Background and purpose:Chimeric antigen receptor T(CAR-T)cell therapy has shown remarkable efficacy in treating hematological and lymphatic system tumors,but its effectiveness in solid tumors is relatively poor,which is partly attributed to target selection.For Ewing sarcoma(ES),CD99 can be a potential target for CAR-T cells.However,due to T cells'endogenous expression of CD99 protein,CAR-T cells targeting CD99 face limitations in their expansion capacity in vitro.This study aimed to identify the optimal conditions for preparing CD99 CAR-T cells by incorporating CD99 knockdown short hairpin RNA(shRNA),optimizing the multiplicity of infection(MOI)for lentiviral transduction,and screening for the best culture medium and container for CAR-T cell expansion.Methods:shRNA sequences were screened to enhance the expansion capacity of CD99 CAR-T cells.Different MOI,culture media,and containers were used to assess CAR-T cell transduction efficiency,cell viability,proliferation capacity,specific killing ability,and interferon-γ(IFN-γ)release levels under various conditions,in order to identify the optimal cell preparation conditions.Results:The expansion level of KO-CD99 CAR-T cells obtained through shRNA knockdown was significantly higher than that of CD99 CAR-T cells[(16.40±0.40)vs(6.33±1.53),P<0.01].The optimal expansion effect was observed when the transduction MOI was between 0.25 and 1.0,and OptiVitro was used as the culture medium.CAR-T cells cultured in ventilated flasks exhibited significantly higher expansion rates compared to cells cultured in bags[MOI=0.25:(50.23±3.32)vs(13.02±4.82);MOI=0.50:(49.96±0.83)vs(18.25±2.88);MOI=1.00:(48.27±5.08)vs(13.16±6.26);P<0.01],with better cell phenotype and higher specific killing ability.Conclusion:KO-CD99 CAR-T cells obtained through shRNA technology can achieve stable expansion.Based on the optimization of expansion conditions,KO-CD99 CAR-T cells exhibit superior expansion capacity and a higher proportion of memory T cells when the MOI is between 0.25 and 1.00,OptiVitro is used as the culture medium,and ventilated flasks are used as the culture container.These findings lay a solid foundation for further clinical trials of CD99 CAR-T cell therapy for ES.

In recent years, mass spectrometry has been widely used to study membrane protein structure and function. However, the application of mass spectrometry to study integral membrane protein is limited because there are many hydrophobic amino acids in the trans-membrane domain of integral membrane protein to cause low sequence coverage detected by LC-MS/MS. Therefore, we used vitamin K epoxide reductase (VKORC1), a human integral membrane protein, as a model to optimize the digestion conditions of chymotrypsin, and developed an in-gel digestion method of chymotrypsin to improve sequence coverage of membrane protein by mass spectrometry. By exploring the effects of calcium concentration, pH value and buffer system on the percentage of sequence coverage, number of total detected and types of unique peptide, and the size of unique peptide, sequence coverage and peptide diversity could be considered under condition of Tris-HCl buffer with 5-10 mmol/L calcium ion concentration and pH value 8.0-8.5. This method could make the sequence coverage of membrane protein to reach more than 80%. It could be widely used in the study of membrane protein structure and function, identification of interaction site between membrane proteins, and identification of binding site between membrane protein and small molecular drug.
Amino Acid Sequence ; Chromatography, Liquid ; Chymotrypsin/metabolism* ; Digestion ; Humans ; Membrane Proteins ; Tandem Mass Spectrometry ; Trypsin ; Vitamin K Epoxide Reductases

Objective To investigate the influence of risperidone on differentiation of 3T3‐L1 pre‐adipocytes .Methods 3T3‐L1 pre‐adipocytes were induced to differentiate into mature adipocytes by adopting the classic hormone cocktail method and observed by the oil red O staining .Meanwhile ,the inducing medium was added with risperidone for studying its influence on 3T3‐L1 pre‐adi‐pocytes differentiation .Results 3T3‐L1 pre‐adipocytes were successfully differentiated into the mature adipocytes ,0 .1 ,1 ,10μmol/L risperidone all could inhibit the differentiation of 3T3‐L1 pre‐adipocytes .Conclusion Risperidone can inhibit the differentiation of 3T3‐L1 pre‐adipocytes .

Objective To compare the effects between percutaneous transhepatic gallbladder drainage (PTGD) plus delayed laparoscopic cholecystectomy (LC) in comparison with emergency LC for elderly patients with acute cholecystitis.Methods From June 2011 to December 2014,the clinical data of elderly patients with acute cholecystitis receiving PTGD plus LC and emergency LC were retrospectively studied.Results In this study 38 patients received PTGD plus LC,59 patients received emergency LC.Patients in PTGD + LC group had longer operative time (67 ± 14) min and higher conversion rate (5 cases)than those in LC group (51 ± 13) min,1 case (t =5.741,x2 =5.057,P ＜ 0.05),but had quicker bowel function recovery time (24.5 ±6.4) h,shorter hospital stay (4.2 ± 1.8) d,less complications (3 cases)than those inLC group (27±5.2) h,(6.2±1.9) d,17 cases (t =2.11,t=5.165,x2 =6.18,P＜0.05).Conclusions Percutaneous transhepatic gallbladder drainage plus delayed cholecystectomy is safe for elderly patients with acute cholecystitis.

Objective To prepare the schizophrenia mouse model by using peritoneal injection of dizocilpine maleate(MK‐801) , then to study the peripheral blood miRNA‐132 abnormal expression in schizophrenia mouse model .Methods Adult male Kunming mice(KM) were selected and randomly divided into the normal saline group(control group) and MK‐801 group(experimental group) .The behaviors changes were observed .Real‐time PCR method was used to detect the miRNA‐132 expression in peripheral blood .Results (1) Compared with the control group ,the stereotyped behavior score in the experimental group was increased sig‐nificantly(P<0 .05) ,the spontaneous activity was increased significantly(P<0 .05) ,showing that the up‐right number was signifi‐cantly decreased ,while the walking‐grid number was significantly increased(P< 0 .05) ,which was consistent with the manifesta‐tions of schizophrenia mouse model .(2)Compared with the control group ,the miRNA‐132 expressed in the experimental group was down‐regulated ,the difference was statistically significant(P<0 .05) .Conclusion miRNA‐132 is abnormally expressed in blood of schizophrenia mouse .

[ ABSTRACT] AIM:To investigate the effect of HOX transcript antisense RNA ( HOTAIR) on the migration and invasion abilities of liver carcinoma HepG 2 cells.METHODS:The expression of phosphoinositide-3-kinase regulatory sub-unit 3 (PIK3R3) in the liver cancer and normal liver tissues was detected by immunohistochemistry .The efficiency of gene silencing of HOTAIR or PIK3R3 by LV3-shHOTAIR or LV3-shPIK3R3 was determined by qPCR and Western blot .The mi-gration and invasion abilities of HepG 2 cells after silencing of HOTAIR and PIK3R3 were measured by wound healing assay and Transwell Matrigel invasion assay .The expression of miR-214 after silencing of HOTAIR and PIK3R3 was analyzed by qPCR.The expression of HOTAIR and PIK3R3 in the HepG2 cells was also evaluated by qPCR after transfected with miR-214 mimics or miR-214 inhibitor .Dual-luciferase reporter assay system was used to determine the regulatory effect of miR-214 on HOTAIR and PIK3R3 expression.RESULTS:PIK3R3 expression increased significantly in the liver cancer tissues compared with normal liver tissues .The abilities of invasion and metastasis of hepatocellular carcinoma were reduced after silencing of HOTAIR and PIK3R3.miR-214 expression was increased when silencing of HOTAIR and PIK3R3 was per-formed.HOTAIR and PIK3R3 expression was reduced after transfection with miR-214 mimics.HOTAIR and PIK3R3 ex-pression was increased after transfection with miR-214 inhibitor.The results of dual-luciferase reporter assay test showed that miR-214 directly regulated HOTAIR and PIK3R3 transcription activity .CONCLUSION: HOTAIR regulates the ex-pression of PIK3R3 through miR-214, thus promoting the migration and invasion abilities in the liver cancer cells .

Objective:To observe the effect of Gemcitabine ( GEM) on the viability and apoptosis of non-small cell lung cancer HCC827 in vitro. Methods:The cell viability,apoptosis and cell cycle of HCC827 cells induced by Gemcitabine were detected with cell counting kit-8 assay (CCK-8),Annexin V-FITC/PI staining and flow cytometry. The expression of Bcl-2 protein of cells treated with GEM was examined by Western blot assay. Results: There was significant inhibition effect on HCC827 cells treated with 0. 1-1 000 ng/ml of GEM,which can promote the occurrence of HCC827 cell apoptosis and arrest cell in the S phrase. The apoptosis induced by GEM was accompanied with the down regulation of Bcl-2 protein. Conclusion: GEM can inhibit the cell viability and induce the HCC827 cell apoptosis and S phrase arrest. Its cell dead type was apoptosis,which was related with the expression of Bcl-2 protein.

Objective To analyze the risk factors of pulmonary embolism complications following laparoscopic bile duct exploration.Methods The data of 201 cases undergoing laparoscopic bile duct exploration from June 2002 to December 2014 were retrospectively analyzed.Fourteen risk factors of pulmonary cmbolism complications were evaluated by univariate analysis using no conditional binary Logistic regression.Results 23 patients developed pulmonary embolism complications (11.4％).Age (≥60y),Diabetes mellitus,coronary heart disease,history of cerebral infarction,preoperative shock,time of operation,ambulation after operation (≥3 d) were significant risk factors of pulmonary embolism complications (P ＜0.05).Multivariate Logistic regression analysis identified diabetes mellitus,coronary heart disease,history of cerebral infarction,time of operation,ambulation after operation (≥ 3 d) as independent risk factors of pulmonary embolism complications (P ＜ 0.05).Conclusions Diabetes mellitus,coronary heart disease,history of cerebral infarction,time of operation,ambulation after operation (≥ 3 d) are independent risk factors for pulmonary embolism complications following laparoscopic bile duct exploration.

Objective To study the expression of P57kip2 and its clinical significance in hilar bile duct adenocarcinoma.Methods The expressions of P57kip2 in hilar bile duct adenocarcinoma tissues (37 cases) and normal bile duct tissues (32 cases) were determined by immunohistochemical SP methods.The relations between the expression levels of P57kip2 with clinicopathologic parameters were analyzed.Results The positive rate of P57kip2 was 43.2％ (16/37) in bile duct adenocarcinoma,while it was 87.5％ (28/32) in normal duct tissues (P ＜0.01).The expression level of P57kip2 in adenocarcinoma showed no significant association with gender,age or CA19-9 level (P ＞0.05),but they were significantly related with lymph node metastasis,invasion and degree of differentiation (P ＜ 0.05).Conclusions P57kip2 is associated with the occurrence and development of hilar bile duct adenocarcinoma.It may play an important role in invasion and metastasis of hilar bile duct adenocarcinoma.The P57 protein can be used as an index to diagnose hilar bile duct adenocarcinoma.

Objective To study the clinical efficacy of splenectomy plus porta-azygous devascularization in the treatment of portal hypertension using either the hand-assisted laparascopic (HALS) or the laparoscopic approaches.Methods The clinical data of 30 cases of portal hypertension treated between January 2008 and June 2012 were enrolled in this study.16 patients underwent HALS and 14 patients underwent traditional laparoscopy approach.Results All the operations were successfully accomplished.The mean operation time of the HALS group was significantly shorter than the traditional laparoscopic group (270 min vs 350 min,P＜0.05).Intra-operative blood loss of the HALS group was significantly less than the traditional laparoscopic group (250 ml vs 370 ml,P＜0.05).The median overall costs were significantly lower in the HALS group (30000 RMB vs 35000 RMB,P＜0.05).There were no significant differences in the time to return to oral diet,the length of hospital stay,and the time to passage of flatus.The complication rates between the two groups were similar.Conclusion Hand-assisted laparoscopic surgery had the advantages over conventional laparoscopic surgery in minimal invasiveness,safety,and quicker postoperative recovery.