The effects of oxygen partial pressure on cryopreservation of the cells with organ preservation solution were explored. Hypoxic UW solution was made by purging the UW solution with argon. The pig proximal tubule epithelial cells (LLC-PK1 cells) were cryopreserved in hypoxic UW solution (Ar-UW group) or standard UW solution (UW group) at 4 degrees C for 48 h. Trypan blue staining and LDH detection were performed to evaluate the injury of the cells. The results showed that the oxygen partial pressure in Ar-UW group was significantly declined from 242+/-6 mmHg to 83+/-10 mmHg. After cryopreservation at 4 degrees C for 48 h, LDH leakage rate and Trypan blue-stained rate in Ar-UW group were (11.3+/-3.4)% and (10.5+/-4.7)%, respectively, which were significantly lower than in UW group [(49.5+/-6.9)% and (47.6+/-9.3)% respectively, both P<0.01]. It was concluded that lower oxygen partial pressure of UW solution was more beneficial to the cryopreservation of LLC.
Adenosine ; Allopurinol ; Cell Hypoxia ; Cell Line ; Cryopreservation ; Cryoprotective Agents ; Epithelial Cells/*cytology ; Glutathione ; Insulin ; Kidney Tubules, Proximal/cytology ; Organ Preservation Solutions ; Oxygen/pharmacology ; Raffinose ; Swine ; Tissue Preservation/methods

The experience in diagnosis and treatment of bleeding complications in severe acute pancreatitis (SAP) by transcatheter arterial embolization was summarized. The clinical data of 19 SAP patients complicated with intra-abdominal bleeding in our hospital from Jan. 2000 to Jan. 2003 were analyzed retrospectively and the therapeutic outcome of TAE was evaluated statistically. The results showed that the short-term successful rate of hemostasis by TAE was 89.5% (17/19), the incidence of re-bleeding after TAE was 36.8% (7/19) and the successful rate of hemostatis by second TAE was 71.4% (5/7). It was concluded that the intra-abdominal bleeding in SAP was mainly caused by the rupture of erosive/infected pseudoaneurysm. Mostly, the broken vessels were splenic artery and gastroduodenal artery; In terms of emergence hemostatis, TAE is the most effective method. Surgical hemostasis is necessary if hemostasis by TAE is failed or re-bleeding occurs after TAE.
Aneurysm, False/diagnosis ; Aneurysm, False/etiology ; Aneurysm, False/therapy ; *Embolization, Therapeutic/methods ; Hemoperitoneum/diagnosis ; Hemoperitoneum/etiology ; Hemoperitoneum/*therapy ; Pancreatic Pseudocyst/diagnosis ; Pancreatic Pseudocyst/etiology ; Pancreatic Pseudocyst/therapy ; Pancreatitis, Acute Necrotizing/*complications ; Pancreatitis, Acute Necrotizing/therapy ; Retrospective Studies

Objective To study and evaluate the diagnostic and differencial diagnostic value of detection of human telomerase reverse transcriptase(hTERT) mRNA and telomerase activity in pancreatic juice of (pancreatic) cancer(PC) .Methods hTERT mRNA expression and telomerase activity in pancreatic juice in 24 cases of (pancreatic) cancer and 14 cases of chronic pancreatitis(CP) were detected and compared.Results Expression of hTERT mRNA was detected in 87 .5% (21/24) of PC cases and 21.4%(3/14) of CP cases(P

Objective To sum up the experience on diagnosis and treatment of intraabdominal bleeding in severe acute pancreatitis (SAP). Methods The clinical data of 37 SAP patients with bleeding complications from Jan 1999 to Jan 2003 were analyzed retrospectively. The causes of bleeding were of erosive (5 cases), infected (20 cases), intra operative/postoperative (10 cases) and coagulopathy (2 cases). Results The mortality was 10% (2/19) for transcatheter arterial embolization (TAE), 30% (7/23) for laparotomy, 50% (2/4) for conservative thepary. The cause related mortality was 0 (0/5), 30% (6/20), 40% (4/10) and 50% (1/2) for erosive, infection, intra/post operative, and coagulopathy respectively. Conclusion In SAP most intra abdominal bleedings are from erosive/infective pseudoaneurysms of splenic artery or/and gastroduodenal artery. The preferred diagnosis methods were CT and selective angiography which often give definite diagnosis. TAE is most effective therapy, when it fails, a surgery is necessary.

The proliferation and differentiation of pancreatic duct epithelial cells in remnant pancreas during regeneration after partial pancreatectomy in rats were studied, and the source of pancreatic stem cells was characterized. Partial (90 %) pancreatectomy was performed on 4- to 5-week-old Sprague-Dawley rats, and different duct epithelial cells and acinar cells were detected by immunohistrochemical stain method and scored using 5-bromo-2'-deoxyuridine (BrdU) labeling index (LI) at various time points after partial pancreatectomy. It was found that at 24 h after partial pancreatectomy proliferation started in the main, large and small duct cells, and persisted in small duct cells to day 5. There was significant difference between the experimental group and the control group (P<0.001). Acinar cells positive for BrdU were greatly increased and reached the peak LI on day 5. The destroyed lobular architecture almost totally recovered on day 7, and the newly islet cells appeared around the pancreatic ducts. These results suggest that regeneration after partial pancreatectomy is involved in proliferation and differentiation of pancreatic stem cells, and pancreatic stem cells may locate in the pancreatic ductules.

In order to investigate the expression of Notch-1 in rats with acute pancreatitis (AP) and its relation with apoptosis of pancreatic cells, mild AP (MAP) and severe AP (SAP) models were established by retrograde injection of different concentrations of sodium taurocholae into pancreatic duct. The apoptosis index and the expression of Notch-1 protein and mRNA were detected by using TUNEL, Western blot and real-time PCR in MAP and SAP at different time intervals. The results showed that in MAP group the apoptosis index was significantly elevated during 4 to 24 h after induction of pancreatitis, but there was no significant difference among different time intervals. In SAP group, the apoptosis index reached the peak at 4th h after induction of pancreatitis, then gradually declined. There was significant difference in apoptosis index between MAP and SAP groups. Starting from 4th after induction of pancreatitis, the expression of Notch-1 in both MAP and SAP group was increased at different time intervals, but that in SAP group was significantly higher than in MAP group (P<0.05). The expression of Notch in MAP and SAP groups reached the peak at 12th and 8th h respectively after induction of pancreatitis. It was concluded that there was significant difference in apoptosis index and the Notch-1 expression in different types of AP. The overexpression of Notch-1 could aggravate AP by inhibiting the apoptosis of pancreatic cells.

Objective To investigate the effects of down-regulating uncoupling protein-2 (UCP-2) expression on acute damage to fatty liver cells and explore a new target for the donor liverwith steatosis. Methods Primary fatty liver cells were isolated from C57BL/6J-ob/ob transgenic miceby two-step collagenase perfusion method. RNAi lentivirus vector targeting mouse UCP-2 gene wasused to knock down the UCP-2 gene in the steatosis hepatocytes (the experimental group). Emptylentivirus vector was transfected into the steatosis hepatocytes cells as the control group. Under thefluorescence microscopy, the transfection efficiency was tested. Real time PCR was used to determinethe effect of RNAi. After the transfected cells were treated with TNF-α for 24 h, apoptosis wasanalyzed by flow cytometry using PI staining. Activation of caspase3 was detected by Western blot.Resalts The expression of UCP-2 gene was inhibited effectively, and the knockdown rate of UCP-2gene was 75%. The apoptosis rate in the experimental group was (4.97±0.25)%, significantlylower than in the control group [(21.13±1.28)%, p<0.05 ]. Activation 'of caspase3 in theexperimental group was also weaker than in the control group. Conclusion Inhibiting the expression ofUCP-2 can attenuate the injury of fatty liver cells.

To investigate the protein and mRNA expression of pancreas/duodenal homeobox-1 (PDX-1), a transcription factor as a marker for pancreatic stem cells, in pancreatic ductal cells of rats after partial (90%) pancreatectomy and evaluated the significance of the PDX-1 expression. Western blot and Reverse transcriptase-polymerase chain reaction (RT-PCR) were used to detect the expression of PDX-1 protein and mRNA respectively. PDX-1 protein was only faintly detected in pancreatic ductal cells on the day 1 after partial pancreatectomy. On the day 2 and 3 after operation in operation group, a 2-3 fold increased PDX-1 protein was observed, corresponding to the characteristic 42-kD protein in Western blot. There was significant difference between operation group and sham-operation group (P<0.05). PDX-1 protein expression on the day 5 and 7 after operation had already been no difference from control group (P>0.05). RT-PCR revealed the PDX-1 mRNA expression showed no significant difference between operation group at various time points and sham-operation group (P> 0.05). These results indicate that there was overexpression of PDX-1 in the cells of pancreatic epithelium during the regeneration of remnant pancreas after partial pancreatectomy in adult rats, suggesting the pancreatic stem cells in pancreatic ductal epithelial cells are involved in the regeneration of remnant pancreas and the expression of PDX-1 in ductal cells was regulated posttranscription.
Epithelial Cells/metabolism ; Homeodomain Proteins/*biosynthesis ; Homeodomain Proteins/genetics ; Pancreatectomy/methods ; Pancreatic Ducts/cytology ; Pancreatic Ducts/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Rats, Sprague-Dawley ; Trans-Activators/*biosynthesis ; Trans-Activators/genetics

Objective To investigate the operational technique of laparoscopic splenectomy and pericardial devascularization (LSPD) and evaluate the clinical efficiency of this method for the treatment of portal hypertension.Methods With the new understanding of anatomical space around the spleen,the cardia and the fundus,two gaps and two tunnels can be created in LSPD.Retrospective analysis was made on the clinical data of patients who underwent LSPD from Jun 2013 to Mar 2015.The operative time,intraoperative blood loss,postoperative hospital stay,conversion rate and postoperative complication rate were measured.Results A total of 189 cases underwent surgery successfully,including 34 cases of splenomegaly and 21 cases of severe esophageal varices.The operative time was (125 ± 52) min,intraoperative blood loss (58 ± 32) ml,postoperative hospital stay (7.5-2.1) d.There were 4 conversion cases in this study.Conclusions The splenic pedicle and stomach pedicle can be safely dissected with the two gaps and two tunnels principle,which makes LSPD safe and convenient.

ObjectiveTo construct recombinant adenovirus vector containing human pancreatic and duodenal homeobox factor 1 (PDX1) and detect its expression in human umblical cord mesenchymal stem cells (HUCMSCs). MethodsPDX1 obtained by BgⅢ/XhoI enzyme digestion from pUC57-PDX1 was ligated into the recombinant shuttle vector pShuttle-GFP-CMV to obtain the recombinant shuttle plasmid pShuttle-GFP-CMVPDX1. pShuttle-GFP-CMV- PDX1 was shifted to pAdxsi vector to obtain pAdxsi-GFP-PDX1 virus plasmid. The recombinant plasmid was packaged and amplified in 293 cells. The expression of PDX1 gene and protein in HUCMSCs was detected by fluorescence microscopy, RT-PCB, immunofluorescence, immunohistochemistry, and Western Blot. ResultsPDX1 gene was inserted correctly into shuttle plasmid and the recombinant adenovirus vector was successfully constructed according to the results of sequence and enzyme digestion identification. The adenovirus was effectively transfected into HUCMSCs. RT-PCR verified that PDX1 mRNA was positively expressed in HUCMSCs. Expression of PDX1 protein in the nuclear of HUCMSCs was found by immunofluorescence assay, immunohistochemistry and Western Blot. ConclusionThe adenovirus vector containing PDX1 gene is successfully constructed and effectively expressed in HUCMSCs.