体外诱导人肾间质成纤维细胞成骨分化  

Induction of osteogenic differentiation of human renal fibroblasts in vitro

作　　者：严一杰 黎承杨[1] 邓耀良[1] 曾国华[2] 陶芝伟[1] 刘云龙[1] 孙春[1] 王扬[1] 

机构地区：[1]广西医科大学第一附属医院泌尿外科,广西南宁530021 [2]广州医科大学第一附属医院泌尿外科,广东广州510120

出　　处：《中国病理生理杂志》2015年第12期2259-2264,共6页Chinese Journal of Pathophysiology

基　　金：广西自然科学基金资助项目(No.2011GXNSFA018177);广东省泌尿外科重点实验室(No.2010A060801016)资助

摘　　要：目的：观察在体外培养条件下,使用成骨诱导培养液或不同浓度钙离子诱导人肾间质成纤维细胞发生成骨分化的效果,初步探讨肾脏Randall斑形成可能的细胞机制。方法：体外培养人肾间质成纤维细胞,实验分为5组：成骨诱导组（加成骨诱导液）、CaⅠ组（加0.5 mmol/L Ca2＋液）、CaⅡ组（加1.5 mmol/L Ca2＋液）、CaⅢ组（加2.5 mmol/L Ca2＋液）和对照组（加PBS）。各组细胞分别培养至第1、3、6、9天时,采用MTT法检测细胞活力;诱导至第9天时,用细胞钙茜素红染色液和钙钴法磷酸酶染色液对各组细胞进行染色,观察钙结节形成和碱性磷酸酶的表达情况。另外,分别采用real-time PCR和Western blot检测各组细胞不同时间点Runt相关转录因子2（Runx2）的mRNA和蛋白表达水平。结果：在1.5 mmol/L和2.5 mmol/L Ca2＋浓度条件下细胞的活力明显受抑制。细胞染色结果证实成骨诱导液组和钙离子组均可以见到典型的红色钙结节和黑色块状的硫化钴沉淀物。成骨诱导组细胞Runx2的mRNA和蛋白相对表达量（0~9 d）逐渐升高（P〈0.05）;诱导至第9天时,3个钙离子组细胞Runx2的mRNA和蛋白表达呈浓度依赖性升高（P〈0.05）。结论：在体外培养环境下,人肾成纤维细胞可以在成骨诱导培养液的刺激作用下发生成骨分化;另外,在高钙离子环境,人肾成纤维细胞也发生了类似的成骨分化。肾乳头组织中的成纤维细胞在高钙离子等因素的作用下发生成骨分化,这可能是肾脏Randall斑形成的细胞学基础。AIM： To investigate the effects of osteogenic induction media and the medias containing different concentration of calcium on the induction of osteogenic differentiation of human renal fibroblasts in vitro. METHODS： Cultured human renal fibroblasts were divided into 5 groups in this experiment： osteogenic induction group（ osteogenic induction media）,CaⅠgroup（ 0. 5 mmol / L Ca2 ＋media）,CaⅡgroup（ 1. 5 mmol / L Ca2 ＋media）,Ca Ⅲ group（ 2. 5 mmol / L Ca2 ＋media） and control group（ PBS）. The cell activity in each groups was measured by MTT assay. At 9th day,the cell calcium Alizarin red S staining and alkaline phosphatase（ ALP） Gomori calcium cobalt staining were performed respectively to observe the formation of calcium nidus and the expression of ALP. In addition,the expression of Runt-related transcription factor 2（ Runx2） at mRNA and protein levels was determined by real-time PCR and Western blot,respectively. RESULTS： The remarkable positive signs which represented the formation of calcium nidus and the deposit of calcium objects in all experiment groups were observed. The mRNA and protein expression of Runx2 in osteogenic induction group increased in accordance with the induction time. Compared with control group,the mRNA and protein expression of Runx2 in the CaⅠ ~ Ⅲ groups increased gradually in a calcium concentration dependent manner at the 9th induction day. CONCLUSION： Human renal interstitial fibroblasts show the potential activity in osteogenic differentiation induced by osteogenic induction media or high level calcium in vitro,which may be account for the cytological formation of the Randall＇s plaque in the kidney.

关 键 词：人肾成纤维细胞 成骨分化 肾乳头钙化斑 高钙尿症 肾结石 

分 类 号：R363[医药卫生—病理学]