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Objective:To explore the expression of miR-216a in patients with acute pancreatitis (AP) associated with acute lung injury (ALI) and its influence on endothelial cells permeability.Methods:40 AP patients admitted in Department of Gastroenterology of the First Affiliated Hospital of Navy Medical University from December 2015 to March 2016 were collected and were classified into AP with ALI (AP-ALI group, n=13) and AP without ALI (AP group, n=27) according to the presence or absence of ALI. 8 normal volunteers were enrolled in the control group. Blood samples were collected and the plasma samples were separated. Plasma RNA was extracted. miR-216a level in plasma was detected by RT-PCR. Plasma exosomes were extracted by exosome extraction kit and identified by the electron microscopy. Exosome RNA was extracted. miR-216a level in exosome was detected by RT-PCR. Plasma exosomes of AP-ALI patients were co-cultured with human umbilical vein endothelial cells (HUVEC, AP-ALI-HUVEC group) and anti-miR-216a transfected HUVECs (AP-ALI-anti-miR-216a HUVEC group) for 24 hours, respectively, and untreated HUVECs served as control group. Trans-endothelium electrical resistance (TEER) was measured by Millicell Ers-2 epithelial volt-ohmmeter to evaluate the cell permeability. Results:RT-PCR results showed that the expression level of plasma miR-216a in AP-ALI group (14.45±1.64) was significantly higher than that in AP group (11.08±1.6) and the control group (5.37±1.54) ( P<0.01). Under electron microscope, plasma exosomes were goblet like vacuoles, with the size of about 50-90 nm. The plasma exosomal miR-216a level in the AP-ALI group (14.03±1.58) was significantly higher than that in the AP group (10.86±1.31) and the control group (5.01±0.79), and the difference was statistically significant ( P<0.01). The resistance value of HUVEC in the control group was referred as 1, and the resistance ratio of HUVEC in AP-ALI-HUVEC group was 0.74±0.04, which was significantly lower than that of HUVEC in AP-ALI-anti-miR-216a HUVEC group (1.02±0.08), the difference was statistically significant ( P<0.01). Conclusions:miR-216a was highly expressed in plasma exosomes of AP patients with ALI. miR-216a can increase endothelial cell permeability, which may be associated with ALI during AP.
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Objective To assess the value of carcinoembryonic antigen (CEA) level,liquid based cytology examination and combining 2 methods in predicting advanced pancreatic cystic neoplasms (PCNs).Methods The clinical data of 78 patients pathologically confirmed with PCN who underwent surgical resection after EUS-FNA and cyst fluid analysis in Shanghai Changhai Hospital,from January 2006 to June 2017 were collected and analyzed,including 32 (A-PCNs) patients and 46 non A-PCNs patients.The comparisons on the CEA level in the cyst fluid and liquid based cytology between the two groups were performed.ROC curve for CEA level in cyst fluid was applied and under curve area was calculated.Sensitive,specificity and accuracy were applied to assess the diagnosis value of 2 methods in predicting A-PCNs.Results In 35 patients,the difference on cyst fluid CEA level was statistically significant between 9 A-PCNs and 26 non A PCNs patients) [(1419.9 ± 1416.9) μg/L vs (316.0 ± 475.2) μg/L,P =0.049].Based on ROC curve,CEA ＞ 418.9 ng/ml could help to predicting A-PCNs with the sensitivity of 85.7％,specificity of 73.1％,and accuracy of 75.8％ as the cutoff value,and the area under ROC curve was 0.863.Liquid based cytology were performed in 27 A-PCNs patients and 33 non A PCNs patients,and the positive rate had statistical difference between 2 groups (48.1 vs 9.1％,P =0.001).The sensitivity,specificity and diagnostic accuracy for liquid-based cytology for diagnosing A-PCNs were 48.1％,90.9％,and 55.1％.Cyst fluid CEA combined with liquid based cytology can effectively diagnose A-PCN,and the sensitivity,specificity,and diagnostic accuracy were 100％,64.7％ and 76.0％.Conclusions Liquid-based cytology and cyst fluid CEA level were useful in predicting A-PCNs to a certain degree.Combining 2 methods could improve the sensitivity and accuracy in predicting A-PCNs.
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Objective To investigate the effect of CYP3A5 on the proliferation of pancreatic cancer cells and its underlying mechanisms.Methods The protein expression of CYP3A5 in five pancreatic cancer cell lines BxPC-3,FG,MDA28,8902 and PANC1 was detected by Western blotting.The PANC1 cells with the lowest protein expression of CYP3A5 and the BxPC-3 cells with highest expression of CYP3A5 were transfected with CYP3A5 overexpression plasmid and CYP3A5 targeted-siRNA (siRNA-CYP3A5),respectively.CCK-8 and cloning formation assay were used to investigate the role of CYP3A5 overexpression and knockdown in the proliferation of pancreatic cancer cells.The changes of the protein and mRNA expression of cell cycle regulating gene cyclin E,cyclin D1 and apoptosis related gene Bcl-2 were detected by Western blotting and PCR,respectively.Results CYP3A5 protein expression in PANC1 cells increased significantly after the transfection of CYP3A5 overexpression plasmid (1.66 ± 0.14 to 1,P =0.0021),which greatly decreased in BxPC-3 cells transfected with siRNA CYP3A5 (0.18 ± 0.02 to 1,P ＜0.0001).A450 values of the CYP3A5 overexpression group and the empty plasmid group in PANC1 cells cultured for 48 and 72 h were 1.36 ±0.05 vs 1.15 ± 0.03,2.1 ± 0.09 vs 1.42 ± 0.03,respectively,which were significantly higher in CYP3A5 overexpression group than empty plasmid group,and the differences were statistically significant (P value ＜ 0.005 or 0.001).The A450 values of BxPC-3 cells in CYP3A5-siRNA transfected group and siRNA-NC transfected group were 0.62 ±0.01 vs 0.77 ± 0.03、0.83 ± 0.01 vs 1.18 ± 0.02,respectively,which in The CYP3A5-siRNA transfection group was significantly lower than that of siRNA-NC transfection group,and the difference was statistically significant (P ＜ 0.05 or ＜ 0.001).The clone formation rate of PANC1 cells in the overexpression group was (19.33 ± 0.58)％,which was significantly higher than that in the empty plasmid group (9.67±0.63) ％,and the clone formation rate in CYP3A5-siRNA group was (8.5± 0.8)％,which was significantly lower than that of group siRNA-NC (16± 0.6)％,and the differences were statistically significant (P ＜0.01).The protein expression of cyclin D1 in CYP3A5 overexpression PANC1 cells was 2.00 ± 0.11,which was obviously higher than 1.00 in empty plasmid group (P ＜0.01).The protein expression of cyclin D1 in siRNA CYP3A5 BxPC cells was 0.45 ±0.04,which was obviously lower than 1.00 in siRNA NC group,and the difference was statistically significant (P＜0.01).However,CYP3A5 overexpression or inhibition did not influence the relative expression of cyclin D1 mRNA and cyclin E,Bcl-2 pretein expression.Conclusions CYP3A5 can promote the proliferation of pancreatic cancer cells by up-regulating cyclin D1 protein expression.
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Objective To investigate the role of fibromodulin (FMOD) in the xidative stress induced activation of pancreatic stellate cells (PSCs).Methods Lentivirus containing ShRNA targeting FMOD (sh-FMOD) was transfected into PSCs,and then the prooxidant menadione (MND) was used to treat PSCs for 24 h (MND + sh-FMOD group).Lentivirus transfected PSCs cells treated by a equal volume of DMSO served as shFMOD group,parent cells as control group and PSCs treated by MND as MND group.RT-PCR were used to detect the mRNA expression of the markers of activated PSCs including α-SMA,Col3α1,Col1α1,TIMP1 and α1-integrin.Chronic pancreatitis (CP) rat model was induced by DBTC infusion into the tail vein.Immunohistochemical (IHC) staining was used to detect the protein expression of FMOD,FN,α-SMA,SOD and MDA in normal pancreatic tissue and CP tissue.Results FMOD mRNA expression of the PSCs in FMOD group was obviously lowerer than that in control group (0.16 ±0.03 vs 1),and the difference was statistically significant (P ＜ 0.01),indicating that FMOD was successfully silenced.The mRNA expression of FMOD,α-SMA,Col3α1,Col1α1,TIMP1 and α1-Integrin of PSCs in MND group was obviously higher than those in control group,which in MND + sh-FMOD group was lower than those in MND group,and the difference was statistically significant (P ＜0.05 or ＜0.01).Compared with those in normal pancreatic tissue,the protein expression of FMOD,α-SMA,SOD and MDA in CP tissue was up-regulated,and the difference was statistically significant (all P ＜ 0.05).Conclusions Oxidative stress can facilitate the activation of PSCs through the induction of fibromodulin expression.
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We studied the effect of the Mycobacterium tuberculosis prokaryotic ubiquitin-like protein-proteasome system on mono-resistant to rifampin resistance to M.tuberculosis.A resazurin-based assay was employed to evaluate minimum inhibitory concentration (MIC) and comparative research on mono-resistant to rifampin MTB with Pup,Dop,PafA,Mpa genes expression and deletion of the difference.Above testing strains,respectively,carbonyl cyanide chlorobenzene hydrazone (CCCP),reserpine (RP),verapamil (VP)and chlorpromazine (CPZ) were tested.We compared and analyzed the change of rifampicin MICs on the various strains.Compared with rifampin resistant MTB,overexpression of Pup,Dop,PafA and Mpa genes were able to make monorifampicin of M.tuberculosis to enhance resistance to rifampin.Deletion of Pup gene,Mpa gene,Dop gene,PafA gene significantly decreased the resistance to rifampicin alone MTB,and the P value was ＜0.05.Results indicated that 4 kinds of efflux pump inhibitors can reduce the degree of rifampin MIC in different strains.Through the factorial analysis,there were some interactions between MTB and PPS efflux pump inhibitors,and the P value was ＜0.05.MTB PPS has influence on mono-rifampin resistance to MTB and it may regulate the efflux pathway related protein to influence its resistance.
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Objective To evaluate the effects of endoscopic ultrasonography ( EUS) combined with cyst fluid analysis, cytology, histopathology based on endoscopic ultrasound guided fine needle aspiration ( EUS?FNA) on pancreatic cystic lesions ( PCLs) . Methods Clinical data of 45 patients were analyzed ret?rospectively from January 2006 to April 2015 including MRI, EUS?FNA, MRCP. The results of conventional imaging and EUS?FNA were compared based on postoperative pathology. Results There were 8 cases of non tumor pancreatic cystic lesions, 4 cases of serous cystadenoma (SCN), 11 cases of mucinouscystadenoma ( MCN) , 8 cases of intraductal papillary mucinous tumor ( IPMN ) , 5 cases of solid pseudopapillary papilloma ( SPN) , 9 cases of non pancreatic cystic lesions ( nPCLs) . The diagnostic accuracy rate of tradi?tional imaging tests ( B?ultrasound, CT, MRI, MRCP ) and EUS?FNA were 42?2% ( 19/45 ) and 77?8%(35/45) (P<0?05). The diagnostic value of EUS?FNA of PCLs was higher than that of traditional imaging tests, with specificity, sensitivity, positive predictive value ( PPV) and negative predictive value ( NPV) of 58?3% ( 7/12 ) , 97?0% ( 32/33 ) , 86?5%( 32/37 ) , 87?5% ( 7/8 ) and 41?7% ( 5/12 ) , 87?9%(29/33), 80?6% (29/36), 55?6% (5/9),respectively. Conclusion EUS?FNA is more valuable than traditional imaging tests for the diagnosis of PCLs.