PDF(Pubmed)
Abstract：
Previous studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration.
This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice.
Five-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.
In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.
MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.
This study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice.
Five-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.
In the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.
MiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.
摘要：
以前的研究报道，各种miRNA参与自身免疫性疾病，但miRNAs在自身免疫性甲状腺炎(AIT)中的潜在调控机制有待进一步探索。
本研究旨在进一步验证miR-326通过在NOD中使用尾静脉和甲状腺注射的慢病毒基因递送通过Ets-1调节Th17/Treg平衡来促进AIT。H-2h4小鼠。
五周大的NOD。将H-2h4小鼠随机分为尾静脉注射组和甲状腺注射组,每个接受mmu-miR-326海绵（LV-海绵）或慢病毒载体对照。将小鼠分为尾静脉注射：治疗性LV-ctrl，治疗性LV海绵，预防性LV-ctrl，和预防性LV海绵组。对照组饲喂高碘水，不静脉注射。通过甲状腺苏木素和伊红(HE)染色和ELISA检测甲状腺淋巴细胞浸润和血清TgAb值,分别。RT-PCR检测Ets-1和淋巴细胞计数,西方印迹,和流式细胞术。免疫荧光法检测甲状腺CD4+IL-17a+细胞和CD4+Ets-1+细胞,ELISA检测血清细胞因子。
在尾静脉注射组中，LV海绵组的甲状腺炎症评分和血清TgAb滴度明显低于对照组和LV-ctrl组，而LV海绵组小鼠脾脏中Ets-1蛋白表达增加。此外，通过流式细胞术测试，LV海绵组的Th17/Treg比率下降，而预防性LV海绵组的Th17/Treg比率显着下降（P=0.036）。免疫荧光显示,在LV海绵组中，CD4+IL-17a+细胞显著降低(P=0.001),而LV海绵组CD4+Ets-1+细胞显著增加(P=0.029)。LV海绵组血清IL-17/IL-10显著降低(P<0.05)。在甲状腺注射组中，与LV-ctrl组相比，LV-海绵组的甲状腺炎症评分和血清TgAb滴度显着降低（P<0.05）。此外,在LV海绵组中，CD4+IL-17a+细胞减少,而通过免疫荧光评估，抑制组中CD4Ets-1细胞显着增加。此外，与甲状腺注射相比，尾静脉注射LV海绵导致甲状腺炎中TgAb水平低得多。
MiR-326靶向治疗可能是AIT的一种有希望的方法。此外,尾静脉注射可能比甲状腺注射达到更好的干预效果。
本研究旨在进一步验证miR-326通过在NOD中使用尾静脉和甲状腺注射的慢病毒基因递送通过Ets-1调节Th17/Treg平衡来促进AIT。H-2h4小鼠。
五周大的NOD。将H-2h4小鼠随机分为尾静脉注射组和甲状腺注射组,每个接受mmu-miR-326海绵（LV-海绵）或慢病毒载体对照。将小鼠分为尾静脉注射：治疗性LV-ctrl，治疗性LV海绵，预防性LV-ctrl，和预防性LV海绵组。对照组饲喂高碘水，不静脉注射。通过甲状腺苏木素和伊红(HE)染色和ELISA检测甲状腺淋巴细胞浸润和血清TgAb值,分别。RT-PCR检测Ets-1和淋巴细胞计数,西方印迹,和流式细胞术。免疫荧光法检测甲状腺CD4+IL-17a+细胞和CD4+Ets-1+细胞,ELISA检测血清细胞因子。
在尾静脉注射组中，LV海绵组的甲状腺炎症评分和血清TgAb滴度明显低于对照组和LV-ctrl组，而LV海绵组小鼠脾脏中Ets-1蛋白表达增加。此外，通过流式细胞术测试，LV海绵组的Th17/Treg比率下降，而预防性LV海绵组的Th17/Treg比率显着下降（P=0.036）。免疫荧光显示,在LV海绵组中，CD4+IL-17a+细胞显著降低(P=0.001),而LV海绵组CD4+Ets-1+细胞显著增加(P=0.029)。LV海绵组血清IL-17/IL-10显著降低(P<0.05)。在甲状腺注射组中，与LV-ctrl组相比，LV-海绵组的甲状腺炎症评分和血清TgAb滴度显着降低（P<0.05）。此外,在LV海绵组中，CD4+IL-17a+细胞减少,而通过免疫荧光评估，抑制组中CD4Ets-1细胞显着增加。此外，与甲状腺注射相比，尾静脉注射LV海绵导致甲状腺炎中TgAb水平低得多。
MiR-326靶向治疗可能是AIT的一种有希望的方法。此外,尾静脉注射可能比甲状腺注射达到更好的干预效果。
