与肾小管上皮转分化相关的miRNA的初步筛选

发布时间：2018-03-30 12:30

本文选题：大鼠肾小管上皮细胞　切入点：肾小管上皮转分化　出处：《浙江医学》2014年17期

【摘要】：目的观察miRNA在大鼠肾小管上皮细胞(NRK-52E)发生上皮-肌成纤维细胞转分化(EMT)过程中的变化,筛选与肾小管上皮细胞转分化相关的miRNA。方法体外培养NRK-52E,用5 ng/ml转化生长因子-13 1(TGF-13 1)分别作用0、3、6、12、24、48 h,以0 h为空白组(BC组),余为TGF-β1组。倒置相差显微镜观察细胞形态学变化;real-time PCR法检测细胞内α-平滑肌肌动蛋白(α-SMA)、E钙蛋白(E-cad)、1型胶原(Col 1)和纤连蛋白(FN)的mRNA表达水平;Western blot法检测细胞内α-SMA的表达水平;ELISA法检测上清液中FN的含量;茎环实时荧光定量PCR法检测6条miRNA在细胞内的表达水平。结果与BC组比较,TGF-β1组部分细胞失去原有的鹅卵石形态,转变成成纤维细胞特有的梭形;细胞内α-SMA、Col I和FN的mRNA表达上调,E-cad的mRNA表达下调,细胞内α-SMA表达量增多,细胞上清液中FN含量升高(均P0.05);miR-30d、miR-132和miR-21的表达上调,miR-200b、miR-192和miR-10b的表达下调(均P0.05)。结论 TGF-β1可诱导NRK-52E发生EMT。miR-30d、miR-132、miR-21、miR-200b、miR-192和miR-10b可能参与大鼠肾小管上皮细胞EMT,值得进一步研究。
[Abstract]:Objective to observe the changes of miRNA in the process of epithelial-myofibroblast transdifferentiation in rat renal tubular epithelial cell line NRK-52. Methods NRK-52E was cultured in vitro and treated with 5 ng/ml transforming growth factor (TGF- 13 1(TGF-13 1) for 48 h, respectively. The cells were treated with 0 h as blank group and TGF- 尾 1 as control group. The morphology of cells was observed by inverted phase contrast microscope. The expression of 伪 -SMA in the supernatant was detected by real-time PCR assay. The expression of 伪 -SMA in the supernatant was detected by Elisa and the expression level of 伪 -SMA in the supernatant was detected by Elisa. The expression level of 伪 -SMA in the supernatant was determined by Elisa. The expression level of 伪 -SMA in the supernatant was determined by Elisa method and the expression level of 伪 -SMA in the supernatant was determined by Elisa. The expression level of 伪 -SMA in the supernatant was determined by Elisa. Results compared with BC group, some cells of TGF- 尾 1 group lost their cobblestone morphology and transformed into fusiform of fibroblast. The expression of 伪 -SMA-Col I and FN mRNA up-regulated the expression of E-cad mRNA and increased the expression of 伪 -SMA in cells. The content of FN in supernatant was increased (both P0.05 miR-30dnmiR-132 and miR-21 up-regulated the expression of miR-200bnmiR-192 and miR-10b (both P0.05A). Conclusion TGF- 尾 1 can induce NRK-52E to produce EMT.miR-132miR-132miR-2miR-200bmiR-192 and miR-10b may be involved in EMTs of rat renal tubular epithelial cells.
【作者单位】：温州医科大学附属第一医院肾内科;温州医科大学附属第二医院心血管内科;温州医科大学微生物免疫学教研室;
【基金】：温州市科技计划项目(Y20080116) 浙江省自然科学基金资助项目(Y12H050017) 浙江省医药卫生科技计划项目(No.2010KYA135)
【分类号】：R363

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