A sample preparation method was developed to simulate the process of intracellular metabolites metabonomics analysis of Escherichia coli. The Escherichia coli cell was firstly quenched with cold sodium chloride solution ( 0. 85 %，precooled at -80℃ for 15 min).The quenched bacterial cell was treated by using the technique of vacuum freeze-drying and liquid nitrogen freezing combined with ultrasonic processing to increase cell membrane penetrability. Finally，a cold aqueous solution of methanol (MeOH:H2O, 1:1，V/ V, 4 ℃) was used as extraction solvent to extract metabolites. In the present research，flow cytometry and OD value recovery were performed to evaluate the degree of cell damage caused by quenching at single cell level and at integral level respectively. The tested results indicated that the degree of damage to cells caused by cold sodium chloride solution was less than 5%. The peak quantity and the total ion intensity detected by LC-TOF in low collision energy were used to evaluate extraction effects. Three different cell membrane penetrability modes and 4 kinds of extraction solvents were investigated and compared.The results showed that the technique of liquid nitrogen freezing combined with ultrasonic processing for cell membrane penetrability and a cold aqueous solution of methanol (MeOH/H2O，1:1，V/V, 4℃) for extraction of metabolites had the best extraction effect(peak quantity was greater than or equal to 105，and total ion intensity was in the range of 106-107).Therefore，in this work，the freeze drying，grinding with liquid nitrogen and ultrasonic extraction were combined to extract metabolites. In this way，it effectively promoted cell lysis and improved the efficiency of extraction. The result of synthetic analysis showed that the method proposed here could meet the requirements of the metabonomics analysis of Escherachaa coli.

[摘 要] 目的：通过构建表达IL-12的小鼠CAR-T细胞，探讨经尾静脉将其输注于小鼠体内建立细胞因子释放综合征（CRS）模型的方法。方法：构建基于靶向鼠源CD19的CAR分子，包装逆转录病毒载体并感染小鼠T细胞构建mCD19-CAR-T、mCD19/IL-12-CAR-T细胞。通过构建小鼠体内胰腺癌Panc02-CD19细胞移植瘤模型，检测mCD19/IL-12-CAR-T细胞的抗肿瘤活性，ELISA法检测两种CAR-T细胞IL-12和IFN-γ分泌水平；经小鼠尾静脉输注mCD19/IL-12-CAR-T 细胞构建CAR-T细胞CRS小鼠模型，流式细胞术检测小鼠血清中IL-6、MCP-1、IL-1、IL-10、TNF-α、IFN-γ等细胞因子的含量，H-E染色法观察荷瘤小鼠肝、脾、肺和肾的病理组织学变化。结果：经过培养扩增的mCD19/IL-12-CAR-T细胞能有效分泌IL-12，CAR阳性率达（56.9±5.4）%；与非靶细胞Panc02或靶细胞Panc02-CD19共培养时，均能高分泌IFN-γ。成功构建小鼠胰腺癌Panc02-CD19细胞移植瘤模型，经小鼠尾静脉注射1×106个mCD19/IL-12-CAR-T细胞能显著抑制移植瘤的生长，但未能诱发严重CRS；输注2×106个mCD19/IL-12-CAR-T细胞后，小鼠出现体质量减轻、血清炎性因子水平升高、组织损伤，最终导致死亡等一系列典型CRS表现。结论：成功构建IL-12-CAR-T细胞诱发的小鼠CRS模型，其稳定性好、重复性高，具有广泛的应用前景。

目的：通过向C57Bl/6J小鼠腹腔注射IFN-γ腺病毒（Ad-mIFN-γ）建立细胞因子释放综合征（CRS）的动物模型。方法：构建Ad-mIFN-γ及对照Ad-lacZ腺病毒载体，分别以MOI=100体外转染小鼠腹腔巨噬细胞，流式细胞术检测其对细胞mIFN-γ分泌的影响。将40只雌性C57Bl/6J小鼠按随机数字表法分为对照组、载体对照组、病毒低、中、高剂量组（每组8只），分别向各组小鼠腹腔注射PBS（200 μL）、Ad-lacZ（2×107 PFU/只）、Ad-mIFN-γ（5×106 PFU/只）、Ad-mIFN-γ（1.5×107 PFU/只）和Ad-mIFN-γ（2×107 PFU /只）。每日观测小鼠的体质量及生存情况；第3天时采用流式细胞术检测小鼠外周血和脾内单核细胞（CD11b+）、巨噬细胞（CD11b+/CD86+）比例，免疫荧光染色法检测脾内CD11b+的单核细胞比例；第9天时采用流式细胞术检测小鼠血清中细胞因子的分泌水平；第14天，采用颈椎脱臼法处死小鼠，H-E染色法观察小鼠肝、脾、肺和肾的病理和组织学变化。结果： Ad-mIFN-γ体外感染小鼠腹腔巨噬细胞，在第3天检测到巨噬细胞分泌mIFN-γ达到峰值（118.34±2.90）pg/mL，并在一周内持续高分泌mIFN-γ，Ad-lacZ对照组IFN-γ分泌水平较低后，第3天时为（0.17±0.08）pg/mL。小鼠腹腔注射Ad-mIFN-γ后，在14 d内病毒低、中剂量组无小鼠死亡，病毒高剂量组小鼠体质量持续减轻（P<0.001）；第3天，病毒高剂量组小鼠外周血和脾组织内单核细胞、巨噬细胞比例较对照组和中剂量组均显著增加（P<0.05或P<0.01）；第9天，病毒低、中、高剂量组小鼠血清中mIFN-γ、IL-6、单核细胞趋化蛋白-1（MCP-1）、IL-1、TNF-α等细胞因子的水平均显著升高（P<0.001）；10 d内病毒高剂量组小鼠死亡率达100%。组织病理检测可见病毒高剂量组小鼠的肝、脾、肺、肾组织有明显损伤。结论： Ad-mIFN-γ体外感染小鼠原代腹腔巨噬细胞后，可以快速分泌mIFN-γ；腹腔注射高剂量（2×107 PFU/只）Ad-mIFN-γ导致小鼠出现CRS典型表现，可作为CAR-T细胞治疗诱发CRS的动物模型。