Ying Nie1, Bang-kun Yang, An-qun Sheng, Wei-xi Zhang2, Chang-chong Li. 1. Department of Pediatrics, Taihe Hospital, Hubei College of Medicine, Shiyan 442000, China. 2. Department of Pediatric Pulmonology, Yuying Children's Hospital, Wenzhou Medical University, Wenzhou 325027, China. Email:zhangweixi112@163.com.
Abstract
OBJECTIVE: To explore whether the signal pathways of phosphoinositide 3-kinase (PI3K) and Notch can realize coordinated regulation on the activation and proliferation of CD4(+)T lymphocytes. METHODS: Male BALB/c mice were randomly divided into control and asthma groups. Then the murine model of asthma was established by the method of ovalbumin (OVA) challenge. The CD4(+)T lymphocytes were isolated by magnetic activated cell sorter (MACS) and then activated with phytohaemagglutinin (PHA) (10 µg/ml) and IL-2 (1000 U/ml) for 6 h. Those cells were then divided into Group A: without any treatment; Group B: treatment with PI3K inhibitor (LY294002); Group C: treatment with Notch inhibitor (gamma-secretase inhibitor, DAPT); Group D: treatment with PI3K inhibitor and Notch inhibitor. The protein and transcription levels of Cyclin A, Cyclin D1 and P27(kip1) of CD4(+)T lymphocytes were assessed by flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The results of flow cytometry showed that the purity of MACS-isolated CD4(+)T lymphocytes was 90.0% ± 5.2% and the survival rate 94.8% ± 3.2%. The protein (28.0% ± 3.5%, 14.9% ± 3.4%) and mRNA levels (0.55 ± 0.16, 1.38 ± 0.42) of Cyclin A and Cyclin D1 in CD4(+)T lymphocytes of asthma group were significantly higher than those of the control group (13.4% ± 3.5%, 7.7% ± 1.8% and 0.32 ± 0.10, 0.92 ± 0.37) (P = 0.002, 0.036 and P = 0.007, 0.042). The protein and mRNA levels (23.3% ± 3.9% and 0.16 ± 0.03) of P27(kip1) of asthma group were significantly lower than those of control group (37.5% ± 5.8% and 0.32 ± 0.03, P = 0.006 and P = 0.000). The protein and mRNA levels of Cyclin D1 in groups A, B, C and D-treated CD4(+)T lymphocytes were 12.2% ± 3.7%, 7.3% ± 3.0%, 8.1% ± 2.3%, 4.2% ± 1.7% and 1.71 ± 0.44, 1.07 ± 0.31, 1.21 ± 0.32 and 0.62 ± 0.20 respectively; groups B, C and D decreased markedly compared with group A (all P < 0.01) while group D decreased significantly compared with groups B and C (all P < 0.05). The protein levels of P27(kip1) in groups A, B, C and D were 22.9% ± 3.0%, 31.6% ± 5.3%, 28.4% ± 5.6% and 44.6% ± 2.8% respectively; group B was significantly higher than that of group A (P = 0.016) while group D was significantly higher than those of groups A, B and C (P = 0.003, 0.004, 0.000). Meanwhile P27(kip1) mRNA levels in each group were 0.16 ± 0.07, 0.36 ± 0.09, 0.63 ± 0.08 and 0.99 ± 0.21 respectively; groups B, C and D were much higher than that of group A (P = 0.016, 0.000, 0.000) while group D was significantly higher than those of groups B and C (P = 0.000, 0.023). The protein and mRNA levels of CylinA showed no statistical significance among different experimental groups (all P > 0.05). CONCLUSION: The signal pathways of PI3K and Notch may coordinately up-regulate the expression of positive regulatory factor cylinD1 and down-regulation the expression of negative regulatory factor P27(kip1) of CD4(+)T lymphocytes.
OBJECTIVE: To explore whether the signal pathways of phosphoinositide 3-kinase (PI3K) and Notch can realize coordinated regulation on the activation and proliferation of CD4(+)T lymphocytes. METHODS: Male BALB/c mice were randomly divided into control and asthma groups. Then the murine model of asthma was established by the method of ovalbumin (OVA) challenge. The CD4(+)T lymphocytes were isolated by magnetic activated cell sorter (MACS) and then activated with phytohaemagglutinin (PHA) (10 µg/ml) and IL-2 (1000 U/ml) for 6 h. Those cells were then divided into Group A: without any treatment; Group B: treatment with PI3K inhibitor (LY294002); Group C: treatment with Notch inhibitor (gamma-secretase inhibitor, DAPT); Group D: treatment with PI3K inhibitor and Notch inhibitor. The protein and transcription levels of Cyclin A, Cyclin D1 and P27(kip1) of CD4(+)T lymphocytes were assessed by flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The results of flow cytometry showed that the purity of MACS-isolated CD4(+)T lymphocytes was 90.0% ± 5.2% and the survival rate 94.8% ± 3.2%. The protein (28.0% ± 3.5%, 14.9% ± 3.4%) and mRNA levels (0.55 ± 0.16, 1.38 ± 0.42) of Cyclin A and Cyclin D1 in CD4(+)T lymphocytes of asthma group were significantly higher than those of the control group (13.4% ± 3.5%, 7.7% ± 1.8% and 0.32 ± 0.10, 0.92 ± 0.37) (P = 0.002, 0.036 and P = 0.007, 0.042). The protein and mRNA levels (23.3% ± 3.9% and 0.16 ± 0.03) of P27(kip1) of asthma group were significantly lower than those of control group (37.5% ± 5.8% and 0.32 ± 0.03, P = 0.006 and P = 0.000). The protein and mRNA levels of Cyclin D1 in groups A, B, C and D-treated CD4(+)T lymphocytes were 12.2% ± 3.7%, 7.3% ± 3.0%, 8.1% ± 2.3%, 4.2% ± 1.7% and 1.71 ± 0.44, 1.07 ± 0.31, 1.21 ± 0.32 and 0.62 ± 0.20 respectively; groups B, C and D decreased markedly compared with group A (all P < 0.01) while group D decreased significantly compared with groups B and C (all P < 0.05). The protein levels of P27(kip1) in groups A, B, C and D were 22.9% ± 3.0%, 31.6% ± 5.3%, 28.4% ± 5.6% and 44.6% ± 2.8% respectively; group B was significantly higher than that of group A (P = 0.016) while group D was significantly higher than those of groups A, B and C (P = 0.003, 0.004, 0.000). Meanwhile P27(kip1) mRNA levels in each group were 0.16 ± 0.07, 0.36 ± 0.09, 0.63 ± 0.08 and 0.99 ± 0.21 respectively; groups B, C and D were much higher than that of group A (P = 0.016, 0.000, 0.000) while group D was significantly higher than those of groups B and C (P = 0.000, 0.023). The protein and mRNA levels of CylinA showed no statistical significance among different experimental groups (all P > 0.05). CONCLUSION: The signal pathways of PI3K and Notch may coordinately up-regulate the expression of positive regulatory factor cylinD1 and down-regulation the expression of negative regulatory factor P27(kip1) of CD4(+)T lymphocytes.