C3aR激动剂对小鼠原代肾小管上皮细胞表型的影响

发布时间：2018-07-05 08:34

本文选题：肾小管上皮细胞 + 原代培养　；参考：《医学研究生学报》2017年01期

【摘要】：目的细胞转分化(EMT)过程中的关键事件包括上皮细胞蛋白(E-钙黏蛋白)的表达下调,细胞活动性增加,细胞基质生成的增多等。文中构建鼠原代肾小管上皮细胞培养及鉴定的方法,探究C3aR活化是否能够引起小管上皮细胞发生上皮-间质细胞转分化。方法用鼠肾小管节段进行肾小管上皮细胞原代培养,以免疫细胞化学及免疫荧光染色对上皮细胞进行鉴定。实验分为正常对照组,C3aR激动剂设5个浓度组:0.1、1、100、500及2000 ng/m L组,另设3个时间组:6、12及24 h组。利用Real-time PCR检测肾小管上皮细胞中E-钙黏蛋白、α-SMA及collagen I的表达水平变化;利用Western blot检测E-钙黏蛋白、α-SMA蛋白表达水平;用鬼笔环肽对上皮细胞行骨架染色,观察细胞骨架形态变化。结果与正常对照组比较,C3aR激动剂组(500 ng/m L、刺激48 h)抑制E-钙黏蛋白表达[(0.950±0.901)vs(0.650±0.221),P0.05],上调α-SMA[(1.380±0.062)vs(1.600±0.103),P0.05]和collagen I表达(P0.05),且各自表达趋势与C3aR激动剂呈时间和剂量依赖关系。细胞骨架染色结果显示,随着C3aR激动剂刺激时间的延长,上皮细胞内由激动蛋白构成的应力纤维生成逐渐增多。结论成功构建了小鼠肾小管上皮细胞原代培养及鉴定的方法;C3aR活化能够促进肾小管上皮细胞发生上皮-间质细胞转分化,并在肾纤维化的发生发展中起一定作用,预示C3aR可能成为肾疾病治疗的新靶点。
[Abstract]:Objective the key events in the process of cell transdifferentiation (EMT) include down-regulation of epithelial cell protein (E-cadherin), increase of cell activity and increase of cell matrix production. In order to investigate whether C3aR activation can induce epithelial-interstitial cell transdifferentiation in primary rat renal tubular epithelial cells culture and identification methods were constructed in this paper. Methods the primary culture of rat renal tubular epithelial cells was carried out. The epithelial cells were identified by immunocytochemistry and immunofluorescence staining. The experiment was divided into normal control group, C3aR agonist group and control group. The rats were divided into 5 groups: 0. 01 ng/m / L and 2000 ng/m / L, respectively. Real-time PCR was used to detect the expression of E-cadherin, 伪 -SMA and collagen I in renal tubular epithelial cells, Western blot was used to detect the expression of Ecadherin and 伪 -SMA, and cytoskeleton staining was performed to observe the morphological changes of the cytoskeleton. Results compared with the control group, the C3aR agonist group (500 ng/m / L, stimulated for 48 h) inhibited the expression of E-cadherin [(0.950 卤0.901) vs (卤0.221) P 0.05], up-regulated 伪 -SMA [(1.380 卤0.062) vs (1.600 卤0.103) P 0.05] and collagen I (P0.05), and showed a time-and dose-dependent relationship between the expression of 伪 -SMA and C3aR agonist. Cytoskeleton staining showed that with the prolongation of the stimulation time of C3aR agonist, the formation of stress fibers composed of excitins in epithelial cells increased gradually. Conclusion the method of primary culture and identification of mouse renal tubular epithelial cells was successfully constructed. The activation of C3aR can promote the epithelial-interstitial cell transdifferentiation of renal tubular epithelial cells and play a role in the development of renal fibrosis. It suggests that C 3 ar may be a new target for the treatment of renal diseases.
【作者单位】：南方医科大学金陵医院(南京军区南京总医院)国家肾脏疾病临床医学研究中心全军肾脏病研究所;
【基金】：国家自然科学基金(81370828)
【分类号】：R692

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