SPF级6周龄Balb/c雄性小鼠60只,随机分为3组,每组20只,适应饲养1周后进行动物试验。雌激素+Gh—relin试验组:前2周隔日腹腔注射苯甲酸雌二醇注射液(含苯甲酸雌二醇0.1mg)0.05mL/只,后2周按每日腹腔注射Ghrelin(含Ghrelin60μg)0.1mL/只;雌激素+生理盐水对照组:前2周隔日腹腔注射苯甲酸雌二醇注射液(含苯甲酸雌二醇0.1mg)0.05mL/只,后2周按每日腹腔注射0.9%生理盐水0.1mI,/只;生理盐水对照组:前2周按0.05mL/只隔日腹腔注射0.9%生理盐水,后2周按0.1mL/只每日腹腔注射0.9%生理盐水。动物试验结束后,统计小鼠胸腺指数,应用光镜观察胸腺形态学变化,采用实时荧光定量PCR法检测胸腺中12种细胞因子mRNA表达量的变化。结果显示,注射Ghrelin后,雌二醇诱导的小鼠胸腺萎缩在形态学上基本恢复到正常水平,胸腺中白细胞介素1(IL-1)、IL-2、IL-4、IL-6、IL-12、干扰素(IFN-γ)、白血病抑制因子(LIF)、抑瘤素(OSM)、干细胞因子(SCF)、胸腺体液因子(THF)、肿瘤坏死因子(TNF-α)mRNA含量显著降低(P〈0.05),而IL-7mRNA含量稍微升高(P〉O.05)。结果表明,Ghrelin对雌激素诱导的小鼠胸腺萎缩具有逆转作用,其机制可能是:一方面通过抑制IL-6、OSM、LIF、SCF等细胞因子的表达,从而促进胸腺细胞的增殖;另-方面通过抑制IL-1、IL-2、IL-4、IL12、THF等细胞因子的表达,从而减少TNF—α和IFN-γ的分泌,进而抑制胸腺细胞的凋亡。
Sixty 6-week-old SPF Balb/c male mice were randomly divided into three groups of 20 each. After raised for one week, we started the animal tests. Estrogen + ghrelin experimental group,the mice were intraperitoneally injected with estradiol benzoate (0.05 mL/0.1 mg)every other day for two weeks,the late two weeks administrated with ghrelin(0. 1 mL/60 μg) in the same way; Estrogen+saline control group,the mice were injected as above,the late two weeks administrated with 0.9% physiological saline(0.1 mL per mouse) in the same way; Saline control group,the mice were intraperitoneally injected with 0. 9% physiological saline(0. 05 mL per mouse) every other day for two weeks, the late two weeks administrated with 0.9% physiological saline(0.1 mL per mouse) in the same way. In the end of the animal tests,we calculated the thymus index and observed the morphology of thymus by light microscope and detected the mRNA expression levels of 12 thymic cytokines by real-time PCR.The results showed that after administration with ghrelin, the estradiol-induced atrophic thymus almost returned to normal in morphology,and the expression levels of interleukin(IL)-1 ,IL-2,IL- 4, IL-6, IL-12, interferon( IFN)-γ, leukemia inhibitory factor ( LIF), oncostatin M ( OSM), stem cell factor(SCF), thymus humoral factor (THF), tumor necrosis factor (TNF)-α mRNA in thymus were significantly decreased(P 〈 0.05 ), while IL-7 slightly increased ( P 〈 0.05 ). The results re- vealed that there are two possible mechanisms to reverse estrogen-induced mouse thymus atrophy by ghrelin. On the one hand, the thymocyte proliferation was promoted by inhibiting the expression of IL-6,OSM,LIF,SCF etc. On the other hand,the expressions of TNF-α and IFN-γ were reduced by decreasing the production of IL-1, IL-2, IL-4, IL-12 etc, which inhibited thymocyte apoptosis.