Ca2＋／CaN-NFATc信号通路在哮喘大鼠淋巴细胞增殖和活化中的作用

目的探讨Ca2＋／CaN-NFATc信号通路在哮喘大鼠淋巴细胞活化、增殖中的作用。方法哮喘组和CsA（cyclospoin A，CaN的抑制物，环孢菌素A）干预组大鼠以卵蛋白溶液致敏及激发，对照组以生理盐水致敏及激发，无菌分离脾淋巴细胞，加PHA—P（终浓度为5μg／ml）培养24h，CsA干预组同时加入CsA稀释液（终浓度为1．0μg／ml）。检测淋巴细胞内Ca2＋]i浓度、CaN活性、去磷酸化NFATc蛋白和CyclinE蛋白的表达、细胞周期各时相分布及上清液中IL-4和IL-2的水平。结果与对照组相比，哮喘组淋巴细胞Ca2＋／CaN-NFATc的活性（81．21±14．39）V8（63．66±9．02）]增加（P〈0．05）；CyclinE蛋白的表达量（0．9327±0．0370）VS（0．8374±0．0637）]增加（P〈0．01），处于G0／G1期的淋巴细胞比例（89．3300±3．3850）VS（94．1260±1．4389）]减少，s期（7．8600±2．8241）VS（4．0270±1．8650）]及S＋G2／M期比例（10．6700±3．3850）V8（5．8740±1．4389）]增加（P均〈0．01））；上清液中IL4水平（1．55±0．19）pg／ml vs（0．99±0．12）pg／m1]及IL-4／IL-2比值（0．81±0．12）vs（0．49±0．49）]增加（P均〈0．01）。CsA可使哮喘大鼠淋巴细胞Ca2＋／CaN—NFATc的活性（47．19±7．16）下降（P〈0．05）；CyclinE蛋白的表达量（0．6840±0．0485）减少（P〈0．01），使处于S期（4．8600±1．9595）和S＋G2／M期（7．9900±1．9405）的淋巴细胞比例减少，G0／G1期比例（92．2100±1．9267）增加（P均〈0．01），细胞增殖受到抑制；使上清液中IL-4（0．47±0．09）pg／ml水平减少（P〈0．01），IL-4／IL-2比值下降（0．78±0．20）；淋巴细胞Ca2＋／CaN-NFATc的活性与IL4的水平及CyclinE蛋白的表达量均呈正相关（r=0．711，P〈0．01和r=0．749，P〈0．01）。结论哮喘大鼠淋巴细胞Ca2＋／CaN-NFATc的活性增加；其活性的增加可通过促进IL4的产生和分泌，导致Th1／Th2的失衡，也可通过促进Cyclin E蛋白的表达而引起淋巴细胞的增殖。

Role of the Ca2 +/CaN-NFATc signal pathway in the proliferation and activation of lymphocyte in asthmatic rats

Objective To evaluate the effect of the activity of Ca2＋/CaN-NFATc on the activation and proliferation of lymphocyte in asthmatic rats. Methods The rats of the asthma group and the CsA group were sensitized and challenged by ovalbumin. So did the control group with saline instead. Lympho- cyte was separated from spleen and cultured for 24 hours, and PHA-p （5μg/ml） was added to the culture medium in every group , CsA （1.0 μg,/ml） was added to the CsA group, respectively. The concentration of Ca2 ＋ ] i , the activity of CaN, the protein expression of dephosphorylated NFATc and Cyclin E in T lym- phocyte were assayed. The level of IL-4 and IL-2 in culture supernatants was measured, and the cell cycle distribution of lymphocyte was analyzed. Results When compared to the control group, the activity of Ca2＋/CaN-NFATc （81.21±14. 39） vs （63.66 ±9.02） ] was increased and the protein expression of CyclinE （0. 9327 ±0. 0370） vs （0. 8374 ±0. 0637） ] was higher in Lymphocyte of the asthma group （ P 〈0. 05, P 〈0. 01, respectively）. The percentage of lymphocyte in the S phase （7. 8600 ±2. 8241） vs （4. 0270 ±1. 8650） ] and S ± G2/M phase （ 10. 6700 ± 3. 3850） vs （5. 8740 ± 1. 4389） ] was higher; however, the percentage of G0/G1 phase （ 89. 3300 ± 3. 3850） vs （ 94. 1260 ± 1. 4389 ） ] was lower in asthma group （ all P 〈 0. O1 ）. The level of IL-4 （ 1.55 ± 0. 19） pg/ml vs （0. 99 ± 0. 12 ） pg/ml ] and the IL-4/IL-2 ratio （0. 81 ±0. 12） vs（0, 49 ±0. 49） ] in culture supernatants of the asthma group were high- er than those of the control group （ all P 〈 0.01 ）. While the activity of Ca2 ＋ / CaN-NFATc （47.19 ± 7.16） was decreased and the protein expression of Cyclin E （0. 6840 ± 0. 0485 ） was reduced in lymphocyte in CsA group（ all P 〈 0. 01 ）, the percentage of lymphocyte in the S phase （4. 8600 ± 1. 9595） and S ＋ GJM phase （7. 9900 ± 1. 9405） was decreased and the percentage of G0/G1 phase （92. 2100 ± 1. 9267） was increased （ all P 〈 0. 05 ） , and the level of IL-4 （0.47 ± 0.09 ） pg/ml and the ratio of IL-4/ IL-2 （0. 78 ± 0. 20） was lower in culture supernatants in the CsA group than that of the asthma group（all P 〈0.01 ）. There was a positive correlation between the protein expression of dephosphorylated NFATe and the protein expression of CyclinE in Lymphocyte, so did between the protein expression of NFATc and the level of IL-4 in culture supernatants（ r = 0.711, P 〈 0.01 and r = 0. 749, P 〈 0.01. respectively）. Conclusions The activity of Ca2 ＋ / CaN-NFATc was increased in lymphocyte of the asthmatic rats. Its increasing might result in the imbalance of Th1/Th2 by promoting the expression of IL-4 and might lead to the proliferation of lymphocyte by promoting the Cyclin E expression.