Objective To construct recombinant human butyrylcholinesterase(rhBChE)knock-in goat fetal fibroblast cell lines(GFFs)by using clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated homology-directed repair mechanism for subsequent production of goat expressing rhBChE.Methods The efficient sgRNA sites targeting goat[3-casein(CSN2)gene were designed and screened,and the targeting efficiency of the sgRNA in goat mammary epithelial cells(GMECs)was confirmed by electro-transfection,flow sorting,and sequencing of PCR products.The red fluorescent reporter gene homology repair vector(P2A-mCherry)targeting the sgRNA was constructed,and then the integration and expression efficiency was detected by flow cytometry.The rhBChE homology repair vector(P2A-rhBChE)targeting the sgRNA of CSN2 gene was constructed in GFFs,the rhBChE positive cell clones were obtained via electro-transfection and flow sorting,and the rhBChE knock-in cell lines was identified by sequencing of PCR products.Results The sgRNA4 was identified as an efficient target of goat CSN2 gene,which could be also used for targeted integration of other genes.Three rhBChE knock-in cell lines were successfully constructed.Conclusion The rhBChE knock-in GFFs targeting goat CSN2 gene lays the foundation for the production of mammary bioreactors expressing rhBChE.

Objective:To evaluate the inflammatory factors effects of early endoscopic therapy for elderly patients with acute biliary pancreatitis (ABP) and its clinical efficacys.Methods:206 elderly patients with ABP admitted from Jan. 2010 to Dec. 2019 were divided into observation group (102 cases) and the control group (104 cases) according to treatment method. The observation group received endoscopic retrograde cholangiopancreatography (ERCP) , endoscopic sphincterotomy (EST) and endoscopic naso-biliary drainage (ENBD) , while the control group received conventional treatment. Clinical symptoms, changes of inflammatory factors, complications and prognosis were observed in each group.Results:CRP, SAA, IL-6, IL-8 and TNF-a after treatment were significantly lowered than those before treatment ( P<0.05) . In addition, the levels of CRP, SAA, IL-6, IL-8 and TNF-a in the observation group were significantly lower than those in the control group ( P<0.05) . The time to abdominal pain extinction, time to fever cessation, hospital stay in observation group were (3.92±1.54) , (3.63±1.41) , and (14.35±2.46) d, significantly less than those in the control group [ (5.81±1.72) , (5.45±2.13) , (19.37±3.12) d, P<0.05]. APACHE Ⅱ score of the observation group was (10.02±2.67) point after treatment, significantly lower than that in the control group [ (12.35±3.62) point, t=4.42, P<0.05]. The incidence of complications in the observation group was 10.78% after treatment, significantly lower than that in the control group [ (24.03%) , χ2=6.27, P< 0.05]. The mortality in the observation group was 1.96%, lower than 4.81% in the control group, with no statistical significance. Conclusion:Early endoscopic therapy is safe and highly effective for elderly patients with ABP, with the advantages of shorter hospital stay, quicker subsided inflammation, and lower incidence of complications.

Objective To investigate the relationship between the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,to observe the inhibitory effect of 5-Aza-CdR on the growth of gastric cancer cells,to observe the effect of demethylation on the expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells,and to explore the molecular mechanism of gastric cancer.Methods Real-time PCR was used to detect the expression of ASPP2 mRNA in two gastric cancer cells and normal gastric epithelial cells.BSP was used to detect the methylation of ASPP2 gene in two gastric cancer cells and normal gastric epithelial cells.CCK-8 was used to detect the growth inhibition rate of gastric cancer cells treated with 5-Aza-CdR of different concentrations,then they were used to detect expression of ASPP2 mRNA and the methylation of ASPP2 gene in gastric cancer cells again after the demethylation.Results ① The expression of ASPP2 mRNA in MKN-45 cells was significantly lower than that in GES-1 cells(P＜0.01).The expression of ASPP2 mRNA in MGC-803 cells was significantly lower than that in GES-1 cells (P＜0.01).There was no significant difference in MGC-803 cells and MKN-45 cells(P＞0.05).② The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in GES-1 cells (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that in GES-1 cells (P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly higher than that in MGC-803 cells (P＜0.01).③ At the same time,the growth inhibition rate of each 5-Aza-CdR concentration group increased as the drug concentration depended.4.The expression of ASPP2 mRNA in MKN-45 cells was significantly higher than that before treatment (P＜0.01),the expression of ASPP2 mRNA in MGC-803 cells was not significantly different from that before treatment(P＞0.05).The methylation rate of ASPP2 in MKN-45 cells was significantly lower than that before treatment (P＜0.01).The methylation rate of ASPP2 in MGC-803 cells was not significantly different from that before treatment (P＞0.05).Conclusion ① Abnormal hypermethylation of ASPP2 gene in MKN-45 cells may be a molecular mechanism of decreased ASPP2 mRNA expression.② 5-Aza-CdR can inhibit the growth of MKN-45 and MGC-803 cells,and it can enhance the expression of ASPP2 mRNA in MKN-45 cells.Reversal of methylation in the promoter region of ASPP2 gene is the possible mechanism.③ Abnormal hypermethylation of the promoter region of ASPP2 gene may lead to silencing of mRNA expression that may be associated with gastric cancer.

Objective To investigate the methods for prevention and treatment of unexpected hemorrhage during operation of biliary tract.Methods The clinical data of 28 patients with intraoptrative hemorrhage from Jan.2006 to Dec.2016 in our hospital were retrospectively analyzed.Results The cause of introoperative hemorrhage included:iatrogenic biliary injuries in 14 cases (50％),cholelithasis complicated biliary infection in 9 cases (32.1％),bleeding in gall bladder bed in 3 patients (10.7％),and other causes in 2 cases (7.1％).The intraoperative blood loss was 600 to 3000 ml.According to the specific location of bleeding,the amount of blood loss,the cause of bleeding and other conditions,the individual treatment was given.26 were cured and 2 died of hemorrhagic shock and MODS.Conclusions The major causes of hemorrhage during operation of biliary tract comprise iatrogenic biliary injuries,biliary infection,and biliary lithiasis.The reasonable treatment of intraoperative hemorrhage should be based on the concrete bleeding etiologies.

Objective To investigate the clinical characteristics and perioperative management of 1aparoscopic cholecystectomy(LC) in the elderly with gallbladder diseases. Methods From January 2006 to December 2016,the clinical data of two hundred and sixteen elderly patients with LC in the Sixth Affiliated People's Hospital of Jinshan Branch were analysed retrospectively. Results The operations of LC were successfully done in 206 patients ( 95. 8%) and 9 cases ( 4. 2%, 9/216 ) underwent conversion to open cholecystectomy for various reasons. Postoperative complications occurred in 22 cases (10. 2%,22/216). 2 cases died within 1 weeks after the operation,and the remaining patients recovered from the hospital. Conclusion Adequate perioperative management,strict surgical indications and operative skills are the necessary conditions for the successful implementation of LC in the elderly.

Objective To explore the etiological factors,clinical characteristic and diagnosis of nonacute biliary pancreatitis (NABP) and acute biliary pancreatitis (ABP).Methods The Clinical data of 152 patients with NABP and 206 patients with ABP from January 2004 to December 2014 in the Hepatobiliary Surgery Department of Jinshan Branch of the Sixth People's Hospital of Shanghai were analyzed retrospectively.Results There were no statistically significant differences in terms of the Ranson score,blood amylase and C reactive protein (CRP) between two groups (P ＞ 0.05).The incidences rate of hepatic insufficiency,renal insufficiency and encephalopathy were 35.5％ (54/152),25.6％ (39/152) and 8.5％ (13/152) in the NABP group,and 25.7％(53/206),12.1％(25/206) and 3.3％(7/206) in the ABP group,with significant difference between the two groups (x2 =4.01,10.89,4.41;P ＜ 0.05).Conclusion The key to reduce the complications and improve the cure rate is to make clear the etiology of NABP and ABP and to take active and effective treatment for the cause of the disease.

Objective To evaluate the effect of ultrasound-guided interscalene brachial plexus block and nerve stimulator on operation around shoulder joints. Methods Sixty cases of patients scheduled for operation around shouler joints were randomly divided into group A and B , with 30 cases in each group. Patients in group A underwent interscalend brachial plexus block guided by nerve stimulator with 0.5% ropivacaine and those in group B interscalend brachial plexus block guided by ultrasound and nerve stimulator with 0.5% ropivacaine 30 mL. The number of puncture, the effect of anesthesia and complication of anesthesia in both groups were recorded. Results The number of puncture of group B was less than that of group A (P﹤0.05) while the effect of anesthesia and the duration of anesthesia of group B were similar to those of group A (P﹥0.05). There was no serious complications in two groups. Conclusion With definite anesthesia effect and less pain , ultrasound-guided interscalene brachial plexus block and nerve stimulator is simple and safe for operation around shouler joints.

Objective To summary clinical experience of laparoscopic treatment of cholecystitis with cholecystolithiasis complicated,in order to provide reference for clinical difficult laparosco-pic resection of gallbladder.Methods Reviewing the operation treatment of laparoscopic cholecystectomy on 94 complex cases of resection during January 2008 to December 2014.Results The 94 patients included 17 cases with severe adhesion around gallbladder,9 cases with gallbladder atrophy,39 cases with gallbladder ampulla and cystic duct stone incarceration,11 cases with acute gangrenous cholecystitis with gallbladder stones,14 cases with gallbladder triangle anatomy is not clearing and 19 cases with gallbladder stones with schistosomial cirrhosis or liver volume variation.Some cases were with the two or more kinds gallbladder stone.There were 91 cases with successful implementation of the laparoscopic cholecystectomy,4 of 91 cases were treated with subtotal cholecystectomy,and 3 cases were converted to open cholecystectomy.There were no cases with serious complications or even deaths.Conclusion Regarding of the complex gallbladder stones appear different situation in operation,we can complete the operation by using different operation method and treatment method in laparoscopic.

Objective To investigate the correlation and expression of PTEN, matrix metalloproteinase-9 (MMP-9),and vascular endothelial growth factor-C(VEGF-C)in colorectal cancer.Methods The expression of PTEN, MMP-9 and VEGF-C in 102 cases of colorectal cancer tissues and 30 cases of normal colorectal tissues was detected using immunohistochemical SP method.Their correlations with cliniopathologic features were analyzed. Results The positive expression of PTEN in colorectal cancer was significantly lower than that in the controls (54.9%vs 100%, P<0.01).Both MMP-9 and VEGF-C were over expressed compared with the controls(83.3%vs 21.7%and 69.6%vs 16.7%respectively, P<0.01).The expression of PTEN was negatively correlated with that of MMP-9 and VEGF-C, and the expression of MMP-9 was positively correlated with that of VEGF-C ( P<0.05).The expressions of the three proteins in colorectal cancer tissues were not associated with age, gender, or tumor size.There were significant associations between the expression of these proteins and lymph node metastasis, distant metastasis, differentiation, and TNM staging(P<0.05).The 1, 3 and 5-year survival rates of the patients with positive PTEN were significantly higher than those of the patients with negative PTEN(P<0.05).The 1, 3 and 5-year survival rates of the patients with positive MMP-9 and VEGF-C were significantly lower than those of the patients with negative MMP-9 and VEGF-C( P<0.05) .Conclusions The decrease in the expression of PTEN and increase in MMP-9 and VEGF-C are closely correlated to the development of colorectal cancer.Their expressions may be favorable markers and useful prognostic indicators in the patients with colorectal cancer.

Objective To investigate the relationship between peri-menopausal women blood lipid level and the gallstone disease in order to provide theoretical basis for the prevention of gallbladder stone.Methods Seventy-two patients with gallbladder calculus from Department of Hepatobiliary Surgery Jinshan District Central Hospital of Shanghai and 52 healthy women coming for physical examination as the control group.Participants were divided into five age groups (20-29,30-39,40-49,50-59 and 60-69 years old) and two age stages (young period (30-40 years old) and peri-menopausal period (45-55 years old)),total cholesterol (TC),triglyceride (TG),high density lipoprotein (HDL-C),low-density lipoproteins (LDL-C) and Body Mass Index (BMI) were determined and compared between the stone group and the control group at different age stages.Results In comparison of the menopausal transition period,the stone group had significantly lower blood TC,HDL-C and LDL-C levels than the control group (TC:(4.58 ± 0.27) mmol/L vs.(5.15 ± 0.26) mmol/L,t=3.112,P ＜0.01 ;HDL-C:(1.17 ±0.11) mmol/L vs.(1.40 ±0.08) mmol/L,t =3.351,P ＜0.01 ;LDL-C:(2.71 ± 0.30) mmol/L vs.(3.15 ± 0.26) mmol/L,t =2.437,P ＜ 0.05).For the young period,there were no significant difference on blood lipid level and BMI was observed between the stone group and the control group (P ＞ 0.05).The fat HDL-C level in blood was significantly lower in the stone group in the menopausal transition period than in the young period ((1.17 ± 0.11) mmol/L vs.(1.33 ± 0.07) mmol/L,t =2.455,P ＜ 0.05).The control group had significantly higher peri-menopausal TC,TG,LDL-C levels in blood and BMI than those in the control group young period (P ＜ 0.05) ; And HDL-C level,no significant difference (TC:(5.15 ± 0.26)mmol/L vs.(4.47 ± 0.34) mmol/L,t =3.175,P ＜ 0.01 ; TG:(1.88 ± 0.39) mmol/L vs.(1.10 ± 0.24)mmol/L,t=3.066,P＜0.01;LDL-C:(3.15 ±0.26) mmol/L vs.(2.71 ±0.31) mmol/L,t =2.261,P＜0.05;BMI:(24.75±0.99) kg/m2vs.(21.73±1.11) kg/m2,t=4.217,P＜0.01).Conclnsion For perimenopausal (45-55 years old) females,decline of serum TC,HDL-C and LDL-C levels may be related to the formation of cholesterol stone.Lower HDL-C level is closely associated with formation of cholesterol stone,indicating that it may be one of the measures to prevent gallbladder stone by adjusting blood fat in perimenopausal women.