Abstract:Objective:To inverstigate the effect and safety of refractory intrahepatic and extrahepatic bile duct stones treated by choledochoscope holmium laser combined with electrohydraulic lithotripsy.
Methods :The cllnical data of 67 cases of postoperative intrahepatic and extrahepatic bile duct stones treated with combined endoscopic holmium laser and electrohydraulic lithotripsy were analysed retrospectively.
Results:After 1 to 7 times of biliary endoscopic holmium laser electrohydraulic lithotripsy treatment, 65 patients had complete removal of all residual stones with the success rate of 97%, and without complications.
Conclusions:After holmium laser combined with electrohydraulic lithotripsy,the effect of choledochoscopic removal of intrahepatic and extrahepatic bile duct residual stones can be greatly improved It is a safe method for treatment of refractory intraand extrahepatic bile duct stones.

Abstract:Objective:To research the clinical value of preoperative insertion of nasobiliary tube for after laparoscopic〖JP〗 exploration of common bile duct (CBD) and replacement of Ttube drainage with primary suture of (BD).
Methods:A retrospective analysis of clinical data of 102 cases of laparoscopic common bile duct exploration due to choledocholithiasis. The clinical effects of 55 cases of preoperative insertion of nasobiliary tube and primary suture of common bile duct(Nasobiliary group) were compared with 47 cases of Ttube drainage(Ttube group).
Results:Both of the groups successfully completed operation without postoperative bile leakage. The extubation time of nasobiliary group was (5.3±1.8)d. The postoperative hospitalization (7.2±1.6)d, time to pass flatus（20.3±5.8）d, which were all shorter than those of Ttube group (29.6±6.3d, and 15.3±3.8, respectively), with significant differences. There were no significant differences in operation time and complications (P>0.05) betweent the 2 groups (P<0.01).
Conclusions:Preoperative insertion of nasobiliary tube and primary suture of common bile duct can prevent not only bile leakage caused by increased biliary pressure after primary suture, but also completely eliminates complications caused by Ttube,and can shortens hospital stay. It is worth widespresd application.

Abstract:Objective:To explore the operation methods, intraoperative treatment, measures for prevention and treatment of complications of laparoscopic cholecystectomy (LC) for acute cholecystitis.
Methods :Two hundred and serentytwo patients with acute cholecystitis from March 2003 to April 2007 were analyzed retrospectively. We performed total laparoscopic cholecystectomy in 260 cases and subtotal laparoscopic cholecystectomy with cholecystomucoclasis of remnant gallbladder in 12 cases.
Results:None of 272 cases was converted to open operation.There was no mortality, and no bile duct injury or major bleeding.
Conclusions:Laparoscopic cholecystectomy is a safe and effective method for acute cholecystitis and it has the advantages of minimal trauma,quick recovery and less blood loss.

Abstract:Objective:To investigate the clinical significance of the treatment of intrahepatic bile duct stones with fiber choledochoscope combined with intraoperative ultrasound.
Methods :The clinical data of 185 cases with intrahepatic bile duct stone were analysed retrospectively, included 96 cases with stone removal by fiber choledochoscope intraand postoperatively（choledochoscope group）, and 89 cases with stone removal by fiber choledochoscope combined with intraoperative ultrasound (combined group).
Results:The rateo of bile duct retained stones in choledochoscope group (9.38%) was higher than that in combined group (2.08%)（P=0.041）, and the rate of bile duct recurrent stones in choledochoscope group (12.34%) was higher than that in combined group (3.61%)（P=0.036）.There was no significant differences between the two groups in the occarrence of cholangitis (P=0.087), but 3 cases of severe cholangitis required reoperation in choledochoscope group.
Conclusions:Fiber choledochoscope combined with intraoperative ultrasound can decrease the rate of bile duct retained stones and bile duct recurrent stones, and can improve the therapeutic effect.

Abstract:Objective:To investigate the diagnosis and treatment of calculus in neck and duct of gallbladder during laparoscopic operation.
Methods :The clinical data of 87 cases of calculus in neck and duct of gallbladder were analyzed retrospectively.
Results:There were 83 cases of cholecystolithiasis and calculus in neck and duct of gallbladder, and 4 cases of cholecystolithiasis and calculus in neck and duct of gallbladder and with common duct stones. Of them, 61 cases of calculus in neck and duct of gallbladder were detected during operation. Sixtynine cases had single stone in duct of gallbladder, and 18 had multistones. All of the 87 cases had successful laparoscopic surgery,and among them, 81 cases had laparoscopic cholecystectomy(LC), and 6 had LC and laparoscopic bile duct exploration and T tube drainage. There were no complications and all were cured and discharged.
Conclusions:During laparoscopic operation,it is imporant to pay high attention to and identify stones in neck and duct of gallbladder and by flexibly applying the basic principles and strategies of LC, one can successfully manage stones in neck snd duct of gallbladder.

Abstract:Objective:To explore the causation,diagnosis and management of iatrogenic bile duct injury(BDI) of laparoscopic cholecystectomy (LC).
Methods :A total of 1 656 patients undergoing laparoscopic cholecystectomy in our department during the last 7 years were included in this study for retrospective analysis.
Results:There were 274 patients with gallbladder polyps,168 patients with acute calculous cholecystitis and 1214 patients with chronic calculous cholecystitis.There were 15 BDIs associated with LC(0.91%).A total of 8 BDI patients were diagnosed during cholecystectomy. The remaining 7 BDI patients were diagnosed postoperatively.The intraoperative diagnosis of BDI was made on the discovery of bile leakage or double biliary stump during cholecystectomy.Clinical features, diagnostic abdominocentesis and imaging findings formed the basis of diagnosis of BDI postoperatively. One BDI patient was treated by repairing the injuried common bile duct with a T-tube drinage. Four BDI patients were treated by end-to-end anastomosis of injuried bile duct, and one of the four patients was reoperated with Roux-en-Y hepaticojejunostomy because of bile leakage. The remaining 10 BDI patients were treated by Roux-en-Y hepaticojejunostomy, and good results were achieved in all of these patients.
Conclusions:There is no relationship between the etiology of gallbladder disease and BDI during laparoscopic cholecystectomy. Good results can be achieved if BDI is diagnosed early and treated properly during or after operation. Roux-en-Y hepaticojejunostomy is the primary operation method for treating BDI.

Abstract:Objective:To investigate the relationship between motor abnormality of sphincter of Oddi (SO) and hepatolithiasis.
Methods :The hepatolithiasis rabbit models were established in adult rabbits(n=64) which were randomly divded into 2 groups:control group （n＝16）and experimental group(n=48),including 2 wk,4 wk and 8 wk group（n＝16 per group）. During the formation process of hepatolithiasis, bile character and ratio of stone formation were both observed, then the myoelectric waves of SO were recorded.Finally, The SO segment was sliced and observed by transmissional electron microscope.
Results:The rate of true stone formation in group 8 wk（68.75％,11/16）was higher than that in group 4wk (12.50％,2/16)and in group 2wk (0.00％,0/16). Compared with the control group, there was significant decrease of both SO myoelectric peak amplitude and SO myoelectric frequency in the experimental group. Electron microscopy showed that SO cells in control group and 2wk group had no change, but the 4wk and 8wk groups underwent changes including fibrolysis of myofilament, increase of collagen fibers, fusion and thickening of microfilament, decrease and array disorder of macula densa, swelling of chondrosome and endoplasmic reticulum, widening of perinuclear space and deviation and pyknosis of nucleus, and formation of inclusion body in cytoplasm; and the degeneration degree in 8wk group was obvious than that in group 4wk.
Conclusions:The functional abnormality of SO may contribute important effects on the formation of hepatolithiasis. Fibrous degeneration of SO maybe the pathogenesis of functional abnormality of SO.

Abstract:Objective:To investigate the effects of atorvastatin on the proliferation and invasion of human cholangiocarcinoma QBC939 cells, and explore the possible mechanisms.
Methods :The human cholangiocarcinoma QBC939 cell line was incubated with different concentrations of atorvastatin for 48h. MTT assay for cell growth and viability，Boyden chamber for invasion and motility assays, and semiquantitative RT-PCR to determine mRNA of p27，MMP-9 and RhoC.
Results:The results showed that atorvastatin inhibited growth and proliferation of QBC939 cells, with a time- and dose-dependent inhibition, and at an IC50 value of 25 umol/L,inhibition was most marked at 48 h. Also, atorvastatin-treated（10 μmol/L，25 μmol/L，50 μmol/L for 48 h） cells were less motile and invasive.Semiquantitative RT-PCR analysis showed that atorvastatin treatment reduced MMP-9 expression and upregulated the cyclin-dependent kinase inhibitor p27 in QBC939 cells, but RhoC was not significantly altered between the experimental groups.
Conclusions:Atorvastatin can inhibit proliferation and invasion of human cholangiocarcinoma QBC939 cells.

Abstract:Objective:To explore the roles of peribiliary vascular plexus(PBP) on regeneration of intrahepatic bile ducts after warm ischemia-reperfusion injury and the possible meshanism of PBP on bile ducts sensitive to warm ischemia-reperfusion injury of transplanted liver.
Methods :Male SD rats were used in this experiment. The animals were divided into three groups: shame operation group（S group）, warm ischemia of grafts for 0 min group (W0 group),and warm ischemia of grafts for 10 min group (W10 group). Seven time points were determined as 0 h,6,24 h,and 3,7,14,30 d post-reperfusion. Six animals were used per time-point. ALT, GGT, and TBIL of serum were determined.Serial liver sections were made and were immunolocalized by CK19 and FⅧ-R Ag to assess the co-localization of bile ducts and microvessels. Bile ducts were identified by CK19 and microvessels were identified by FⅧ-R Ag.
Results:In W10 group, the level of plasma GGT and total bilirubin was elevated with a increase longer than in W0 group. The portal inflammation, damage of bile ducts,and fibrosis area of portal area of W10 group were significantly more seriously than those in W0 and S groups. Under electron microscope, micro-thrombosis in microvessels were observed in W10 group. Furthermore, in W10 group, the number of intraportal microvessels at 6h and 24 h post-reperfusion was markedly decreased compared to that of W0 and S groups. In W10 group, although cholangiocytes showed significant regeneration at 24 h post-reperfusion, the number of intraportal microvessels and bile ducts with accompanying microvessels was not higher than that of W0 and S groups at 14 to 30 days after reperfusion.
Conclusions:Warm ischemia-reperfusion injury of liver grafts can cause microthrombosis of peribilary vascular plexus and induce increased damage of vascular endothelial cells and the later can cause disturbance of microcirculation of bile ducts and delayed regcneration of bile ducts.These changes may be important causes of vulnerability of bile ducts to warm ischemia-reperfusion injury.

Abstract:Objective:To establish tetracycline-controlled inducible system (Tet-on) in HepG2 cell for further research of the function of related gens.
Methods :The HepG2 cells were transfected with pWHE146 vector by using liposome transfection reagent. The transfected cells were selected in medium containing G418, and G418-resistant clones were isolated. All individual G418-resistant clones were screened by transient transfection with plasmid pTRE-hyg-luc for clones with low background and high induction of luciferase in response to Dox.
Results:One HepG2 cell line, which exhibited high levels of induction（154,106 times）and high gene expression levels, was obtained.
Conclusions:The HepG2 cell line can be used to highly express eukaryotic gene and this Tet-on system is available for use in eukaryotic gene function studies.

Abstract:Objective:To explore the inhibitory effects of ω-3 polyunsaturated fatty acid on the proliferation of hepatocellular carcinoma cells.
Methods :The proliferation of hepatocellular carcinoma cells(HepG2) was assessed by MTT, immunohistochemical method of PCNA and growth curve respectively. Apoptosis was detected by fluorescent staining, transmission electron microscopy and flow cytometry.
Results:The proliferation of HepG2 was inhibited significantly by ω-3 polyunsaturated fatty acid with a dosage-dependent(range from 15 to 75 μg/mL, P＜0.05) and time-dependent manner. Under transmission electron microscopy and fluorescent microscopy, some HepG2 cells underwent typical apoptosis after 48 h incubated with ω-3 polyunsaturated fatty acid. Compared with the controls, the apoptosis index was significantly increased after 48 h incubated with ω-3 polyunsaturated fatty acid (P＜0.05).
Conclusions:ω-3 polyunsaturated fatty acid could suppress the proliferation of HepG2 cells and induce apoptosis.

Abstract:Objective:To explore the effect of CXCR7shRNA lentiviral vector on CXCR7 expression of human colon cancer cell line HT-29.
Methods :Three siRNA interference sequences targeting CXCR7 gene were designed. Double-stranded shRNA hairpins were synthetized and separately cloned into plVTHM vector digested by double-enzyme so as to recombinate 3 shuttle-plasmids of plVTHM shRNA1, plVTHM shRNA2, plVTHM shRNA3. Then these recombinant plasmids were transformed into DH5α competent cells. After Amp screening positive clones, plasmid was taken from line for subsequent identification by enzyme-digestion and DNA sequencing analysis. The titer of lentivirus was determined after 293T cells were cotransfected with plVTHM shRNA and lentiviral packaging plasmids. The 3 kinds of recombinant lentiviruses and negative control lentiviruses were used to infect HT-29 cells and then CXCR7mRNA and protein were detected by real-time PCR and Western blotting, respectively.
Results:Identification of enzyme-digestion and DNA sequencing analysis confirmed that the 3 CXCR7shRNA sequences were successfully inserted into the lentiviral vectors with the titers of concentrated virus 6×107TU/mL, 4×107TU/mL and 5×107TU/mL, respectively. CXCR7 expression in HT-29 cells was significantly decreased at both mRNA and protein levels compared with the non-transfected and negative control vector (P<0.05). The LV-CXCR7shRNA-2 and LV-CXCR7shRNA-3 played a significant role in reducing mRNA by 72% and 67%, protein by 68% and 55% (P<0.05), respectively; the silent effect showed no significant difference between the nontransfected and negative control vector (P>0.05).
Conclusions:The 3 lentiviral shRNA expression vectors targeting CXCR7 gene were successfully constructed and can effectively downregulate the expression of CXCR7 in HT-29 cells.Thus it provid stable cell transfection vector for further study of CXCR7 gene-targeted gene therapy on colon cancer

Abstract:Objective:To explore the effect and the mechanism of TREM-1vshRNA in the form of sepsis in mice caused by live bacteroides fragilis and the expression of TNF-α, IL-1β, IL-6 and IL-8.
Methods :Male mice（BALB/c） were randomly divided into 4 groups: positive control （group C）, saline（group S）,TREM-1vshRNA(group T) and GFPvshRNA control vshRNA (group G) groups. Bacteroides fragilis (25mg/ kg) was injected via caudal veins in each group to establish the murine septic model. The expression of TREM-1, tumor necrosis factor alpha (TNF-α), IL-1β,lL-6, IkBa, pDAP12 and NF-κBp65 in spleen was observed in each group.
Results:In comparison with group C there was marked elevation of expession of TREM-1mRNA and protein in group T (P＜0.01).Compared to group C there was marked elevation of TNF-α, IL-1β and IL-6 content in splenic cells of groups T,G and S (P＜0.01).The level of TNF-α, IL-1β and IL-6 in splenic cells of group T was markedly lower than that of groups S and G (P＜0.01).In splenic cells of group S,pDAP12 and NF-κBP 65 protein expression was markedly increased compared to group T; the expression of 1κBa protein in splenic cells of groups was decreased.
Conclusions:The interference of TREM-1 signal pathway by TREM-1vshRNA may inhibit the genetic transcriptions of inflammatory factors, such as TNF-α,IL-1β ,IL-6 by downregulating the expression of DAP12, so to stablize the compound of IkBa/NF-κB, can inhibit the inflammatory reaction induced by Bacteroides fragilis.

Abstract:Objective:To analyze the clinicopathologic factors related to the prognosis of gallbladder carcinoma.
Methods :A retrospective analysis was conducted on 61 cases of advanced gallbladder carcinoma who were operated on from 2001 to 2006 in Nanjing First Hospital.
Results:According to the 6th UICC/AJCC TNM classification and staging of gallbladder carcinoma, of the 61 patients, 8 patients were stage IB, 22 were IIA, 11 were IIB, 14 were III and 6 were IV. Palliative cholecystectomy and/or bile duct drainage procedures were performed in 24 patients; removal of gallbladder plus regional lymph nodes and fatty tissue dissections were performed in 14 cases; radical resection was performed in 7 patients; removal of gallbladder and part of liver in combination with resection of bile duct (radical operatian) was performed in 13 patients; 1 patient had extensive radical operation including pancreaticoduodenectomy. In 37 patients, 18 had positive lymph node metastasis. Fifty-two patients were followed up, 41（67.2%） patients were alive at 6 months after operation, 26（42.6%）were alive at 12 months, 10（16.4%） were alive at 24 months, and 7 cases were alive at 36 months, 1 patient is still alive 5 years after operation; 51 patients died within 2 years after operation. The symptoms of recurrence in 31 patients included progressing jaundice (21 cases), metastasis in liver (14 cases), and refractory ascites (12 cases). Five patients died of non- bile duct disease.
Conclusions:The prognosis of gallbladder cancer has a close relationship with the clinical stage and method of resection.

Abstract:Objective:To explore the therapeutic effect of choledochojejunostomy with a subcutaneous blind loop of interposed jejunum for treating postoperative residual biliary stones.
Methods :The clinical data of 72 cases of choledochojejunostomy with subcutataneous blind loop of interposed jejunum were retrospectively analysed.
Results:After exploration of distal common bole duct, and when a No. 8-10 bougie could pass through the sphincter of Oddi, a 10-15 cm segment of jejunum with its vascular pedicle was mobilized, and an end to-side choledochojejunal anastomosis was performed at the proximal side of the jejunal segment and its distal end was converted to a blind loop and fixed subcutaneously to relevant abdominal wall. A T-tube for external drainage was inserted across the choledochojejunal anastomosis and exited through the blind jejunal loop.Seventy-two patients were cured without any complication or mortality.
Conclusions:The procedure is simple and minimally invasive, so that it could be carried out very easily and quickly by general surgeons with basic choledochojejunostomy skill.

Abstract:Objective:To study the prevention and management of early post-cholecystectomy jaundice.
Methods :The dinical data of 43 cases of jaundice occurring within one week after simple open cholecystectomy in 11 226 cases were retrospectively analyzed.
Results:The incidence of jaundice at one week after operation was 0.38% in simple open cholecystectomy, among them, medical jaundice accounted for 18 cases, and surgical jaundice for 25cases. Ultrasound,ERCP,MRCP,liver enzyme profile and reoperation confirmed the folowing: Extra-hepatic bile duct or the right hepatic duct was ligated in 9 cases,common bile duct residual stone in 6,partial gallbladder or gallbladder duct stone in 4,biliary leak in 4, cholangitic hepatitis in 4,hepatitis B or posthepatitis cirrhosis in 5,icterus after blood transfusion in 2,hemobilia in 2, ligation of right branch of hepatic artery in 2, and icterus of unknown causes in 5 cases. Reoperation was done in 8 cases, with one mortality.
Conclusions:Detailed case history,adequate examination before operation,and careful operative technique are the essential prerequisites to prevent early post-cholecystectomy jaundice. Early post-cholecystectomy jaundice should be treated according to the different causes.