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Objective To analysis the clinical features of dorsal pancreas agenesis ( DPA) and the associated diabetes, pancreatitis and other congenital organ malformations.Methods Chinese databases of Sinomed, CQVIP and CNKI using the term of short pancreas, pancreas agenesis, bulbar pancreas and dorsal pancreas, and English databases of PubMed using the term of dorsal pancreas agenesis, short pancreas and pancreas hypoplasia were searched.The clinical manifestation, pancreatic head characteristics and associations with diabetes, pancreatitis and other congenital organ malformations were analyzed.Results Six related publications from Chinese databases were searched and 21 patients were included with 2 cases excluded.Sixty-one publications from English database were searched and 71 patients were included.Thus, a total of 91 patients with DPA were analyzed.Abdominal pain was the most common manifestation, which was reported by 61.5% of the patients. 15.3% patients were identified during regular physical examination. Other manifestations including jaundice, fatigue, abdominal discomfort and diabetes were rare.After removing cases with insufficient information, 39 patients (61.9%) carried abnormal pancreatic head.Prevalence of diabetes or impaired glucose tolerance was 56.7% and the percentage of insulin-dependent diabetes in patients with abnormal glycaemia was 47.3%(n=18).20 patients (26.7%) were associated with pancreatitis, including 15 patients (75.0%) with acute pancreatitis, 1 patient (5.0%) with recurrent pancreatitis, and 4 patients (20.0%) with chronic pancreatitis. Thirty-three patients ( 36.2%) suffered other congenital organ malformations, including 21 patients (63.6%) with splenic malformation, 8 patients (24.2%) with heart malformation, and 17 patients (51.5%) with multi-organs malformations like gastrointestinal malformation, azygos vein and inferior cava vena fusion, duodenal and biliary atresia and renal absence.Conclusions The main diagnostic criteria of DPA was the absence of dorsal pancreatic duct.Diabetes was the most common complication followed by pancreatitis.
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Objective To silence the beclin1 gene expression by using RNA interference technology in AR42J rat pancreatic acinar cells,and build a stable AR42J line silencing beclin1.Methods Three kinds of shRNA targeting rat beclin1 mRNA and negative control shRNA were designed and synthesized,and were inserted into the plasmids GV112,respectively.The recombinant plasmids were named as p-sh-Beclin1-1,psh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC.Lipo3000 was used to transfect the recombinant plasmid into AR42J cells,the expression of beclin1 mRNA were detected by RT-PCR to screen for the most efficient silencing plasmid,and then it was packaged into lentiviral (LV).AR42J cells were infected with LV and screened by puromycin.Beclin1 mRNA and protein expression was determined by RT-PCR and Western blot.Results The recombinant plasmid was confirmed by agarose gel electrophoresis and sequencing showed that shRNA sequences were in line with expectations.The beclin1 mRNA inhibition rates of AR42J cells after p-sh-Beclin1-1,p-sh-Beclin1-2,p-sh-Beclin1-3 and p-shRNA-NC transfection were (17.8 ± 4.0) ％,(30.6 ± 2.8) ％,(45.8 ± 7.7) ％,(7.0 ± 11.8) ％,respectively.The inhibition rates of three p-sh-Beclin1 transfection cells were significantly higher than that in non-transfection cells,and the difference was statistically significant (P ＜ 0.05).While the inhibition rate of p-shRNA-NC transfection cells was not significantly different from that of non-transfection cells,p-sh-Beclin 1-3 with highest rate of inhibition was packaged by LV,and infected AR42J cells,then puromycin was applied to screen,inhibition rate of beclin mRNA expression in LV infection cells was (86.1 ± 1.2) ％,and the protein expression inhibition rate was (87.9 ± 2.8) ％,and the difference between infection and non-infection groups was statistically significant (P ＜ 0.05).Conclusions The stable AR42J line silencing beclin1 is successfully established,which can provide a new cell model for future research of the role of beclin1 in the pathogenesis of acute pancreatitis.
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Objective To investigate the changes and significance of autophagy in rats with experimental acute necrosis pancreatitis (ANP).Methods According to method of random number,18 rats were randomly divided into control group,ANP group,ANP+rapamycin (RAP) group.The ANP rat model was established by intraperitoneal injection of 20％ L-arginine.The rats of ANP+RAP group were intraperitoneal injected with RAP 1.2 mg/kg at 30 minutes before modeling.The rats of control group were intraperitoneal injected with 0.9％ NaCl solution.The blood was drawed from the hearts nine hours after modeling for subsequent experiments.Serum levels of trypsinogen activation peptide (TAP),interleukin (IL-1),IL-6 and tumor necrosis factor (TNF) α were measured with enzyme-linked immunosorbent assay.The pancreatic tissues were pathologically scored.Autophagy-related structures in rat pancreatic acinar cells were observed by transmition electron microscopy.The expression of autophagy marker microtuble assciated protein 1 light chain 3 (LC3)-Ⅱ and Beclin-1 at mRNA and protein level were measured by quantitative real-time polymerase chain reaction (qRT-PCR),Western bloting and immunohistochemistry.The single factor analysis of variance was used for mean comparison among groups.Results A rat model of ANP was successfully established.Histopathological score of pancreas acinar cell necrosis of ANP+RAP group (2.19±1.38) was higher than that of ANP group (0.97±0.68),and the difference was statistically significant(F=33.75,P＜0.05).The results of Western blotting indicated that the protein expression of LC3-Ⅱ and Beclin-1 in ANP group (35.25±2.68 and 49.40±5.28)were higher than those in control group (1.54±0.16 and 0.78±0.06),furthermore the expressions in ANP+RAP group(123.53±3.21 and 76.41±3.80) were higher than those in ANP group,and the differences were statistically significant(F=2 045.54,326.87,both P＜0.01).Immunohistochemistry results also indicated that the LC3Ⅱ and Beclin-1 expression at protein level of ANP+RAP group (7 570.63±4 357.67 and 3 418.09±2 035.78) were higher than those of ANP group (1 926.53±1 414.44 and 536.11±403.10),and the differences were statistically significant (F=39.83,41.58,both P＜0.01).The expression of Beclin-1 at mRNA level of ANP group (107.12±29.10) was statistically higher than that of control group(7.01 ±3.39),and the difference was statistically significant (F=3.61,P＜0.05),but the expression of ANP+RAP group (97.63 ± 65.38)was no significant difference compared with ANP group.However,the expression of LC3-Ⅱ at mRNA level of ANP+ RAP group (4.37 ± 1.67) was statistically higher than that of ANP group (1.76 ± 1.59),and the difference was statistically significant(F=16.10,P＜0.05),but the expression of ANP group was no significant difference compared with control group (1.51 ±0.95).The result of electron microscopy showed that autophagy related structures increased in ANP group compared with that of control group,which of ANP+RAP group was more.The serum levels of TAP,IL-1 and IL-6 of ANP + RAP group were (36.47 ± 1.71) pmol/L,(122.88± 26.67) pg/mL and (107.39±13.95) pg/mL,which were all higher than those of ANP group ((25.63 ± 6.05) pmol/L,(98.06 ±9.29) pg/mL and (86.16± 7.20) pg/mL),and the differences were statistically significant (F=116.71,50.45,79.67; all P＜0.01).There was no significant difference in TNFα between ANP+ RAP group ((140.80±60.82) pg/mL) and ANP group ((105.23±6.95) pg/mL,F=14.76,P＞0.05).Conclusions Autophagy increased in rats with ANP.Promoting autophagy could significantly activate trypsinogen,aggravate pancreatic injury and increase inflammation reaction,which indicated that autophagy might involve in the pathogenesis of ANP through trypsinogen activation.
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Objective To establish a stable AR42J cell line expressing EGFP LC3.Methods The EGFP LC3 overexpressed Lentivirus was constructed and transfected into pancreatic acinar cells (AR42J) of rats.The rats with Lentiviral EGFP transfection were treated as negative control.The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The EGFP LC3 protein expression in the stable cell lines were analyzed by Western blot.The cells were treated with thapsigargin to establish endoplasmic reticulum stress model,and the LC3,PERK protein expressions were detected by Western blot.Results The transfection efficiency of Lentiviral EGFP LC3 of AR42J cell was ＞ 85％,which could achieve stable passage.The expression of LC3 mRNA of AR42J cells transfected with Lentiviral EGFP LC3 was 9.14 ±0.32 folds higher than that of negative control,which had no expression of LC3 protein,only EGFP expression.However,compared with non-transfection group,the LC3 mRNA expression in EGFP group was not significantly different.Conclusions A pancreatic acinar cell line (AR42J) of rat stably expressing EGFP LC3 protein is successfully constructed.And it may provide a new model for further research of the relationship between acute pancreatitis and autophagy.
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Background:Damage of intestinal mucosal barrier is a key factor in the development and progress of acute pancreatitis(AP),and is closely related with the prognosis of the disease. Aims:To investigate the protective effect and possible mechanism of mucoprotective agent teprenone on intestinal mucosal barrier in rats with experimental AP. Methods:Forty-five adult male Sprague-Dawley rats were randomly divided into normal control group(n = 5),AP model group(n = 20)and teprenone treated group(n = 20). AP model was established by subcutaneous injection of cerulein at abdominal wall. Rats in treated group were intervened with teprenone intragastrically before and after model establishment. ELISA was used for measurement of serum interleukin-1(IL-1),IL-6,tumor necrosis factor-α(TNF-α)and amylase;histopathological and ultrastructural changes of small intestinal mucosa were observed by light microscope and transmission electron microscope;Western blotting was used to detect the expressions of tight junction protein occludin and ZO-1. Results:Serum levels of IL-1,IL-6,TNF-α and amylase in AP model group were significantly higher than those in normal control group(P < 0. 05),accompanied by necrosis and exfoliation of small intestinal villus,widening of intercellular tight junctions and downregulation of occludin and ZO-1 expression. While in teprenone treated group,serum levels of proinflammatory cytokines and amylase were significantly decreased as compared with AP model group(P < 0. 05),the villus of small intestine remained intact,and dense tight junctions were observed. Expressions of occludin and ZO-1 in teprenone treated group were upregulated. Conclusions:Teprenone may protect against intestinal mucosal barrier injury in AP model rats by upregulating tight junction protein expression.
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Objective To observe the changes of zymophagy during experimental acute pancreatitis (AP) induced by caerulein.Methods Pancreatic acinar cell line AR42J cells were cultured in 6-well plates till 90％ confluent and then divided into AP group and control group.Caerulein (1 × 10-8 mol/L) was added into AP group to establish AP cell model,and 1640 cell culture medium was added into control group.After caerulein treated for one,four,six,eight,12 and 24 hours,cells and cell culture supernatant were collected.The levels of cytokine interleukin (IL)-1,tumor necrosis factor (TNF)α,trypsinogen activation (TAP) and amylase were measured with enzyme-linked immunosorbent assay (ELISA) method.The expression of LC3 and Beclin1 at mRNA of each group were detected by reverse transcription-polymerase chain reaction (RT-PCR).The LC3B protein level of each group were detected by Western blotting.The changes of autophagosome and zymophagosome were observed by transmission electron microscopy.The difference between AP group and control group was analyzed by analysis of variance.Results The level of IL-1,TNFα,amylase and TAP in cell culture supernatant of control group was (18.83±7.10) pg/mL,(14.20±3.79) pg/mL,(10.03±2.85) U/L and (39.48±8.62) pg/mL,respectively.Those of AP group significantly increased at first hour ((62.13±11.25) pg/mL,F=3.32,P＜0.01 ; (30.98±7.11) pg/mL,F=3.05,P＜0.05; (25.06±6.82) U/L,F=2.90,P＜0.05 and (128.51± 18.30) pg/mL),F=2.62,P＜0.01,at fourth or sixth hour reached peak (IL-1 at fourth hour:(71.96± 15.82) pg/mL,F=7.25,P＜0.01;TNFα at sixth hour:(39.92±8.94) pg/mL,F=4.93,P＜0.05; amylase at fourth hour:(28.83 ± 8.31) U/L,F=2.06,P＜0.05; TAP at fourth hour:(146.29± 29.36) pg/mL,F=0.14,P＜0.01) and then gradually decreased.At fourth and sixth hour,the expression of LC3 at mRNA level in AP group was 3.18±0.82,1.71±0.14,respectively,while the expression of Beclin-1 rnRNA at first,fourth hour was 2.44±0.34 and 4.13±0.30,all of them were significantly increased compared with those of control group (0.21±0.04 and 0.30±0.08,LC3 mRNA F=0.79、0.06; Beclin mRNA F=2.31、0.36,all P＜ 0.05).There were no significant differences at other time points.The numbers of autophagosome and zymophagosome of AP group were significantly higher than those of control group under transmission electron microscopy.Conclusion Zymophagy occurred during AP cell model induced by caerulein,which suggested that zymophagy might involve in the mechanism of AP.
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Objective To evaluate the value of medical treatment in the management of SAP.Methods From January 2000 to December 2011,a total of 1064 cases out of 931 SAP patients were admitted and retrospectively analyzed.The etiologies,severity score,complication rates,therapies,effectiveness and costs of those SAP cases were summarized.Results There were 559 males and 372 females with a mean age of (51 ± 15)years old.The main cause was biliary tract disease (58.3％),followed by fat-rich diet (31.2％),hyperlipidemia (13.6％) and alcohol (7.1％).At the time of admission,95.5％ of SAP patients presented with level D disease according to Balthazar CT severity index,26.0％ had a Ranson score ≥3 and 30.1％ had an APACHE Ⅱ score ≥ 8.There were 42.7％ cases complicated with systemic inflammatory response syndrome (SIRS).Acute lung injury and acute respiratory distress syndrome (ARDS),acute kidney injury,shock or heart failure,acute liver dysfunction,and diffuse intravascular clotting (DIC)occurred in 24.0％,8.1％,5.4％,3.2％,and 1％ of all patients,respectively.Other complications of SAP included abdominal cavity bleeding (n =17),pseudocyst bleeding (n =9),pancreatic abscess (n =78) and gastrointestinal fistula (n =33).Totally 25 (2.3％) patients died in hospital and 36 (3.4％) patients were discharged against advice,with an overall treatment success rate of 94.3％.The mean hospital stay was (23.7 ± 19.2) d,and the average cost was 52.3 thousands of RMB.Conclusions A comprehensive treatment pathway relying on medical treatment,focusing on organ function support and assisted by miniinvasive intervention may improve the treatment success rate of SAP,which is worth of further application.
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To observe the alteration in the expression of DNA repair enzymes hOGG1 and hMYHalpha and the change in 8-OHdG levels in the HBx gene-transfected cells HepG2/HBx and to explore the mechanisms of the HBV-associated hepatocellular carcinoma, the gene-transfected cells HepG2/HBx which stably expressed HBx was established, and the effect of HBx on the cell cycle and proliferation of HepG2 was examined. By using the beta-actin as the interior control, real-time polymerase chain reaction (Real-time qPCR) was employed to quantitatively detect the expression of DNA repair enzymes hOGG1 and hMYHalpha in the HepG2/HBx, the control cells HepG2 and HepG2 transfected with pcDNA3.1 vector (HepG2/pDNA3.1). The 8-OHdG levels were determined by HPLC/ECD in the established gene-transfected cells HepG2/HBx and the control cells HepG2 and HepG2/pcDNA3.1. Our results showed that the expression of DNA repair enzyme hMYHalpha in the HepG2/HBx (0.021+/-0.007) was significantly lower than that of HepG2 (0.099+/-0.041) (P0.05). The 8-OHdG level in the HepG2/HBx was significantly higher than that in HepG2 and HepG2/pcDNA3.1 (P<0.05). It is concluded that HBx gene may inhibit the expression of DNA repair enzyme hMYHalpha mRNA to impair the ability to repair the intracellular DNA oxidative damage, to increase the oxidative DNA-adduct 8-OHdG and to affect the nucleotide excision repair function, thus participate in the occurrence and development of hepatocellular carcinoma.
Subject(s)
DNA Glycosylases/genetics , DNA Glycosylases/metabolism , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Hep G2 Cells , RNA, Messenger/genetics , RNA, Messenger/metabolism , Trans-Activators/geneticsABSTRACT
Objective To study the effects of the HBV x gene (HBx) on the biological characteristics and the expression of DNA repair enzyme hMTH1 mRNA of the L02/HBx transgene cell model. Methods Light microscopy was used to observe the morphologic characteristics of gene-transfected cell strain Lff2/HBx that stably expressed the HBx protein and the control groups of L02 and L02/PcDNA3.1. The changes of L02/HBx on the proliferation, cell cycle and apoptosis were observed by MTT assays and flow cytometry analysis respectively. Moreover, the malignant transformation of L02/HBxwas assayed by colony formation in soft agar and the expression of DNA repair enzyme hMTH1 mRNA was assayed in each group by real-time qPCR. Results Inversion phase contrast microscope showed that the morphologic characteristics of L02/HBx had changed obviously compared with control groups. The MTT showed that L02/HBx proliferated more quickly and flow cytometry analysis indicated that HBx could accelerate the progression of cell cycle and inhibit apoptosis. Colony formation in soft agar demonstrated that the rate of colony formation of L02/HBx was remarkably higher than the L02 and the L02/peDNA3. 1 cells (P<0. 05). The real-time qPCR detection showed that the expression of hMTH1 mRNA in L02/HBx was significantly higher than that in the control groups ( P < 0. 05 ). Conclusion HBx could play an important role in the malignant transformation of L02/HBx and the over expression of hMTH1 mRNA.