不同浓度雌二醇对去卵巢小鼠T淋巴细胞凋亡的影响及作用机制

目的 研究不同浓度雌二醇(E2)对去卵巢小鼠脾脏T淋巴细胞凋亡的影响并探讨其作用机制.方法 分离出小鼠脾脏T淋巴细胞,分为10组,分别为青年组、假手术组、去卵巢对照组、去卵巢+E2(10-11mol/L)组、去卵巢+E2(10-10mol/L)组、去卵巢+E2(10-8mol/L)组、去卵巢+E2(10-6mol/L)组、去卵巢+E2(10-10mol/L)+雌激素受体拮抗剂IC1182 780(10-7mol/L)组、去卵巢+E2(10-10mol/L)+1-甲基-4-苯基-四氢吡啶(MPP,10-6mol/L)组、去卵巢+E2(10-10mol/L)+四氢化吡咯二硫代氨基甲酸酯(PDTC,10-6mol/L)组.流式细胞仪检测各组细胞凋亡率,Westernblot方法检测各组细胞雌激素受体(ER)α、ERβ、Bax、Bcl-2蛋白表达及细胞核中P65蛋白水平.结果 去卵巢对照组细胞凋亡率为(19.4±2.5)%,高于青年组(14.6±2.4)%和假手术组(14.5±2.3)%,Bcl-2、核内P65蛋白水平分别为0.25±0.05、0.09±0.01,低于青年组(0.40±0.07、0.15±0.02)和假手术组(0.38±0.05、0.15±0.02),均P<0.01;去卵巢+E2(10-10mol/L)组凋亡率为(16.6±1.8)%,低于去卵巢对照组(P<0.05),Bel-2及核内P65蛋白水平高于去卵巢对照组(P<0.01),而去卵巢+E2(10-8mol/L,10-6mol/L)组凋亡率高于去卵巢对照组(分别为P<0.05、P<0.01).去卵巢对照组ERα、ERβ蛋白水平分别为0.23±0.01、0.22±0.03,低于青年组(0.27±0.02、0.29±0.04)和假手术组(0.28±0.03、0.29±0.02),去卵巢+E2(10-11mol/L、10-10mol/L、10-8mol/L)组ERα、ERβ蛋白水平均高于去卵巢对照组,而去卵巢+E2(10-mol/L)组(0.09±0.01、0.14±0.02)低于去卵巢对照组(均P<0.01).去卵巢+E2(10-10mol/L)+IC1182 780组及去卵巢+E2(10-10mol/L)+PDTC组凋亡率与去卵巢对照组差异无统计学意义,去卵巢+E2(10-10mol/L)+MPP组凋亡率与去卵巢+E2(10-10mol/L)组比较,差异无统计学意义.结论 去卵巢小鼠T淋巴细胞凋亡增多,E2对去卵巢小鼠T淋巴细胞的凋亡呈剂量依赖的双相调节作用,生理范围内可部分逆转去卵巢小鼠T淋巴细胞凋亡,而超过生理范围则增加其凋亡.生理剂量E2可能通过ERβ及核因子-kB途径抑制去卵巢小鼠的T淋巴细胞凋亡.

Effects of different doses of estradiol on apoptosis of T lymphocytes in ovariectomized mice and their possible mechanisms

Objective To investigate the effects of different doses of estradiol on apoptosis of T lymphocytes from spleens in ovariectomized mice and explore the possible mechanisms.Methods The mice splenic T lymphocytes were isolated and divided into ten groups: young group, sham- ovariectomized group, ovariectomized group, ovariectomized plus estradiol(10-11, 10-10, 10-8 and 106groups, ovariectomized plus estradiol (10-10mol/L) plus 1CI182 780 (10-7tool/L) group, ovariectomized plus estradiol(10-10mol/L) plus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPP, 10-6mol/L) group, ovariectomized plus estradiol(10-10mol/L) plus pyrroline dithiocarbamate(PDTC, 10-6mol/L) group. The apoptosis rates were determined by flow eytometry using Annexin V-FITC/ PI and the protein levels of ERa, ERβ, Bax, Bcl-2 and P65 were detected by Western blot. Results The apoptosis rate of ovariectomized group was(19. 4±2.5)%, which was higher than that of young group [(14.6±2.4%) 3 and sham-ovariectomized group [p (14.5±2.3)%], and the levels of Bcl-2 and nuclear P65 were lower than the young group [(0. 25±0. 05, 0. 09±0. 01) vs. (0. 40± 0.07,0. 15±0. 02), P<0.01]. The ovariectomized plus estradiol (10-10tool/L) group had lower apoptosis rate and higher Bcl-2 and P65 levels compared to the ovariectornized group[(16.6±1.8)% vs.(19.4±2.5)%,P<0.05;0.36±0.03 vs. 0.25～0.05, 0.14±0.01 vs. 0.09±0.01, P< 0. 01)], while the ovariectomized plus estradiol(10-8mol/L, 10-6tool/L) groups had higher apoptosis rates than the ovariectomized group[(22. 55±2. 5)% vs. (19. 4±2. 5)% ,P<0. 05;(27.8±3.1)% vs. (19.4 4±2. 5)%,P<0. 01, respectively]. The 2protein levels of ERa and ERβ of ovariectomized group were 0. 23±k0.01 and 0. 22±0. 03, respectively, which were lower than those of young((0. 27±0. 02) and (0. 29±0.04)] and sham-ovariectomized group [(0. 28±0. 03) and (0. 29±0.02)]. The ovariectomized plus estradiol(10-1110-1010-8tool/L) groups had higher while ovariectomized plus estradiol(10-6mol/L) group had lower ERα and ERβ protein levels (0. 09±0. 01,0. 14±0.02) than the ovariectomized group(P<0. 01). There was no significant difference between ovariectomized plus estradiol(10-10mol/L) plus ICI182 780 group or ovariectomized plus estradiol(10-10tool/L) plus PDTC group and ovariectomized group [(19.4±1.6)% vs. (19.4±2. 5)%, (21.0±2. 9)% vs. (19.4d±2. 5)%, P>0. 05). There were also no significant difference between ovariectomized plus estradiol(10-10mol/L) plus MPP group and ovariectomized plus estradiol (10-10mol/L) grou p[(16.9±2.2)% vs. (16.6±1.8)%, P>0.05]. Conclusions The ovariectomy of mice leads to increased apoptosis rates of splenic T lymphocytes. The effects of estradiol on the apoptosis of T lymphocytes in ovariectomized mice are dependent on doses: physiological dose of estradiol inhibits while higher dose of estradiol exacerbats the apoptosis of T lymphocytes in ovariectomized mice.Physiological dose of estradiol may act on Rice T lymphocytes via ERβ and NFkB signaling.