催乳素对葡萄球菌肠毒素A诱导T细胞凋亡的影响

Effects of prolactin on T cell apoptosis induced by staphylococcal enterotoxin A

目的:观察作为重要细胞因子的催乳素在T细胞活化诱导凋亡过程中的作用。方法:实验于2005-03/2006-06在校级实验室新乡医学院免疫学实验室完成。实验材料:正常人外周血由新乡医学院第一附属医院提供,葡萄球菌肠毒素A(sigma)。实验方法:①T细胞活化诱导凋亡模型的建立:取健康人外周血5mL,加入等体积淋巴细胞分离液,采用密度梯度离心法获得淋巴细胞。完全1640培养基重悬细胞,加入葡萄球菌肠毒素A(SEA),置于37℃、体积分数为0.05的CO2培养,至第4天,800r/min离心10min,以新鲜完全1640培养基换液,尽可能除去SEA。培养液中加入人白细胞介素2继续培养2周待用。②催乳素干预:将获得细胞离心并弃去培养液,用新鲜完全1640培养基重悬,调整细胞密度为5×109L-1,加入24孔板中,分别加入4mg/LSEA诱导细胞凋亡,同时加入20,300,1000μg/L催乳素,以不加催乳素的为对照组。③实验评估:于培养0,16,24h时采用MTT法检测T细胞增殖情况。培养16h时用流式技术检测细胞凋亡情况,并通过琼脂糖凝胶电泳检测T细胞凋亡DNA,并采用流式细胞术检测T细胞膜表面Fas和FasL,WesternBlot法检测T细胞内凋亡相关蛋白Bcl-2和Bax。结果:①MTT法检测T细胞增殖:不同质量浓度催乳素干预组在16,24h的T细胞数量较对照组明显增多[吸光度值:对照组:0.65±0.02,0.54±0.04;20μg/L催乳素组:0.81±0.04,0.71±0.11;300μg/L催乳素组:1.14±0.10,1.22±0.10;1000μg/L催乳素组:1.12±0.12,1.22±0.12,P<0.01]。0～24h内300,1000μg/L催乳素组T细胞数量呈上升趋势(P<0.01),20μg/L催乳素组T细胞数量呈下降趋势(P<0.01)。②流式细胞术检测T细胞凋亡率:培养16h时,与对照组比较,各质量浓度催乳素干预组T细胞凋亡率降低[(46.80±9.21)%,(31.40±7.03)%,(17.50±4.29)%,(10.20±3.84)%,P<0.05]。③流式细胞术检测T细胞膜表面Fas、FasL:与对照组比较,各质量浓度催乳素干预组Fas和FasL细胞阳性率均下降[Fas:(88.0±17.3)%,(43.0±13.1)%,(22.0±6.7)%,(25.0±8.2)%,P<0.01;FasL:(46.0±17.5)%,(33.0±11.4)%,(31.0±14.0)%,(28.0±13.8)%,P<0.05]。④WesternBlot检测T细胞Bcl-2、Bax:与对照组比较,20μg/L催乳素组Bax和Bcl-2表达均无明显差异(P>0.05),300μg/L,1000μg/L催乳素组Bax表达明显降低(P<0.05),Bcl-2表达明显升高(P<0.05)。结论:在葡萄球菌肠毒素A诱导的T细胞活化诱导凋亡过程中,催乳素以细胞因子的身份,可通过抑制Fas、FasL和Bax表达,提高Bcl-2表达,来抑制T细胞凋亡,维持T细胞增殖。

AIM： To study the effects of prolactin as an important cytokine on T cell apoptosis in activation induced cell death （AICD）. METHODS： The experiment was carried out in the Immunology Laboratory of Xinxiang Medical College from March 2005 to June 2006. （1）Establishment of the T cell AICD model： 5 mL normal human peripheral blood mononuclear cells （PBMC） provided by the First Hospital affiliated to Xinxiang Medical College was collected, and equal volume lymphocyte isolation agent was added to isolate the lymphocytes by gradient centrifugation. Cells were resuspensed by RPMI1640 and staphylococcal enterotoxin A （SEA） was added, then the cells were cultured in 0.05 volume fraction CO2 at 37 ℃. After 4 days, the cells were collected by centrifugalization with 800 r/min for 10 minutes, and washed by fresh RPMI1640 to remove SEA. Then human interleukin-2 （IL-2） was added to the medium for another 2 weeks before use. （2） Intervention of prolactin： The cells were harvested by centrifugation, and resuspensed by fresh RPMI1640. After the cell density was modulated to 5×10^9 L^-1, the cells were seeded to 24-well plate. Prolactin of 20, 300, and 1 000 μg/L were applied to the AICD model after 4 mg/L SEA were added again to induce the apoptosis of T ceils. Cells cultured without prolactin served as control. （3）When the cells were cultured for another 0, 16 and 24 hours, the proliferation of T cells was detected by MTT, and the apoptotic rate of T cells was tested by flow cytometry and apoptotic DNA peak was also tested by agarose gel electrophoresis at 16 hours. Meanwhile, the level of Fas and FasL of T cells was detected by flow cytometry, and the expression of Bax and Bcl-2 of T cells was measured with Western Blot. RESULTS： （1）MTT detection showed that the proliferative ability of T cells treated with prolactin was significantly higher than that of control group at 16 and 24 hours [A value： control group： 0.65±0.02, 0.54±0.04; 20 μg/L prolactin group： 0.81±0.04, 0.71±0.11; 300 μg/L prolactin treated group： 1.14±0.10, 1.22±0.10; 1 000 μg/L prolactin group： 1.12±0.12, 1.22±0.12, P〈 0.01]. There was an increase of proliferation in T cells treated with 300 and 1 000 μg/L prolactin at 0 and 24 hours （P 〈 0.01）, and a decrease with 20 μg/L prolactin （P 〈 0.01）. （2）There was a decrease in apoptotic rate of T cells treated with prolactin compared with control group at 16 hours by flow cytometric analysis [（46.80±9.21）%, （31.40±7.03）%, （17.50±4.29）%, （10.20±3.84）%, P 〈 0.05]. （3）Positive cell rate of Fas and FasL in each prolactin group was decreased compared with control group [Fas： （88.0±17.3）%, （43.0±13.1）%, （22.0±6.7）%, （25.0±8.2）%, P 〈 0.01; FasL： （46.0±17.5）%, （33.0±11.4）%, （31.0±14.0）%, （28.0±13.8）%, P 〈 0.05]. （4）There was no significant difference in the expression of Bax and Bcl-2 between 20 μg/L prolactin group and control group with Western Blot method （P 〉 0.05）, but a remarkable decrease of the Bax expression and a marked increase of the Bcl-2 expression in 300 and 1 000 μg/L prolactin groups .compared with control group （P 〈 0.05）. CONCLUSION： In the process of T cells AICD induced by SEA, prolactin, as a cytokine, inhibits T cell apoptosis, and maintains the proliferative ability by inhibiting the expression of Fas, FasL and Bax, and promoting the expression of Bcl-2.