CAJ | 학술논문

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 =0.000、0.023).A、B、C、D各组CD4+T淋巴细胞Cyclin A蛋白和mRNA表达水平差异均无统计学意义(均P＞0.05).结论 PI3K信号通路与Notch信号通路通过共同促进CD4+T淋巴细胞内的正性调控因子Cyclin D1表达的上调和负性调控因子P27 kip1表达的下调,而起到协同调控哮喘CD4+T淋巴细胞的活化及增殖的作用. 目的探討燐酯酰肌醇3激酶(PI3K)和Notch信號通路對哮喘小鼠CD4+T淋巴細胞活化及增殖的協同調控作用.方法以卵清白蛋白(OVA)緻敏激髮法製備16隻小鼠哮喘模型,按隨機數字錶法分為對照組和哮喘組各8隻,免疫磁珠法分離哮喘小鼠脾髒原代CD4+T淋巴細胞.在細胞水平分組:未榦預組(A組),PI3K抑製劑組(LY294002)(B組),Notch抑製劑組(γ-分泌酶抑製劑,DAPT)(C組),PI3K抑製劑+Notch抑製劑組(D組),均以10 μg/ml的植物血凝素和1000 U/ml的白細胞介素(IL)-2刺激增殖6h後進行榦預;用流式細胞術檢測CD4+T淋巴細胞內的細胞週期蛋白(Cyclin)A、Cyclin D1和P27kip1的蛋白錶達,反轉錄-PCR測定各指標的mRNA錶達.結果分選後CD4+T淋巴細胞純度達90.0％±5.2％,CD4+T淋巴細胞的存活率為94.8％±3.2％.哮喘組小鼠脾髒CD4+T淋巴細胞Cyclin A、Cyclin D1蛋白(28.0％±3.5％、14.9％±3.4％)和mRNA(0.55±0.16、1.38±0.42)錶達水平均顯著高于對照組(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),而哮喘組P27 kip1蛋白和mRNA錶達水平(23.3％±3.9％和0.16±0.03)均顯著低于對照組(37.5％±5.8％和0.32 ±0.03,P=0.006和P=0.000).A、B、C、D各榦預組哮喘CD4+T淋巴細胞Cyclin D1蛋白和mRNA錶達水平分彆為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組均顯著低于A組(均P＜0.叭),D組均較B、C組下降更為顯著(均P＜0.05).A、B、C、D各組P27 kip1蛋白錶達水平分彆為22.9％±3.0％、31.6％±5.3％、28.4％±5.6％、44.6％±2.8％,B組顯著高于A組(P=0.叭6),D組均顯著高于A、B、C組(P =0.003、0.004、0.000);mRNA錶達水平分彆為0.16 ±0.07、0.36±0.09、0.63±0.08、0.99±0.21,B、C、D組均顯著高于A組(P=0.016、0.000、0.000),D組均顯著高于B、C組(P =0.000、0.023).A、B、C、D各組CD4+T淋巴細胞Cyclin A蛋白和mRNA錶達水平差異均無統計學意義(均P＞0.05).結論 PI3K信號通路與Notch信號通路通過共同促進CD4+T淋巴細胞內的正性調控因子Cyclin D1錶達的上調和負性調控因子P27 kip1錶達的下調,而起到協同調控哮喘CD4+T淋巴細胞的活化及增殖的作用.
목적 탐토린지선기순3격매(PI3K)화Notch신호통로대효천소서CD4+T림파세포활화급증식적협동조공작용.방법 이란청백단백(OVA)치민격발법제비16지소서효천모형,안수궤수자표법분위대조조화효천조각8지,면역자주법분리효천소서비장원대CD4+T림파세포.재세포수평분조:미간예조(A조),PI3K억제제조(LY294002)(B조),Notch억제제조(γ-분비매억제제,DAPT)(C조),PI3K억제제+Notch억제제조(D조),균이10 μg/ml적식물혈응소화1000 U/ml적백세포개소(IL)-2자격증식6h후진행간예;용류식세포술검측CD4+T림파세포내적세포주기단백(Cyclin)A、Cyclin D1화P27kip1적단백표체,반전록-PCR측정각지표적mRNA표체.결과 분선후CD4+T림파세포순도체90.0％±5.2％,CD4+T림파세포적존활솔위94.8％±3.2％.효천조소서비장CD4+T림파세포Cyclin A、Cyclin D1단백(28.0％±3.5％、14.9％±3.4％)화mRNA(0.55±0.16、1.38±0.42)표체수평균현저고우대조조(13.4％±3.5％、7.7％±1.8％화0.32±0.10、0.92±0.37)(P=0.002、0.036화P=0.007、0.042),이효천조P27 kip1단백화mRNA표체수평(23.3％±3.9％화0.16±0.03)균현저저우대조조(37.5％±5.8％화0.32 ±0.03,P=0.006화P=0.000).A、B、C、D각간예조효천CD4+T림파세포Cyclin D1단백화mRNA표체수평분별위12.2％±3.7％、7.3％±3.0％、8.1％±2.3％、4.2％±1.7％화1.71 ±0.44、1.07 ±0.31、1.21 ±0.32、0.62±0.20,B、C、D조균현저저우A조(균P＜0.팔),D조균교B、C조하강경위현저(균P＜0.05).A、B、C、D각조P27 kip1단백표체수평분별위22.9％±3.0％、31.6％±5.3％、28.4％±5.6％、44.6％±2.8％,B조현저고우A조(P=0.팔6),D조균현저고우A、B、C조(P =0.003、0.004、0.000);mRNA표체수평분별위0.16 ±0.07、0.36±0.09、0.63±0.08、0.99±0.21,B、C、D조균현저고우A조(P=0.016、0.000、0.000),D조균현저고우B、C조(P =0.000、0.023).A、B、C、D각조CD4+T림파세포Cyclin A단백화mRNA표체수평차이균무통계학의의(균P＞0.05).결론 PI3K신호통로여Notch신호통로통과공동촉진CD4+T림파세포내적정성조공인자Cyclin D1표체적상조화부성조공인자P27 kip1표체적하조,이기도협동조공효천CD4+T림파세포적활화급증식적작용.
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 decreased significantly compared with groups B and C (all P ＜ 0.05).The protein levels of P27kip1 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 P27kip1 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 P27kip1 of CD4 +T lymphocytes.