过表达Klotho抑制RAW264.7细胞向破骨细胞分化

发布时间：2018-06-04 10:50

本文选题：Klotho + 破骨细胞　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:探讨转染Klotho基因(KL)的表达对可溶性核因子κB配体的受体活化因子(sRANKL)诱导的RAW264.7单核巨噬细胞向破骨细胞分化的影响。方法:实验设计分为高表达KL组(Ad-KL组)、空载体组(AdGFP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情况;实时荧光定量PCR(RT-PCR)和蛋白质印迹法(Western blot)检测KL在各组的表达情况;抗酒石酸酸性磷酸酶(TRAP)染色检测KL的表达对sRANKL诱导RAW264.7向破骨细胞分化的形态学影响;RT-PCR和蛋白质印迹法检测各组破骨细胞成熟标志物TRAP、组织蛋白酶K(CTSK)的相对表达情况。结果:Ad-KL成功转染RAW264.7细胞,与Ad-GFP组和空白对照组比较,Ad-KL组细胞KL mRNA表达水平显著升高且蛋白相对表达水平显著增加(P0.01);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表达降低且蛋白相对表达水平也明显降低(P0.01)。结论:KL的表达可以抑制s RANKL诱导RAW264.7向破骨细胞的分化过程。
[Abstract]:Aim: to investigate the effect of Klotho gene transfection on the differentiation of RAW264.7 mononuclear macrophages into osteoclasts induced by soluble nuclear factor 魏 B ligand receptor activating factor (sRANKL). Methods: the experimental design was divided into two groups: high expression KL group (Ad-KL group), empty vector group (AdGFP group) and blank control group (control group). The transfection of recombinant adenovirus into RAW264.7 was observed by inverted fluorescence microscope. The expression of KL in each group was detected by real-time quantitative PCR RT-PCR and Western blot. The expression of KL in osteoclasts induced by sRANKL was detected by tartrate-resistant acid phosphatase assay. RT-PCR and Western blotting were used to detect the relative expression of osteoclast maturation marker TRAPK and cathepsin CTSKK in osteoclasts. Results compared with Ad-GFP group and blank control group, the expression of KL mRNA in Ad-KL group was significantly higher than that in Ad-KL group, and the relative protein expression level was significantly increased in Ad-KL group. The results 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, and that in Ad-KL group was significantly higher than that in Ad-GFP group and blank control group. Compared with Ad-GFP group and blank control group, the expression of TRAP and CTSK mRNA in Ad-KL group was decreased and the relative expression level of protein was also significantly decreased in Ad-KL group. Conclusion the expression of KL can inhibit the differentiation of RAW264.7 into osteoclasts induced by s RANKL.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R580

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