miRNA-21在茵陈提取物干预大鼠糖尿病肾病中的作用及机制

发布时间：2018-06-07 01:05

本文选题：miRNA-21 + 糖尿病肾病　；参考：《吉林大学》2017年硕士论文

【摘要】：糖尿病肾病(Diabetic nephropathy,DN)是由糖尿病(Diabetes mellitus,DM)引起的一种较为常见的慢性并发症,是导致终末期肾病的主要危险因素之一。MicroRNAs(miRNAs)是一类大小约为20-25个核苷酸长度,具有调控功能的内源性非编码RNAs。目前关于miRNA在各种疾病中具体作用机制的研究,尤其是miRNA在肾脏相关疾病中的机制研究尚不完善。茵陈为菊科植物滨蒿或茵陈蒿的地上干燥部分,目前对于茵陈与DN的相关研究还很少,大多研究见于与茵陈相关的复方。本课题组前期实验证实茵陈提取物对DN大鼠肾脏具有一定的保护作用,并从细胞外基质降解的角度进行了机制方面的探讨。本研究在前期实验的基础上延长了HACE对肾脏的干预作用,检测了两个时间段(8周和16周)HACE的干预作用,并从miRNAs水平探讨茵陈提取物在干预大鼠糖尿病肾病中的可能机制,为茵陈防治DN提供新的理论基础与依据。本研究采用单次腹腔注射链脲佐菌素(Streptozotocin,STZ)(55mg/kg)复制DN大鼠模型,并灌胃给予茵陈提取物(HACE)(5g/kg体重)进行治疗,分别在给药8周及16周后检测大鼠血液及尿液中各项相关指标,观察给药后各组大鼠肾脏功能及肾脏组织形态学的改变。miRNA芯片检测各组大鼠肾脏mi RNAs表达谱,在差异表达高于1.74倍(log2|Fold change|0.8)的高丰度(信号800)miRNAs中筛选出7个高表达的miRNAs,运用Realtime PCR进行验证。结合芯片结果、Realtime PCR验证结果及相关文献,我们选择miRNA-21进行进一步功能研究,并利用mi RNAs靶基因预测分析数据库预测miRNA-21的靶基因,最后利用Western Blot技术及免疫组化技术检测靶基因在各组大鼠肾组织中的表达情况。结果显示,模型组多项肾功能指标出现明显变化、肾脏出现不同程度病变,在给予HACE治疗后部分指标明显减轻,病理变化也有不同程度的改善,且随着给药时长的增加,其改善作用也随之增强。比较分析正常组和模型组大鼠肾脏miRNAs差异表达谱,共筛选出35个差异表达的miRNAs;比较分析HACE组和模型组大鼠肾脏miRNA的差异表达谱,共筛选出17个差异表达的miRNAs。Realtime PCR验证7个筛选出的阳性miRNAs,发现给药8周后,与正常组比较,模型组mir-1306-3p、mir-672-5p及mir-3550表达明显下调(Ρ0.01或Ρ0.05);给予hace治疗后mir-672-5p明显上调(Ρ0.05)。给药16周后,与正常组比较,模型组mir-21-5p表达明显上调(Ρ0.01),mir-1306-3p、mir-672-5p及mir-466d表达明显下调(Ρ0.01或Ρ0.05);给予hace治疗后mir-21-5p、mir-1306-3p表达明显下调(Ρ0.05),mir-672-5p表达明显上调(Ρ0.05)。综合分析给药8周及16周后realtimepcr实验结果,造模后出现差异变化且给予hace后趋于正常的mirnas有mir-21-5p及mir-672-5p。mirna靶基因预测软件(targetscan、pictar、microrna.org)预测mirna-21的靶基因,发现smad7在三个数据库中均有出现,分析基因序列发现smad7基因有7个碱基位点与mirna-21互补配对。预测结合位点自由能分析软件rnahybrid验证发现mirna-21有较大的概率调控smad7的表达。利用kegg数据库对所有预测到的靶基因进行功能分析,发现可富集到tgf-β1/smad信号传导途径。westernblot技术及免疫组化技术对tgf-β1/smad通路蛋白(tgf-β1、smad2/3、smad7)进行检测,结果显示模型组中tgf-β1、smad2/3表达量明显上升(p0.05),给予hace治疗后其表达量明显下降(p0.05)。与正常组比较,smad7含量明显下降(p0.01),给予hace治疗后smad7表达量明显上升(p0.01)。根据研究结果得出结论:1.茵陈提取物可以降低糖尿病肾病大鼠血脂水平,增强肾功能,抑制其肾脏病理改变,进而减轻肾脏的损伤。2.糖尿病肾病大鼠中多个差异表达的mirna参与了dn的发展进程,其中mirna-21的表达变化较为明显。mirna-21可以通过调节smad7、tgf-β1、smad2/3蛋白的表达调控tgf-β1/smad通路,从而参与dn的发生发展。3.茵陈提取物对糖尿病肾病大鼠不同阶段多个mirnas表达均有影响,其中mirna-21的表达变化尤为明显。茵陈提取物同时可抑制大鼠肾脏tgf-β1、smad2/3的表达,增加smad7的表达,提示其对糖尿病肾病大鼠的肾脏保护作用可能与mirna-21介导的tgf-β1/smad通路有关。创新点:1.首次发现茵陈提取物可影响糖尿病肾病大鼠的肾脏mirnas表达谱。2.首次提出茵陈提取物干预大鼠糖尿病肾病可能与mirna-21介导的TGF-β1/Smad通路有关。
[Abstract]:Diabetic nephropathy (DN) is a common chronic complication caused by diabetes (Diabetes mellitus, DM). It is one of the major risk factors leading to end-stage renal disease,.MicroRNAs (miRNAs) is a class of 20-25 nucleotides in size. The endogenous non coded RNAs. with regulatory function is currently on miRNA. Research on specific mechanisms of action in all kinds of diseases, especially the mechanism of miRNA in kidney related diseases, is not perfect. The dry part of Artemisia Artemisia or Artemisia Artemisia is rare, and most of the studies have been found on the compound of DN related to Artemisia. A protective effect of the extract on the kidney of DN rats was carried out, and the mechanism was discussed from the angle of extracellular matrix degradation. This study extended the intervention effect of HACE on the kidney on the basis of earlier experiments, and detected the dry preconditioning of HACE in two time periods (8 and 16 weeks), and explored the extract of Artemisia from the level of miRNAs. The possible mechanism of interfering with diabetic nephropathy in rats was provided to provide a new theoretical basis and basis for the prevention and control of DN. In this study, a single intraperitoneal injection of Streptozotocin (STZ) (55mg/kg) was used to replicate the DN rat model, and the gastric perfusion (HACE) (5g/ kg weight) was given to the rats for 8 weeks and 16 weeks after administration. The changes of renal function and renal histomorphology in each group after administration were observed. The MI RNAs expression profiles of kidney in each group were detected by.MiRNA chip, and 7 high expression miRNAs were screened in the high abundance (log2|Fold change|0.8) high abundance (log2|Fold change|0.8) miRNAs, and Realtime PCR was used. On the basis of the results of the chip, the results of Realtime PCR verification and the related literature, we chose miRNA-21 for further functional study, and used the MI RNAs target gene to predict the target gene of miRNA-21, and finally used Western Blot technology and immunohistochemical technique to detect the expression of target genes in the kidney tissues of each group. The results showed that the renal function indexes of the model group changed obviously, the kidney appeared different degrees of pathological changes, some of the indexes were obviously reduced after the treatment of HACE, and the pathological changes were also improved in different degrees. With the increase of time, the improvement of the renal function was also enhanced. The difference of miRNAs difference between the normal group and the model group was compared and analyzed. A total of 35 differential expressions of miRNAs were screened, and the differential expression profiles of miRNA in the kidney of HACE and model rats were compared and analyzed, and 17 differentially expressed miRNAs.Realtime PCR were screened to verify 7 screened positive miRNAs. After 8 weeks, the expression of mir-1306-3p, mir-672-5p and mir-3550 in the model group was obviously lower than that of the normal group. The expression of mir-672-5p was significantly up (0.05) after the treatment of hace. After 16 weeks of administration, the expression of mir-21-5p in the model group was obviously up regulated (0.01), and the expression of mir-1306-3p, mir-672-5p and mir-466d was obviously down (0.01 or 0.05); mir-21-5p, mir-1306-3p expression (0.05), mir-672-5p table after HACE treatment. A comprehensive analysis of the results of the realtimepcr experiment after 8 and 16 weeks of administration, the difference in the realtimepcr test after the 8 and 16 weeks, and the mir-21-5p and mir-672-5p.mirna target gene prediction software (targetscan, pictar, microrna.org) for the prediction of the target gene of miRNA-21 after the modeling, and the discovery of Smad7 in three databases. At present, the analysis gene sequence found that the Smad7 gene has 7 base sites and miRNA-21 complementary pairs. The prediction of the binding site free energy analysis software rnahybrid verification found that miRNA-21 has a larger probability of regulating the expression of Smad7. Using the KEGG database, the function of all the predicted target genes is analyzed, and it is found that the tgf- beta 1/smad signal transduction can be enriched. The tgf- beta 1/smad pathway protein (tgf- beta 1, smad2/3, Smad7) was detected by means of.Westernblot and immunohistochemistry. The results showed that the expression of tgf- beta 1 and smad2/3 increased significantly (P0.05), and the expression of smad2/3 was significantly decreased (P0.05) after the treatment of hace. The expression of D7 was significantly increased (P0.01). According to the results of the study, the results of the study concluded that: 1. the extract of Herba capillaris can reduce the blood lipid level of diabetic nephropathy rats, strengthen the renal function, inhibit the renal pathological changes, and then reduce the renal injury of.2. diabetic nephropathy rats, the miRNA participates in the development of DN, in which the expression of miRNA-21 is expressed. .mirna-21 can regulate the tgf- beta 1/smad pathway by regulating the expression of Smad7, tgf- beta 1, smad2/3 protein, thus participating in the occurrence and development of DN, and the extract of.3. on the multiple miRNAs expressions in different stages of diabetic nephropathy rats, and the expression of miRNA-21 is particularly obvious. The extract of Artemisia capillaris can inhibit rats at the same time. The expression of tgf- beta 1 and smad2/3 in the kidney increases the expression of Smad7, suggesting that the renal protection of diabetic nephropathy rats may be related to the tgf- beta 1/smad pathway mediated by miRNA-21. Diabetic nephropathy may be related to miRNA-21 mediated TGF- beta 1/Smad pathway.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R285.5

【参考文献】