重组脂联素对自然流产模型小鼠妊娠结局及Th17/Treg平衡的影响

Effect of recombinant adiponectin on pregnancy outcome and Th17/Treg balance in spontaneous abortion mice model

目的分析重组脂联素对自然流产(SA)模型小鼠妊娠结局、辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的影响。方法选择SPF级CBA/J雌性小鼠与DAB/2、BALB/c雄性小鼠建立正常妊娠模型(CBA/J雌鼠与BALB/c雄鼠交配)与SA模型(CBA/J雌鼠与DBA/2雄鼠交配),SA模型小鼠分为模型组与重组脂联素组。重组脂联素组自妊娠第5天开始腹腔注射重组脂联素10μg(kg·d),连续9d;模型组同步注射等量生理盐水;正常妊娠组不作任何处理。妊娠2周后处死各组小鼠,计算胚胎吸收率。苏木精-伊红(HE)染色观察各组小鼠滋养细胞形态改变。酶联免疫吸附法(ELISA)测定各组小鼠血清脂联素、Th17相关细胞因子白细胞介素-17(IL-17)及Treg相关细胞因子白细胞介素-10(IL-10)、转化生长因子(TGF)-β1水平。取小鼠脾脏组织,流式细胞术检测Th17/Treg细胞平衡的变化。实时荧光定量聚合酶链反应法(RT-PCR)测定Th17细胞转录因子RORγt及Treg细胞转录因子FOXP3mRNA水平。结果与正常妊娠组比较,模型组小鼠胚胎吸收率上升(P<0.05);与模型组比较,重组脂联素组胚胎吸收率降低(P<0.05);正常妊娠组、重组脂联素组胚胎吸收率比较差异无统计学意义(P>0.05);与正常妊娠组比较,模型组、重组脂联素组血脂联素、IL-10、TGF-β1水平降低,IL-17水平及IL-17/TGF-β1值上升(P<0.05),脾脏淋巴细胞Th17细胞比率、Th17/Treg值、RORγtmRNA上升,Treg细胞比率、FOXP3mRNA降低(P<0.05);与模型组比较,重组脂联素组血清脂联素、IL-10、TGF-β水平上升,IL-17水平及IL-17/TGF-β1值降低(P<0.05),脾脏淋巴细胞Th17细胞比率、Th17/Treg值、RORγtmRNA降低,Treg细胞比率、FOXP3mRNA上升(P<0.05)。结论重组脂联素可能通过调节Th17/Treg免疫平衡改善SA模型小鼠妊娠结局,维持妊娠早期免疫耐受。

Objective To analyze the effect of recombinant adiponectin on pregnancy outcome and helper T cell 17(Th17)/regulatory T cell(Treg)balance in spontaneous abortion(SA)mice model.Methods SPF grade CBA/J female mice and DAB/2,BALB/c male mice were chosen to establish normal pregnancy models(CBA/J female mice mated with BALB/c male mice)and SA models(CBA/J female mice mated with DBA/2 male mice).SA model mice were divided into model group and recombinant adiponectin group.The recombinant adiponectin group were injected intraperitoneally with 10μg(kg·d)of recombinant adiponectin for 9 consecutive days since the 5th day of pregnancy.The model group were injected with the same amount of normal saline synchronously,and the normal pregnancy group were not given any treatment.The mice in each group were sacrificed after 2 weeks of gestation to calculate the embryo resorption rate.The hematoxylin-eosin(HE)staining method was used to observe morphological changes of trophoblast cells in each group.The enzyme linked immunosorbent assay(ELISA)was used to determine serum adiponectin,Th17-related cytokines interleukin-17(IL-17),Treg-related cytokines interleukin-10(IL-10)and transforming growth factor(TGF)-β1 levels.The spleen tissues were collected to detect changes in Th17/Treg balance by flow cytometry.The real-time fluorescence quantitative polymerase chain reaction(RT-PCR)was used to determine Th17 cell transcription factor RORγt and Treg cell transcription factor FOXP3 mRNA levels.Results The embryo resorption rate was higher in the model group than the normal pregnancy group(P<0.05),and lower in the recombinant adiponectin group than the model group(P<0.05).There was no statistically significant difference in embryo resorption rate between the normal pregnancy group and the recombinant adiponectin group(P>0.05).Compared with normal pregnancy group,blood adiponectin,IL-10 and TGF-β1 decreased while IL-17 and IL-17 TGF-β1 increased in model group and recombinant adiponectin group(P<0.05).The splenic lymphocyte Th17 cell ratio,Th17/Treg ratio and RORγt mRNA increased,while Treg cell ratio and FOXP3 mRNA decreased(P<0.05).Compared with the model group,serum adiponectin,IL-10 and TGF-β1 increased,while IL-17 and IL-17/TGF-β1 decreased in the recombinant adiponectin group(P<0.05).Splenic lymphocyte Th17 cell ratio,Th17/Treg ratio and RORγt mRNA decreased,while Treg cell ratio and FOXP3 mRNA increased(P<0.05).Conclusion Recombinant adiponectin may improve the pregnancy outcome of SA model mice and maintain the immune tolerance in early pregnancy by regulating Th17/Treg balance.