)calculi has always challenged the therapeutic endoscopist.Extracorporeal shockwave lithotripsy(ESWL)is an excellent tool for patients with large pancreatic and CBD calculi that are not amenable to routine endotherapy.Pancreatic calculi in the head and body are targeted by ESWL,with an aim to fragment them to<3 mm diameter so that they can be extracted by subsequent endoscopic retrograde cholangio-pancreatography(ERCP).In our experience,complete clearance of the pancreatic duct was achieved in 76% and partial clearance in 17%of 1006 patients.Short-term pain relief with reduction in the number of analgesics ingested was seen in 84%of these patients.For large CBD calculi,a nasobiliary tube is placed to help target the calculi,as well as bathe the calculi in salinea simple maneuver which helps to facilitate fragmenta-tion.The aim is to fragment calculi to<5 mm size and clear the same during ERCP.Complete clearance of the CBD was achieved in 84.4%of and partial clearance in 12.3%of 283 patients.More than 90%of the patients with pancreatic and biliary calculi needed three or fewer sessions of ESWL with 5000 shocks being de-livered at each session.The use of epidural anesthesia helped in reducing patient movement.This,together with the better focus achieved with newer third-gen-eration lithotripters,prevents collateral tissue damage and minimizes the complications.Complications in our experience with nearly 1300 patients were minimal,and no extension of hospital stay was required.Similar rates of clearance of pancreatic and biliary calculi with minimal adverse effects have been reported from the centers where ESWL is performed regularly.In view of its high efficiency,non-invasive nature and low complication rates,ESWL can be offered as the first-line therapy for selected patients with large pancreatic and CBD calculi.

Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells

Human pluripotent stem cells represent a potentially unlimited source of functional pancreatic endocrine lineage cells. Here we report a highly efficient approach to induce human embryonic stem （ES） cells and induced pluripo- tent stem （iPS） cells to differentiate into mature insulin-producing cells in a chemical-defined culture system. The differentiated human ES cells obtained by this approach comprised nearly 25% insulin-positive cells as assayed by flow cytometry analysis, which released insulin/C-peptide in response to glucose stimuli in a manner comparable to that of adult human islets. Most of these insulin-producing cells co-expressed mature β cell-specific markers such as NKX6-1 and PDX1, indicating a similar gene expression pattern to adult islet β cells in vivo. In this study, we also demonstrated that EGF facilitates the expansion of PDXl-positive pancreatic progenitors. Moreover, our protocol also succeeded in efficiently inducing human iPS cells to differentiate into insuIin-producing ceils. Therefore, this work not only provides a new model to study the mechanism of human pancreatic specialization and maturation in vitro, but also enhances the possibility of utilizing patient-specific iPS cells for the treatment of diabetes.

Presence of CCK-A, B receptors and effect of gastrin and cholecystokinin on growth of pancreatobiliary cancer cell lines

AIM: To investigate the effects of gastrin and cholecystokinin (CCK) and their specific antagonists on the growth of pancreatic and biliary tract cancer cell lines. METHODS: Five pancreatic and 6 biliary cancer cell lines with 2 conrtol cells were used in this study. Cell proliferation study was done using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) test and direct cell count method. Reverse transcription-polymerase chain reaction (RT-PCR) and slot blot hybridization were performed to examine and quantify the expression of hormonal receptors in these cell lines. RESULTS: SNU-308 showed a growth stimulating effect by gastrin-17, as did SNU-478 by both gastrin-17 and CCK-8. The trophic effect of these two hormones was completely blocked by specific antagonists (L-365, 260 for gastrin and L-364, 718 for CCK). Other cell lines did not respond to gastrin or CCK. In RT-PCR, the presence of CCK-A receptor and CCK-B/gastrin receptor mRNA was detected in all biliary and pancreatic cancer cell lines. In slot blot hybridization, compared to the cell lines which did not respond to hormones, those that responded to hormones showed high expression of receptor mRNA. CONCLUSION: Gastrin and CCK exert a trophic action on some of the biliary tract cancers.

In Vitro Lethal Effect of Photodynamic Therapy on Human Pancreatic Cancer Cells and Its Major Influencing Factors

OBJECTIVE To investigate the in vitro lethal effect of photo- dynamic therapy （PDT） using the photosensitizer hematoporphyrin on the human pancreatic cancer cell line Panc-1, the major influencing factors and the mechanisms of treatment. METHODS Three factors--the time needed for photosensitizer and cell incubation, the photosensitizer concentration （PhoC） and the exposure dose （ExpD）--were examined with different levels of these factors. Optical density （OD） was used as a measure of CCK-8 in the experiment, and was converted to the rate of cell survival. The separate effect of each factor on the photodynamic action was studied, and the interactions were investigated. The effects of different incubation times and PhoC levels on the fluorescence intensity （FI） of the intracellular photosensitizer were determined, and the mechanisms of these factors leading to the therapeutic effects of PDT discussed. RESULTS An increase in the photosensitizer and cell incubation time, an increase of PhoC, and enhancement of the ExpD, produced a corresponding decrease in the rate of Panc-1 cell survival after PDT （P 〈 0.05）. PDT achieved its maximum lethal effects 16 h after starting the incubation, with a PhoC of 10 mg/L and an ExpD of 20 J/cm2; at these levels a synergistic interaction between PhoC and the ExpD occurred, decreasing the cell survival rate （P 〈 0.05）. Neither simple administration of photosensitizer without ExpD （0 J/cm2） or illumination in the absence of PhoC （0 mg/L） affected the rate of cell survival （P 〉 0.05）. With an increase of PhoC and lengthening of the incubation time, the FI of the intracellular photosensitizer accordingly increased （P 〈 0.05）, and attained its maximum value at a PhoC of 10 mg/L and 36 h after the incubation. With an increase of PhoC, the FI of the photosensitizer, hematoporphyrin, in the solution increased progressively at first and then decreased （fluorescence quenching）. CONCLUSION PDT with the photosensitizer hematoporphyrin has clear lethal effects on the human pancreatic cancer cell line Panc-1, but the presence of a photosensitizer and laser irradiation by themselves do not have independent lethal effects. The three influencing factors--the time for photosensitizer and cell incuba- tion, PhoC and ExpD--correlate positively with the PDT response, within certain limits. Beyond these limits, the PDT response does not significantly increase. The main mechanism of the PDT response lies in the effect of these factors on the level of the intracellular photosensitizer and the fluorescence quenching of the photosensitizer. A synergistic effect exists between PhoC and ExpD.

Recombinant adenoviral vector expressing the tumor necrosis factor-related apoptosis-inducing ligand gene suppresses human pancreatic cancer growth

Objective: To investigate the antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene transfection mediated by adenovirus into human pancreatic carcinoma cell line Panc-1, and the mechanisms involved in this effect. Methods: TRAIL gene was transfected into pancreatic cancer cell line Panc-1 by an adenovirus vector (Ad-TRAIL). Level of TRAIL mRNA expression was determined using RT-PCR, and TRAIL protein synthesis was evaluated with Western blot. Cell-growth activities were determined by MTT assay. The bystander effect was observed by co-culturing the Panc-1 cells with the transfected TRAIL gene at different ratios. Apoptosis in pancreatic cancer cells was detected by flow cytometry. Procaspase-8 and procaspase-3 were determined by Western blot. Results: The stable overexpression of TRAIL was de-tected in Panc-1 cells transfected by Ad-TRAIL. Ad-TRAIL significantly inhibited of cell viability of Panc-1 cells. Furthermore, co-culture of cancer cells transfected with TRAIL with that nontransfected resulted in the cell death of both cells by bystander effect. Moreover, the percentage of apoptotic cells was significantly higher in the Ad-TRAIL-treatment group compared to the control groups (P < 0.01). And there was a diminished amount of procaspase-8 and procaspase-3 after infection with Ad-TRAIL. Conclusion: The overexpression of TRAIL gene in Panc-1 cells by Ad-TRAIL exerts its antitumor effects, and the mechanisms involved in this effect may be proapoptosis and bystander effect.

Resection of non-cystic adenocarcinoma in pancreatic body and tail

AIM: To report the outcome of Chinese patents with non-cystic adenocarcinoma in pancreatic body and tail (NCAPBT) after resection and to discuss its surgical strategy. METHODS: Resection of NCAPBT was performed in eight Chinese patients with complete clinical-pathological data in our hospital from January 2000 to May 2004. The surgical strategy was explored by analyzing the results of these patients. RESULTS: The resection rate of NCAPBT in patients without back pain was higher than that in patients with back pain (66.67% vs 20%, 2/3 vs 1/5). The prognosis in the group receiving palliative resection was poorer than that in the group receiving curative resection. The median survival time was 12 mo in the curative resection group and 6 mo in the palliative resection group, respectively. CONCLUSION: The overall survival time of the Chinese patients with NCAPBT is dismal. The Chinese patients after curative resection of NCAPBT have a longer survival time. The Chinese NCAPBT patients with back pain trend to have a lower curative resection rate, but back pain should not be considered a contraindication for curative resection.

Construction of a lentiviral vector for RNA interference of human VIM gene and its silencing effect in pancreatic cancer cells

Objective： To construct a lentiviral expression vector for RNA interference （RNAi） of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods： Three pairs of human VIM gene short hairpin RNA（shRNA） sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides （dsOligo） were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction （PCR） and DNA sequencing analysis. Real-time PCR and Westemblotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells. The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 calls were validated by real-time PCR and Western-blotting. Results： An effective Lv-VIM-shRNA was successfully constructed. The titer of lentivirus was determined on 2× 10^9TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion： An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.

Dickkopf3 overexpression inhibits pancreatic cancer cell growth in vitro

AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2'deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.