PI3K和Notch信号通路对哮喘小鼠CD4＋T淋巴细胞活化及增殖的协同调控作用  

Coordinated regulation of PI3K and Notch signal pathways in the activation and proliferation of CD4＋T lymphocytes in asthma mice

作　　者：聂颖[1] 杨邦坤[2] 盛安群 张维溪[3] 李昌崇[3] 

机构地区：[1]湖北医药学院附属太和医院儿科三病区,湖北省十堰市442000 [2]湖北医药学院附属太和医院神经外科二病区,湖北省十堰市442000 [3]温州医科大学附属育英儿童医院呼吸科

出　　处：《中华医学杂志》2013年第44期3532-3536,共5页National Medical Journal of China

基　　金：国家自然科学基金（81100015）;浙江省自然科学基金（Y2090327）

摘　　要：目的 探讨磷酯酰肌醇3激酶（PI3K）和Notch信号通路对哮喘小鼠CD4＋T淋巴细胞活化及增殖的协同调控作用.方法 以卵清白蛋白（OVA）致敏激发法制备16只小鼠哮喘模型,按随机数字表法分为对照组和哮喘组各8只,免疫磁珠法分离哮喘小鼠脾脏原代CD4＋T淋巴细胞.在细胞水平分组：未干预组（A组）,PI3K抑制剂组（LY294002）（B组）,Notch抑制剂组（γ-分泌酶抑制剂,DAPT）（C组）,PI3K抑制剂＋Notch抑制剂组（D组）,均以10 μg/ml的植物血凝素和1000 U/ml的白细胞介素（IL）-2刺激增殖6h后进行干预;用流式细胞术检测CD4＋T淋巴细胞内的细胞周期蛋白（Cyclin）A、Cyclin D1和P27kip1的蛋白表达,反转录-PCR测定各指标的mRNA表达.结果 分选后CD4＋T淋巴细胞纯度达90.0％±5.2％,CD4＋T淋巴细胞的存活率为94.8％±3.2％.哮喘组小鼠脾脏CD4＋T淋巴细胞Cyclin A、Cyclin D1蛋白（28.0％±3.5％、14.9％±3.4％）和mRNA（0.55±0.16、1.38±0.42）表达水平均显著高于对照组（13.4％±3.5％、7.7％±1.8％和0.32±0.10、0.92±0.37）（P=0.002、0.036和P=0.007、0.042）,而哮喘组P27 kip1蛋白和mRNA表达水平（23.3％±3.9％和0.16±0.03）均显著低于对照组（37.5％±5.8％和0.32 ±0.03,P=0.006和P=0.000）.A、B、C、D各干预组哮喘CD4＋T淋巴细胞Cyclin D1蛋白和mRNA表达水平分别为12.2％±3.7％、7.3％±3.0％、8.1％±2.3％、4.2％±1.7％和1.71 ±0.44、1.07 ±0.31、1.21 ±0.32、0.62±0.20,B、C、D组均显著低于A组（均P＜0.叭）,D组均较B、C组下降更为显著（均P＜0.05）.A、B、C、D各组P27 kip1蛋白表达水平分别为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.叭6）,D组均显著高于A、B、C组（P =0.003、0.004、0.000）;mRNA表达水平分别为0.16 ±0.07、0.36±0.09、0.63±0.08、0.99±0.21,B、C、D组均显著高于A组（P=0.016、0.000、0.000）,D组均显著高于B、C组（P 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 P27kip1 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 P27kip1 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 decre

关 键 词：哮喘 小鼠 CD4阳性T淋巴细胞 磷酯酰肌醇3激酶 NOTCH 

分 类 号：R725.6[医药卫生—儿科]