The human proinsulin gene was introduced a cleanage site for furin with two point mutation by polymerase chain reaction. The mutated gene was subcloned to the retroviral vector, and transferred into HepG2 hepatocarcinoma cells. Mature insulin can be expressed and secreted into supernatant by transferred HepG2 cells. The site mutation method is concise and with no misproduction.

AIM:To explore the recombined human proinsulin gene containing glucose reaction element(GLRE) expression in transfected CBRH7919 cells.METHODS: The packaged retrovirus encoding genetically modified human proinsulin PLXSN-(GLRE)3-BP-1MpINS3 and PLXSN-(GLRE)3-BP-1MpINS2 were transfected into CBRH7919 cells.Insulin values in cells after transient and steady expression screened by G418 at different glucose levels were detected.Chromosome DNA was isolated from transfected and untransfected cells and polymerase chain reaction(PCR) was performed.PCR products were analyzed by electrophoresis.RESULTS: 38 h after transfection,at the glucose levels of 0-25 mmol/L,the levels of insulin produced by cells including PLXSN-(GLRE)3-BP-1MpINS3 were(3.57?0.21)U/L,(5.30?0.20)U/L,(16.27?0.87)U/L,(23.23?1.12)U/L,respectively(P

Objective to investigate the feasibility of laparoscopic radical resection for hilar cholangiocarcinoma.Methods From June 2016 to June 2018,30 patients with hilar cholangiocarcinoma (HCC) underwent total laparoscopic radical resection.Results Surgery was successfully performed in all cases without conversion,15 cases underwent hilar bile duct resection,and hilar lymph node dissection,14 cases underwent left hemi-hepatectomy and caudate lobectomy,and 1 case underwent extrahepatic bile duct resection.The operation time was 258-336 min,(286.8 ± 18) min,intraoperative bleeding was 60-210 ml (139.7 ±38.0) ml.Postoperative bile fistula occurred in 5 cases,healed conservatively after 5 days,stress ulcer in 1 case,and intestinal anastomotic bleeding in 1 case,were all cured by themselves.Postoperative hospitalization was 7-12 days (mean 8.9 d).After 6-12 months follow-up,3 cases died of recurrent and metastatic cancer after 6 months of operation,27 cases were alive.Conclusion Total laparoscopic radical resection of hilar cholangiocarcinoma is safe and feasible.

Objective To investigate the application of purse string suture and the end-to-side invagination pancreaticojejunostomy in pancreaticoduodenectomy (PD).Methods Clinical data of 175 cases who were admitted in Cangzhou Central Hospital and underwent pancreaticoduodenectomy because of malignant tumor from December 2012 to December 2016 were retrospectively analyzed.According to the texture of pancreas in the operation,the patients were divided into 2 groups.Purse string suture and the end-to-side invagination pancreaticojejunostomy was performed in study group.Duct-to-mucosa pancreaticojejunostomy was conducted in control group.The operation time,the time of pancreaticojejunostomy,the amount of intraoperative bleeding,the incidence of postoperative pancreatic fistula,postoperative recovery time of gastrointestinal function,the average length of hospital stay and the like were comparative analyzed between the two groups.Results There was no significant difference on gender,age,primary diagnosis,preoperative total serum bilirubin,alanine aminotransferase and serum albumin levels between the two groups.The pancreatic texture of the study group was softer than that of the control group,the pancreatic duct diameter in the study group was significantly smaller than that in the control group [(2.0 ± 0.9) mm vs (3.4 ± 1.3) mm],the time of pancreaticojejunostomy in the study group was significantly shorter than that in the control group [(13 ± 4) min vs (17-± 7) min],the incidence of postoperative pancreatic fistula in the study group was significantly lower than that in the control group(5.88％ vs 15.56％),and the differences were statistically significant (all P ＜0.05).There was no significant difference on mean operative time,mean blood loss,postoperative gastrointestinal recovery time and average hospital stay between the two groups.1 cases with postoperative abdominal bleeding occurred in the study group,and the bleeding was successfully stopped by secondary surgery.Conclusions Purse string suture and the end-to-side invagination pancreaticojejunostomy was simple,safe and effective,which had a advantage of reducing the time of pancreaticojejunostomy and the incidence of postoperative pancreatic fistula.

Objective To investigate the clinical features of Menetrier disease and the related treatment progresses.Methods The clinical data of 7 patients with Menetrier disease who were admitted to Cangzhou Central Hospital from Jan 1997 to Mar 2018 were retrospectively analyzed.Results There were 3 males and 4 females.The main clinical manifestations were abdominal pain (n =5),loss of appetite (n =4),nausea and vomiting (n =2),weight loss (n =4),edema (n =3),hypoalbuminemia (n =6) and anemia (n =6).All patients had typical imaging findings.Two of the patients underwent non-surgical treatmen with temporary symptom reliefs.Finally,6 patients received surgical treatment including Subtotal gastrectomy in 1 patient,total gastrectomy in 5 patients.All the 6 cases underwent surgery successfully with no serious complications.5 cases were followed-up data until April 2018.All were in good condition,Hemoglobin and albumin levels were within the normal range.Conclusion According to the clinical manifestations and the related auxiliary examinations,this disease is easy to diagnose.Surgical treatment is the only way to cure Menetrier disease.

Objective To investigate the surgical methods and techniques of total laparoscopic resection of primary extraperitoneal pelvic benign neoplasms.Methods The clinical data of 32 patients undergoing laparoscopic resection for primary extraperitoneal pelvic benign neoplasms in Cangzhou Central Hospital from March 2011 to June 2016 were retrospectively analyzed.Results All cases were successfully operated under total laparoscopic resection.The operation time was 95-240 min,with an average of(152 ± 37) min,the amount of blood loss was 30-100 ml,the average was (56 ± 14) ml.Postoperative pathological examination confirmed mature teratoma in 14 cases,lipoma in 6 cases,epidermoid cyst in 7 cases,dermoid cyst in 5 cases.Patients were discharged from hospital 5 to 8 days after surgery,with an average of (6.5 ± 0.7) days.All patients were followed up for 2 to 36 months with no tumor recurrence.Conclusion Laparoscopic surgery is a safe and effective method for primary extraperitoneal pelvic benign neoplasms.

Objective To compare the short-term efficacy of laparoscopic pancreaticoduodenectomy (LPD) with open pancreatoduodenectomy (OPD).Methods The clinical data of 122 LPD patients and 167 OPD patients at our department from 2015 to 2017 were analyzed retrospectively.Results There was no significant difference in demographic,ASA classification,preoperative liver function between the two groups (P ＞0.05).The operative time was (405 ± 109) min vs.(312 ±57) min,liquid diet intake time postoperatively was (5.4 ± 2.6) d vs.(7.9 ± 3.3) d,postoperative hospital stay was (10.4 ± 6.1) d vs.(14.5 ± 8.6) d,with all the difference statistically significant (P ＜ 0.05).Intraoperative blood loss was (290 ± 102) ml vs.(322 ± 75) ml,the number of lymph node dissection in malignant tumor cases was (14.7 ± 5.6) vs.(13.9 ± 7.3),R0 resection rate in malignant tumor cases was 97.9％ (96/98)vs.96.6％ (143/148),the incidence of postoperative complications was 39.3％ (48/122) vs.35.3％(59/167) (all P ＞0.05).All patients were followed up with a median time m group LPD and group OPD was 9 months and 11 months respectively,and the tumor free survival rate was 93.4％ (114/122) and 90.4％ (151/167) at 6 months (P ＞ 0.05).Conclusion LPD has the same safety and radical effect as the traditional OPD surgery,while of less trauma and quicker postoperative recovery.

Objective To assess the value of endovascular management in treating long segment occlusion of femoral-popliteal artery by using retrograde puncture path.Methods The clinical data of a total of 46 patients with long segment occlusion of femoral-popliteal artery,who were admitted to authors' hospital during the period from June 2009 to January 2017 to receive endovascular treatment,were retrospectively analyzed.The patients included 27 males and 19 females,with a mean age of (69±8) years old.Grade Ⅲ of Rutherford classification was seen in 32 patients,grade ⅣV in 8 patients,and grade V in 6 patients.On CT angiography,the mean length of occluded lesion was (17±5) cm.Results In 46 patients of this series,the technical success rate was 100％.The average time used for making the small skin incision and puncturing the artery was (5.2±2.3) min.A 4-F artery sheath was employed in 7 patients,endovascular management with no use of artery sheath was carried out in 39 patients.The guide wire smoothly passed through the occluded segment of femoral-popliteal artery and entered the proximal true lumen in 41 patients.Subintimal arterial flossing with antegrade-retregrade intervention (SAFARI) technique was adopted to establish guide wire working track in 5 patients.On the third postoperative day,the ankle brachial index (ABI) was (0.71±0.12),which was significantly different from preoperative (0.33±0.11),and the difference was statistically significant (t=12.483,P＜0.001).The overall incidence of operation-related complications was 23.9％ (11/46),the incidence of distal arterial puncture-related complications was 4.3％ (2/46).No death occurred in perioperative period.Forty-three patients (93.5％) were followed up for 3-24 months.CTA reexamination performed at 3,6 and 12 months after treatment showed that the primary patency rate of the stent were 91.3％ (4/46),78.3％ (10/46) and 58.7％(19/46) respectively,and the one-year limb salvage rate was 95.7％ (44/46).Conclusion For the treatment of long segment occlusion of femoral-popliteal artery,endovascular management by using retrograde puncture path is technically simple and clinically safe and effective,this technique has certain clinical application value.

Objective To study and prepare the monoclonal antibody library against human FXYD6 functional region,to screen the hybridoma cell lines secreting the monoclonal antibodies against intracellular or extracellular region of human FXYD6,and to identify the biological function of monoclonal antibody against extracellular domain.Methods FXYD6 functional region recombinant protein which did not contain the transmembrane region was prokaryotically expressed,purified,and FXYD6 recombinant protein was used to immunize BALB/c mice.Then splenocytes after immunization were fused with myeloma cells SP2/0.After several rounds of screening and cloning,the hybridomas which secreted the antibodies against the extracellular domain or the intracellular domain of human FXYD6 were established.The antibody specificity and subtype were identified with indirect ELISA,western blot and immunohistochemistry.The monoclonal antibodies against the extracellular domain which recognized the native conformation were screened with flow cytometry.The antibody against extracellular region was prepared with the ascites revulsion method and purified.The affinity constants were measured with indirect ELISA.The function of extracellular monoclonal antibody was detected by HepG2 cell line with high expression of FXYD6.Results The hybridoma cell library which secreted the monoclonal antibody against extracellular domain or the intracellular domain of human FXYD6 was successfully obtained,and extracellular region monoclonal antibodies with the functional blocking were prepared.Conclusion The prepared anti-human FXYD6 extracellular monoclonal antibodies could inhibit HepG2 cell proliferation.