靶向CD33的CAR修饰的NK92细胞对CD33^+急性髓系白血病细胞的杀伤作用

目的:根据抗CD33-sc Fv序列构建CD33-CAR修饰的NK92细胞,探讨其对CD33^+急性髓系白血病(acute myeloid leukemia,AML)细胞的杀伤作用。方法:通过基因合成以及分子克隆技术获得抗CD33-CAR片段,然后将其构建到慢病毒载体上,进行慢病毒包装,将得到的慢病毒转染NK92细胞,用流式细胞术检测细胞转染效率并通过嘌呤霉素筛选得到稳定表达抗CD33-CAR的细胞系CD33-CAR-NK92,利用钙黄绿素释放法检测该细胞的杀伤作用,ELISA法检测细胞因子IFN-γ分泌的变化。结果:成功构建p CDH-CD33-CAR重组慢病毒质粒,慢病毒转导后约18.7%的NK92细胞表达CD33-CAR(命名为CD33-CARNK92细胞),嘌呤霉素筛选后表达CD33-CAR的NK92细胞比例约86.3%。CD33-CAR-NK92细胞对CD33^+AML细胞MOLM-13的杀伤作用明显高于未被基因修饰的NK92细胞(P<0.01),而两者对CD33-肿瘤细胞JURKAT的杀伤作用没有明显差异(P>0.05)。效靶比为2∶1共培养6 h后,经CD33-CAR修饰的NK92细胞相比未被基因修饰的NK92细胞IFN-γ的分泌水平明显升高[(190.97±11.52)vs(88.41±2.75)pg/ml,P<0.01]。结论:CD33-CAR-NK92细胞能特异性识别CD33抗原并杀伤CD33^+AML细胞,其杀伤作用显著高于未被基因修饰的NK92细胞,为进一步开展靶向CD33的CAR-NK92细胞治疗AML的临床转化奠定了实验基础。

Microcalorimetric Study of Energy Metabolism of HSV-2 and FMDV Infection Processes

The metabolic thermogenic power data of the HSV-2 infected HeLa cells and the FMDV infected BHK-21 cells were determined by LKB-2277 bioactivity monitor. The aim of the study was to investigate the difference of the cell metabolism under the action of two different viruses and the effects of hyperthermia and drugs on it. The results illustrated that the metabolic thermogenic power of infected cells was larger than the uninfected ones and there was a significant difference between the metabolism heat released by the two types of infected cells. From the maximal thermal power and total metabolism heat, the infection process was observed to be thermosensitive and could be inhibited by interferon. Our experiments also revealed that 6 month storage of FMDV could attenuate its virulence and infectivity. The study shows that microcalorimetry is a potent tool to investigate the metabolism of virus infection process.