【摘要】：實驗?zāi)康?腎間質(zhì)纖維化是腎臟衰老和多種腎臟疾病進展到終末期腎臟病的病理學(xué)基礎(chǔ)。腎臟上皮-間充質(zhì)轉(zhuǎn)分化是腎間質(zhì)纖維化發(fā)生發(fā)展的重要環(huán)節(jié)。我們前期研究中發(fā)現(xiàn)青年、老年大鼠骨髓來源間充質(zhì)干細胞微泡中miR-133b存在差異表達,并抑制轉(zhuǎn)化生長因子β1( transforming growth factorβ1, TGF-β1)誘導(dǎo)的腎小管上皮細胞轉(zhuǎn)分化。本研究進一步探討miR-133b調(diào)控老年腎間質(zhì)纖維化的作用靶點及機制。實驗方法:采用RT-PCR檢測在不同濃度(0、6、8、10ng/ml)和不同時間(0、24、48、72h) TGF-β1誘導(dǎo)HK2細胞轉(zhuǎn)分化過程中miR-133b的表達變化;雙熒光素酶報告基因檢測miR-133b靶基因。體外實驗觀察轉(zhuǎn)染miR-133bHK2細胞,TGF-β 1刺激下miR-133b靶基因和E-cadherin、α-SMA、FN及Co13A1的mRNA和蛋白表達情況;上調(diào)HK2細胞miR-133b靶基因的表達,通過檢測上述指標進一步證實證實miR-133b抑制HK2細胞EMT的靶基因。構(gòu)建老年小鼠UUO模型,利用轉(zhuǎn)染試劑尾靜脈注射miR-133b,第7、14天檢測腎組織miR-133b和靶基因以及上述指標的蛋白表達和腎間質(zhì)纖維化。實驗結(jié)果:miR-133b的表達隨著TGF-β 1作用濃度的增加和干預(yù)時間的延長而逐漸降低,濃度為8ng/ml干預(yù)時間為48h時miR-133b的表達下調(diào)最明顯(p0.01)。轉(zhuǎn)染miR-133b mimic能下調(diào) CTGF的表達(p0.05),而轉(zhuǎn)染miRNAmimic control對CTGF的表達無影響,雙熒光素酶報告基因檢測證實CTGF為miR-133b的直接作用靶點。轉(zhuǎn)染miR-133b mimic能夠抑制TGF-β 1誘導(dǎo)HK2細胞上皮標記蛋白E-cadherin的下調(diào)(p0.05),以及a-SMA、FN和Co13A1的上調(diào)(p0.05)。過表達靶基因CTGF可以逆轉(zhuǎn)miR-133b對EMT的抑制作用。在注入miRNA-133b過表達轉(zhuǎn)染復(fù)合物后,小鼠腎臟miRNA-133b表達顯著升高(約70-100倍),病理染色、Westernblot和腎功能結(jié)果結(jié)果顯示,轉(zhuǎn)染miR-133b可明顯改善老年小鼠UUO模型的腎小管萎縮和間質(zhì)纖維化,延緩腎功能惡化進程。實驗結(jié)論:miR-133b可靶向抑制CTGF表達,抑制TGF-β1誘導(dǎo)HK2細胞上皮-間質(zhì)轉(zhuǎn)分化,減輕老年UUO小鼠的腎間質(zhì)纖維化,延緩腎功能惡化進程。
[Abstract]:Objective: renal interstitial fibrosis is the pathological basis of renal aging and progression of various renal diseases to end-stage renal disease. Renal epithelial-mesenchymal transdifferentiation is an important link in the occurrence and development of renal interstitial fibrosis. In our previous study, we found that there was a differential expression of miR-133b in bone marrow-derived mesenchymal stem cell microbubbles of young and aged rats, and inhibited the transdifferentiation of renal tubular epithelial cells induced by transforming growth factor 尾 1 (TGF- 尾 1). The aim and mechanism of miR-133b in regulating renal interstitial fibrosis in the elderly were investigated. Methods: RT-PCR was used to detect the expression of miR-133b during the process of HK2 cell transdifferentiation induced by TGF- 尾 1 at different concentrations (0 ~ 6 ~ 8ng / ml) and at different time (n = 72), and double luciferase reporter gene was used to detect miR-133b target gene. The expression of miR-133b target gene, E-cadherin, 偽 -SMA,FN and Co13A1 in miR-133bHK2 cells stimulated by TGF- 尾 1 was observed in vitro. The expression of miR-133b target gene in HK2 cells was upregulated. It was further confirmed that miR-133b inhibited the target gene of EMT in HK2 cells by detecting the above indexes. The UUO model of aged mice was established. The expression of miR-133b, target gene, protein expression and interstitial fibrosis in renal tissue were detected by tail vein injection of miR-133b, on the 14th day after transfection. The results showed that the expression of miR-133b decreased gradually with the increase of TGF- 尾 1 concentration and the prolongation of intervention time, and the down-regulation of miR-133b expression was the most obvious when the concentration of 8ng/ml was 48 h (p0.01). Transfection of miR-133b mimic could down-regulate the expression of CTGF (p0.05), but transfection of miRNAmimic control had no effect on the expression of CTGF. The detection of double luciferase reporter gene confirmed that CTGF was the direct target of miR-133b. Transfection of miR-133b mimic could inhibit the down-regulation of HK2 epithelial marker protein E-cadherin (p0.05) induced by TGF- 尾 1, and the up-regulation of a-SMAFN and Co13A1 (p0.05). Overexpression of target gene CTGF can reverse the inhibitory effect of miR-133b on EMT. After injection of miRNA-133b overexpression transfection complex, the expression of miRNA-133b in the kidney of mice was significantly increased (about 70-100 times). The results of pathological staining, Westernblot and renal function showed that, Transfection of miR-133b could significantly improve renal tubular atrophy and interstitial fibrosis of UUO model in aged mice, and delay the progression of renal function deterioration. Conclusion: miR-133b can inhibit the expression of CTGF, inhibit the epithelial-interstitial transdifferentiation of HK2 cells induced by TGF- 尾 1, alleviate the renal interstitial fibrosis in aged UUO mice, and delay the progression of renal function deterioration.
【學(xué)位授予單位】：中國人民解放軍醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R692

【相似文獻】

相關(guān)期刊論文 前10條

1 萬青松,夏明珠,胡家才;腎間質(zhì)纖維化的分子基礎(chǔ)[J];中國中西醫(yī)結(jié)合腎病雜志;2003年07期

2 姜Z腪，

本文編號：2308010