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 clinical effect of hyperbaric oxygen (HBO) combined with platelet-rich plasma (PRP) in the reconstruction of anterior cruciate ligament injury (ACLI).Methods:A total of 100 ACLI patients who were treated in the Department of Joint Trauma Surgery of Qingdao Jiaozhou Central Hospital from January 2018 to May 2019 were randomly divided into two groups according to the random number table: observation group and control group. Each group had 50 cases. The patients in the control group were only treated with PRP, while the patients in the observation group were treated with hyperbaric oxygen combined with PRP. Then the curative effect of the two groups, Lysholm, Tegner, and IDKC scores of knee joint, serum insulin-like growth factor-1 (IGF-1), bone gla protein (BGP), and matrix metalloproteinase-1 (MMP-1) were observed after 3 consecutive courses of treatment.Results:After 3 consecutive courses of treatment, the total effective rate of the observation group (90.0%) was significantly higher than that of the control group (68.0%) ( P<0.05); the difference was statistically significant. The scores of Lysholm, Tegner, and IDKC of the knee joint in the observation group and the control group were significantly higher than those before treatment, and the scores of Lysholm, Tegner, and IDKC of the knee joint in the observation group were significantly higher than those in the control group ( P<0.05). The contents of IGF-1, BGP, and MMP-1 in the two groups were significantly lower than those before treatment, and those contents of observation group were significantly lower than those of the control group ( P<0.05). Conclusion:HBO combined with platelet-rich plasma can effectively improve the therapeutic effect of ACLI reconstruction, improve the serum contents of IGF-1, BGP, and MMP-1. This treatment, therefore, is worthy of popularization and application in clinic.

Objective:To investigate the clinical effect of hyperbaric oxygen (HBO) combined with platelet-rich plasma (PRP) in the reconstruction of anterior cruciate ligament injury (ACLI).Methods:A total of 100 ACLI patients who were treated in the Department of Joint Trauma Surgery of Qingdao Jiaozhou Central Hospital from January 2018 to May 2019 were randomly divided into two groups according to the random number table: observation group and control group. Each group had 50 cases. The patients in the control group were only treated with PRP, while the patients in the observation group were treated with hyperbaric oxygen combined with PRP. Then the curative effect of the two groups, Lysholm, Tegner, and IDKC scores of knee joint, serum insulin-like growth factor-1 (IGF-1), bone gla protein (BGP), and matrix metalloproteinase-1 (MMP-1) were observed after 3 consecutive courses of treatment.Results:After 3 consecutive courses of treatment, the total effective rate of the observation group (90.0%) was significantly higher than that of the control group (68.0%) ( P<0.05); the difference was statistically significant. The scores of Lysholm, Tegner, and IDKC of the knee joint in the observation group and the control group were significantly higher than those before treatment, and the scores of Lysholm, Tegner, and IDKC of the knee joint in the observation group were significantly higher than those in the control group ( P<0.05). The contents of IGF-1, BGP, and MMP-1 in the two groups were significantly lower than those before treatment, and those contents of observation group were significantly lower than those of the control group ( P<0.05). Conclusion:HBO combined with platelet-rich plasma can effectively improve the therapeutic effect of ACLI reconstruction, improve the serum contents of IGF-1, BGP, and MMP-1. This treatment, therefore, is worthy of popularization and application in clinic.

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 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 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 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 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.