Background and Aims:Complex choledocholithiasis often coexists with stenosis of the ampulla of Vater,which increases the difficulty and complexity of treatment.If only the stones in the bile duct are removed without addressing the ampullary stenosis,the disease is prone to recurrence.Previously,most treatments involved the use of endoscopic retrograde cholangiopancreatography(ERCP)to guide the wire for sphincterotomy and stone extraction,followed by laparoscopic cholecystectomy.However,ERCP has limitations in handling complex cases.In response,our team pioneered a new method of treating choledocholithiasis combined with stenosis of the ampulla of Vater via a transabdominal approach.This study was performed to investigate the feasibility and efficacy of this method,aiming to provide a new therapeutic option for clinical practice.Methods:A randomized controlled study was conducted with 120 patients treated at Chengdu Second People's Hospital from 2021 to 2023 for gallbladder stones and choledocholithiasis with stenosis of the ampulla of Vater.Patients were divided into an observation group and a control group,with 60 cases in each group.The observation group underwent laparoscopic cholecystectomy with choledochotomy for stone extraction,followed by retrograde guidance of duodenal papillary sphincterotomy through the opened bile duct,simultaneously treating gallbladder,bile duct stones,and stenosis of the ampulla of Vater.The control group underwent traditional ERCP approach for sphincterotomy,stone extraction,and laparoscopic cholecystectomy.Perioperative variables were collected for both groups and the surgical outcomes were compared.Results:Among the 120 patients,54 were male and 66 were female.There were no statistically significant differences between the two groups in terms of stone extraction success rate,intraoperative blood loss,postoperative 24-h total bilirubin,direct bilirubin,transaminases,white blood cell count,jaundice relief time,or incidence rates of bile leakage,retroperitoneal bleeding/infection,and severe pancreatitis(all P>0.05).The observation group had significantly shorter average operative time and postoperative hospital stay compared to the control group(98.67 min vs.110.8 min,P<0.05;3.81 d vs.5.61 d,P<0.05).Additionally,the observation group had a significantly lower incidence of postoperative hyperamylasemia and/or hyperlipasemia and mild pancreatitis(1.67%vs.25.00%,P<0.001;0 vs.10%,P=0.027).Conclusion:The novel transabdominal approach is superior to the ERCP approach in terms of reducing surgery time and hospitalization time,and it carries a lower risk of postoperative mild pancreatitis and hyperamylasemia and/or hyperlipasemia.The stone extraction success rate is comparable to that of ERCP,making it a viable alternative treatment option.

Background and Aims:Complex choledocholithiasis often coexists with stenosis of the ampulla of Vater,which increases the difficulty and complexity of treatment.If only the stones in the bile duct are removed without addressing the ampullary stenosis,the disease is prone to recurrence.Previously,most treatments involved the use of endoscopic retrograde cholangiopancreatography(ERCP)to guide the wire for sphincterotomy and stone extraction,followed by laparoscopic cholecystectomy.However,ERCP has limitations in handling complex cases.In response,our team pioneered a new method of treating choledocholithiasis combined with stenosis of the ampulla of Vater via a transabdominal approach.This study was performed to investigate the feasibility and efficacy of this method,aiming to provide a new therapeutic option for clinical practice.Methods:A randomized controlled study was conducted with 120 patients treated at Chengdu Second People's Hospital from 2021 to 2023 for gallbladder stones and choledocholithiasis with stenosis of the ampulla of Vater.Patients were divided into an observation group and a control group,with 60 cases in each group.The observation group underwent laparoscopic cholecystectomy with choledochotomy for stone extraction,followed by retrograde guidance of duodenal papillary sphincterotomy through the opened bile duct,simultaneously treating gallbladder,bile duct stones,and stenosis of the ampulla of Vater.The control group underwent traditional ERCP approach for sphincterotomy,stone extraction,and laparoscopic cholecystectomy.Perioperative variables were collected for both groups and the surgical outcomes were compared.Results:Among the 120 patients,54 were male and 66 were female.There were no statistically significant differences between the two groups in terms of stone extraction success rate,intraoperative blood loss,postoperative 24-h total bilirubin,direct bilirubin,transaminases,white blood cell count,jaundice relief time,or incidence rates of bile leakage,retroperitoneal bleeding/infection,and severe pancreatitis(all P>0.05).The observation group had significantly shorter average operative time and postoperative hospital stay compared to the control group(98.67 min vs.110.8 min,P<0.05;3.81 d vs.5.61 d,P<0.05).Additionally,the observation group had a significantly lower incidence of postoperative hyperamylasemia and/or hyperlipasemia and mild pancreatitis(1.67%vs.25.00%,P<0.001;0 vs.10%,P=0.027).Conclusion:The novel transabdominal approach is superior to the ERCP approach in terms of reducing surgery time and hospitalization time,and it carries a lower risk of postoperative mild pancreatitis and hyperamylasemia and/or hyperlipasemia.The stone extraction success rate is comparable to that of ERCP,making it a viable alternative treatment option.

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.
Clone Cells ; Mutagenesis, Site-Directed ; Mutation ; Plasmids/genetics* ; Polymerase Chain Reaction/methods*

OBJECTIVETo summarize the clinical features, biochemical change and genetic mutations of a neonate with congenital bile acid synthesis disorder type 2.

METHODSClinical features, blood biochemical index, gene analysis and treatment of the patient were reviewed.

RESULTSThe patient presented with the symptoms of jaundice 3 days after birth but without skin itching. Pale stool was noted. Subsequently, he presented with hepatomegaly, blood coagulation disorders, left cochlear nerve damage, liver cirrhosis and remarkable growth retardation. Serum biochemistries showed that bilirubin and transaminase were elevated, while γ -GT and total bile acid was normal. Abdominal ultrasonography indicated decline of gallbladder contraction. Cholangiography showed normal extra- and intrahepatic bile ducts and patent biliary tract. Liver biopsy showed intrahepatic cholestasis. Gene testing has identified a homozygous mutation in AKR1D1 gene.

CONCLUSIONCongenital bile acid synthesis disorder should be suspected when a neonate has presented with jaundice, elevated bilirubin and transaminase, normal or reduced TBA and γ -GT. Genetic testing and urine mass spectrometry analysis can diagnose congenital bile acid synthesis disorder. Early therapy is crucial to patients with congenital bile acid synthesis disorder.

Objective To investigate the clinical efficacy of primary closure in laparoscopic common bile duct exploration (LCBDE).Methods The retrospective cross-sectional study was conducted.The clinicopathological data of 2 429 patients who underwent primary closure in LCBDE in the Second Hospital of Chengdu City from March 1992 to December 2017 were collected.Patients underwent laparoscopic cholecystectomy (LC) + stone extraction using LCBDE or extracorporeal shock wave lithotripsy (ESWL),laparoscopic endoscopic sphincteropapillotomy (LEST) was performed selectively,then underwent laparoscopic endoscopic nasobiliary drainage (LENBD) or laparoscopic transabdominal antegrade-guide common bile duct stent implantation,finally underwent primary closure of common bile duct.Observation indicators:(1) surgical situations;(2) postoperative recovery;(3) postoperative short-term complications;(4) follow-up situation.Follow-up using outpatient examination and telephone interview was performed to observe long-term surgical complications once every 3 months up to 1 year postoperatively.Measurement data with normal distribution were represented as (x) ± s.Measurement data with skewed distribution were described as M (range).Results (1) Surgical situations:of 2 429 patients,2 251 underwent successful stone extraction using LCBDE,relief of the obstruction and primary closure of common bile duct,with depletion of stones;15 underwent stone extraction using conversion to open surgery,with depletion of stones;163 had residual stones or surgery-related complications.Of 2 429 patients,1 144,898,223,110 and 54 were respectively detected in grade N or 0,1,2,3 and 4 of laparoscopic distal of the common bile duct smooth classification (LDSC);599 underwent LEST,367 underwent LENBD,207 indwelled urinary catheter through cystic duct stump,125 underwent laparoscopic transabdominal antegrade-guide common bile duct stent implantation and 1 131 underwent primary closure in LCBDE only.Number of removing the stones,diameter of common bile duct,volume of intraoperative blood loss and operation time was (2.5±0.2)per case,(0.7±0.4)cm,(22.4±2.6)mL and (100±12) minutes,respectively.(2) Postoperative recovery:time of postoperative gastrointestinal function recovery,duration of hospital stay and treatment expenses were respectively (2.5±0.5) days,(7.3± 1.2) days and (2.7±0.3) × 104 yuan.Of 2 429 patients,367 removed nasobiliary catheter at 3-7 days postoperatively,207 removed urinary catheter at 3-6 weeks postoperatively,and 125 removed common bile duct stent through duodenoscope port at 1-4 months postoperatively.(3) Postoperative short-term complications:incidence of postoperative short-term complications in 2 429 patients was 6.711％ (163/2 429).① Ninety-four patients with bile leakage were cured after drainage and symptomatic and supportive treatment.② Of 29 patients with residual stones:25 with residual stones of common bile duct were cured by stone extraction using endoscopic sphincterotomy of duodenal papilla,and 4 with residual stones of intrahepatic bile duct didn't receive treatment.③ One patient died at 15 days after surgery for pancreatic cancer.④ Of 39 with postoperative other complications:2 with postoperative hemorrhage were cured by laparoscopic reoperation;12,19 and 3 were respectively complicated with postoperative inflammatory stenosis of duodenal papilla induced to short-term obstructive jaundice,mild acute pancreatitis and stress ulcer bleeding of upper digestive tract,and they were improved by endoscopy or non-operation treatment;2 with stenosis of bile duct didn't receive treatment;1 had a miss ligation at bending section of front zone of nasobiliary catheter,and ligation was removed by endoscopic retrograde cannulation of the pancreatic at 19 days postoperatively.(4) Follow-up situation:of 2 429 patients,1 749 were followed up for 3-12 months,with a median time of 6 months.During the follow-up,of 1 749 patients,2 had mild stenosis in the primary closure area of common bile duct incision and 1 had stenosis of duodenal papilla,they were not treated,and other patients didn't have related complications.Conclusion Controlling strictly the operative indication,primary closure of common bile duct in LCBDE is safe and feasible,with satisfactory clinical outcomes.

Objective To analyze our experience in primary closure of common bile duct after laparoscopic bile duct exploration.Methods From June 1992 to March 2018,2 740 patients underwent primary closure of common bile duct after laparoscopic common bile duct exploration in the Second People's Hospital of Chengdu.Results The operations were successfully carried out in 2 534 (92.4％) out of 2 740 patients,of whom 15 patients (0.6％) were converted to open common bile duct exploration.Bile leakage occurred in 113 patients (4.1％).Residual stones were found in 29 patients (1.1％).One patient (0.1％) who had a pancreatic carcinoma died on postoperation day 15.48 patients (1.8％) developed other complications.The total postoperative complication rate was 7.0％ (191/2 740).Conclusion In suitable patients,laparoscopic bile duct exploration with primary closure was feasible,safe and effective.

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

Objective To observe the curative effect of lumbar sympathetic nerve injury combined with systemic and local medication in intractable lower limb ischemic pain.Methods21 patients with intractable lower limb ischemic pain accepted lumbar sympathectomy,at the same time foot topical Votalinemulgel used and Combined with anticonvulsants and opioids according to circumstances,the visual analogue scale of resting pain(visual analogue scale,VAS),the Local skin temperaturebefore and after treatment were observed and evaluated.ResultsAfter Lumbar sympathetic nerve block and topical Votalinemulgel,21 patients had pain relief.After treatment, 2 patients had poor effect,the remaining 19 patients had pain relief to VAS<3, 4 patients did not need systemic analgesic drugs,the rest of the patients need systemic analgesic drugs.ConclusionIt is necessary to adopt multimodal comprehensive treatment for intractable lower limb ischemic pain.

To explore the operation methods and indications of the duodenoscopic papillotomy (IEST) with endoscopic nasobiliary drainage (IENBD) for the treatment of duodenal papilla stenosis during the course of common bile duct operation.The clinical data of 219 cases of cholecystolithiasis with choledocholith and the stenosis of papillary underwent endoscopic sphincterotomy (IEST) plus endoscopic nasobiliary drainage (IENBD) in the Second People's Hospital of Chengdu were retrospectively analyzed.It was successful in 198 cases who had the gallbladder and common bile duct stones removed,and endoscopic papillary dissection was performed and the nasobiliary tube was successfully inserted.Nasobiliary drainage was successful in 186 cases (93.9％) of 198 cases.No liquid outflow was observed in nasobiliary drainage in 7 cases (3.5％).Nasal bile duct slipped early in 5 case (2.5％).Primary closure of bile duct incision was completed in 198 cases.It failed in 4 cases (2.0％) who had the bile leakage with primary closure of duct incision.Mild pancreatitis after operation occurred in 3 cases (1.5％).Nose bile duct ligation was performed in 1 case (0.5％).The overall postoperative complication rate was 4.0％ (8/198).IEST + IENBD in open laparotomy was successful in 21 cases.No perforation of intestine and bile duct,bleeding,severe pancreatitis and other complications and death were detected postoperatively in two groups.During the course of laparoscopy and open laparotomy,IEST + IENBD in treating cholecystolithiasis with choledocholith and the stenosis of papillary and primary closure of duct incision after the endoscopic nasobiliary drainage is safe and effective.

Objective To evaluate the clinical efficacy of modified Chen's biliojejunostomy technique in treating hilar cholangiocarcinoma.Methods The clinical data of the patients with hilar cholangiocarcinoma from January 2011 to June 2014 in the PLA general hospital and its first affiliated hospital were retrospective ly studied,and 17 of them underwent modified biliojejunostomy.There were 10 male and 7 female patients with a mean of 65 years old (range 34 ～82).Cases diagnosed as Bismuth-Corlette Type Ⅱ,Ⅲ a,Ⅲ b and Ⅳ were 2,6,4,and 5,respectively.Results Liver segment Ⅳ were resected from 2 patients,segment Ⅳ + Ⅴ from 7 patients and segment Ⅳ + Ⅴ + Ⅰ from 8 patients.No death was observed during the study period.One patient had mild bile leakage,2 patients had cholangititis,and another patient had biliary intestinal anastomotic bleeding.Three patients received resection and reconstruction of the portal vein.Three right hepatic arteries and 1 anterior branch of right hepatic artery were resected in combination with the tumor because of invasion.All the complications were alleviated under conservative treatment.Twelve cases had been followed up for a median time of 16 months (range 3 ～ 24).Two cases had cholangititis intermittently.One case underwent radiotherapy because of local recurrence in 2 years after the surgery.MRCP and relevant enzymes were within the normal range.Conclusion Modified Chen's biliojejunostomy is a simple,effective and safe method,which can be widely used when there are multiple biliary intestinal anastomoses.