miR-146b及miR-503在博来霉素诱导的小鼠肺纤维化中的表达及作用初步研究

发布时间：2018-03-16 09:23

本文选题：间质性肺病　切入点：系统性硬化症　出处：《中南大学》2014年博士论文　论文类型：学位论文

【摘要】：目的：探讨microRNA-146b (miR-146b)及microRNA-503(miR-503)在博来霉素所致肺纤维化(bleomycin-induced pulmonary fibrosis)小鼠肺组织中的表达及其可能的作用机制。方法：①将50只C57BL/6小鼠随机分为三组：对照组、博来霉素肺纤维化组(模型组)、硼替佐米干预组(干预组)；对照组予以气管内灌注生理盐水,造模后1周予以腹腔注射生理盐水(0.1ml)；模型组则气管内滴注博来霉素溶液(3.5mg/kg),造模后1周予以腹腔注射生理盐水(0.1ml),干预组给予气管内滴注博来霉素溶液(方法剂量同模型组),造模后1周予以腹腔注射硼替佐米溶液(120ug/kg,0.1ml);分别于造模后第3周、第4周处死小鼠。②采用肺组织苏木精—伊红染色法(hematoxylin-eosin staining,HE染色)、masson三色染色法及纤维化相关基因检测等方法验证纤维化模型是否构建成功。③提取肺组织总核糖核酸(Ribonucleic Acid, RNA),检测各组小鼠肺组织中miR-146b及miR-503的表达。④利用生物信息学网站(TargetScan, PicTar, MiRanda)及生物医学检索网站PubMed预测miR-146b及miR-503纤维化相关靶基因。⑤利用实时荧光定量聚合酶链式反应(Real-time Polymerase Chain Reaction, RT-PCR)及蛋白质印记(Western-blot)分析所预测的靶基因的表达。结果：①成功构建博来霉素诱导的小鼠肺纤维化小鼠模型。②miR-146b在模型组小鼠中表达较对照组明显上调；miR-503在模型组小鼠中表达较对照组明显下调(P0.01)。③通过生物信息学预测miR-146b纤维化相关靶基因有irak、siah2、klf4、mmp16、notchl及fox03,其中在模型组小鼠中fox03,siah2表达下调(P0.01),与miR-146b表达趋势相反。④通过生物信息学预测miR-503纤维化相关靶基因Smad7,在模型组小鼠肺组织中mRNA水平表达无明显改变。结论：①在博来霉素诱导肺纤维化小鼠肺组织中miR-146b表达上调,miR-503表达下调；②在博来霉素诱导的肺纤维化小鼠肺组织中foxO3、siah2表达下调,而生物信息学预钡foxO3、siah2为miR-146b靶基因；③未发现硼替佐米(120ug/kg)能改善博来霉素诱导的小鼠肺纤维化。
[Abstract]:Aim: to investigate the expression and possible mechanism of microRNA-146b miR-146b and microRNA-503miR-503 in bleomycin-induced pulmonary fibrosis mice induced by bleomycin. Methods 50 C57BL / 6 mice were randomly divided into three groups: control group, bleomycin pulmonary fibrosis group (model group), bortezomib intervention group (intervention group) and control group were given intratracheal instillation of normal saline. In the model group, intratracheal infusion of bleomycin solution (3.5 mg / kg), intraperitoneal injection of normal saline (0.1 ml) one week after modeling, and intratracheal infusion of bleomycin solution (same dose) were given in the intervention group. The model group was treated with intraperitoneal injection of bortezomil solution (120ug- kg / kg) 0.1 ml / ml 1 week after the model was made, respectively at the third week after the model was made. At week 4, the mice were killed by hematoxylin-eosin staininging-HE staining and fibrosis-related gene detection to verify whether the fibrosis model was successfully constructed or not. 3. 3 was used to extract total ribonucleic acid from lung tissue. Detection of miR-146b and miR-503 expression in lung tissues of mice in each group using bioinformatics websites: Target Scan, PicTaran, MiRanda) and biomedical search site PubMed for predicting miR-146b and miR-503 Fibrosis-related target gene .5 using real-time fluorescence quantitative polymerase chain reaction. Real-time Polymerase Chain reaction (RT-PCR) and protein imprinting were used to analyze the expression of target gene. Results the mouse pulmonary fibrosis model induced by bleomycin was successfully constructed. 2miR-146b expression was significantly up-regulated in the model group compared with the control group. The target genes related to miR-146b fibrosis are irakassiah2lf4mmp16notchl and fox03. among them, the expression of fox03 siah2 is down-regulated in model group, contrary to the trend of miR-146b expression, the target gene Smad7 is predicted by bioinformatics, and the mRNA level in lung tissue of model group is predicted by bioinformatics. There was no obvious change in expression. Conclusion the expression of miR-146b in bleomycin-induced pulmonary fibrosis mice is up-regulated and down-regulated in bleomycin induced pulmonary fibrosis mice, and the expression of foxO3osiah2 is down-regulated in bleomycin induced pulmonary fibrosis mice. However, bioinformatics showed that barium foxo _ 3 siah2 was the target gene of miR-146b, but bortezomide 120ugr / kg could improve pulmonary fibrosis induced by bleomycin in mice.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R563

【相似文献】