Guoping Ding

Analysis of serum exosomal microRNAs and clinicopathologic features of patients with pancreatic adenocarcinoma

BackgroundAltered expression of serum microRNAs (miRNAs) have been reported to correlate with carcinogenesis and progression of pancreatic adenocarcinoma (PC), but descriptions of serum exosomal miRNAs in PC are still lacking. This study was designed to evaluate serum exosomal miRNA levels in PC patients and to investigate their relationships with clinicopathologic features and prognosis.MethodsFour miRNAs (miR-17-5p, miR-21, miR-155 and miR-196a) related to PC were selected for examination in our research. Serum miRNA was examined by RT-PCR in a group of 49 patients, including 22 with PCs, 6 with benign pancreatic tumors, 7 with ampullary carcinomas, 6 with chronic pancreatitis and 8 healthy participants. The clinicopathologic data were also collected, and PC patients were classified according to the presence of metastasis, tumor differentiation and advanced stage.ResultsThere were low expressions of exosomal miR-155 and miR-196a in serum samples of PC patients when U-6 was used as a control. Serum exosomal miR-17-5p was higher in PC patients than in non–PC patients and healthy participants. High levels of miR-17-5p were significantly correlated with metastasis and advanced stage of PC. The serum exosomal miR-21 level in PC was higher than that in the normal and chronic pancreatitis groups, but was not significantly correlated with PC differentiation and tumor stage.ConclusionsThere were high expressions of serum exosomal miR-17-5p and miR-21 in PC patients. Examination of serum exosomal microRNA is a useful serum biomarker for PC diagnosis other than serum-free microRNA. It is postulated that exosomal miR-17-5p participates in the progression of PC.

Pancreatic cancer derived exosomes regulate the expression of TLR4 in dendritic cells via miR-203.

MicroRNAs (miRNAs) are aberrant in many human tumors which can be transferred to immune cells by tumor-derived exosomes. Dendritic cells (DCs) play an important role in activation of immune response. However, the effect of tumor-derived exosomes on toll-like receptor (TLR) in DCs remains unclear. We investigated the influence of pancreatic cancer derived exosomes on TLR4, and downstream cytokines via miR-203. Our results showed that miR-203 expressed in panc-1 cells and exosomes, and upregulated in exosomes-treated DCs. TLR4 decreased after treatment of exosomes and miR-203 mimics, while increased in exosomes-treated DCs by miR-203 inhibitors. But the mRNA level of TLR4 was not significantly different between DCs and exosomes-treated DCs. Tumor necrosis factor-α (TNF-α) and interleukin-12 (IL-12) also decreased under treatment of exosomes and miR-203 mimics, both of which increased in exosomes-treated DCs by miR-203 inhibitors. Collectively, pancreatic cancer derived exosomes downregulate TLR4 and downstream cytokines in DCs via miR-203.

Pancreatic cancer-derived exosomes transfer miRNAs to dendritic cells and inhibit RFXAP expression via miR-212-3p

It has been reported tumor-derived exosomes can transfer miRNAs to recipient cells in the tumor microenvironment, promoting tumor invasion and metastasis. The present research aimed to explore how pancreatic cancer (PC) derived exosomal miRNAs inhibited mRNA expression of dendritic cells and induced immune tolerance. Our study revealed that 9 PC-related miRNAs were increased and 208 mRNAs were inhibited in exosome-stimulated dendritic cells (exo-iDCs) compared to immature dendritic cells (iDCs). A target prediction between the 9 miRNAs and 208 mRNAs was performed by bioinformatics database analysis. From the target prediction, it was predicted and validated that regulatory factor X-associated protein (RFXAP), an important transcription factor for MHC II, was inhibited by miR-212-3p transferred from PC-secreted exosomes, resulting in decreased MHC II expression. Moreover, a clinical study showed a negative correlation between miR-212-3p and RFXAP in PC tissue. From these data, we concluded that PC-related miRNAs can be transferred to dendritic cells via exosome and inhibit target mRNA expression. More importantly, PC-derived exosomes inhibit RFXAP expression via miR-212-3p, which decrease MHC II expression and induce immune tolerance of dendritic cells. RFXAP deficiency has never been reported in solid tumors. The functions and mechanisms of RFXAP in tumors deserve future explorations.

Correlation of nitric oxide and other free radicals with the severity of acute pancreatitis and complicated systemic inflammatory response syndrome.

Objectives: To investigate the correlation of nitric oxide (NO) and other free radicals with the severity of acute pancreatitis (AP) and complicated systemic inflammatory response syndrome (SIRS). Methods: Fifty AP patients (24 simple AP patients and 26 patients with AP complicated by SIRS) were involved in the study. Fifty healthy volunteers were included as controls. Acute Physiology and Chronic Health Evaluation II (APACHE II) scores were evaluated, and plasma NO, plasma lipid peroxides, plasma vitamin E, plasma &bgr;-carotene, whole-blood glutathione (GSH), and the activity of plasma GSH peroxidase were measured. Results: Compared with the control group, the APACHE II scores heightened in the AP group, and the SIRS group had the highest APACHE II scores (P < 0.005, P < 0.001, respectively). Plasma NO and plasma lipid peroxides increased with the heightening APACHE II scores, demonstrating a significant linear positive correlation (r = 0.618, r = 0.577, respectively; P < 0.001). Plasma vitamin E, plasma &bgr;-carotene, whole-blood GSH, and the activity of plasma GSH peroxidase decreased with the heightening APACHE II scores, demonstrating a significant linear negative correlation (r = −0.600, r = −0.609, r = −0.559, r = −0.592, respectively; P < 0.001). Conclusions: Nitric oxide and other free radicals take part in the aggravation of oxidative stress and oxidative injury and may play important roles in the pathogenesis of AP and SIRS. It may be valuable to measure free radicals to predict the severity of AP.

Increasing the immune activity of exosomes: the effect of miRNA-depleted exosome proteins on activating dendritic cell/cytokine-induced killer cells against pancreatic cancer

BackgroundTumor-derived exosomes were considered to be potential candidates for tumor vaccines because they are abundant in immune-regulating proteins, whereas tumor exosomal miRNAs may induce immune tolerance, thereby having an opposite immune function.ObjectiveThis study was designed to separate exosomal protein and depleted exosomal microRNAs (miRNAs), increasing the immune activity of exosomes for activating dendritic cell/cytokine-induced killer cells (DC/CIKs) against pancreatic cancer (PC).MethodsPC-derived exosomes (PEs) were extracted from cultured PANC-1 cell supernatants and then ruptured; this was followed by ultrafiltered exosome lysates (UELs). DCs were stimulated with lipopolysaccharide (LPS), PE, and UEL, followed by co-culture with CIKs. The anti-tumor effects of DC/CIKs against PC were evaluated by proliferation and killing rates, tumor necrosis factor-α (TNF-α) and perforin secretion. Exosomal miRNAs were depleted after lysis and ultrafiltration, while 128 proteins were retained, including several immune-activating proteins.ResultsUEL-stimulated DC/CIKs showed a higher killing rate than LPS- and PE-stimulated DC/CIKs.ConclusionsmiRNA-depleted exosome proteins may be promising agonists for specifically activating DC/CIKs against PC.中文概要目的本文通过分离提取无小RNA(miRNA)的外来 体(exosome)刺激树突细胞/细胞因子活化杀伤 细胞(DC/CIKs),激活其对于胰腺癌细胞的免 疫杀伤作用。创新点无miRNA 的exosome 超速离心裂解产物可以通 过激活DC/CIKs 细胞增强其对肿瘤细胞的杀伤 作用。方法通过收集PANC-1 细胞的上清并超速离心提取其 中的exosome。提取的DC 细胞分别通过脂多糖、 肿瘤来源exosome 及无miRNA 的exosome 刺激 后,与CIK 细胞共培养。通过计算增值与杀伤效 率,肿瘤坏死因子-α(TNF-α)及穿孔素的分泌, 比较各组间CIK 细胞对胰腺癌细胞的杀伤作用。结论经无miRNA 的exosome 刺激后的CIK 细胞比其 他两组表现出更高的杀伤效应。实验结果表明无 miRNA 的exosome 蛋白在DC/CIKs 细胞的胰腺 癌治疗中是有相当前景的激动剂。

A modified Jarnagin-Blumgart classification better predicts survival for resectable hilar cholangiocarcinoma

BackgroundPrediction of postoperative survival for hilar cholangiocarcinoma (HCCA) remains difficult although there have been a variety of clinical classification and staging systems. This study was designed to validate and compare some of the major HCCA staging systems in use today. In addition, we sought to build up a new staging system modified from Jarnagin-Blumgart (J-B) classification for HCCA, to predict survival better.MethodsA total of 154 consecutive cases of HCCA including 95 surgical patients between 2005 and 2014 were enrolled in this study. The clinical and pathological data were recorded retrospectively and three commonly used classification methods: Bismuth-Corlette (B-C) classification, TNM staging, and J-B classification were performed to analyze the correlations with resectability and survival. Chi-square test, Kaplan-Meier analysis, and kappa statistics were used to compare and confirm the relationships between the variables and survival.ResultsFor all 154 patients, the resection rate of J-B T1 was 68.6% (48/70), higher than that of J-B T2 (44.8%, P = 0.007). J-B T2 also showed a higher resectability than J-B T3 (19.2%, P = 0.025). There was no significant difference in resectability within the groups B-C type and TNM stages. We set up a new staging system based on J-B classification, tumor differentiation, distant metastasis (N2 or M1 of TNM stage), and resection integrality. The total survival predictive accuracy was 69.5% (kappa = 0.547), higher than that of TNM staging and J-B classification.ConclusionsJ-B classification was more useful than B-C classification, while its value for predicting survival did not exceed TNM staging system. The new staging system, based on J-B classification, provides a better method to stratify HCCA patients during the operation.

Overexpressions of DLL4 and CD105 are Associated with Poor Prognosis of Patients with Pancreatic Ductal Adenocarcinoma

The aim of this study was to investigate the expression of Delta-like ligand 4(DLL4) and Endoglin(CD105) labeled microvessel density(MVD) in pancreatic ductal adenocarcinoma (PDAC) and evaluate their correlation with major clinicopathologic features and patients’ survival. Forty-two pancreatic cancer and 20 normal pancreatic tissues were included in the study. Immunohistochemical staining was employed to assess the expression level of DLL4 both in tumor cells and stromal vascular endothelial cells, as well as CD105 which was used to determine MVD. The relationships of DLL4 and CD105 expression with clinicopathologic parameters and clinical outcome were evaluated. Both DLL4 and CD105-labeled microvessel were observed highly immunostained in PDAC cases, and high expression of DLL4 was positively correlated with MVD. Moreover, the high expression of DLL4 was significantly associated with histological grade, node stage and TNM stage in not only the cancer cells but also stroma; while high expression of CD105 was associated with histological grade, TNM stage, node stage and distant metastasis. In univariant analysis, patients with high expression of DLL4 and CD105 tended to significantly poorer overall survival. Both DLL4 and CD105 were overexpressed in a large proportion of patients with PDAC. The expression of DLL4 was positively correlated with CD105-labeled MVD, indicating DLL4 may involved in angiogenesis. In addition, high DLL4 and CD105 expression correlated with the poor clinical outcome and overall survival in patients with PDAC.

Transumbilical single-port laparoscopic cholecystectomy using traditional laparoscopic instruments: a report of thirty-six cases

ObjectiveTo evaluate the feasibility and safety of the operation of transumbilical single-port laparoscopic cholecystectomy (TSPLC) by traditional laparoscopic instruments and summarize the initial experience.MethodsSixty subjects with cholelithiasis were divided into two groups. One group (36 cases) underwent TSPLC and the control group (24 cases) underwent traditional three-port laparoscopic cholecystectomy (LC). Postoperative complications were observed and operation time, hospital days, visual analogue scale (VAS) after 6 and 24 h of operation, and subject satisfaction score were measured.ResultsTSPLC and traditional LC were performed successfully in the two groups. The operation time in the TSPLC group was significantly longer than that in the control group. There was no statistically significant difference in hospital stay and VAS between the TSPLC and control groups. The subject satisfaction score in the TSPLC group was 91.2, significantly higher than that in the control group (P<0.01). All subjects recovered from the operation and no postoperative complication occurred during the period of two weeks after operation.ConclusionsTSPLC is a feasible and safe method for cholecystectomy, although it may be more time-consuming. However, it is welcomed by patients who are more concerned with cosmetic outcomes. Future studies are needed to confirm its disadvantages and contraindications.

Cholangiocarcinoma-derived exosomes inhibit the antitumor activity of cytokine-induced killer cells by down-regulating the secretion of tumor necrosis factor-α and perforin

ObjectiveThe aim of our study is to observe the impact of cholangiocarcinoma-derived exosomes on the antitumor activities of cytokine-induced killer (CIK) cells and then demonstrate the appropriate mechanism.MethodsTumor-derived exosomes (TEXs), which are derived from RBE cells (human cholangiocarcinoma line), were collected by ultracentrifugation. CIK cells induced from peripheral blood were stimulated by TEXs. Fluorescence-activated cell sorting (FACS) was performed to determine the phenotypes of TEX-CIK and N-CIK (normal CIK) cells. The concentrations of tumor necrosis factor-α (TNF-α) and perforin in the culture medium supernatant were examined by using an enzyme-linked immunosorbent assay (ELISA) kit. A CCK-8 kit was used to evaluate the cytotoxic activity of the CIK cells to the RBE cell line.ResultsThe concentrations of TNF-α and perforin of the group TEX-CIK were 138.61 pg/ml and 2.41 ng/ml, respectively, lower than those of the group N-CIK 194.08 pg/ml (P<0.01) and 3.39 ng/ml (P<0.05). The killing rate of the group TEX-CIK was 33.35%, lower than that of the group N-CIK (47.35% (P<0.01)). The population of CD3+, CD8+, NK (CD56+), and CD3+CD56+ cells decreased in the TEX-CIK group ((63.2±6.8)%, (2.5±1.0)%, (0.53±0.49)%, (0.45±0.42)%) compared with the N-CIK group ((90.3±7.3)%, (65.7±3.3)%, (4.2±1.2)%, (15.2±2.7)%), P<0.01.ConclusionsOur results suggest that RBE cells-derived exosomes inhibit the antitumor activity of CIK cells by down-regulating the population of CD3+, CD8+, NK (CD56+), and CD3+CD56+ cells and the secretion of TNF-α and perforin. TEX may play an important role in cholangiocarcinoma immune escape.中文概要目 的探索胆管癌来源外泌体(TEX)对细胞因子诱导的杀伤细胞(CIK)抗肿瘤活性的影响,并初步探讨其作用机制。创新点首次通过体外实验证明TEX 可引起CIK 抗肿瘤活性下降,且此作用与肿瘤坏死因子α(TNF-α)和穿孔素表达抑制相关。方 法采用超速离心法提取人胆管癌细胞(RBE)来源的外泌体,同时CIK 通过人外周血培养获得。将TEX 负载到CIK 培养体系中作为TEX-CIK 组,不加TEX 的CIK 作为N-CIK 组。流式细胞仪检测两组CIK 细胞表型变化,酶联免疫吸附法(ELISA)检测两组培养基上清液中TNF-α 和穿孔素的浓度, CCK-8 法检测CIK 对RBE 细胞的杀伤活性。结 论TEX 能降低CIK 细胞CD3+、CD8+、NK(CD56+)以及CD3+CD56+比例,并且抑制TNF-α 和穿孔素表达,从而降低CIK 细胞的抗肿瘤效应。

Saline conducted electric coagulation (SCEC): original experience in experimental hepatectomy

ObjectiveTo evaluate the feasibility and superiority of a new coagulating and hemostatic method named “saline conducted electric coagulation (SCEC)”.MethodsThe Peng’ s multifunction operative dissector (PMOD) was modified to enable saline to effuse persistently out of its nib at a constant speed. In a group of six New Zealand rabbits, two hepatic lobes of each rabbits were resected respectively by SCEC and conventional electric coagulation (EC). The features of SCEC were recorded by photo and compared with conventional EC. After 7 d, the coagulating depth was measured in each residual hepatic lobe. Hepatic tissue was dyed by hematoxylin and eosin (HE) and studied under a microscope.ResultsThe coagulating depth increased with the continuation of SCEC time. Hepatectomies were performed successfully, no rabbit died in the perioperative period. The incisal surface of SCEC was gray-white with no red bleeding point. There was a thick solidified layer at the margin and a thin red-white intermittent layer between the solidified layer and normal hepatic tissue at the vertical section of SCEC. The mean coagulating depth of SCEC was 1.8 cm vs. 0.3 cm of conventional EC. Pathological examination showed a mild inflammatory reaction by SCEC.ConclusionsSCEC is a feasible and safe method for surgical hemostasis. As a new technique for liver resection, SCEC shows better coagulating effect and milder inflammatory reaction than conventional EC. Our study shows bloodless liver resection can also be performed by SCEC, especially for liver malignant tumor.