本文選題：miRNA-21 + 糖尿病腎病��； 參考：《吉林大學(xué)》2017年碩士論文

【摘要】：糖尿病腎病(Diabetic nephropathy,DN)是由糖尿病(Diabetes mellitus,DM)引起的一種較為常見(jiàn)的慢性并發(fā)癥,是導(dǎo)致終末期腎病的主要危險(xiǎn)因素之一。MicroRNAs(miRNAs)是一類(lèi)大小約為20-25個(gè)核苷酸長(zhǎng)度,具有調(diào)控功能的內(nèi)源性非編碼RNAs。目前關(guān)于miRNA在各種疾病中具體作用機(jī)制的研究,尤其是miRNA在腎臟相關(guān)疾病中的機(jī)制研究尚不完善。茵陳為菊科植物濱蒿或茵陳蒿的地上干燥部分,目前對(duì)于茵陳與DN的相關(guān)研究還很少,大多研究見(jiàn)于與茵陳相關(guān)的復(fù)方。本課題組前期實(shí)驗(yàn)證實(shí)茵陳提取物對(duì)DN大鼠腎臟具有一定的保護(hù)作用,并從細(xì)胞外基質(zhì)降解的角度進(jìn)行了機(jī)制方面的探討。本研究在前期實(shí)驗(yàn)的基礎(chǔ)上延長(zhǎng)了HACE對(duì)腎臟的干預(yù)作用,檢測(cè)了兩個(gè)時(shí)間段(8周和16周)HACE的干預(yù)作用,并從miRNAs水平探討茵陳提取物在干預(yù)大鼠糖尿病腎病中的可能機(jī)制,為茵陳防治DN提供新的理論基礎(chǔ)與依據(jù)。本研究采用單次腹腔注射鏈脲佐菌素(Streptozotocin,STZ)(55mg/kg)復(fù)制DN大鼠模型,并灌胃給予茵陳提取物(HACE)(5g/kg體重)進(jìn)行治療,分別在給藥8周及16周后檢測(cè)大鼠血液及尿液中各項(xiàng)相關(guān)指標(biāo),觀察給藥后各組大鼠腎臟功能及腎臟組織形態(tài)學(xué)的改變。miRNA芯片檢測(cè)各組大鼠腎臟mi RNAs表達(dá)譜,在差異表達(dá)高于1.74倍(log2|Fold change|0.8)的高豐度(信號(hào)800)miRNAs中篩選出7個(gè)高表達(dá)的miRNAs,運(yùn)用Realtime PCR進(jìn)行驗(yàn)證。結(jié)合芯片結(jié)果、Realtime PCR驗(yàn)證結(jié)果及相關(guān)文獻(xiàn),我們選擇miRNA-21進(jìn)行進(jìn)一步功能研究,并利用mi RNAs靶基因預(yù)測(cè)分析數(shù)據(jù)庫(kù)預(yù)測(cè)miRNA-21的靶基因,最后利用Western Blot技術(shù)及免疫組化技術(shù)檢測(cè)靶基因在各組大鼠腎組織中的表達(dá)情況。結(jié)果顯示,模型組多項(xiàng)腎功能指標(biāo)出現(xiàn)明顯變化、腎臟出現(xiàn)不同程度病變,在給予HACE治療后部分指標(biāo)明顯減輕,病理變化也有不同程度的改善,且隨著給藥時(shí)長(zhǎng)的增加,其改善作用也隨之增強(qiáng)。比較分析正常組和模型組大鼠腎臟miRNAs差異表達(dá)譜,共篩選出35個(gè)差異表達(dá)的miRNAs;比較分析HACE組和模型組大鼠腎臟miRNA的差異表達(dá)譜,共篩選出17個(gè)差異表達(dá)的miRNAs。Realtime PCR驗(yàn)證7個(gè)篩選出的陽(yáng)性miRNAs,發(fā)現(xiàn)給藥8周后,與正常組比較,模型組mir-1306-3p、mir-672-5p及mir-3550表達(dá)明顯下調(diào)(Ρ0.01或Ρ0.05);給予hace治療后mir-672-5p明顯上調(diào)(Ρ0.05)。給藥16周后,與正常組比較,模型組mir-21-5p表達(dá)明顯上調(diào)(Ρ0.01),mir-1306-3p、mir-672-5p及mir-466d表達(dá)明顯下調(diào)(Ρ0.01或Ρ0.05);給予hace治療后mir-21-5p、mir-1306-3p表達(dá)明顯下調(diào)(Ρ0.05),mir-672-5p表達(dá)明顯上調(diào)(Ρ0.05)。綜合分析給藥8周及16周后realtimepcr實(shí)驗(yàn)結(jié)果,造模后出現(xiàn)差異變化且給予hace后趨于正常的mirnas有mir-21-5p及mir-672-5p。mirna靶基因預(yù)測(cè)軟件(targetscan、pictar、microrna.org)預(yù)測(cè)mirna-21的靶基因,發(fā)現(xiàn)smad7在三個(gè)數(shù)據(jù)庫(kù)中均有出現(xiàn),分析基因序列發(fā)現(xiàn)smad7基因有7個(gè)堿基位點(diǎn)與mirna-21互補(bǔ)配對(duì)。預(yù)測(cè)結(jié)合位點(diǎn)自由能分析軟件rnahybrid驗(yàn)證發(fā)現(xiàn)mirna-21有較大的概率調(diào)控smad7的表達(dá)。利用kegg數(shù)據(jù)庫(kù)對(duì)所有預(yù)測(cè)到的靶基因進(jìn)行功能分析,發(fā)現(xiàn)可富集到tgf-β1/smad信號(hào)傳導(dǎo)途徑。westernblot技術(shù)及免疫組化技術(shù)對(duì)tgf-β1/smad通路蛋白(tgf-β1、smad2/3、smad7)進(jìn)行檢測(cè),結(jié)果顯示模型組中tgf-β1、smad2/3表達(dá)量明顯上升(p0.05),給予hace治療后其表達(dá)量明顯下降(p0.05)。與正常組比較,smad7含量明顯下降(p0.01),給予hace治療后smad7表達(dá)量明顯上升(p0.01)。根據(jù)研究結(jié)果得出結(jié)論:1.茵陳提取物可以降低糖尿病腎病大鼠血脂水平,增強(qiáng)腎功能,抑制其腎臟病理改變,進(jìn)而減輕腎臟的損傷。2.糖尿病腎病大鼠中多個(gè)差異表達(dá)的mirna參與了dn的發(fā)展進(jìn)程,其中mirna-21的表達(dá)變化較為明顯。mirna-21可以通過(guò)調(diào)節(jié)smad7、tgf-β1、smad2/3蛋白的表達(dá)調(diào)控tgf-β1/smad通路,從而參與dn的發(fā)生發(fā)展。3.茵陳提取物對(duì)糖尿病腎病大鼠不同階段多個(gè)mirnas表達(dá)均有影響,其中mirna-21的表達(dá)變化尤為明顯。茵陳提取物同時(shí)可抑制大鼠腎臟tgf-β1、smad2/3的表達(dá),增加smad7的表達(dá),提示其對(duì)糖尿病腎病大鼠的腎臟保護(hù)作用可能與mirna-21介導(dǎo)的tgf-β1/smad通路有關(guān)。創(chuàng)新點(diǎn):1.首次發(fā)現(xiàn)茵陳提取物可影響糖尿病腎病大鼠的腎臟mirnas表達(dá)譜。2.首次提出茵陳提取物干預(yù)大鼠糖尿病腎病可能與mirna-21介導(dǎo)的TGF-β1/Smad通路有關(guān)。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R285.5

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 彭睿;查何;周吉;彭惠民;張政;;糖尿病腎病循環(huán)microRNA差異表達(dá)譜的分析[J];西南大學(xué)學(xué)報(bào)(自然科學(xué)版);2014年11期

2 CHIN Francis Y.L.;LEUNG Henry C.M.;YIU S.M.;;Sequence assembly using next generation sequencing data —challenges and solutions[J];Science China(Life Sciences);2014年11期

3 朱柄銘;陳文t，

本文編號(hào)：1988964