小鼠眼眶成纤维细胞TLR4基因静默对甲状腺眼病的效应研究

Inhibition effect of mouse orbital fibroblasts TLR4 gene silencing on the thyroid-associated ophthalmopathy

目的:构建小鼠眼眶成纤维细胞TLR4的shRNA慢病毒干扰载体,研究TL4-/-的成纤维细胞对甲状腺相关性眼病的治疗作用和机制。方法:设计、构建、筛选小鼠眼眶成纤维细胞TLR4基因的最优干扰shRNA表达质粒,选择Gateway方法将质粒导入慢病毒表达载体中,使用重组慢病毒载体感染小鼠眼眶成纤维细胞,研究其对免疫炎性反应的负向调控能力,并在小鼠甲状腺眼病模型中采用沉默成纤维细胞TLR4基因的方法,观察其体内治疗效果。结果:筛选出具有最好基因静默效果的shRNA序列,导入慢病毒载体,病毒滴度为1.5×106TU/m L。转染慢病毒的Balb/c小鼠眼眶成纤维细胞能够负向调控免疫应答,抑制免疫炎症反应。在疾病动物模型中转染了干扰病毒载体实验组小鼠,其眼病发生发展情况均优于对照组。结论:成功获得了小鼠成纤维细胞TLR4-/-shRNA的慢病毒载体,转染了该载体的小鼠眼眶成纤维细胞能够抑制正向免疫应答,可以有效地抑制甲状腺眼病的发展,揭示干扰TLR4表达可能成为防治甲状腺眼病的生物诊疗措施。

AIM：To construct shRNA lentivirus interference vector of mice orbital fibroblasts TLR4 and to research the therapeutic effect and mechanism of TL4^（-/-） fibroblasts on thyroid-associated ophthalmopathy.METHODS：Optimal shRNA interference expression plasmid of mouse orbital fibroblasts TLR4 gene was designed,built,and screened.Then the best shRNA was introduced into lentiviral expression vector by Gateway method and recombinant lentiviral vector was used to infect mouse orbital fibroblasts.Its capability of negative regulating the immune inflammatory response was researched.At last the method of fibroblast TLR4 gene silencing in the model mice of thyroid- associated ophthalmopathy was used,the in vivo therapeutic effect was observed.RESULTS：ShRNA sequences with the best effect of gene silencing were selected and introduced into lentiviral vectors（virus titer was 1.5×10~6TU/mL）.Balb/c mice orbital fibroblasts transfected lentivirus could negatively regulate the immune response,inhibit immune inflammatory response.The proceeding of thyroid- associated ophthalmopathyof the mice transfected TLR4^（-/-） recombinant lentivirus was obviously prior to that of the control mice..CONCLUSION：Mouse fibroblast TLR4^（-/-）siRNA lentiviral vectors are successfully obtained,which has the favourable inhibitory effect on immune inflammatory responses.The recombinant lentivirus could protect the proceeding of thyroid- associated ophthalmopathy,therefore the TLR4 expression interference is a novel potential target for thyroid-associated ophthalmopathy.