Objective To identify,construct and express scFv CD133,verify its biological function.Methods VL and VH were isolated from hybridoma of mAb CD133 by using antibody engineering technology.Its DNA sequencing and CDR were determined.scFv CD133 was then cloned into pET32a,transformed into Origami,induced by IPTG,purified by Ni2+-NTA His resin.Its affinity and specificity were tested by NH4SCN elution and ELISA.Results The size of VL and VH of scFv CD133 was 339 bp and 342 bp,which coded 113 and 114 amino acid separately.Its VL belonged to mouse Igκ chain and VH belonged to mouse IgG heavy chain subtype I.The molecular weight of scFv CD133 was about 27 × 103 which was testified by SDSPAGE and Western blot.Its affinity and specificity were also verified.Conclusion scFv CD133 has been successfully constructed and expressed in Origami,which could supply basis for target therapy of CD+133 cancer stem cell.

Objective To analyze the characteristics and clinical value of time intensity curve (TIC) of contrast-enhanced ultrasound (CEUS) in recurrent small hepatocellular carcinoma (RSHCC) and primary small hepatocellular carrcinoma (PSHCC). Methods Sixty-five cases of RSHCC (all lesions ≤3 cm) were devided into group B1 with 42 cases of RSHCC (≤2 years ) , and group B2 with 23 cases of RSHCC ( > 2 yeras ) and group A invloved 49 cases of PSHCC (all lesions ≤3 cm). Enhancement patterns in arterial, portal and delayed phase were evaluated respectively in three groups through CEUS and analytic software Sonoliver was applied to obtain quantitative features of CEUS in the region of interest. Receiver operating characteristic (ROC) was drawn and the area under curve (AUC) was calculated. Results CEUS showed hyper-enhancement difference in arte-rial phase in group B2 (72.4%) and group A (94.8%)(P′ = 0.008) showed statistical significance, but no sig-nificance was found in enhanced iso in portal phase (P = 0.078). Hypo-enhancement in the delayed phase in group B2 (75.9%), group A (96.6%) and group B1 (95.3%) (P′ = 0.003, P′ = 0.005). TIC showed HT difference (half time of descending) in B2 group, A group and B1 group (P′ = 0.007, P′ = 0.013) indicated statistical significance but RT, TTP, MTT(P = 0.319,P = 0.104, P = 0.461) showed no difference. AUC was 0.841 (half time of descending). Conclusions Enhancement patterns of CEUS (RSHCC) are related to recur-rent time . En hancement patterns of RSHCC (> 2 years ) is not typical so CEUS should be combined with quanti-tative analysis of TIC to provide reference for its treatment and prognosis.

Objective To investigate the value of dynamic contrast?enhanced MRI (DCE?MRI) in the differential diagnosis of glioblastoma and brain metastases. Methods Twenty patients with high grade gliomas and 20 cases patients with brain metastases proved by surgery and pathology were collected, and patients were examined with conventional MRI and DCE?MRI preoperatively. The ROIs were manually placed in solid parts of the tumors and their surrounding tissues to calculate Ktrans, Kep and Ve values. The Ktrans, Kep and Ve values differences for the solid part and surrounding tissues of the two brain tumors were compared by two independent sample t test. The correlation between Ktrans of the solid parts of the two brain tumors and Ktrans, Kep and Ve values of their surrounding tissues were studied by Pearson correlation analysis. Results The Ktrans, Kep and Ve values of glioblastoma were(0.258 ± 0.063)min-1,(0.398 ± 0.082)min-1, 0.632±0.084, the Ktrans, Kep and Ve values of brain metastases were(0.233±0.053)min-1,(0.357±0.042)min-1, 0.672±0.113. There were no significant differences between the glioblastoma and brain metastases for Ktrans, Kep and Ve values(t=-1.354,-1.982, 1.276, all P>0.05). The Ktrans, Kep and Ve values of surrounding tissues of glioblastoma were(0.093±0.032)min-1,(0.411±0.089)min-1, 0.107±0.021, the Ktrans, Kep and Ve values of surrounding tissues of brain metastases were(0.033±0.010)min-1,(0.204±0.045)min-1, 0.069±0.017. The Ktrans, Kep and Ve values of surrounding tissues between glioblastoma and brain metastases had significant difference (t=-7.978,-9.303,-6.203, all P<0.05). The Ktrans of glioblastoma were correlated with Ktrans, Kep and Ve values of their surrounding tissues (r=0.759, 0.464, 0.651, all P<0.05); The Ktrans values of brain metastases had no relationship with Ktrans, Kep and Ve values of their surrounding tissues (P>0.05). Conclusion The DCE?MRI can quantitatively display the microvascular permeability and accurately evaluate the damage of blood?brain barrier of glioblastoma and brain metastases, which has an important value in studying biological characteristics and differential diagnosis of the two brain tumors.

Objective To compare the clinical effect and safety of peroral endoscopic myotomy (POEM) with surgical therapy in treatment of esophageal achalasia. Methods 78 patients diagnosed as esophageal achalasia from January 2012 to October 2014 were enrolled in the study and divided into POME group and Heller group. There were 42 patients in POEM group and 36 patients in Heller group. The clinical symptom remission rate, LES resting pressure, Eckardt scores, complication rate, length of hospital stay and the hospitalization expenses were analyzed between the two groups. Results The patients in POEM group and Heller group both got clinical remissions after the treatment. There was no statistical difference in the rate of complication occurrence, Eckardt scores and LES resting pressure between the two groups. Patients in POEM group had shorter operation time, hospital stay and less expenses compared with the Heller group. Conclusions Compared with Heller group, the POEM group has the similar curative effect in treatment of esophageal achalasia. The POEM as a minimally invasive surgery has the advantages of less pain and trauma, shorter hospital stay, well tolerated and low cost. Therefore, the POEM is worth to be popularized and applied in treating esophageal achalasia.

Objective To evaluate the role of intestinal flora disturbance in development of postoperative cognitive dysfunction (POCD) in aged mice and the relationship with regulatory T cells (Treg) and T helper cells 1/T helper cells 2 (Th1/Th2) in the small intestine.Methods Thirty-six SPF healthy male C57BL/6J mice,weighing 45-50 g,aged 18 months,were divided into 3 groups (n=12 each) using a random number table method:control group (group C),POCD group and POCD plus VSL#3 group (group PV).POCD was induced by abdominal exploration.VSL#3 probiotics was given by intragastric gavage (300 μl per time,once a day) every 24 h for 7 consecutive days starting from the end of surgery in group PV.Morris water maze test was used to assess the cognitive function at day 7 after operation.Orbital venous blood samples were collected after the end of Morris water maze test,and animals were then sacrificed and small intestine and hippocampi were removed for measurement of the percentage of CD4+ CD25+ Foxp3+Treg,TCD4+IFN-γ+Th1 and CD4+IL-4+Th2 in the lamina propria of small intestine and plasma and expression of IL-4 and IFN-γmRNA in the lamina propria of small intestine,plasma and hippocampal tissues,and IL-4 mRNA/IFN-γmRNA ratio was calculated.Results Compared with group C,the percentage of CD4+CD25+Foxp3+Treg and CD4+IL-4+ Th2 in the lamina propria of small intestine and plasma was significantly decreased,the percentage of CD4+ IFN-γ+Th1 in the lamina propria of small intestine and plasma was increased,the expression of IL-4 mRNA in the lamina propria of small intestine,plasma and hippocampal tissues was down-regulated,the expression of IFN-γ mRNA in the lamina propria of small intestine,plasma and hippocampal tissues was up-regulated,IL-4 mRNA/IFN-γ mRNA ratio was decreased,the escape latency and swimming distance were prolonged,and the time spent in the target quadrant was shortened in group POCD (P＜0.05).Compared with group POCD,the percentage of CD4+ CD25+ Foxp3+ Treg and CD4+IL-4+ Th2 in the lamina propria of small intestine and plasma was significantly increased,the percentage of CD4+IFN-γ+Th1 in the lamina propria of small intestine and plasma was decreased,the expression of IL-4 mRNA in the lamina propria of small intestine,plasma and hippocampal tissues was upregulated,the expression of IFN-γmRNA in the lamina propria of small intestine,plasma and hippocampal tissues was down-regulated,IL-4 mRNA/IFN-γmRNA ratio was increased,the escape latency and swimming distance were shortened,and the time spent in the target quadrant was prolonged in group PV (P＜ 0.05).Conclusion Intestinal flora disturbance can promote the development of POCD in aged mice,which is related to the decreased percentage of Treg and Th1/Th2 imbalance in the small intestine.

Objective:To evaluate the influence of goal-directed volume management based on cardiac output index (CI), intrathoracic blood volume index (ITBVI) and extravascular lung water index (EVLWI) in patients undergoing off-pump coronary artery bypass surgery.Methods:Forty patients (ASA 2 to 3 grade) undergoing off-pump coronary artery bypass surgery in Lanzhou University Second Hospital from January 2017 to December 2018 were selected. The patients were divided into 2 groups by random digits table method with 20 cases in each group: study group (goal-directed fluid therapy treatment with CI, ITBVI and EVLWI) and control group (conventional fluid therapy). The control group was given central venous pressure (CVP) monitoring rehydration, and the study group was given PiCCO hemodynamic monitoring indicators. The CVP, CI, ITBVI and EVLWI for fluid management were measured. Accurate assessment of volume status of patients was done. The study group received goal-directed fluid therapy based on CVP, CI, ITBVI and EVLWI, with the goal of CI in the 3.0 to 5.0 L/(min·m 2) range, ITBVI in the 800 to 1 000 ml/m 2 range and EVLWI in the 3.0 to 7.0 ml/kg range. The heart rate, mean arterial pressure (MAP), urine volume, central venous oxygen saturation (ScvO 2), lactic acid and renal function were monitored. The ventilator withdrawal time, hospitalization in ICU, length of stay, incidence of acute pulmonary edema, incidence of acute renal failure, mortality of 30 d after surgery were recorded and compared between the two groups. Results:Tissue perfusion and urine volume of the study group was significantly improved compared with that of control group ( P<0.05). ScvO 2 of the study group was higher than that of the routine group ( P<0.05). The concentration of lactic acid of the study group was lower than that of the routine group ( P<0.05). The incidences of acute pulmonary edema, acute renal insufficiency and mortality of the study group were lower than those of the routine group (5.0% vs. 15.0%, 5.0% vs. 10.0% and 5.0% vs. 15.0%), and there were statistical differences ( P<0.05). The length of stay and hospitalization in ICU were both lower than those in the control group ( P<0.01). Conclusions:Goal-directed fluid therapy based on CI, ITBVI and EVLWI can effectively optimize the cardiac preload of patients undergoing off-pump coronary artery bypass surgery, improve cardiac output, ensure microcirculation perfusion, maintain the balance of oxygen supply and demand, and reduce the incidence of complications and mortality.

Objective:To investigate the mechanism of cathepsin G(CatG) in improving the treatment efficacy of T cells in gliomas.Methods:(1) Clinical data of 397 glioma patients in the glioma database were collected, Kaplan-Meier method was used to perform survival analysis, and the correlation between CTSG and β2-microglobulin ( β2M) mRNA expressions in glioma tissues was analyzed. (2) Glioma stem cell (GSC) 387 and GSC3565 were isolated from glioblastoma and differentiated into differentiated glioma cell (DGC) 387 and DGC3565, respectively; GSC387 was divided into CatG group and CatG inhibitor group, and cells in the CatG group and CatG inhibitor group were cultured with 0.1 μg/μL recombinant human leukocyte antigen (HLA)-A*02:01 and HLA-B*15:01 combined with 4 ng/μL CatG or 10 mol/L CatG inhibitor for 10 min, respectively; the expressions of HLA-A*02:01 and HLA-B*15:01 were detected by Thomas bright blue staining, and the protein expressions of major histocompatibility complex (MHC)-I and MHC-DR were detected by Western blotting. (3) GSC387, GSC3565, DGC387, and DGC3565 were divided into 4 groups, including CatG group, CatG inhibitory group, blank antibody group 1 and blank antibody group 2, respectively; 4 ng/μL CatG, 10 μmol/L CatG inhibitor, blank antibody 1 and blank antibody 2 were added into the cells from the 4 groups for 24 h, and the expression of HLA-ABC was detected by flow cytometry. (4) GSC387, GSC3565, DGC387, and DGC3565 were divided into CatG group and CatG inhibitory group, respectively; luciferase assay was used to detect the influence of CatG in the killing effects of T cells and natural killer cells. Results:(1) The survival rate in patients from CTSG mRNA high expression group was significantly higher than that in patients from CTSG mRNA low expression group, and the survival rate in patients from β2M mRNA low expression group was statistically higher than that in patients from β2M mRNA high expression group ( P<0.05); a negative correlation between CTSG mRNA and β2M mRNA expressions was noted in glioma tissues ( r=-9.160, P=0.000). (2) Thomas bright blue staining showed that the expressions of HLA-A*02:01 and HLA-B*15:01 obviously increased in the CatG group as compared with those in the CatG inhibitor group; Western blotting showed that as compared with the CatG inhibitor group, the CatG group had increased MHC-I expression, and decreased expressions of α and β chains of MHC-DR. (3) Flow cytometry showed that the HLA-ABC expressions in GSC387 and GSC3565 of the CatG group were statistically higher than those in the CatG inhibitor group ( P<0.05). (4) Luciferase assay showed that, as compared with the CatG inhibitor group, the CatG group had statistically higher proportion of T cells killing GSCs ( P<0.05). Conclusion:CatG can improve the immunotherapy efficacy in GSCs, mainly by increasing the MHC-I expression on the cell surface.

[Abstract] Glioblastoma multiforme（GBM）is a malignant tumor of central nervous system with high incidence, aggressive and poor prognosis. Since temozolomide was approved by FDAin 2005, there is no new curative strategy with obvious improvement in therapeutic effect. With the developments in molecular biology, tumor immunology and immunotherapy technology, the discovery of new molecular targets, breakthrough in central immunization exemption theory, and advance in gene transduction and cell technology, GBM immunotherapy ushered in a new breakthrough in immunotherapy. Cellular immunotherapy, presented by chimeric antigen receptor-modified T cell (CAR-T) therapy, has exhibiteditsprominentapplicationprospectinGBMinvitro experimentstargetingEGFRvIII, IL-13Rα2, HER2, erythropoietin-producing hepatocellular carcinomaA2 (EphA2), animal models and early clinical trials. However, the GBM molecular heterogeneity, immunosuppressive microenvironment and blood-brain barrier have presented challenge for CAR-T going into the first-line clinical treatment. Researchers are now exploring key antigens of oncogenic phenotype, designing optimal combination of target antigens to prevent the immune escape, improving CAR-T passing through blood-brain barrier and invading tumor tissue, finding the best route for cell deliver, and optimizing evaluation system for central nervous system (CNS) immunotherapy. It is believed that the breakthrough of CAR-T cell immunotherapy will finally help GBM patients pursuing a beautiful life.

Objective: To investigate the development of hepatocyte senescence during liver fibrogenesis and to explore the effect and possible mechanism of insulin-like growth factor 1 (IGF-1) on hepatocyte senescence and liver fibrosis. Methods: A total of 42 male Sprague Dawley (SD) rats were selected. Eighteen rats were induced by carbon tetrachloride (CCl₄) to establish the rat model of liver fibrosis. On the day 0, six and 28 after the establishment of the model, six rats were executed respectively to analyze the liver fibrosis and hepatocyte senescence in CCl₄-induced liver fibrosis rat models. Twenty-four rats were divided into control group, CCl₄ group, CCl₄+ lentivirus vector (LV-CTR) group and CCl₄+ LV-IGF-1 group, with six rats in each group.The rats were sacrificed on the 28th day after the establishment of the model. The liver tissues were obtained and the inferior vena cava blood was collected to analyze the effect of IGF-1 overexpression on liver fibrosis and hepatocyte senescence. Analysis variance (ANOVA), least significant difference (LSD) and Dunnett T3 test were performed for statistical analysis. Results: Steatohepatitis on the 6th day and early stage of hepatic fibrosis on the 28th day, which indicated the model was successfully established. The results of the effects of IGF-1 overexpression on hepatic fibrosis and hepatocyte senescence showed that on the 28th day, compared with those of control group, both the score of Ishak liver inflammation and necrosis and the score of Ishak liver fibrosis were increased in the CCl₄ group, CCl₄+ LV-CTR group and CCl₄+ LV-IGF-1 group (0, 14.55±1.94, 15.43±2.19 and 10.29±1.47, respectively; 0, 3.51±0.51, 3.21±0.79 and 1.32±0.40, respectively). The area of liver tissues by Masson staining (0.45±0.40, 5.62±1.08, 6.03±0.65 and 2.88±1.54), SA-β-Gal staining (1.75±0.80, 4.28±1.19, 4.92±1.14, 3.11±0.79), p53 (2.02±0.81, 4.36±1.02, 4.72±0.72 and 3.58±0.70) and progerin (0.72±0.40, 4.52±1.01, 4.01±1.25 and 2.66±0.80) all were increased. The levels of serum IGF-1 all were decreased ((632.00±6.04), (503.00±40.42), (508.00±21.94) and (572.40±5.94) ng/L). However the levels of ALT all were increased ((11.20±5.97), (214.00±73.90), (245.00±76.06) and (30.00±5.00) U/L). The relative expression levels of p53 (0.58±0.06, 1.78±0.18, 1.72±0.10 and 1.23±0.22) and progerin (0.12±0.02, 0.78±0.15, 1.32±0.20 and 0.81±0.16) in the primary hepatocytes were increased. The differences were all statistically significant (F=91.674, 90.778, 32.982, 9.726, 10.640, 17.029, 103.910, 30.059, 64.707 and 97.457, all P<0.05). Compared with those of CCl₄+ LV-CTR group, the score of Ishak liver inflammation and necrosis and the score of Ishak liver fibrosis were decreased in the rats′ liver tissues of CCl₄+ LV-IGF-1 group, the areas of Masson staining, SA-β-Gal staining, p53 and progerin in the liver tissues were decreased, the level of serum IGF-1 was increased, the level of ALT was decreased, and the relative expression levels of p53 and progerin in primary hepatocytes both were decreased. The differences were all statistically significant (all P<0.05, respectively). Conclusions Hepatocyte senescence increases in the process of liver fibrosis induced by CCl₄. Overexpression of IGF-1 may alleviate liver injury, improve hepatocyte senescence and liver fibrogenesis by regulating the nuclear p53/progerin pathway.

Objective To investigate the clinicopathological characteristics and prognosis of well-differentiated rectal neuroendocrine tumor(RNET).Methods A retrospective analysis was conducted using the clinical data from 83 patients with well-differentiated RNET from August 2017 to December 2021,including clinical manifestations,endoscopy,endoscopic treatment,postoperative complications,postoperative pathology,follow-up and prognosis.Pathological results according to the 2019 World Health Organization(WHO)Classification of digestive system tumors,83 patients were divided into G1 stage group(72 cases)and G2 stage group(11 cases);Based on the number of tumors in the patient,83 patients were divided into two groups:single RNET group(77 cases)and multiple RNET group(6 cases),the expressions of chromogranin A(CgA),synapsin(Syn)and CD56 were compared among different groups.Results Based on pathological findings in the group,G1 stage group CgA positive rate was significantly higher than that of G2 stage group,the difference was statistically significant(χ2 = 4.23,P = 0.040);Based on the number of tumors,multiple RNET group CgA positive rate was significantly higher than that of single RNET group,the difference was statistically significant(χ2 = 5.74,P = 0.017).It was no significant difference in Syn and CD56 between the two groups(P＞0.050).Conclusion Well-differentiated RNET has no specific clinical manifestations.It is mostly isolated in G1 stage and single RNET.ESD is safe and has a good prognosis,the positive rate of CgA is higher in G1 stage patients,and the positive rate of CgA is higher in patients with multiple RNET.