摘要
目的通过RNA干扰技术沉默小鼠肺组织中Smad3基因,观察其对百草枯(PQ)所致肺间质纤维化是否具有保护作用。方法利用小鼠L929成纤维细胞,体外验证抑制Smad3基因表达的有效shRNA,并进一步将其合成进重组腺病毒。实验小鼠分为三组,在小鼠染毒后2h,气管内分别注入PBS、非靶向重组腺病毒和靶向重组腺病毒。不同时间点(0d,7d,28d)对每组6只小鼠进行处理,留取肺组织标本。提取肺组织中总RNA,Real time PCR检测肺组织中Smad3、I型前胶原mRNA表达,Western Blot检测肺组织细胞核中Smad3蛋白表达,羟脯氨酸检测试剂盒分析肺组织中羟脯氨酸的含量以及肺组织HE染色后观察组织病理学变化。结果同PBS组、非靶向重组腺病毒组相比,靶向重组腺病毒有效抑制了肺组织内Smad3 mRNA和蛋白表达。重组腺病毒介导的shRNA转染进一步抑制了肺组织中I型前胶原mRNA表达和羟脯氨酸的产生,减轻了PQ所致的肺间质纤维化的形成。结论Smad3对百草枯引起的肺间质纤维化有重要作用,RNA干扰抑制肺组织Smad3基因表达可能是一种有效的治疗该病的方法。
Objective To observe whether targeted silencing of Smad3 gene by RNA interference（RNAi） inhibits paraquat（PQ）-induced pulmonary fibrosis in mice. Methods A RNAi method using short hairpin RNAs（sh RNAs） targeting Smad3 was developed. The sh RNAs expression cassettes capable of effectively silencing Smad3 were validated in L929 mouse fibroblasts and transferred into adenovirus vector. Eighteen mice were divided averagely into PBS, non-target adenovirus and target adenovirus（intratracheally administered into mouse lung after 2h of PQ treatment） groups. The total RNA of lung tissues was extracted and the expressions of Smad3 and procollagen typeⅠm RNA were detected by real-time PCR. The expression of Smad3 protein in lung tissues was analyzed by Western blot, the hydroxyproline content in the lung was assayed by a commercially hydroxyproline detection kit. HE staining was used to observe the pathological change of lung. Results Compared with PBS group or non-target group, the recombinant adenovirus mediated sh RNA inhibited the expression of Smad3 m RNA and protein, and downregulated the expression level of procollagen type Ⅰ m RNA and hydroxyproline concentration in lung tissue, and greatly attenuated pulmonary fibrosis caused by PQ. Conclusion Smad3 is involved in the pathogenesis of the pulmonary fibrosis and the inhibition of expression of Smad3 by RNAi might be a feasible therapeutic approach.
出处
《解剖科学进展》
2015年第6期605-608,613,共5页
Progress of Anatomical Sciences
基金
辽宁省科学技术计划项目(2013225303)
