miR-133b调控CTGF表达抑制老年肾间质纤维化的研究
发布时间：2018-11-03 14:05
【摘要】：实验目的:肾间质纤维化是肾脏衰老和多种肾脏疾病进展到终末期肾脏病的病理学基础。肾脏上皮-间充质转分化是肾间质纤维化发生发展的重要环节。我们前期研究中发现青年、老年大鼠骨髓来源间充质干细胞微泡中miR-133b存在差异表达,并抑制转化生长因子β1( transforming growth factorβ1, TGF-β1)诱导的肾小管上皮细胞转分化。本研究进一步探讨miR-133b调控老年肾间质纤维化的作用靶点及机制。实验方法:采用RT-PCR检测在不同浓度(0、6、8、10ng/ml)和不同时间(0、24、48、72h) TGF-β1诱导HK2细胞转分化过程中miR-133b的表达变化;双荧光素酶报告基因检测miR-133b靶基因。体外实验观察转染miR-133bHK2细胞,TGF-β 1刺激下miR-133b靶基因和E-cadherin、α-SMA、FN及Co13A1的mRNA和蛋白表达情况;上调HK2细胞miR-133b靶基因的表达,通过检测上述指标进一步证实证实miR-133b抑制HK2细胞EMT的靶基因。构建老年小鼠UUO模型,利用转染试剂尾静脉注射miR-133b,第7、14天检测肾组织miR-133b和靶基因以及上述指标的蛋白表达和肾间质纤维化。实验结果:miR-133b的表达随着TGF-β 1作用浓度的增加和干预时间的延长而逐渐降低,浓度为8ng/ml干预时间为48h时miR-133b的表达下调最明显(p0.01)。转染miR-133b mimic能下调 CTGF的表达(p0.05),而转染miRNAmimic control对CTGF的表达无影响,双荧光素酶报告基因检测证实CTGF为miR-133b的直接作用靶点。转染miR-133b mimic能够抑制TGF-β 1诱导HK2细胞上皮标记蛋白E-cadherin的下调(p0.05),以及a-SMA、FN和Co13A1的上调(p0.05)。过表达靶基因CTGF可以逆转miR-133b对EMT的抑制作用。在注入miRNA-133b过表达转染复合物后,小鼠肾脏miRNA-133b表达显著升高(约70-100倍),病理染色、Westernblot和肾功能结果结果显示,转染miR-133b可明显改善老年小鼠UUO模型的肾小管萎缩和间质纤维化,延缓肾功能恶化进程。实验结论:miR-133b可靶向抑制CTGF表达,抑制TGF-β1诱导HK2细胞上皮-间质转分化,减轻老年UUO小鼠的肾间质纤维化,延缓肾功能恶化进程。
[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, 伪 -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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
[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, 伪 -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.
【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R692
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1 万青松,夏明珠,胡家才;肾间质纤维化的分子基础[J];中国中西医结合肾病杂志;2003年07期
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