Keqiang Wang1, Yanqiang Hou2, Chenxing Gu3, Dapeng Zhao1, Yanchao Duan4, Zhangshen Ran5, Qinghua Li6, Xiangqi Li7. 1. Department of Clinical Laboratory, The Affiliated Hospital of Taishan Medical University Tai'an, Shandong Province, China. 2. Department of Clinical Laboratory, The First People's Hospital Affiliated to Shanghai Jiaotong University Shanghai, China. 3. Department of Cardiology, The First Affiliated Hospital of General Hospital of Chinese PLA Beijing, China. 4. Department of Hematology, The Affiliated Hospital of Taishan Medical University Tai'an, Shandong Province, China. 5. Department of Medical Examination Center, The Affiliated Hospital of Taishan Medical University Tai'an, Shandong Province, China. 6. Department of Burn and Plastic, The Affiliated Hospital of Taishan Medical University Tai'an, Shandong Province, China. 7. Department of Breast Surgery, The Affiliated Hospital of Taishan Medical University Tai'an, Shandong Province, China.
Abstract
OBJECTIVE: This study aimed to observe the inhibitory effects of mitogen activated protein kinase (MAPK/ERK) specific inhibitor PD98059 on Mtb-Ag activated γδΤ cells and to investigate the role of MAPK in MAPK/ERK pathway of γδΤ cells activated by Mtb-Ag. METHODS: Healthy human peripheral blood mononuclear cell (PBMC) was isolated from the peripheral blood, and then stimulated by phorbol esters (PMA), ionomycin (IM) and Mtb-Ag. PBMC of the experimental group was pretreated by PD98059 of 0, 1, 10 or 100 μmol/L while the control group was given no pretreatment. Flow cytometry was applied to detect the expression of CD69 in γδΤ cells at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment respectively. The influence of PD98059 on the amplification of γδΤ cells was also detected. RESULTS: After stimulated by PMA and IM for 6 hours, the expression of CD69 in γδΤ reached the peak of (99.3±1.09)%, while in the Mtb-Ag stimulation group, it reached the highest level of 75.2% at 24 hours. The level of CD69 in two group was significant difference between the two groups at each time respectively (p<0.05). Pretreated with PD98059 of 0, 1, 10, 100 μmol/L, the level of CD 69 expression in Mtb-Ag stimulated γδΤ was 79±0.8%, 75±0.7%, 54±0.5% and 17±0.2% respectively; In PMA+IM stimulated group, CD69 expression were all more than 98% in different concentration of PD98059. After treated with PD98059 of 0, 1, 10, 100 μmol/L, total cell number increased from 1.5×106 to (10.3±2.5)×106, (9.5±2.1)×106, (5.8±1.8)×106 and (2.1±0.5)×106 respectively. Number of γδΤ cells reached to (6.2±0.9)×106, (5.02±0.8)×106, (2.05±0.5)×106 and (0.41±0.1)×106 respectively. CONCLUSION: γδΤ cells were specifically activated by Mtb-Ag, and the activation was depended on MAPK/ERK pathway. The activation effects of Mtb-Ag on γδΤ cells can be obviously inhibited by PD98059. IJCEP
OBJECTIVE: This study aimed to observe the inhibitory effects of mitogen activated protein kinase (MAPK/ERK) specific inhibitor PD98059 on Mtb-Ag activated γδΤ cells and to investigate the role of MAPK in MAPK/ERK pathway of γδΤ cells activated by Mtb-Ag. METHODS: Healthy human peripheral blood mononuclear cell (PBMC) was isolated from the peripheral blood, and then stimulated by phorbol esters (PMA), ionomycin (IM) and Mtb-Ag. PBMC of the experimental group was pretreated by PD98059 of 0, 1, 10 or 100 μmol/L while the control group was given no pretreatment. Flow cytometry was applied to detect the expression of CD69 in γδΤ cells at 0 h, 6 h, 12 h, 24 h, 48 h and 72 h after treatment respectively. The influence of PD98059 on the amplification of γδΤ cells was also detected. RESULTS: After stimulated by PMA and IM for 6 hours, the expression of CD69 in γδΤ reached the peak of (99.3±1.09)%, while in the Mtb-Ag stimulation group, it reached the highest level of 75.2% at 24 hours. The level of CD69 in two group was significant difference between the two groups at each time respectively (p<0.05). Pretreated with PD98059 of 0, 1, 10, 100 μmol/L, the level of CD 69 expression in Mtb-Ag stimulated γδΤ was 79±0.8%, 75±0.7%, 54±0.5% and 17±0.2% respectively; In PMA+IM stimulated group, CD69 expression were all more than 98% in different concentration of PD98059. After treated with PD98059 of 0, 1, 10, 100 μmol/L, total cell number increased from 1.5×106 to (10.3±2.5)×106, (9.5±2.1)×106, (5.8±1.8)×106 and (2.1±0.5)×106 respectively. Number of γδΤ cells reached to (6.2±0.9)×106, (5.02±0.8)×106, (2.05±0.5)×106 and (0.41±0.1)×106 respectively. CONCLUSION: γδΤ cells were specifically activated by Mtb-Ag, and the activation was depended on MAPK/ERK pathway. The activation effects of Mtb-Ag on γδΤ cells can be obviously inhibited by PD98059. IJCEP