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Patients benefit greatly from early detection of colorectal cancer, but present diagnostic procedures have high costs, low sensitivity, and low specificity. However, it is still difficult to develop a strategy that can effectively detect cancer early using high-throughput blood analysis. Fano resonance-boosted SERS platform label-free serum creates an effective diagnostic system at the point of care. We obtained 220 high-quality SERS serum spectral datasets from 88 healthy volunteers and 132 patients with colorectal cancer. The biomarker detected in serum was further evaluated using 100 colorectal cancer tissues and adjacent normal intestinal tissues collected from West China Biobanks, West China Hospital, Sichuan University. The results showed that in 97 out of 100 paired samples, the biomarkers were successfully detected using the SERS platform. This demonstrates that Fano resonance-based SERS is highly effective for diagnosing colorectal cancer., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Objective: To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC)., Methods: A total of 20 wild-type C 57 BL /6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L 1 (+/+) mice and 14 PD-L 1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors., Results: The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P <0.05). According to the results of the PD-L 1 gene mouse experiment, the control groups of PD-L 1 (+/+) mice and PD-L 1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L 1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L 1 (+/+) NEC mouse ( P <0.05). In addition, serum interleukin (IL)-10 in PD-L 1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L 1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1β all increased by more than 25% (all P <0.05)., Conclusion: PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L 1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To improve the apoptosis/necrosis In vitro model of ascites-induced acute pancreatitic (AP) acinar cells by using the co-culture system and mixed ascites technology for the first time. Furthermore, we compared this improved model with cerulein and cerulein+LPS models, and observed the effects of three models on acinar apoptotic/necrotic-related indicators., Methods: The In vitro cultured rat acinar cells AR42J were divided in four groups: control group (medium+PBS), cerulein group (medium+10 nmol/L cerulein), cerulein+LPS group (medium+10 nmol/L cerulein+10 mg/L LPS) and improved ascites group. In the improved ascites group, the ascites of sodium taurocholate-induced rat model was mixed and added into the co-culture system to stimulated In vitro cultured homogenous acinar cells. The co-culture system was set as follows: the chambers with the pore size of 1 μm were placed in the cultue plate, and the culture medium and mixed ascites were respectively added to the culture plate and the chamber at a ratio of 1:1. The acinar cells in each group were collected after 24 h stimulation. The apoptotic/necrotic rates, the expressions of apoptosis/necrosis related proteins [B-cell lymphoma protein 2 (Bcl-2), Bcl-2-associated X protein (Bax) and receptor interacting protein 1 (RIP1)], and the ultra-structure of acinar cells were detected by flow cytometry, Western blot and transmission electron microscopy (TEM)., Results: The acinar cells in the improved ascites model were mainly characterized by necrosis; compared with the other 3 groups, the apoptosis rate and necrosis rate were both up-regulated, RIP1 and BAX protein expression levels were up-regulated, and Bcl-2 protein was down-regulated. TEM results showed the organelle structure of acinar cells was destroyed, and the cell membrane was degraded in the improved ascites model. Compared with the control group, apoptosis rate of acinar cells in the cerulein and cerulein+LPS models were increased and necrosis rate were not changed. The expression of pro-apoptotic protein Bax was increased, while the expression level of RIP1 was not significantly increased. TEM results showed that in cerulein group and cerulein+LPS group, the chromatin of the cells was condensed into a mass, the cytoplasm was degraded and the cell membrane was intact, showing typical apoptosis characteristics., Conclusion: Compared with cerulein and cerulein +LPS models, which mainly focus on apoptosis of acinar cells and applied to mild acute pancreatitis study, the improved ascites model mainly focuses on the necrosis of acinar cells and is a good model for studying acinar cell necrosis and severe acute pancreatitis., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Science Edition).)

Acute pancreatitis is an inflammatory condition that may lead to multisystemic organ failure with considerable mortality. Recently, resolvin D1 (RvD1) as an endogenous anti-inflammatory lipid mediator has been confirmed to protect against many inflammatory diseases. This study was designed to investigate the effects of RvD1 in acute pancreatitis and associated lung injury. Acute pancreatitis varying from mild to severe was induced by cerulein or cerulein combined with LPS, respectively. Mice were pretreated with RvD1 at a dose of 300 ng/mouse 30 min before the first injection of cerulein. Severity of AP was assessed by biochemical markers and histology. Serum cytokines and myeloperoxidase (MPO) levels in pancreas and lung were determined for assessing the extent of inflammatory response. NF-κB activation was determined by Western blotting. The injection of cerulein or cerulein combined with LPS resulted in local injury in the pancreas and corresponding systemic inflammatory changes with pronounced severity in the cerulein and LPS group. Pretreated RvD1 significantly reduced the degree of amylase, lipase, TNF-α, and IL-6 serum levels; the MPO activities in the pancreas and the lungs; the pancreatic NF-κB activation; and the severity of pancreatic injury and associated lung injury, especially in the severe acute pancreatitis model. These results suggest that RvD1 is capable of improving injury of pancreas and lung and exerting anti-inflammatory effects through the inhibition of NF-κB activation in experimental acute pancreatitis, with more notable protective effect in severe acute pancreatitis. These findings indicate that RvD1 may constitute a novel therapeutic strategy in the management of severe acute pancreatitis., (Copyright © 2016 the American Physiological Society.)

Objective: To design and construct a lentiviral vector containing shRNA against rat glucose-regulated protein 78 gene (GRP78), and to establish rat pancreatic acinar cell line with stable knockdown of GRP78 expression., Methods: Constructed three plasmid expression vectors coding shRNA against GRP78, and converted them into lentiviral particles using three plasmid package systems. Then, AR42J cells were transduced with produced lentiviral particles. The green fluorescent protein (GFP) positive cells were selected by fluorescence-activated cell sorting (FACS), the GRP78 gene mRNA and protein expression were detected by real-time PCR and Western blot., Results: The DNA sequencing showed that the lentiviral vectors containing shRNA against GRP78 gene were constructed correctly. After the transduction, highly efficient transduction (> 85%) of lentivirus in AR42J cells was observed by fluorescent microscopy and FACS. Quantitative real-time PCR showed that GRP78 mRNA expression in AR42J cells was suppressed by LVshGRP78-1, LVshGRP78-2 and LVshGRP78-3 lentivirus about 69.4% +/- 1.42%, 74.7% +/- 1.69% and 86.6% +/- 1.73% as compared with that of the untreated cells (P < 0.05), while LV-Non Target had no significant effect on the GRP78 mRNA level (P > 0.05). Western blot showed that the suppressed effect of LVshGRP78 lentivirus on GRP78 protein was consistent with GRP78 mRNA., Conclusion: The results demonstrated that lentiviral vectors containing the shRNA against GRP78 gene were successfully constructed, which could stably knock down the GRP78 expression in AR42J cells. This study will provide a new cell model for further study of the role of GRP78 in the pathogenesis of acute pancreatitis.

Background/aims: To study the potential role of the 78kDa glucose regulated protein (GRP78) in the pathogenesis of acute pancreatitis (AP) in vitro., Methodology: AR42J cells were stimulated by cerulein or cerulein plus lipoplysaccharide (LPS). The severity of pancreatic inflammation was evaluated by amylase, lipase, TNF-a, and IL-6. Apoptosis was determined by flow cytometry; the expressions of apoptotic genes, GRP78 and the downstream molecules were determined by real-time quantitative PCR and Western blot., Results: After cerulein stimulation, the levels of amylase, lipase, TNF-a and IL-6 were all increased, with a more pronounced increase after cerulein plus LPS stimulation. Apoptosis was different in two cell models, high apoptosis in cerulein group; whereas cerulein plus LPS induced relatively less apoptosis. Apoptotic gene expressions revealed more pronounced increase in the cerulein group than those in cerulein plus LPS group. The expressions of GRP78 and downstream molecules were different in two cell models. GRP78 expression was down-regulated in cerulein group and upregulated in cerulein plus LPS group., Conclusions: GRP78 expression was associated with apoptosis and the severity of cerulein-induced pancreatic inflammation, indicating that GRP78 might prevent apoptosis in pancreatic acinar cells thereby deteriorating the severity of AP.

Objective: To design and construct a lentiviral vector containing shRNA against rat myeloid differentiation protein 88 gene (MyD88), and to establish rat pancreatic ductal cell line with stable knockdown of MyD88 expression., Methods: Constructed two plasmid expression vectors coding shRNA against MyD88 and converted them into lentiviral particles using three-plamid package system, named as Lenti-sh MyD88-1 and LentishMyD88-2. Rat pancreatic ductal cell, ARIP were divided into untreated group, Lenti-Non Target group, LentishMyD88-1 and LentishMyD88-2 treated groups, and transduced with corresponding lentiviral particles. The GFP positive cells were selected by fluorescence-activated cell sorting and puromycin, the MyD88 gene silencing efficiency was detected by real-time RT-PCR., Results: After the transduction, we observed highly efficient transduction (reach to 100%) of lentivirus in ARIP cells by fluorescent microscopy and FACS. Quantitative RT-PCR showed that Lenti-shMyD88-1 reduced MyD88 mRNA expression by about 82.73% +/- 1.203% in ARIP cells, and LentishMyD88-2, reduced 75.56% +/- 1.176% as compared with that of the untreated cells (P < 0.05)., Conclusion: The results demonstrated that lentivector containing the short hairpin RNA expression cassette specifically targeting MyD88 (pLVshMyD88-1,-2) were successfully constructed, which could stably knock down the MyD88 expression in ARIP cells. This study finally provided a new and stable cell model for the study of MyD88's function in acute pancreatitis.

Objective: To investigate the expression of microRNA(miR)-21 and miR-125 in colorectal cancer (CRC) and its relationship with clinicopathological features., Methods: Quantitative real-time PCR was applied to examine the expression of miR-21 and miR-125 in 100 primary CRC specimens which were diagnosed and operated in West China Hospital between 2006 and 2007, in comparison with the corresponding normal mucosa specimens. The relationship between the expression of miRNAs and clinicopathological features was analyzed., Results: The expression of miR-21 in CRC was up-regulated by 2.3 times compared to normal mucosa (P =0.025), while the expression of miR-125 was down-regulated by 3.3 times in comparison with normal mucosa (P =0.005). Furthermore, the expression of miR-21 was related to TNM stage (P =0.028) and local invasion (P =0.023). On the other hand, no significant relationship was found between the expression of miR-125 and clinicopathological features (P >0.05)., Conclusion: The over-expression of miR-21 may play a role in the development and progression of CRC, while miR-125 may not be related to the pathogenesis of CRC.

Objective: To research the reliable method for the isolation and culture of Kupffer cell in BALB/c mouse by mixed enzyme., Methods: Kupffer cells were isolated from liver by in situ perfusion and digestion with 0.1% IV collagenase, 0.2% pronase and 0.01% Dnase I, and by percoll density gradient centrifugation. Kupffer cells were identified by fluorescence microscope, immunohistochemistry and cell endocytosis effect., Results: Kupffer cells were isolated successfully with high purity, the yield of (2-3) 10(6)/per mouse liver and the identification that 0.4% trypan blue indicated that the cells survival rate and purity were more than 96% and more than 92% respectively. The shape of Kupffer cell appeared to be multiplicity, irregularity, polygon and multiangular. Kupffer cells showed lysozyme positive by immunohistochemistry staining. And particles of India ink were found in cytoplasm., Conclusion: Here described technique for isolation and culture of Kupffer cells is simple and reliable, and can be used for preparing Kupffer cells with high yield, activity and purity.