Nie Ying, Yang Bang-kun, Sheng An-qun, Zhang Wei-xi, Li Chang-chong
Department of Pediatrics, Taihe Hospital, Hubei College of Medicine, Shiyan 442000, China.
Department of Pediatric Pulmonology, Yuying Children's Hospital, Wenzhou Medical University, Wenzhou 325027, China. Email:
Zhonghua Yi Xue Za Zhi. 2013 Nov 26;93(44):3532-6.
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.
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).
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).
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.
探讨磷酸肌醇3激酶（PI3K）和Notch信号通路能否对CD4(+)T淋巴细胞的活化和增殖实现协同调控。
将雄性BALB/c小鼠随机分为对照组和哮喘组。然后采用卵清蛋白（OVA）激发法建立小鼠哮喘模型。通过磁珠分选法（MACS）分离CD4(+)T淋巴细胞，并用植物血凝素（PHA）（10 μg/ml）和白细胞介素-2（IL-2）（1000 U/ml）激活6小时。将这些细胞分为A组：未作任何处理；B组：用PI3K抑制剂（LY294002）处理；C组：用Notch抑制剂（γ-分泌酶抑制剂，DAPT）处理；D组：用PI3K抑制剂和Notch抑制剂处理。采用流式细胞术和逆转录聚合酶链反应（RT-PCR）评估CD4(+)T淋巴细胞中细胞周期蛋白A（Cyclin A）、细胞周期蛋白D1（Cyclin D1）和P27(kip1)的蛋白及转录水平。
流式细胞术结果显示，MACS分选的CD4(+)T淋巴细胞纯度为90.0%±5.2%，存活率为94.8%±3.2%。哮喘组CD4(+)T淋巴细胞中Cyclin A和Cyclin D1的蛋白水平（28.0%±3.5%，14.9%±3.4%）及mRNA水平（0.55±0.16，1.38±0.42）显著高于对照组（13.4%±3.5%，7.7%±1.8%及0.32±0.10，0.92±0.37）（P = 0.002，0.036及P = 0.007，0.042）。哮喘组P27(kip1)的蛋白及mRNA水平（23.3%±3.9%及0.16±0.03）显著低于对照组（37.5%±5.8%及0.32±0.03，P = 0.006及P = 0.000）。A、B、C、D组处理的CD4(+)T淋巴细胞中Cyclin D1的蛋白水平分别为12.2%±3.7%、7.3%±3.0%、8.1%±2.3%、4.2%±1.7%，mRNA水平分别为1.71±0.44、1.07±0.31、1.21±0.32、0.62±0.20；B、C、D组与A组相比显著降低（均P < 0.01），而D组与B、C组相比显著降低（均P < 0.05）。A、B、C、D组P27(kip1)的蛋白水平分别为22.9%±3.0%、31.6%±5.3%、28.4%±5.6%、44.6%±2.8%；B组显著高于A组（P = 0.016），而D组显著高于A、B、C组（P = 0.003，0.004，0.000）。同时，各组P27(kip1)的mRNA水平分别为0.16±0.07、0.36±0.09、0.63±0.08、0.99±0.21；B、C、D组显著高于A组（P = 0.016，0.000，0.000），而D组显著高于B、C组（P = 0.000，0.023）。不同实验组间CyclinA的蛋白及mRNA水平无统计学意义（均P > 0.05）。
PI3K和Notch信号通路可能协同上调CD4(+)T淋巴细胞正性调节因子CyclinD1的表达，下调负性调节因子P27(kip1)的表达。
