过表达Klotho抑制小鼠RAW264.7细胞向破骨细胞分化

发布时间：2018-02-24 17:04

本文关键词： Klotho 破骨细胞 RAW.细胞抗酒石酸酸性磷酸酶　出处：《细胞与分子免疫学杂志》2017年06期 　论文类型：期刊论文

【摘要】：目的探讨过表达Klotho(KL)对可溶性核因子κB受体激活蛋白配体(sRANKL)诱导小鼠RAW264.7细胞向破骨细胞分化的影响。方法实验分为过表达KL组(Ad-KL组)、空载体组(Ad-GFP组)与空白对照组。倒置荧光显微镜观察重组腺病毒转染RAW264.7细胞情况,实时荧光定量(qRT-PCR)和Western blot法检测KL的mRNA或蛋白水平;抗酒石酸酸性磷酸酶(TRAP)染色检测过表达KL对sRANKL诱导RAW264.7细胞向破骨细胞分化的形态学影响;qRT-PCR和Western blot法检测各组破骨细胞成熟标志物TRAP、组织蛋白酶K(CTSK)的mRNA或蛋白水平。结果与Ad-GFP组和空白对照组比较,Ad-KL组细胞KL mRNA和蛋白水平显著升高;TRAP染色显示,Ad-KL组TRAP阳性细胞数量显少于Ad-GFP组和空白对照组,体积也更小;与Ad-GFP组和空白对照组比较,Ad-KL组TRAP和CTSK mRNA和蛋白水平也明显降低。结论过表达KL抑制RAW264.7细胞向破骨细胞分化。
[Abstract]:Objective to investigate the effect of overexpression of Klothotrol (KL) on the differentiation of RAW264.7 cells into osteoclasts induced by soluble nuclear factor 魏 B receptor activator ligand (sRANKL). Methods the cells were divided into two groups: overexpression KL group (Ad-KL group), empty vector group (Ad-GFP group) and blank control group (control group). The transfection of recombinant adenovirus into RAW264.7 cells was observed by inverted fluorescence microscope. The mRNA or protein levels of KL were detected by real-time fluorescence quantitative analysis (qRT-PCR) and Western blot. Morphological effects of overexpression of KL on differentiation of RAW264.7 cells into osteoclasts induced by sRANKL by tartrate-resistant acid phosphatase assay and Western blot assay for detection of mRNA or protein levels of osteoclast maturation marker TRAPK. Results compared with Ad-GFP group and blank control group, the levels of KL mRNA and protein in Ad-KL group were significantly higher than those in Ad-KL group. The results of trap staining showed that the number of TRAP positive cells in Ad-KL group was significantly lower than that in Ad-GFP group and blank control group. Compared with Ad-GFP group and blank control group, the levels of TRAP and CTSK mRNA and protein in Ad-KL group were significantly decreased. Conclusion overexpression of KL inhibits the differentiation of RAW264.7 cells into osteoclasts.
【作者单位】：重庆医科大学附属第一医院老年病科;重庆市急救医疗中心老年科;
【基金】：国家自然科学基金(30672212) 重庆市医学科研计划项目(2013-2-029) 重庆市卫生计生委医学科研项目(2016MSXM080) 重庆市渝中区科技计划项目(20150139) 国家临床重点专科建设项目[国卫办医函[2013]544号]
【分类号】：R580

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