Objective:To discuss the construction of a NOD-signal regulatory protein α(SIRPA)gene knock-in mouse model based on clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein(Cas)9 technology,and to provide the reference for the establishment of more advanced humanized mouse models.Methods:Based on CRISPR/Cas9 technology,the NOD-background SIRPA gene was knocked into the BALB/c mouse fertilized eggs,and homozygous mice with stable genotypes(SIRPA-KI mice)were obtained through expansion and breeding for experiments;PCR primers were designed,and mouse genotypes were identified by nucleic acid electrophoresis.The mice were divided into C57BL/6 group,BALB/c group,and SIRPA-KI group according to their strains,and 3 mice with similar ages were randomly selected from each group for experiments.Mature bone marrow-derived macrophages were co-incubated with human CD47-Fc fusion protein,stained with Streptavidin PE/Cy7,and the mean fluorescence intensity(MFI)of PE/Cy7 was detected by flow cytometry to compare the ability of SIRPA in the mice from various groups to bind human CD47 Fc.Each mouse was intravenously injected with 2×10? carboxyfluorescein diacetate,succinimidyl ester(CFSE)-labeled human red blood cells,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vivo phagocytosis assay.One BALB/c mouse and one SIRPA-KI mouse were randomly selected to induce mature bone marrow-derived macrophages,and the phagocytosis of human red blood cells by macrophages in various groups was detected by in vitro phagocytosis assay.Results:BLAB/c SIRPANOD/NOD homozygous mice were successfully obtained.The flow cytometry results showed that compared with C57BL/6 group,the MFI of the mice in BALB/c group was significantly increased(P<0.01);compared with C57BL/6 group and BALB/c group,the MFI of the mice in SIRPA-KI group was significantly increased(P<0.01).The in vivo phagocytosis assay results showed that the macrophages in C57BL/6 group exhibited the fastest overall clearance rate of human red blood cells;at 6 h of macrophage phagocytosis,compared with SIRPA-KI group,the residual percentage of the human red blood cells in C57BL/6 group was significantly decreased(P<0.01)and was closed to 0%;compared with SIRPA-KI group,the residual percentage of the human red blood cells in BALB/c group was significantly decreased(P<0.05).The in vitro phagocytosis assay results showed that the macrophages in BALB/c group significantly phagocytosed the human red blood cells,with a high phagocytic index of 30.7%;compared with BALB/c group,the phagocytic index of the macrophages in SIRPA-KI group was significantly decreased(P<0.01).Conclusion:The study successfully constructs a mouse model with enhanced affinity for human CD47 and significantly inhibited phagocytosis of human red blood cells by knocking the NOD-background SIRPA gene into the BALB/c strain using CRISPR/Cas9 technology,providing a superior human cell xenotransplantation efficiency.

BACKGROUND: Glutamine synthetase (GS) and arginase 1 (Arg1) are widely used pathological markers that discriminate hepatocellular carcinoma (HCC) from intrahepatic cholangiocarcinoma; however, their clinical significance in HCC remains unclear. METHODS: We retrospectively analyzed 431 HCC patients: 251 received hepatectomy alone, and the other 180 received sorafenib as adjuvant treatment after hepatectomy. Expression of GS and Arg1 in tumor specimens was evaluated using immunostaining. mRNA sequencing and immunostaining to detect progenitor markers (cytokeratin 19 [CK19] and epithelial cell adhesion molecule [EpCAM]) and mutant TP53 were also conducted. RESULTS: Up to 72.4% (312/431) of HCC tumors were GS positive (GS+). Of the patients receiving hepatectomy alone, GS negative (GS-) patients had significantly better overall survival (OS) and recurrence-free survival (RFS) than GS+ patients; negative expression of Arg1, which is exclusively expressed in GS- hepatocytes in the healthy liver, had a negative effect on prognosis. Of the patients with a high risk of recurrence who received additional sorafenib treatment, GS- patients tended to have better RFS than GS+ patients, regardless of the expression status of Arg1. GS+ HCC tumors exhibit many features of the established proliferation molecular stratification subtype, including poor differentiation, high alpha-fetoprotein levels, increased progenitor tumor cells, TP53 mutation, and upregulation of multiple tumor-related signaling pathways. CONCLUSIONS GS- HCC patients have a better prognosis and are more likely to benefit from sorafenib treatment after hepatectomy. Immunostaining of GS may provide a simple and applicable approach for HCC molecular stratification to predict prognosis and guide targeted therapy.
Humans ; Carcinoma, Hepatocellular/metabolism* ; Sorafenib/therapeutic use* ; Liver Neoplasms/metabolism* ; Glutamate-Ammonia Ligase/metabolism* ; Hepatectomy ; Retrospective Studies ; Prognosis ; Neoplasm Recurrence, Local/surgery*

Objective To compare salvage liver transplantation (SLT) with othotopic liver transplantation (OLT) in treatment of hepatocellular carcinoma.Methods A systematic literature search of PubMed,Embase,Cochrane Library,CBM,CNKI and Wanfang Med Online was performed from their dates of establishment to October 2017.The results were screened,data extracted and then analyzed with Stata 14.Results 23 studies with 4 161 patients were selected,including 579 patients in the SLT group and 3 582 patients in the OLT group.Compared with OLT,SLT was associated with a longer operative time (SMD =0.56,95％CI:0.29～0.83),higher intraoperative blood loss (SMD=1.56,95％CI:0.63～2.49),an increased risk of postoperative bleeding (OR =1.84,95％CI:1.08 ～ 3.14),a poorer overal survival rate (HR =1.29;95％CI:1.11～1.49) and disease free survival rate (HR=1.88;95％CI:1.26～2.81).The differences were all significant (all P＜0.05).The biliary complications (OR=1.25;95％CI:0.79～1.98),vascular complications (OR=1.41;95％CI:0.69～2.89),sepsis (OR=1.10;95％CI:0.60～ 1.99),acute rejection (OR =1.25;95％ CI:0.69 ～ 2.28) and perioperative mortality (OR =1.60;95 ％ CI:0.94 ～ 2.70) rates were not significantly different (all P＞0.05).Conclusions OLT is a better treatment strategy for patients with transplantable hepatocellular carcinoma (HCC) compared with SLT.However,severe organ limitation,and feasibility and safety of surgery make SLT a better option for patients with HCC recurrence after liver resection.

Objective To quantitatively evaluate the diagnostic value of blood oxygen leveldependent (BOLD) MRI in the diagnosis of different degrees of liver warm ischemia-reperfusion injury (WIRI) in rabbits and evaluate the intervention effect of liposomal prostaglandin E1 (Lipo-PGE1).Methods Seventy healthy adult New Zealand white rabbits were randomly divided into sham -operated group (A0),thermal ischemic groups (A1～A3) and intervention groups (A4～A6).All experimental rabbits were scanned by routine MR and BOLD MRI after 6-hour reperfusion.R2* images were calculated by two radiologists.The levels of alanine aminotransferase (ALT),asparate aminotransferase (AST) and lactate dehydrogenase (LDH) were examined.And liver pathological sectioning was performed.All data were processed by one-way,Spearman's correlation and receiver operating characteristic curve analyses.Results The intraclass correlation coefficient (ICC) was 0.805 of two measurements suggesting that the repeatability of the outcome was decent.R2* values among sham-operated,thermal ischemia and intervention groups were statistically significant (P＜0.05).R2 * values in sham-operated and ischemia groups were statistically significant (P＜0.05).As warm ischemia time elapsed,R2* value showed a rising trend.R2* values in sham-operated and intervention groups were statistically significant (P＜0.05).R2* values of sham-operated group at the same timepoint of thermal ischemia and intervention groups were statistically significant (P＜0.05).Under the same ischemic time,R2* values of intervention groups were smaller than those of thermal ischemia groups.With the prolongation of ischemia time,reduction of R2* values became more pronounced.However,it did not reach the level of A0 group.R2* values were significantly positively correlated with ALT,AST and LDH (r＞0.5,P＜0.05).ROC analysis indicated that R2* had an excellent diagnostic performance.Conclusions BOLD MRI may be applied for noninvasive assessment of liver ischemia-reperfusion injury in different degrees.Lipo-PGE1 alleviates ischemia -reperfusion injury and BOLD MRI can evaluate the relieving degree of Lipo-PGE1.

Objective To explore the predictive value of serum uric acid on new-onset cholelithiasis.Methods The retrospective cohort study was conducted.The data of 97 469 subjects who participated health examination at the Kailuan General Hospital Affiliated to the North China University of Science and Technology,Kailuan Linxi Hospital,Kailuan Zhaogezhuang Hospital,Kailuan Tangjiazhuang Hospital,Kailuan Fan'gezhuang Hospital,Kailuan Lyujiatuo Hospital,Kailuan Jinggezhuang Hospital,Kailuan Linnancang Hospital,Kailuan Qianjiaying Hospital,Kailuan Majiagou Hospital and Kailuan Branch Hospital from June 2006 to December 2015 were collected.Epidemiological investigation,anthropometric parameters and biochemical indicators were collected.All the subjects were allocated into 4 groups according to squartiles of serum uric acid:24 140 with serum uric acid ＜232 μmol/L in the Q1 group,24 473 with 232 μmol/L≤ serum uric acid ＜282 μmol/L in the Q2 group,24 382 with 282 μmol/L≤ serum uric acid ＜338 μmol/L in the Q3 group and 24 474 with serum uric acid ≥ 338 μmol/L in the Q4 group.Observation indicators:(1) comparisons of clinical characteristics among the 4 groups;(2) incidence of cholelithiasis in the 4 groups;(3) effects of serum uric acid on the new-onset cholelithiasis:① the dose-response relationship between serum uric acid and the risk of cholelithiasis,② comparisons of the fitting degree of serum uric acid on the cholelithiasis model,③ effects of different serum uric acid levels on incidence of cholelithiasis after stratification by sex,④ serum uric acid of different gender on the boxplots,⑤ effects of different serum uric acid levels on the incidence of cholelithiasis after stratification by age.Measurement data with normal distribution were expressed as (x)±s,and comparisons among groups were analyzed using the one-way ANOVA.Measurement data with skewed distribution is expressed by M (Q),and comparisons among groups were analyzed using the nonparametric Krustal-willis test.Count data were represented by percentage,and comparisons among groups were analyzed using chi-square test.The incidences of cholethiasis in 4 groups of different serum uric acid were calculated by person-year incidence.Restrictive cubic spline regression was used to calculate the dose-response relation between the continuous variable and the risks of new-onset cholelithiasis and 95％ confidence interval (CI).COX regression model was used to analyze the hazard ratio (HR) and 95％ CI of different serum uric acid levels on new-onset cholelithiasis.Likelihood ratio test and akaike information criterion (AIC) were used to calculate the fitting degree of serum uric acid on new-onset cholelithiasis model.Boxplots were used to describe serum uric acid in different genders.Results (1) comparisons of clinical characteristics among the 4 groups:sex (male),age,body mass index (BMI),systolic pressure,diastolic pressure,fasting plasma glucose (FPG),total cholesterol (TC),triglyceride (TG),high sensitive C-reactive protein,diabetes,hypertension,smoking,drinking and physical exercise were 15 162,(50± 11) years,(24±3)kg/m2,(123±21)mmHg (1 mmHg=0.133 kPa),(82± 12)mmHg,(5.6±2.0) mmol/L,(4.8±1.2) mmol/L,1.14 mmol/L (range,0.81-1.63 mmol/L),0.70 mmol/L (range,0.23-2.23 mmol/L),2 537,9 415,4575,2380,2 649 in the Q1 group,19 079,(51±12) years,(25±3)kg/m2,(130±21)mmHg,(83±12) mmHg,(5.5 ± 1.7) mmol/L,(4.9 ± 1.2) mmol/L,1.20 mmol/L (range,0.86-1.76 mmol/L),0.71 mmol/L (range,0.28-1.98 mmol/L),2 287,10 124,6 918,3 649,3 288 in the Q2 group,21 132,(52±13)years,(25±3)kg/m2,(132±21)mmHg,(84±12)mmHg,(5.5±1.6)mmol/L,(5.0±1.2) mmol/L,1.29 mmol/L (range,0.91-1.94 mmol/L),0.80 mmol/L (range,0.30-2.06 mmol/L),2 027,10 755,8 259,4 730,3 958 in the Q3 group,22 651,(53± 14) years,(26± 3) kg/m2,(134± 21) mmHg,(85±12)mmHg,(5.4±1.5)mmol/L,(5.1±1.2)mmol/L,1.54 mmol/L (range,1.05-2.35 mmol/L),1.02 mmol/L (range,0.43-2.50 mmol/L),1 981,12 082,9 562,6 209,4 758 in the Q4 group,respectively,with statistically significant differences among the 4 groups (x2 =7 624.63,F=279.93,961.91,330.84,271.40,38.25,353.18,H =3 406.30,912.23,x2 =108.15,590.49,2567.07,2 209.21,760.15,P＜0.05).(2)Incidence of cholelithiasis in the 4 groups:97 469 participants were followed up for 592 922 person-year,4 270 participants had new-onset cholelithiasis,with a total person-year incidence of 7.20 thousand person / year.The person-year incidence were respectively 6.34 (971/153 205 * 1 000),6.91 (1 034/149 686 * 1 000),7.44 (1 090/146 549 * 1 000),8.19 (1 175/143 482 * 1 000) thousand person / year in Q1,Q2,Q3 and Q4 group.(3) Effects of serum uric acid on the new-onset cholelithiasis.① The dose-response relationship between serum uric acid and the risk of cholelithiasis:restricted cubic spline regression showed a linear relationship between continuous serum uric acid,logarithmic transformated serum uric acid and the risk of cholelithiasis (x2 =11.74,8.01,P＜0.05).② Comparisons of the fitting degree of serum uric acid on the cholelithiasis model:adjusted for sex,age,BMI,TC,TG,diabetes,hypertension,smoking,drinking and physical exercise,risks of new-onset cholelithiasis increased in Q3 and Q4 groups compared with Q1 group (HR=1.10,1.12,95％CI:1.01-1.20,1.03-1.23,P＜0.05).The-2Log L and AIC value of multivariate model,serum uric acid+multivariate model were 92 532.39,92 550.39 and 92 525.35,92 549.35,respectively,with a statistically significant difference (x2=7.04,P ＜ 0.05).③ Effects of different serum uric acid levels on incidence of cholelithiasis after stratification by sex:in female participants,adjusted for age,BMI,TC,TG,diabetes,hypertension,smoking,drinking and physical exercise,risk of new-onset cholelithiasis in Q1 group was not statistically significant different from that in Q2,Q3,Q4 group (HR=1.06,1.15,1.09,95％CI:0.88-1.28,0.93-1.34,0.91-1.31,P＞0.05).In male participants,adjusted for age,BMI,TC,TG,diabetes,hypertension,smoking,drinking and physical exercise,risks of new-onset cholelithiasis in Q2,Q3 and Q4 groups were increased compared with Q1 group (HR=1.17,1.24,1.30,95％CI:1.06-1.30,1.12-1.37,1.18-1.44,P＜0.05).④ Serum uric acid of different gender on the boxplots:in female participants,the level of serum uric acid was (249 ± 61) μmol/L,(235±50)μmol/L,(231±56) μmol/L,(250±66) μmol/L,(266±75) μmol/L,(281±81) μmol/L,(298±76) μmol/L,(379±86)μmol/L respectively in the group of 18-27 years old,28-37 years old,38-47 years old,48-57 years old,58-67 years old,68-77 years old,78-87 years old,88-97 years old after stratified by 10 years old.In male participants,the level of serum uric acid was respectively (310±76)μmol/L,(298 ±75) μmol/L,(298±74) μmol/L,(294±74) μmol/L,(302±78) μmol/L,(311 ±80) μmol/L,(322±80) μmol/Land (330±75)μmol/L after participants stratified by 10 years old.⑤ Effects of different serum uric acid levels on the incidence of cholelithiasis after stratification by age:in participants with age ≤ 60 years old,adjusted for sex,BMI,TC,TG,diabetes,hypertension,smoking,drinking and physical exercise,risk of new-onset cholelithiasis in the Q2 and Q3 groups were not increased compared with Q1 group (HR=1.05,1.10,95％CI:0.94-1.17,0.99-1.23,P＞0.05),however,risk of new-onset cholelithiasis was increased in the Q4 group (HR =1.15,95％CI:1.02-1.28,P＜0.05).In participants with age ＞ 60 years old,adjusted for sex,BMI,TC,TG,diabetes,hypertension,smoking,drinking and physical exercise,risk of new-onset cholelithiasis in the Q2 groups was not increased compared with Q1 group (HR=1.16,95％CI:0.99-1.36,P＞0.05),however,risks of new-onset cholelithiasis were increased in the Q3 and Q4 groups (HR =1.19,1.21,95％CI:1.02-1.40,1.04-1.41,P＜ 0.05).Conclusion Elevated serum uric acid is an independent risk factor for the new-onset cholelithiasis.

OBJECTIVETo observe the clinical efficacy of auricular point sticking on prevention and treatment of gastrointestinal complications after gynecological laparoscopic operation of general anesthesia, and to explore whether it is achieved by regulating the secretion of plasma motilin (MTL).

METHODSSixty patients who received selective gynecological laparoscopy under general anesthesia were randomly assigned into an observation group and a control group, 30 patients in each one. The patients in the observation group were treated with auricular point sticking at each morning and night, 30 min before anesthesia, revival after surgery and 24 h after surgery. The adhesive fabric with vaccaria seeds was pressed at shenmen (TF), wei (CO), benmen (CO), jiaogan (AH) and pizhixia (AT) for 3 to 5 min until the sensation of sourness, distension and numb appeared. The treatment was given for one week. The patients in the control group were treated only with similar adhesive fabric at auricular points at identical time points; each auricular point was pressed for 3 to 5 min. The anus exhaust time, defecating time and borborygmus were recorded; the level of plasma MTL was tested 30 min before anesthesia, 24 h after o-peration and 48 h after operation; the occurrence of nausea and vomiting from the end of operation to the end of treatment were also recorded.

RESULTSCompared with the control group, the occurrence of nausea after operation was reduced in the observation group (<0.05), and the anus exhaust time and defecating time were shortened (both<0.05), and the recovery of borborygmus was improved (<0.05). The levels of MTL 24 h and 48 h after surgery were higher than those before operation in the two groups (all<0.05); The levels of MTL 24 h and 48 h after surgery in the observation group were significantly lower than those in the control group (both<0.05).

CONCLUSIONSThe assist of auricular point sticking could reduce the occurrence of nausea-vomiting and accelerate the recovery of gastrointestinal function in gynecological laparoscopic operation under general anesthesia, which is likely to be related with the inhibition on excess secretion of MTL.

Objective To evaluate the effect of transcutaneous electric acupoint stimulation (TEAS)on prognosis in the patients at high risk of postoperative pulmonary complications. Methods Sixty American Society of Anesthesiologists physical statusⅡorⅢpatients of both sexes, aged 65-72 yr, with body mass index of 18-25 kg∕m2, scheduled for elective thoracoscopic and laparoscopic radical resection of e-sophageal cancer, were divided into placebo control group(group C, n=30)and TEAS group(n=30). In group TEAS, bilateral Zusanli(ST36), Hegu(LI4)and Feishu(BL13)acupoints were stimulated for 30 min(disperse-dense waves, frequency 2∕100 Hz, intensity of currents 8-12 mA)starting from 30 min be-fore anesthesia induction.In group C, electrodes were placed on the same acupoints before anesthesia induc-tion, but no current was given.General anesthesia was performed in two groups.Before one-lung ventilation, at 30 min and 2 h of one-lung ventilation and at 1 h after operation(T1-4), blood samples were drawn from the radial artery for blood gas analysis, and oxygenation index and alveolar-arterial oxygen partial pressure difference were calculated.Blood samples were collected from the internal jugular vein at T1-3and 24 h after surgery(T5)for determination of plasma tumor necrosis factor-alpha, interleukin-6(IL-6)and IL-10 con-centrations.The development of postoperative pulmonary complications, time for removal of drainage tube and length of hospital stay were recorded. Results Compared with group C, the oxygenation index at T2-4and plasma IL-10 concentrations at T3were significantly increased, the alveolar-arterial oxygen partial pressure difference at T2,3, plasma tumor necrosis factor-alpha concentrations at T2,3, plasma IL-10 concentrations at T3,5and incidence of postoperative pulmonary complications were decreased, and the time for removal of drainage tube and length of hospital stay were shortened in group TEAS(P<0.05). Conclusion TEAS can improve prognosis in the patients at high risk of postoperative pulmonary complications.

Objective:To explore the cooperation and clinical significance of HIF-1α,ALDH1 and Hedgehog signaling pathway in the activation of cancer stem cell(CSC) in triple negative breast cancer(TNBC).Methods: ALDH1+(Aldehyde dehydrogenase1)breast cancer stem cells and ALDH1-breast cancer cells were selected from MDA-MB-231 cells by magnetic activated cell sorting system(MACS),qRT-PCR method was employed to analyze the expression differences of HIF-1α and Hedgehog signaling molecules Sonic hedgehog(SHH),patched1(PTCH1),Smoothened(SMO) and Glioma-associated oncogene homoglog1(GLI1) in ALDH1+ breast cancer stem cells and ALDH1-breast cancer cells.Immunohistochemical method was applied to study the expressions of HIF-1α and ALDH1 and the relationships among HIF-1α,ALDH1 and Hedgehog signaling molecules in TNBC.Results: The expressions of HIF-1α mRNA,SMO mRNA and GLI1 mRNA in ALDH1+ breast cancer stem cell were higher than those in ALDH1-breast cancer cell(P all<0.05).The positive expression rates of HIF-1α were 90.0% and 70.0%,and the positive rates of ALDH1 were 93.3 % and 66.7 % in TNBC and non-TNBC,respectively(P all<0.05).Spearman rank correlation analysis showed that the expression of HIF-1α was positively related with that of ALDH1 in TNBC(r=0.53,P<0.01).HIF-1α expression was correlated with lymph node metastasis and TNM stage(P all<0.05),ALDH1 expression was correlated with histological grade and TNM stage(P all<0.05).In addition,the expression of HIF-1α was positively related with that of Hedgehog signaling molecules SHH(r=0.584,P<0.01),SMO(r=0.467,P<0.01) and GLI1(r=0.439,P<0.05),the expression of ALDH1 was positively related with that of SHH(r=0.426,P<0.05) and GLI1(r=0.394,P<0.05).Conclusion: HIF-1α and Hedgehog signaling pathway were activated in ALDH1+ breast cancer stem cell.HIF-1α,ALDH1 and Hedgehog molecules may cooperate with each other to activate breast CSC to promote the malignant progression of TNBC.

Objective T o observe the changes of cystathionine β-synthase (C B S ) expression in the cere-bral cortex after brain contusion at different tim es. Methods A n experim ental m odel of traum atic brain injury (T B I) in m ice w as established by an im proved w eight-drop device. T hen W estern blotting and im m unohistochem ical exam ination w ere used to detect the C B S expression in cerebral cortex around in-jury at different tim e points (1 h, 6 h, 12 h, 1 d, 2 d, 3 d, 7 d). Results T he results of W estern blotting revealed that the expression level of C B S w as dow n-regulated and reached its low est level at the 3rd days after injury, and then restored to norm al level after 7 days. T he results of im m unohistochem istry show ed that C B S w as present in the norm al brain cortex. C B S expression gradually decreased at the 3rd days after injury, and then restored to norm al level after 7 days. Conclusion C B S has the potential to be a reference index for tim e estim ation after brain contusion in forensic practice.

Hypoxic-ischemic brain damage is a serious complication of perinatal asphyxia. Enriched environment is the living condi-tions for the promotion of feeling, cognition and behavior. Enriched environment may promote the repairing of hypoxic-ischemic brain dam-age in term of praxiology, morphology and molecular biology, that enriched environment may regulate the secretion and release of various factors, and enhance synaptic plasticity, induce neuron regeneration, inhibit its apoptosis, and result in ethological and morphological recov-ery.