[摘 要] 目的：制备双特异性CAR-T（bsCAR-T）细胞，观察其对表达表皮生长因子Ⅲ型突变阳性（EGFRvⅢ+，简称vⅢ+）和CD133+胶质瘤干细胞的靶向杀伤作用。方法：基于前期研制的vⅢ/CD133双特异性微抗体和二代CAR构建的双特异性CAR（bsCAR），制备慢病毒载体转染人外周血T细胞，FCM和WB法检测bsCAR转染效率和表达水平。bsCAR-T细胞和vⅢ+/CD133+ U87胶质瘤干细胞共培养，乳酸脱氢酶（LDH）释放实验、IFN-γ分泌实验检测其特异性杀伤作用和对IFN-γ分泌的促进作用。制备裸鼠vⅢ+/CD133+ U87干细胞移植瘤模型检测bsCAR-T细胞对移植瘤生长的抑制作用。结果：vⅢscFv和CD133scFv通过重叠PCR无缝连接入二代CAR表达框（S-vⅢscFv/CD133scFv-Hinge-TM-CD137-CD3z）中，然后克隆入pCDH-MSCV-MCS-EF1-copGFP载体的EcoRⅠ和BamHⅠ位点（pbsCAR）。3种质粒（pVSV-G、pCMV-dR8.9和pbsCAR）共转染HEK293T细胞制备慢病毒载体，转染外周血T细胞，FCM检测bsCAR表达率为71.1%，WB法结果显示bsCAR表达正确。bsCAR-T细胞和vⅢ+/CD133+ U87干细胞共培养检测结果显示，bsCAR-T细胞对胶质瘤干细胞具有特异性杀伤作用，与效靶比呈正比；IFN-γ分泌量为（2 350.6±92） pg·mL-1，明显高于对照组（P<0.01）。裸鼠移植瘤动物模型显示，bsCAR-T细胞在体内具有明显的移植瘤抑制作用（P<0.01）。结论： bsCAR-T细胞能够特异性靶向杀伤vⅢ+/CD133+胶质瘤干细胞，实验结果为促进实体瘤的细胞免疫治疗提供了实验依据。

ObjectiveTo investigate the influencing factors for the clinical effect of emergency endoscopic therapy in the treatment of patients with acute-on-chronic liver failure and gastroesophageal variceal bleeding. MethodsA total of 51 patients with acute-on-chronic liver failure and gastroesophageal variceal bleeding who underwent emergency endoscopic therapy in Beijing Shijitan Hospital and The Fifth Medical Center of Chinese PLA General Hospital from January 2016 to December 2018 were enrolled, among whom 26 had successful hemostasis and 25 had failed hemostasis. The two groups were compared in terms of general information, varices grade and bleeding manifestations under endoscope, blood biochemical parameters, ultrasound findings, Child-Pugh class, and Model for End-Stage Liver Disease (MELD) score, and the influencing factors for the outcome of hemostasis were analyzed. The t-test or the Mann-Whitney U test was used for comparison of continuous data between two groups, and the chi-square test was used for comparison of categorical data between two groups; the logistic regression model was used to perform the multivariate analysis. ResultsOf all patients, 26 achieved successful hemostasis, with a success rate of hemostasis of 51%. There were no significant differences between the two groups in sex, age, etiology of liver cirrhosis, presence or absence of liver cancer, presence or absence of portal vein thrombosis, bleeding for the first time or not, white blood cell count, hemoglobin, platelet count, prothrombin time activity, alanine aminotransferase, total bilirubin, albumin, cholinesterase, MELD score, and bleeding site and bleeding manifestations under gastroscope (all P＞0.05). Compared with the failed hemostasis group, the successful hemostasis group had a significantly longer course of disease (t=2.760, P=0.008) and significantly larger portal vein diameter and diameter of varicose veins under endoscope (t=-4.847, χ2=-6.590, both P＜0.05), and the failed hemostasis group had a significantly higher proportion of patients with Child-Pugh class C disease than the successful hemostasis group (χ2=5.684, P=0.017). Course of liver cirrhosis (odds ratio ［OR］=0.913, 95% confidence interval ［CI］: 0.838-0.994, P＜0.05), portal vein diameter (OR=1.925,95%CI: 1.516-2.443, P＜0.05), and diameter of varicose veins (OR=23.254, 95%CI: 2.250-240.352, P＜0.05) were independent influencing factors for the clinical effect of endoscopic hemostasis. ConclusionThere is a relatively low success rate of emergency endoscopic hemostasis in patients with acute-on-chronic liver failure, and course of liver cirrhosis, portal vein diameter, and diameter of varicose veins are independent influencing factors for the clinical effect of endoscopic hemostasis.