小鼠抗人CD28单抗对Jurkat细胞辐射敏感性的影响及机制研究OA

Objective:To research the effect and mechanism of mouse anti-human CD28 mAb on radiosensitivity of Jurkat cells,providing reference for radiation therapy of acute lymphoblastic leukemia.Methods:①6E8 mAb was prepared via ascites induc-tion,affinity purified using Protein A immunochromatography,and its activity was confirmed by flow cytometry;②Effect of 6E8 mAb on in vitro viability of Jurkat cells was detected by CCK8 assay;③Cells were grouped into blank group,6E8 group(5 μg/mL),γ-ray irradiation at 3 Gy+IgG group(5 μg/mL),γ-ray irradiation at 6 Gy+IgG group(5 μg/mL),γ-ray irradiation at 3 Gy+GM-27197AB group(5 μg/mL),γ-ray irradiation at 6 Gy+GM-27197AB group(5 μg/mL),γ-ray irradiation at 3 Gy+6E8 group(5 μg/mL)and γ-ray irradiation at 6 Gy+6E8 group(5 μg/mL);④Apoptosis of cells in each experimental group was detected by flow cytometry,respec-tively;⑤DNA damage of cells in each experimental group was detected by immunofluorescence;⑥Label-free proteomics was used to compare differences in protein expression between 6E8 mAb and IgG isotype control groups in Jurkat cells,and parallel reaction moni-toring(PRM)was used for verification;⑦Differential proteins screened by mass spectrometry were subjected to GO annotation and KEGG enrichment analysis.Results:①Concentration of purified 6E8 mAb was 2.4 mg/mL,which positive binding rate to Daudi tumor cells was 92.3%;②6E8 mAb(5 μg/mL)was non-toxic to Jurkat cells and did not affect cell viability;③Compared with IgG group,6E8 mAb increased apoptosis rate of Jurkat cells induced by γ-radiation(P<0.01);④Compared with IgG control group,6E8 mAb significantly increased degree of radiation-induced DNA damage;⑤Compared with IgG group,139 differentially expressed proteins were identified in 6E8 group(59 up-regulated,80 down-regulated).PRM-based quantitative verification aligned with proteomics results;⑥GO analysis of differentially proteins mainly involved in mRNA catabolism,apoptosis signaling pathway regulation,DNA binding regulation and other biological processes.Differential proteins were mainly located in ribosome subunits,ribosomes,mitochondria and other organelles.In terms of molecular functions,most of them were involved in regulating structural components of ribosomes and activity of enzyme inhibitors and so on.KEGG revealed that differential proteins were involved in apoptosis-related pathways in Jurkat cells.Such as apoptosis,autophagy,cellular senescence,GnRH,Hippo,MAPK,nucleotide excision repair,EGFR,VEGF,etc.Conclusion:6E8 mAb alters protein expression profile and apoptotic pathway by specifically binding to CD28 molecule on cell mem-brane surface of Jurkat,enhances radiation sensitivity,and promotes cell apoptosis.